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Sample records for detect enteroaggregative escherichia

  1. Enteroaggregative Escherichia coli in Daycare

    DEFF Research Database (Denmark)

    Hebbelstrup Jensen, Betina; Stensvold, Christen R.; Struve, Carsten

    2016-01-01

    Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier...... and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0-6 years were followed in a cohort study, in the period between 2009...

  2. [Detection of virulence genes of the enteroaggregative pathotype in Escherichia coli strains isolated from groundwater sources in the province of Chaco, Argentina].

    Science.gov (United States)

    Lösch, Liliana S; Gariboglio Vázquez, María L; Rivas, Marta; Merino, Luis A

    2015-01-01

    Groundwater is an important source of drinking water for many communities in Northern Argentina; particularly, in the province of Chaco, where about 14% of households use this natural resource. Enteroaggregative Escherichia coli is an emerging pathogen whose global importance in public health has increased in recent years. Despite the significant risk of disease linked to contaminated water exposure, the prevalence of E. coli pathotypes in aquatic environments is still not so well defined. The aim of the present study was to detect the presence of typical enteroaggregative E. coli through the recognition of its virulence factors aap, AA probe and aggR by molecular techniques. A total of 93 water samples from different small communities of Chaco were analyzed. E. coli was identified in 36 (38.7%) of the tested samples. Six strains isolated from different samples harbored the studied genes. Of these 6 isolates, 3 carried the aap gene, 2 the AA probe and the last one the combination of aap/aggR genes. The prevalence of E. coli isolates harboring enteroaggregative virulence genes in groundwater sources was 6.4%. This work represents the first contribution to the study of the presence and distribution of virulence genes of EAEC in groundwater sources in this region of Argentina. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  3. Enteroaggregative Escherichia coli: An Emerging Enteric Food Borne Pathogen

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    P. Kaur

    2010-01-01

    Full Text Available Enteroaggregative Escherichia coli (EAEC are quite heterogeneous category of an emerging enteric pathogen associated with cases of acute or persistent diarrhea worldwide in children and adults, and over the past decade has received increasing attention as a cause of watery diarrhea, which is often persistent. EAEC infection is an important cause of diarrhea in outbreak and non-outbreak settings in developing and developed countries. Recently, EAEC has been implicated in the development of irritable bowel syndrome, but this remains to be confirmed. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence (AA to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa with a “stacked-brick” adherence phenotype, which is related to the presence of a 60 MDa plasmid (pAA. At the molecular level, strains demonstrating the aggregative phenotype are quite heterogeneous; several virulence factors are detected by polymerase chain reaction; however, none exhibited 100% specificity. Although several studies have identified specific virulence factor(s unique to EAEC, the mechanism by which EAEC exerts its pathogenesis is, thus, far unknown. The present review updates the current knowledge on the epidemiology, chronic complications, detection, virulence factors, and treatment of EAEC, an emerging enteric food borne pathogen.

  4. Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

    DEFF Research Database (Denmark)

    Jønsson, Rie; Struve, Carsten; Boisen, Nadia

    2015-01-01

    Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from....... Transformation to a nonadherent E. coli HB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found the agg5A gene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant....

  5. Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli.

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    Kong, Haishen; Hong, Xiaoping; Li, Xuefen

    2015-08-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that causes acute and persistent diarrhea in children and adults. While the pathogenic mechanisms of EAEC intestinal colonization have been uncovered (including bacterial adhesion, enterotoxin and cytotoxin secretion, and stimulation of mucosal inflammation), those of severe extraintestinal infections remain largely unknown. The recent emergence of multidrug resistant EAEC represents an alarming public health threat and clinical challenge, and research on the molecular mechanisms of resistance is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli

    DEFF Research Database (Denmark)

    Hebbelstrup Jensen, Betina; Olsen, Katharina E P; Struve, Carsten

    2014-01-01

    , reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children...... occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive......Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission...

  7. Genetic virulence profile of enteroaggregative Escherichia coli strains isolated from Danish children with either acute or persistent diarrhea

    DEFF Research Database (Denmark)

    Jensen, Betina Hebbelstrup; Poulsen, Anja; Rasmussen, Stig Hebbelstrup Rye

    2017-01-01

    Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however...

  8. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    BACKGROUND: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  9. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Minh, Van Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Background: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  10. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; van Minh, Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E.

  11. Intracellular Expression of the Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

    OpenAIRE

    Sui, Bao Quan; Dutta, Pinaki R.; Nataro, James P.

    2003-01-01

    The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a serine protease autotransporter that acts as an enterotoxin and cytotoxin. When applied to epithelial cells in culture, purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibers and electrophysiologic changes. Although it has been hypothesized that Pet has an intracellular site of action, evidence for this is...

  12. Distribution of serine protease autotransporters of Enterobacteriaceae in typical and atypical enteroaggregative Escherichia coli.

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    Andrade, Fernanda B; Abreu, Afonso G; Nunes, Kamila O; Gomes, Tânia A T; Piazza, Roxane M F; Elias, Waldir P

    2017-06-01

    Enteroaggregative Escherichia coli (EAEC) is an agent of acute and persistent diarrhea worldwide, categorized in typical or atypical subgroups. Some EAEC virulence factors are members of the serine protease autotransporters of Enterobacteriaceae (SPATE). The presence of SPATE-encoding genes of different E. coli pathotypes was searched in a large collection of EAEC strains, and a possible association between SPATEs and E. coli phylogroups was investigated. Among 108 typical and 85 atypical EAEC, pic was the most prevalent gene, detected in 47.1% of the strains, followed by sat (24.3%), espI (21.2%), pet (19.2%), sepA (13.5%), sigA (4.1%), eatA (4.1%), vat (1.0%), espP and tsh, detected in one strain (0.5%) each; while epeA and espC were not detected. Phylogenetic analysis demonstrated that 39.9% of the strains belonged to group A, 23.3% to B1, 10.9% to B2, 7.8% to D, 8.8% to E and 1.5% to F. The majority of the SPATE genes were distributed in typical and atypical strains without association with any phylogroup. In addition, pic and pet were strongly associated with typical EAEC and sepA was detected in close association with atypical EAEC. Our data indicate that SPATEs may represent important virulence traits in both subgroups of EAEC. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Molecular characterization and antibiotic resistance of enterotoxigenic and entero-aggregative Escherichia coli isolated from raw milk and unpasteurized cheeses

    Directory of Open Access Journals (Sweden)

    Mojtaba Bonyadian

    2014-04-01

    Full Text Available The aim of this study was to determine the occurrence of enterotoxigenic and enteroaggregative Escherichia coli strains and antibiotic resistance of the isolates in raw milk and unpasteurized cheese. Out of 200 samples of raw milk and 50 samples of unpasteurized cheeses, 96 and 24 strains of E. coli were isolated, respectively. Polymerase chain reaction (PCR was used to detect the genes encoding heat-stable enterotoxin a (STa, heat-stable enterotoxin b (STb, heat labile toxin (LT and enteroaggregative heat-stable toxin1 (EAST1. Twelve out of 120 (10.00% isolates harbored the gene for EAST1, 2(1.66% isolates were detected as producing STb and LT toxins and 12 (10.00% strains contained STb and EAST1 genes. None of the strains contain the STa gene. All of the strains were tested for antibiotic resistance by disk diffusion method. Disks included: ciprofloxacin (CFN, trimetoprim-sulfamethoxazole (TSX, oxytetracycline (OTC, gentamicin (GMN, cephalexin (CPN, nalidixic acid (NDA and nitrofurantoin (NFN, ampicillin (AMP, neomycin (NEO and streptomycin (STM. Among 120 isolated strains of E. coli, the resistance to each antibiotics were as follows: OTC100%, CPN 86.00%, NDA 56.00%, NFN 42.00%, GMN 30.00%, TSX 28.00%, CFN 20%, AM 23.40% and STM 4.25%. None of the isolates were resistant to NEO. The present data indicate that different resistant E. coli pathogens may be found in raw milk and unpasteurized cheese. It poses an infection risk for human and transferring the resistant factors to microflora of the consumers gut.

  14. Clonality, virulence and antimicrobial resistance of enteroaggregative Escherichia coli from Mirzapur, Bangladesh

    DEFF Research Database (Denmark)

    Chattaway, Marie Anne; Day, Michaela; Mtwale, Julia

    2017-01-01

    Purpose. This study investigates the virulence and antimicrobial resistance in association with common clonal complexes (CCs) of enteroaggregative Escherichia coli (EAEC) isolated from Bangladesh. The aim was to determine whether specific CCs were more likely to be associated with putative...... virulence genes and/or antimicrobial resistance.Methodology. The presence of 15 virulence genes (by PCR) and susceptibility to 18 antibiotics were determined for 151 EAEC isolated from cases and controls during an intestinal infectious disease study carried out between 2007-2011 in the rural setting...... between the presence of virulence or antimicrobial resistance genes in isolates of EAEC from cases versus controls. However, when stratified by clonal complex (CC) one CC associated with cases harboured more virulence factors (CC40) and one CC harboured more resistance genes (CC38) than the average...

  15. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam.

    Science.gov (United States)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-09-09

    Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in 'backyard' farms in south-east Asia. Faecal samples collected from 204 chicken farms; 204 farmers and 306 age- and gender-matched individuals not exposed to poultry farming were plated on MacConkey agar plates with and without antimicrobials being supplemented. Sweep samples obtained from MacConkey agar plates without supplemented antimicrobials were screened by multiplex PCR for the detection of the stx1, stx2 and aggR genes. One chicken farm sample each (0.5 %) contained the stx1 and the aggR gene. Eleven (2.4 %) human faecal samples contained the stx1 gene, 2 samples (0.4 %) contained stx2 gene, and 31 (6.8 %) contained the aggR gene. From 46 PCR-positive samples, 205 E. coli isolates were tested for the presence of stx1, stx2, aggR, wzx O104 and fliC H4 genes. None of the isolates simultaneously contained the four genetic markers associated with E. coli O104:H4 epidemic strain (aggR, stx2, wzx O104 and fliC H4 ). Of 34 EAEC, 64.7 % were resistant to 3(rd)-generation cephalosporins. These results indicate that in southern Vietnam, the human population is a more likely reservoir of aggR and stx gene carrying E. coli than the chicken population. However, conditions for transmission of isolates and/or genes between human and animal reservoirs resulting in the emergence of highly virulent E. coli strains are still favorable, given the nature of'backyard' farms in Vietnam.

  16. Susceptibility patterns of enteroaggregative Escherichia coli associated with traveller's diarrhoea: emergence of quinolone resistance.

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    Vila, J; Vargas, M; Ruiz, J; Espasa, M; Pujol, M; Corachán, M; Jiménez de Anta, M T; Gascón, J

    2001-11-01

    Enteroaggregative Escherichia coli (EAggEC) isolates were identified as a cause of traveller's diarrhoea in 50 (9%) of 517 patients and their antimicrobial susceptibility was determined. Molecular epidemiological characterisation and investigation of the mechanisms of acquisition of quinolone resistance among nalidixic acid-resistant EAggEC strains was performed. Seventeen (34%) of 50 patients needed antimicrobial therapy, because of persistence of symptoms in nine cases and the severity of symptoms in eight cases. Ampicillin and tetracycline resistance was high, whereas chloramphenicol and co-trimoxazole showed moderate activity and amoxicillin plus clavulanic acid, nalidixic acid and ciprofloxacin showed very good activity. Resistance to nalidixic acid was demonstrated in three isolates, two from patients who had travelled to India. In all three strains the resistance was linked to mutations in the gyrA gene alone or in both gyrA and parC genes. Although ciprofloxacin shows excellent in-vitro activity and could be useful in the treatment of traveller's diarrhoea in patients travelling abroad, it may not be useful in patients who have journeyed to India or to Mexico.

  17. Virulent Bacteriophages Can Target O104:H4 Enteroaggregative Escherichia coli in the Mouse Intestine

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    Maura, Damien; Galtier, Matthieu; Le Bouguénec, Chantal

    2012-01-01

    In vivo bacteriophage targeting of enteroaggregative Escherichia coli (EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophages in vivo for the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers. PMID:23006754

  18. Evaluation of Prevalence, Homology and Immunogenicity of Dispersin among Enteroaggregative Escherichia coli Isolates from Iran.

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    Asadi Karam, Mohammad Reza; Rezaei, Ali Akbar; Siadat, Seyed Davar; Habibi, Mehri; Bouzari, Saeid

    2017-01-01

    Diarrhea, caused by enteroaggregative Escherichia coli (EAEC), is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin (antiaggregation protein, aap) is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients. PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated. The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed. Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections.

  19. Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine.

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    Maura, Damien; Galtier, Matthieu; Le Bouguénec, Chantal; Debarbieux, Laurent

    2012-12-01

    In vivo bacteriophage targeting of enteroaggregative Escherichia coli (EAEC) was assessed using a mouse intestinal model of colonization with the O104:H4 55989Str strain and a cocktail of three virulent bacteriophages. The colonization model was shown to mimic asymptomatic intestinal carriage found in humans. The addition of the cocktail to drinking water for 24 h strongly decreased ileal and weakly decreased fecal 55989Str concentrations in a dose-dependent manner. These decreases in ileal and fecal bacterial concentrations were only transient, since 55989Str concentrations returned to their original levels 3 days later. These transient decreases were independent of the mouse microbiota, as similar results were obtained with axenic mice. We studied the infectivity of each bacteriophage in the ileal and fecal environments and found that 55989Str bacteria in the mouse ileum were permissive to all three bacteriophages, whereas those in the feces were permissive to only one bacteriophage. Our results provide the first demonstration that bacterial permissivity to infection with virulent bacteriophages is not uniform throughout the gut; this highlights the need for a detailed characterization of the interactions between bacteria and bacteriophages in vivo for the further development of phage therapy targeting intestinal pathogens found in the gut of asymptomatic human carriers.

  20. Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

    Science.gov (United States)

    Maura, Damien; Morello, Eric; du Merle, Laurence; Bomme, Perrine; Le Bouguénec, Chantal; Debarbieux, Laurent

    2012-08-01

    Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  1. Clonality, virulence and antimicrobial resistance of enteroaggregative Escherichia coli from Mirzapur, Bangladesh.

    Science.gov (United States)

    Chattaway, Marie Anne; Day, Michaela; Mtwale, Julia; White, Emma; Rogers, James; Day, Martin; Powell, David; Ahmad, Marwa; Harris, Ross; Talukder, Kaisar Ali; Wain, John; Jenkins, Claire; Cravioto, Alejandro

    2017-10-01

    This study investigates the virulence and antimicrobial resistance in association with common clonal complexes (CCs) of enteroaggregative Escherichia coli (EAEC) isolated from Bangladesh. The aim was to determine whether specific CCs were more likely to be associated with putative virulence genes and/or antimicrobial resistance. The presence of 15 virulence genes (by PCR) and susceptibility to 18 antibiotics were determined for 151 EAEC isolated from cases and controls during an intestinal infectious disease study carried out between 2007-2011 in the rural setting of Mirzapur, Bangladesh (Kotloff KL, Blackwelder WC, Nasrin D, Nataro JP, Farag TH et al.Clin Infect Dis 2012;55:S232-S245). These data were then analysed in the context of previously determined serotypes and clonal complexes defined by multi-locus sequence typing. Overall there was no association between the presence of virulence or antimicrobial resistance genes in isolates of EAEC from cases versus controls. However, when stratified by clonal complex (CC) one CC associated with cases harboured more virulence factors (CC40) and one CC harboured more resistance genes (CC38) than the average. There was no direct link between the virulence gene content and antibiotic resistance. Strains within a single CC had variable virulence and resistance gene content indicating independent and multiple gene acquisitions over time. In Bangladesh, there are multiple clonal complexes of EAEC harbouring a variety of virulence and resistance genes. The emergence of two of the most successful clones appeared to be linked to either increased virulence (CC40) or antimicrobial resistance (CC38), but increased resistance and virulence were not found in the same clonal complexes.

  2. IS3 profiling identifies the enterohaemorrhagic Escherichia coli O-island 62 in a distinct enteroaggregative E. coli lineage

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    Okeke Iruka N

    2011-03-01

    Full Text Available Abstract Background Enteroaggregative Escherichia coli (EAEC are important diarrhoeal pathogens that are defined by a HEp-2 adherence assay performed in specialist laboratories. Multilocus sequence typing (MLST has revealed that aggregative adherence is convergent, providing an explanation for why not all EAEC hybridize with the plasmid-derived probe for this category, designated CVD432. Some EAEC lineages are globally disseminated or more closely associated with disease. Results To identify genetic loci conserved within significant EAEC lineages, but absent from non-EAEC, IS3-based PCR profiles were generated for 22 well-characterised EAEC strains. Six bands that were conserved among, or missing from, specific EAEC lineages were cloned and sequenced. One band corresponded to the aggR gene, a plasmid-encoded regulator that has been used as a diagnostic target but predominantly detects EAEC bearing the plasmid already marked by CVD432. The sequence from a second band was homologous to an open-reading frame within the cryptic enterohaemorrhagic E. coli (EHEC O157 genomic island, designated O-island 62. Screening of an additional 46 EAEC strains revealed that the EHEC O-island 62 was only present in those EAEC strains belonging to the ECOR phylogenetic group D, largely comprised of sequence type (ST complexes 31, 38 and 394. Conclusions The EAEC 042 gene orf1600, which lies within the EAEC equivalent of O-island 62 island, can be used as a marker for EAEC strains belonging to the ECOR phylogenetic group D. The discovery of EHEC O-island 62 in EAEC validates the genetic profiling approach for identifying conserved loci among phylogenetically related strains.

  3. Genetic Virulence Profile of Enteroaggregative Escherichia coli Strains Isolated from Danish Children with Either Acute or Persistent Diarrhea

    DEFF Research Database (Denmark)

    Hebbelstrup Jensen, Betina; Poulsen, Anja; Hebbelstrup Rye Rasmussen, Stig

    2017-01-01

    Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however, their ro......Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however......, their roles in acute and persistent diarrhea have not been previously investigated. The aim of this study was to identify specific EAEC virulence genes associated with duration and type of diarrhea in Danish children. We aimed to improve the current diagnostics of EAEC and enable targeting of strains...... with an expected severe disease course. Questionnaires answered by parents provided information regarding duration of diarrhea and presence of blood or mucus. A total of 295 EAEC strains were collected from children with acute (≤7 days) and persistent diarrhea (≥14 days) and were compared by using multiplex PCR...

  4. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

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    The Shiga toxigenic Escherichia coli O104:H4 bares the characteristics of both enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga...

  5. Enteroaggregative Shiga toxin-producing Escherichia coli of serotype O104:H4 in Belgium and Luxembourg

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    K. De Rauw

    2014-09-01

    Full Text Available In 2011, a large outbreak of infections caused by Shiga toxin-producing Escherichia coli (STEC O104:H4 occurred in Germany. This exceptionally virulent strain combined virulence factors of enteroaggregative E. coli (EAggEC and STEC. After the outbreak only a few sporadic cases of infection with this rare serotype were reported, most of which were related to travel to the Middle East or North Africa. Here we describe two cases of enteroaggregative STEC (Agg-STEC O104:H4 infection that occurred in Belgium in 2012 and 2013 respectively. In both cases travel in a Mediterranean country preceded the infection. The first strain was isolated from the stool of a 42-year-old woman presenting bloody diarrhoea, who had travelled to Tunisia the week before. The second case involves a 14-year-old girl who, upon her return from Turkey to Belgium, suffered from an episode of bloody diarrhoea and haemolytic uraemic syndrome. Extended typing of the isolates with pulsed field gel electrophoresis revealed that the strains were closely related, though not exactly the same as the 2011 outbreak strain. This report supports the previously made hypothesis that Agg-STEC has a human reservoir and might be imported by travellers coming from an area where the pathogen is endemic. Furthermore, it emphasizes the concern that these bacteria may cause future outbreaks as evenly virulent O104:H4 isolates seem to be widespread.

  6. An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK.

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    Timothy J Dallman

    Full Text Available Following a large outbreak of foodborne gastrointestinal (GI disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75% of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.

  7. [Stx2a-producing enteroaggregative Escherichia coli O104:H4-ST678. Microbiological diagnostic already, for this and other STEC/VTEC serotypes!].

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    Blanco, Jorge

    2012-02-01

    A Stx2a-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, with virulence features common to the enteroaggregative E. coli pathotype, was reported as the cause of the recent 2011 outbreak in Germany. In addition, the German outbreak strain was found to possess several virulence factors of extra-intestinal pathogenic E. coli and to have acquired resistance to numerous antibiotics, including third-generation cephalosporins, owing to several plasmid-borne genes encoding TEM-1 and CTX-M-15 β-lactamases. There are only a few reports of serotype O104:H4, which is very rare in humans, and has never been detected in animals or food. Once the serotype of the German outbreak strain became known, specific molecular methods were developed for its detection based on conventional and real-time PCR. Data from Galicia suggest that, per year in Spain, STEC O157:H7 is responsible for more than 500 cases of infection, and non-O157 for more than 2,000. A microbiological diagnosis for O104:H4, O157:H7 and other STEC serotypes is required in Spanish hospitals. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  8. Characterization and biofilm forming ability of diarrhoeagenic enteroaggregative Escherichia coli isolates recovered from human infants and young animals.

    Science.gov (United States)

    Vijay, Deepthi; Dhaka, Pankaj; Vergis, Jess; Negi, Mamta; Mohan, Vysakh; Kumar, Manesh; Doijad, Swapnil; Poharkar, Krupali; Kumar, Ashok; Malik, Satyaveer Singh; Barbuddhe, Sukhadeo Baliram; Rawool, Deepak B

    2015-02-01

    Enteroaggregative Escherichia coli (EAEC) is an important pathotype that causes infection in humans and animals. EAEC isolates (n=86) recovered from diarrhoeal cases in human infants (37) and young animals (49) were characterized as 'typical' and/or 'atypical' EAEC strains employing PCR for virulence associated genes (cvd432, aaiA, astA, pilS, irp2, ecp, pic, aggR, aafA, aggA, and agg3A). Besides, biofilm formation ability of human and animal EAEC isolates was assessed using microtiter plate assay. In addition, the transcriptional profile of biofilm associated genes (fis and ecp) was also evaluated and correlated with biofilm formation assay for few selected EAEC isolates of human and animal origins. Overall, a diverse virulence gene profile was observed for the EAEC isolates of human and animal origins as none of the EAEC isolates revealed the presence of all the genes that were targeted. Nine 'typical' EAEC isolates were identified (6 from humans and 3 from animals) while, the majority of the isolates were 'atypical' EAEC strains. Isolation and identification of three 'typical' EAEC isolates from animals (canines) appears to be the first report globally. Further, based on the observations of the biofilm formation assay, the study suggested that human EAEC isolates in particular were comparatively more biofilm producers than that of the animal EAEC isolates. The fis gene was highly expressed in majority of 'typical' EAEC isolates and the ecp gene in 'atypical' EAEC isolates. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili

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    Araújo Ana CG

    2010-02-01

    Full Text Available Abstract Background Enteroaggregative Escherichia coli (EAEC are enteropathogenic strains identified by the aggregative adhesion (AA pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea. Results During an epidemiologic study focusing on infantile diarrhea, aggregative C. freundii (EACF and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated. Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P traA were capable of forming bacterial aggregates only in the presence of EACF. Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili. In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc. Conclusions Putative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.

  10. Diffuse and enteroaggregative patterns of adherence of Escherichia coli isolated from stools of children in northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Scaletsky Isabel Cristina Affonso

    2001-01-01

    Full Text Available Childhood diarrheal diseases remain highly endemic in northeastern Brazil. The attributable fraction of all diarrheal diseases among children less than 2 years of age due to Escherichia coli was examined in a 2-year prospective study in two large urban centers of Brazil. Between May 1997 and June 1999, fecal E. coli isolates from 237 children with diarrhea (217 acute and 20 persistent cases and 231 children without diarrhea (controls attending two hospitals in Northeast Brazil were tested for their pattern of adherence to HEp-2 cells and for colony hybridization with DNA probes specific for the six pathotypes of diarrheagenic E. coli. Enteroinvasive E. coli, enterotoxigenic E. coli and enterohemorrhagic E. coli were not isolated from any children. Diffusely adherent E. coli (DAEC and enteroaggregative E. coli (EAEC were the most frequent isolates with similar frequencies from children with or without diarrhea. Atypical EPEC (EAF-negative strains were isolated with similiar frequency from both cases (5.5% and controls (5.6%. Enteropathogenic E. coli (typical EPEC strains, characterized by localized adherence pattern of adherence, hybridization with the EAF probe, and belonging to the classical O serogroups, were significantly associated with diarrhea (P = 0.03. These E. coli strains associated with diarrhea accounted for 9% of all children with diarrhea. Collectively, in Northeast Brazil, E. coli strains comprise a small proportion of severe diarrhea prevalence in children.

  11. Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

    Science.gov (United States)

    Carvalho, Eunice B; Maga, Elizabeth A; Quetz, Josiane S; Lima, Ila F N; Magalhães, Hemerson Y F; Rodrigues, Felipe A R; Silva, Antônio V A; Prata, Mara M G; Cavalcante, Paloma A; Havt, Alexandre; Bertolini, Marcelo; Bertolini, Luciana R; Lima, Aldo A M

    2012-08-11

    Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

  12. Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli.

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    Badreddine Douzi

    Full Text Available The type VI secretion system (T6SS is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp and terminated by a trimeric valine-glycine repeat protein G (VgrG component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.

  13. Enteroaggregative Escherichia coli O78:H10, the cause of an outbreak of urinary tract infection

    DEFF Research Database (Denmark)

    Olesen, Bente; Scheutz, Flemming; Andersen, Rebecca L

    2012-01-01

    In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according...

  14. Genetic Virulence Profile of EnteroaggregativeEscherichia coliStrains Isolated from Danish Children with Either Acute or Persistent Diarrhea.

    Science.gov (United States)

    Hebbelstrup Jensen, Betina; Poulsen, Anja; Hebbelstrup Rye Rasmussen, Stig; Struve, Carsten; Engberg, Jørgen H; Friis-Møller, Alice; Boisen, Nadia; Jønsson, Rie; Petersen, Randi F; Petersen, Andreas M; Krogfelt, Karen A

    2017-01-01

    Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however, their roles in acute and persistent diarrhea have not been previously investigated. The aim of this study was to identify specific EAEC virulence genes associated with duration and type of diarrhea in Danish children. We aimed to improve the current diagnostics of EAEC and enable targeting of strains with an expected severe disease course. Questionnaires answered by parents provided information regarding duration of diarrhea and presence of blood or mucus. A total of 295 EAEC strains were collected from children with acute (≤7 days) and persistent diarrhea (≥14 days) and were compared by using multiplex PCR targeting the genes sat, sepA, pic, sigA, pet, astA, aatA, aggR, aaiC, aap, agg3/4C, ORF3, aafA, aggA, agg3A, agg4A , and agg5A . Furthermore, the distribution of EAEC genes in strains collected from cases of bloody, mucoid, and watery diarrhea was investigated. The classification and regression tree analysis (CART) was applied to investigate the relationship between EAEC virulence genes and diarrheal duration and type. Persistent diarrhea was associated with strains lacking the pic gene ( p = 0.002) and with the combination of the genes pic, sat , and absence of the aggA gene ( p = 0.05). Prolonged diarrhea was associated with the combination of the genes aatA and astA ( p = 0.03). Non-mucoid diarrhea was associated with strains lacking the aatA gene ( p = 0.004). Acute diarrhea was associated with the genes aggR, aap , and aggA by individual odds ratios. Resistance toward gentamicin and ciprofloxacin was observed in 7.5 and 3% of strains, respectively. Multi-drug resistance was observed in 38% of strains. Genetic host factors have been associated with an increased risk of

  15. Genetic Virulence Profile of Enteroaggregative Escherichia coli Strains Isolated from Danish Children with Either Acute or Persistent Diarrhea

    Directory of Open Access Journals (Sweden)

    Betina Hebbelstrup Jensen

    2017-05-01

    Full Text Available Enteroaggregative Escherichia coli (EAEC is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however, their roles in acute and persistent diarrhea have not been previously investigated. The aim of this study was to identify specific EAEC virulence genes associated with duration and type of diarrhea in Danish children. We aimed to improve the current diagnostics of EAEC and enable targeting of strains with an expected severe disease course. Questionnaires answered by parents provided information regarding duration of diarrhea and presence of blood or mucus. A total of 295 EAEC strains were collected from children with acute (≤7 days and persistent diarrhea (≥14 days and were compared by using multiplex PCR targeting the genes sat, sepA, pic, sigA, pet, astA, aatA, aggR, aaiC, aap, agg3/4C, ORF3, aafA, aggA, agg3A, agg4A, and agg5A. Furthermore, the distribution of EAEC genes in strains collected from cases of bloody, mucoid, and watery diarrhea was investigated. The classification and regression tree analysis (CART was applied to investigate the relationship between EAEC virulence genes and diarrheal duration and type. Persistent diarrhea was associated with strains lacking the pic gene (p = 0.002 and with the combination of the genes pic, sat, and absence of the aggA gene (p = 0.05. Prolonged diarrhea was associated with the combination of the genes aatA and astA (p = 0.03. Non-mucoid diarrhea was associated with strains lacking the aatA gene (p = 0.004. Acute diarrhea was associated with the genes aggR, aap, and aggA by individual odds ratios. Resistance toward gentamicin and ciprofloxacin was observed in 7.5 and 3% of strains, respectively. Multi-drug resistance was observed in 38% of strains. Genetic host factors have been associated with an increased risk

  16. Genetic Virulence Profile of Enteroaggregative Escherichia coli Strains Isolated from Danish Children with Either Acute or Persistent Diarrhea

    Science.gov (United States)

    Hebbelstrup Jensen, Betina; Poulsen, Anja; Hebbelstrup Rye Rasmussen, Stig; Struve, Carsten; Engberg, Jørgen H.; Friis-Møller, Alice; Boisen, Nadia; Jønsson, Rie; Petersen, Randi F.; Petersen, Andreas M.; Krogfelt, Karen A.

    2017-01-01

    Enteroaggregative Escherichia coli (EAEC) is frequently found in diarrheal stools worldwide. It has been associated with persistent diarrhea, weight loss, and failure to thrive in children living in developing countries. A number of important EAEC virulence genes are identified; however, their roles in acute and persistent diarrhea have not been previously investigated. The aim of this study was to identify specific EAEC virulence genes associated with duration and type of diarrhea in Danish children. We aimed to improve the current diagnostics of EAEC and enable targeting of strains with an expected severe disease course. Questionnaires answered by parents provided information regarding duration of diarrhea and presence of blood or mucus. A total of 295 EAEC strains were collected from children with acute (≤7 days) and persistent diarrhea (≥14 days) and were compared by using multiplex PCR targeting the genes sat, sepA, pic, sigA, pet, astA, aatA, aggR, aaiC, aap, agg3/4C, ORF3, aafA, aggA, agg3A, agg4A, and agg5A. Furthermore, the distribution of EAEC genes in strains collected from cases of bloody, mucoid, and watery diarrhea was investigated. The classification and regression tree analysis (CART) was applied to investigate the relationship between EAEC virulence genes and diarrheal duration and type. Persistent diarrhea was associated with strains lacking the pic gene (p = 0.002) and with the combination of the genes pic, sat, and absence of the aggA gene (p = 0.05). Prolonged diarrhea was associated with the combination of the genes aatA and astA (p = 0.03). Non-mucoid diarrhea was associated with strains lacking the aatA gene (p = 0.004). Acute diarrhea was associated with the genes aggR, aap, and aggA by individual odds ratios. Resistance toward gentamicin and ciprofloxacin was observed in 7.5 and 3% of strains, respectively. Multi-drug resistance was observed in 38% of strains. Genetic host factors have been associated with an increased risk of EAEC

  17. Comparative sequence analysis of enteroaggregative Escherichia coli heat-stable enterotoxin 1 identified in Korean and Japanese Escherichia coli strains.

    Science.gov (United States)

    Seo, Dong Joo; Choi, SunKeum; Jeon, Su Been; Jeong, Suntak; Park, Hyunkyung; Lee, Bog-Hieu; Kim, Geun-Bae; Yang, Soo-Jin; Nishikawa, Yoshikazu; Choi, Changsun

    2017-02-21

    The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Association of vitamin D status with incidence of enterotoxigenic, enteropathogenic and enteroaggregative Escherichia coli diarrhoea in children of urban Bangladesh.

    Science.gov (United States)

    Ahmed, A M S; Soares Magalhaes, R J; Long, K Z; Ahmed, T; Alam, Md A; Hossain, Md I; Islam, Md M; Mahfuz, M; Mondal, D; Haque, R; Mamun, A A

    2016-08-01

    To evaluate the association between vitamin D status and diarrhoeal episodes by enterotoxigenic (ETEC), enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli in underweight and normal-weight children aged 6-24 months in urban Bangladesh. Cohorts of 446 normal-weight and 466 underweight children were tested separately for ETEC, EPEC and EAEC from diarrhoeal stool samples collected during 5 months of follow-up while considering vitamin D status at enrolment as the exposure. Cox proportional hazards models with unordered failure events of the same type were used to determine diarrhoeal risk factors after adjusting for sociodemographic and concurrent micronutrient status. Vitamin D status was not independently associated with the risk of incidence of ETEC, EPEC and EAEC diarrhoea in underweight children, but moderate-to-severe retinol deficiency was associated with reduced risk for EPEC diarrhoea upon adjustment. Among normal-weight children, insufficient vitamin D status and moderate-to-severe retinol deficiency were independently associated with 44% and 38% reduced risk of incidence of EAEC diarrhoea, respectively. These children were at higher risk of ETEC diarrhoea with vitamin D deficiency status when adjusted for micronutrient status only. This study demonstrates for the first time that normal-weight children with insufficient vitamin D status have a reduced risk of EAEC diarrhoea than children with sufficient status. Moderate-to-severe deficiency of serum retinol is associated with reduced risk of EPEC and EAEC diarrhoea in underweight and normal-weight children. © 2016 John Wiley & Sons Ltd.

  19. The effect of sub-minimum inhibitory concentration of ciprofloxacin concentrations on enteroaggregative Escherichia coli and the role of the surface protein dispersin

    Energy Technology Data Exchange (ETDEWEB)

    Mortensen, Ninell P [ORNL; Fowlkes, Jason Davidson [ORNL; Trevino-Dopatka, Sonia [ORNL; Maggart, Michael J [ORNL; Boisen, Nadia [University of Virginia School of Medicine; Doktycz, Mitchel John [ORNL; Nataro, James [University of Virginia School of Medicine; Allison, David P [ORNL

    2011-01-01

    Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

  20. Effects of sub-minimum inhibitory concentrations of ciprofloxacin on enteroaggregative Escherichia coli and the role of the surface protein dispersin

    Energy Technology Data Exchange (ETDEWEB)

    Fowlkes, Jason Davidson [ORNL; Doktycz, Mitchel John [ORNL; Allison, David Post [ORNL

    2011-01-01

    Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

  1. Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

    Science.gov (United States)

    Bartelt, Luther A; Bolick, David T; Mayneris-Perxachs, Jordi; Kolling, Glynis L; Medlock, Gregory L; Zaenker, Edna I; Donowitz, Jeffery; Thomas-Beckett, Rose Viguna; Rogala, Allison; Carroll, Ian M; Singer, Steven M; Papin, Jason; Swann, Jonathan R; Guerrant, Richard L

    2017-07-01

    Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

  2. Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    García-Heredia, Alam; García, Santos; Merino-Mascorro, José Ángel; Feng, Peter; Heredia, Norma

    2016-10-01

    The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the EnteroaggregativeEscherichia coliStrain 042.

    Science.gov (United States)

    Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

    2018-01-01

    Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

  4. Bacterial-epithelial contact is a key determinant of host innate immune responses to enteropathogenic and enteroaggregative Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Lindsey A Edwards

    Full Text Available BACKGROUND: Enteropathogenic (EPEC and Enteroaggregative (EAEC E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains. METHODOLOGY: Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence. RESULTS: Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti-inflammatory response. CONCLUSION: Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbe's ability to down

  5. A role for Tn6029 in the evolution of the complex antibiotic resistance gene loci in genomic island 3 in enteroaggregative hemorrhagic Escherichia coli O104:H4.

    Directory of Open Access Journals (Sweden)

    Piklu Roy Chowdhury

    Full Text Available In enteroaggregative hemorrhagic Escherichia coli (EAHEC O104 the complex antibiotic resistance gene loci (CRL found in the region of divergence 1 (RD1 within E. coli genomic island 3 (GI3 contains blaTEM-1, strAB, sul2, tet(AA, and dfrA7 genes encoding resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim respectively. The precise arrangement of antibiotic resistance genes and the role of mobile elements that drove the evolutionary events and created the CRL have not been investigated. We used a combination of bioinformatics and iterative BLASTn searches to determine the micro-evolutionary events that likely led to the formation of the CRL in GI3 using the closed genome sequences of EAHEC O104:H4 strains 2011C-3493 and 2009EL-2050 and high quality draft genomes of EAHEC E. coli O104:H4 isolates from sporadic cases not associated with the initial outbreak. Our analyses indicate that the CRL in GI3 evolved from a progenitor structure that contained an In2-derived class 1 integron in a Tn21/Tn1721 hybrid backbone. Within the hybrid backbone, a Tn6029-family transposon, identified here as Tn6029C abuts the sul1 gene in the 3'-Conserved Segment (-CS of a class 1 integron generating a unique molecular signature that has only previously been observed in pASL01a, a small plasmid found in commensal E. coli in West Africa. From this common progenitor, independent IS26-mediated events created two novel transposons identified here as Tn6029D and Tn6222 in 2011C-3493 and 2009EL-2050 respectively. Analysis of RD1 within GI3 reveals IS26 has played a crucial role in the assembly of regions within the CRL.

  6. Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    Science.gov (United States)

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-04-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

  7. Escherichia coli O104:H4 Pathogenesis: an Enteroaggregative E. coli/Shiga Toxin-Producing E. coli Explosive Cocktail of High Virulence.

    Science.gov (United States)

    Navarro-Garcia, Fernando

    2014-12-01

    A major outbreak caused by Escherichia coli of serotype O104:H4 spread throughout Europe in 2011. This large outbreak was caused by an unusual strain that is most similar to enteroaggregative E. coli (EAEC) of serotype O104:H4. A significant difference, however, is the presence of a prophage encoding the Shiga toxin, which is characteristic of enterohemorrhagic E. coli (EHEC) strains. This combination of genomic features, associating characteristics from both EAEC and EHEC, represents a new pathotype. The 2011 E. coli O104:H4 outbreak of hemorrhagic diarrhea in Germany is an example of the explosive cocktail of high virulence and resistance that can emerge in this species. A total of 46 deaths, 782 cases of hemolytic-uremic syndrome, and 3,128 cases of acute gastroenteritis were attributed to this new clone of EAEC/EHEC. In addition, recent identification in France of similar O104:H4 clones exhibiting the same virulence factors suggests that the EHEC O104:H4 pathogen has become endemically established in Europe after the end of the outbreak. EAEC strains of serotype O104:H4 contain a large set of virulence-associated genes regulated by the AggR transcription factor. They include, among other factors, the pAA plasmid genes encoding the aggregative adherence fimbriae, which anchor the bacterium to the intestinal mucosa (stacked-brick adherence pattern on epithelial cells). Furthermore, sequencing studies showed that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga toxin-producing EAEC O104:H4 strain that caused the German outbreak. This article discusses the role these virulence factors could have in EAEC/EHEC O104:H4 pathogenesis.

  8. Diarrheagenic Escherichia coli and acute and persistent diarrhea in returned travelers

    NARCIS (Netherlands)

    Schultsz, C.; van den Ende, J.; Cobelens, F.; Vervoort, T.; van Gompel, A.; Wetsteyn, J. C.; Dankert, J.

    2000-01-01

    To determine the role of diarrheagenic Escherichia coli in acute and persistent diarrhea in returned travelers, a case control study was performed. Enterotoxigenic E. coli (ETEC) was detected in stool samples from 18 (10.7%) of 169 patients and 4 (3.7%) of 108 controls. Enteroaggregative E. coli

  9. Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    Vanessa Bueris

    2007-11-01

    Full Text Available We identified different diarrheagenic (DEC Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC, Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC, Enterotoxigenic E. coli (ETEC and Enteroaggregative E. coli (EAEC to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients and 18.7% (35 patients in the diarrhea group (1,020 patients and the control group (187 patients, respectively. The most frequently isolated pathotype was EAEC (10.7%, followed by atypical EPEC (9.4%, ETEC (3.7%, and STEC (0.6%. Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

  10. X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

    Energy Technology Data Exchange (ETDEWEB)

    Domingo Meza-Aguilar, J. [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Fromme, Petra [Department of Chemistry and Biochemistry, Arizona State University, Physical Sciences BLDG D-102, Tempe, AZ 85287 (United States); Torres-Larios, Alfredo [Instituto de Fisiología Celular UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Mendoza-Hernández, Guillermo [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); Hernandez-Chiñas, Ulises [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Arreguin-Espinosa de los Monteros, Roberto A. [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); and others

    2014-03-07

    Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

  11. Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food and In Vitro.

    Science.gov (United States)

    Böhnlein, Christina; Kabisch, Jan; Meske, Diana; Franz, Charles M A P; Pichner, Rohtraud

    2016-11-01

    In 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rare Escherichia coli serotype, O104:H4, that shared the virulence profiles of Shiga toxin-producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food or in vitro, were compared with those of E. coli O157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable than E. coli O157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward several E. coli strains, including serotype O157 strains, could be determined. Our study suggests that EHEC/EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead of E. coli O157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential. In 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive

  12. Molecular detection and antimicrobial resistance of diarrheagenic Escherichia coli strains isolated from diarrheal cases

    International Nuclear Information System (INIS)

    Aslani, Mehdi M.; Salmanzadeh-Ahrabi, S.; Jafari, F.; Zali, Reza M.; Mani, M.; Alikhani, Yousef M.

    2008-01-01

    Objective was to identify and classify Iranian isolates of diarrheagenic Escherichia coli (E. coli) on the basis of presence of virulence genes and to determine antibiotic susceptibility of isolated strains. The current cross-sectional study was conducted in 2005 at the Pasteur Institute, Tehran, Iran. One hundred and ninety-three diarrheagenic E. coli isolated from diarrheal patients in different regions of Iran were included in current study. Virulence factors genees for diarrheagenic E. coli were detected by polymerase chain reaction. Of the 193 diarrheagenic E. coli detected by PCR, 86(44.5%) were Shiga toxin-producing E. coli (STEC), 74 (38.4%) enteropathogenic E. coli (EPEC), 19 (9.8%) enteroaggregative E. coli and 14 (7.3%) enterotoxigenic E. coli isolates. Susceptibility to 12 clinically important antimicrobial agents was determined for 193 strains of diarrhheagenic E. coli. A high incidence of resistance to tetracycline (63%), ampicillin (62%), streptomycin (56%), amoxicillin/clavulanic acid (44.5%), trimetoprim/sulphamethoxazole (39.5%) and cephalothin (37%) was observed. The STEC and EPEC strains with high resistance to tetracycline and ampicillin but highly susceptible to quinolones are among the most important causative agent of diarrhea in Iran. This study suggests that antimicrobial resistance is wide spread among E. coli strains colonizing Iranian patients. Guidelines for appropriate use of antibiotics in developing countries require updating. (author)

  13. FTIR nanobiosensors for Escherichia coli detection

    Directory of Open Access Journals (Sweden)

    Stefania Mura

    2012-07-01

    Full Text Available Infections due to enterohaemorrhagic E. coli (Escherichia coli have a low incidence but can have severe and sometimes fatal health consequences, and thus represent some of the most serious diseases due to the contamination of water and food. New, fast and simple devices that monitor these pathogens are necessary to improve the safety of our food supply chain. In this work we report on mesoporous titania thin-film substrates as sensors to detect E. coli O157:H7. Titania films treated with APTES ((3-aminopropyltriethoxysilane and GA (glutaraldehyde were functionalized with specific antibodies and the absorption properties monitored. The film-based biosensors showed a detection limit for E. coli of 1 × 102 CFU/mL, constituting a simple and selective method for the effective screening of water samples.

  14. Detection of diarrheagenic Escherichia coli by multiplex PCR

    Directory of Open Access Journals (Sweden)

    A Hegde

    2012-01-01

    Full Text Available Background: Diarrheagenic E.coli (DEC are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus forEnteropathogenic E.coli (EPEC, hlyA for Enterohemorrhagic E.coli (EHEC, elt and stla for Enterotoxigenic E.coli (ETEC, CVD432 for Enteroaggregative E.coli (EAEC and ial for Enteroinvasive E.coli (EIEC. Results: In 200 children with diarrhea 52 (26% DEC infections were found. Among 100 controls 8 (8% DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%and controls (5,5%, followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5% and 3 (1.5% of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.

  15. Development of PCR primers based on a fragment from randomly amplified polymorphic DNA for the detection of Escherichia coli O157:H7/NM.

    Science.gov (United States)

    Lin, Chien-Ku; Lin, Jia-Chi

    2007-06-01

    Serotype O157:H7 of EHEC is by far the most prevalent serotype associated with haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). Although PCR methods aimed on the detection of genes associated with the pathogenicity of Escherichia coli O157:H7 have been reported, tests allowing the direct identification of this serotype are rare. In this study, we used RAPD-PCR tests to analyze strains of E. coli O157:H7 serotype, strains of non-pathogenic E. coli, and strains of other pathotypes, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enteroaggregation E. coli (EAggEC). One RAPD fragment co-shared by serotype O157:H7 strains was observed when 10-mer primer termed as OPQ3 was used. After sequencing this fragment, three primers were designed and combined to form two PCR primer pairs. These two primer pairs were highly specific to the strains belonging to E. coli O157:H7/NM (non-motile).

  16. Enterobacteria identification and detection of diarrheagenic Escherichia coli in a Port Complex

    Directory of Open Access Journals (Sweden)

    Clarissa Frota Macatrão Costa

    2014-09-01

    Full Text Available The Port Complex of Maranhão (PCM is the second largest port complex in Brazil, receiving ships with large volumes of ballast water. To evaluate the microbiological quality of its waters, physicochemical parameters (pH and salinity, the number of coliforms (thermotolerants and totals, and the presence of enterobacterias and diarrheagenic Escherichia coli strains were analyzed. In order to identify the presence of E. coli virulence genes target regions of the stx, elt, est, aggR, CVD432, ipaH and eae nucleotide sequences were studied. The presence of totals and thermotolerants coliforms were positive. Analyzing the salinity parameter, a significant increase in total coliforms was observed during the rainy season. We identified the species Escherichia coli, Proteus mirabilis, Citrobacter freundii, Proteus vulgaris, Klebsiella pneumoniae, Klebsiella ozaenae, Morganella morganii, Enterobacter cloacae and Edwardsiella tarda. Out of the 51 E. coli isolated, two were positive for the elt gene and one was positive for the CVD432 sequence, features of enterotoxigenic and enteroaggregative strains, respectively. This study reveals that the PCM is contaminated by enterobacteria and diarrheagenic E.coli thus providing evidence regarding the risk of these bacteria being carried by ships to other countries, and draws attention to the input of fecal bacteria brought by ships in the port waters of Maranhão.

  17. Enterobacteria identification and detection of diarrheagenic Escherichia coli in a Port Complex.

    Science.gov (United States)

    Costa, Clarissa Frota Macatrão; Monteiro Neto, Valério; Santos, Bruno Rafael de Carvalho; Costa, Bruno Rafael Rabelo; Azevedo, Alexandre; Serra, Josilene Lima; Mendes, Hermínio Benítez Rabello; Nascimento, Adenilde Ribeiro; Mendes, Mariana Bonfim Pinto; Kuppinger, Oliver

    2014-01-01

    The Port Complex of Maranhão (PCM) is the second largest port complex in Brazil, receiving ships with large volumes of ballast water. To evaluate the microbiological quality of its waters, physicochemical parameters (pH and salinity), the number of coliforms (thermotolerants and totals), and the presence of enterobacterias and diarrheagenic Escherichia coli strains were analyzed. In order to identify the presence of E. coli virulence genes target regions of the stx, elt, est, aggR, CVD432, ipaH and eae nucleotide sequences were studied. The presence of totals and thermotolerants coliforms were positive. Analyzing the salinity parameter, a significant increase in total coliforms was observed during the rainy season. We identified the species Escherichia coli, Proteus mirabilis, Citrobacter freundii, Proteus vulgaris, Klebsiella pneumoniae, Klebsiella ozaenae, Morganella morganii, Enterobacter cloacae and Edwardsiella tarda. Out of the 51 E. coli isolated, two were positive for the elt gene and one was positive for the CVD432 sequence, features of enterotoxigenic and enteroaggregative strains, respectively. This study reveals that the PCM is contaminated by enterobacteria and diarrheagenic E.coli thus providing evidence regarding the risk of these bacteria being carried by ships to other countries, and draws attention to the input of fecal bacteria brought by ships in the port waters of Maranhão.

  18. An integrated microsystem with dielectrophoresis enrichment and impedance detection for detection of Escherichia coli

    Science.gov (United States)

    An integrated microsystem device with matched interdigitated microelectrode chip was fabricated for enrichment and detection of Escherichia coli O157:H7. The microsystem has integrated with positive dielectrophoresis (pDEP) enrichment and in situ impedance detection, whose total volume is only 3.0 ×...

  19. Pathogenic Escherichia coli and food handlers in luxury hotels in Nairobi, Kenya.

    Science.gov (United States)

    Onyango, Abel O; Kenya, Eucharia U; Mbithi, John J N; Ng'ayo, Musa O

    2009-11-01

    The epidemiology and virulence properties of pathogenic Escherichia coli among food handlers in tourist destination hotels in Kenya are largely uncharacterized. This cross-sectional study among consenting 885 food handlers working in nine luxurious tourist hotels in Nairobi, Kenya determined the epidemiology, virulence properties, antibiotics susceptibility profiles and conjugation abilities of pathogenic Escherichia coli. Pathogenic Escherichia coli was detected among 39 (4.4%) subjects, including 1.8% enteroaggregative Escherichia coli (EAEC) harboring aggR genes, 1.2% enterotoxigenic Escherichia coli (ETEC) expressing both LT and STp toxins, 1.1% enteropathogenic Escherichia coli (EPEC) and 0.2% Shiga-like Escherichia coli (EHEC) both harboring eaeA and stx2 genes respectively. All the pathotypes had increased surface hydrophobicity. Using multivariate analyses, food handlers with loose stools were more likely to be infected with pathogenic Escherichia coli. Majority 53.8% of the pathotypes were resistant to tetracycline with 40.2% being multi-drug resistant. About 85.7% pathotypes trans-conjugated with Escherichia coli K12 F(-) NA(r) LA. The carriage of multi-drug resistant, toxin expressing pathogenic Escherichia coli by this population is of public health concern because exposure to low doses can result in infection. Screening food handlers and implementing public awareness programs is recommended as an intervention to control transmission of enteric pathogens.

  20. Changing Diagnostic Methods and Increased Detection of Verotoxigenic Escherichia coli, Ireland

    OpenAIRE

    Rice, Thomas; Quinn, Noreen; Sleator, Roy D.; Lucey, Brigid

    2016-01-01

    The recent paradigm shift in infectious disease diagnosis from culture-based to molecular-based approaches is exemplified in the findings of a national study assessing the detection of verotoxigenic Escherichia coli infections in Ireland. The methodologic changes have been accompanied by a dramatic increase in detections of non-O157 verotoxigenic E. coli serotypes.

  1. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli▿

    Science.gov (United States)

    Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K.; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M.; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R.; Tarr, Phillip I.; Vats, Abhay

    2008-01-01

    We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform. PMID:18550738

  2. Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli.

    Science.gov (United States)

    Hill, Joshua; Beriwal, Shilpa; Chandra, Ishwad; Paul, Vinod K; Kapil, Aarti; Singh, Tripti; Wadowsky, Robert M; Singh, Vinita; Goyal, Ankur; Jahnukainen, Timo; Johnson, James R; Tarr, Phillip I; Vats, Abhay

    2008-08-01

    We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

  3. Detection of Escherichia albertii from chicken meat and giblets.

    Science.gov (United States)

    Maeda, Eriko; Murakami, Koichi; Sera, Nobuyuki; Ito, Kenitiro; Fujimoto, Shuji

    2015-07-01

    Escherichia albertii occasionally causes food-borne outbreaks of gastroenteritis in humans; however, little is known about the vehicle of transmission. To screen retail chicken products for the presence of E. albertii, 104 retail chicken products were investigated. Portions of enrichment cultures that were PCR-positive for E. albertii (n=3) were sub-cultured on agar medium. Only 2 strains obtained from 2 chicken giblet samples were identified as E. albertii by multi locus sequence typing. Antimicrobial susceptibility testing showed that 1 strain was resistant to streptomycin and sulfisoxazole. Both strains harbored the virulence genes cdt and eae. This study is the first description of E. albertii isolation from retail food, suggesting that chicken products are a potential vehicle of E. albertii transmission.

  4. Detection of Florfenicol Resistance Genes in Escherichia coli Isolated from Sick Chickens

    OpenAIRE

    Keyes, Kathleen; Hudson, Charlene; Maurer, John J.; Thayer, Stephan; White, David G.; Lee, Margie D.

    2000-01-01

    Florfenicol is an antibiotic approved for veterinary use in cattle in the United States in 1996. Although this drug is not used in poultry, we have detected resistance to florfenicol in clinical isolates of avian Escherichia coli. Molecular typing demonstrated that the florfenicol resistance gene, flo, was independently acquired and is plasmid encoded.

  5. Detection of Florfenicol Resistance Genes in Escherichia coli Isolated from Sick Chickens

    Science.gov (United States)

    Keyes, Kathleen; Hudson, Charlene; Maurer, John J.; Thayer, Stephan; White, David G.; Lee, Margie D.

    2000-01-01

    Florfenicol is an antibiotic approved for veterinary use in cattle in the United States in 1996. Although this drug is not used in poultry, we have detected resistance to florfenicol in clinical isolates of avian Escherichia coli. Molecular typing demonstrated that the florfenicol resistance gene, flo, was independently acquired and is plasmid encoded. PMID:10639375

  6. Evaluation of Eight Different Cephalosporins for Detection of Cephalosporin Resistance in Salmonella enterica and Escherichia coli

    NARCIS (Netherlands)

    Aarestrup, F.M.; Hasman, H.; Veldman, K.T.; Mevius, D.J.

    2010-01-01

    This study evaluates the efficacy of eight different cephalosporins for detection of cephalosporin resistance mediated by extended spectrum beta-lactamases (ESBL) and plasmidic AmpC beta-lactamases in Salmonella and Escherichia coli. A total of 138 E. coli and 86 Salmonella isolates with known

  7. Review: Biosensors for the detection of Escherichia coli | Maas ...

    African Journals Online (AJOL)

    The supply of safe potable water, free from pathogens and chemicals, requires routine analyses and the application of several diagnostic techniques. Apart from being expensive, many of the detection methods require trained personnel and are often time-consuming. With drastic climate changes, severe droughts, ...

  8. Prevalence of diarrheogenic Escherichia coli and rotavirus among children from Botucatu, São Paulo State, Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigues J.

    2002-01-01

    Full Text Available In a one-year prospective study carried out to define the role of rotavirus and Escherichia coli in local childhood diarrhea, we determined the prevalence of both agents in 54 diarrheic children attending a health center in Botucatu. Diarrheogenic E. coli (DEC strains were characterized by O:H serotyping, a search for virulence genetic markers, and assays of adherence to HEp-2 cells. Except for enteroaggregative E. coli (EAEC, no other DEC category was detected in the children's stools. Both EAEC and rotavirus were isolated from 22 of the 54 (41.0% diarrheic children as single agents or in combination with other enteropathogens. However, when considering the presence of a single agent, EAEC was dominant and isolated from 20.4% of the patients, whereas rotavirus was detected in 14.8%. These results indicate that rotavirus and EAEC play a significant role as agents of childhood diarrhea in the local population.

  9. Atypical Enteropathogenic Escherichia coli Secretes Plasmid Encoded Toxin

    Directory of Open Access Journals (Sweden)

    Rita C. Ruiz

    2014-01-01

    Full Text Available Plasmid encoded toxin (Pet is a serine protease originally described in enteroaggregative Escherichia coli (EAEC prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

  10. Comprehensive Characterization of Escherichia coil O104 : H4 Isolated from Patients in the Netherlands

    NARCIS (Netherlands)

    Ferdous, Mithila; Zhou, Kai; de Boer, Richard F.; Friedrich, Alexander W.; Kooistra-Smid, Mirjam; Rossen, John W. A.

    2015-01-01

    In 2011, a Shiga toxin producing Enteroaggregative Escherichia coil (EAEC Stx2a+) 0104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolyticuremic syndrome (HUS) in Germany. In 2013, E. col O104:H4 isolates were obtained from a patient with HUS and her friend showing only

  11. A direct detection of Escherichia coli genomic DNA using gold nanoprobes

    Directory of Open Access Journals (Sweden)

    Padmavathy

    2012-02-01

    Full Text Available Abstract Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to

  12. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5 ' nuclease PCR assay

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization...... of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5' nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays...

  13. Rapid detection of Escherichia coli O157:H7 using tunneling magnetoresistance biosensor

    Science.gov (United States)

    Wu, Yuanzhao; Liu, Yiwei; Zhan, Qingfeng; Liu, J. Ping; Li, Run-Wei

    2017-05-01

    A rapid method for the sensitive detection of bacteria using magnetic immunoassay, which are measured with a tunneling magnetoresistance (TMR) sensor, is described. For the measurement of Escherichia coli O157:H7 (E. coli O157:H7) bacteria, the target was labeled by magnetic beads through magnetic immunoassay. The magnetic beads produce a weak magnetic fringe field when external field is applied, thus induce the magnetoresistance change of TMR sensor. A detection limit of 100 CFU/mL E. coli O157:H7 bacteria in 5 hours was obtained. With its high sensitive and rapid detection scheme based on the TMR biosensor, the detection system is an excellent candidate suitable and promising for food safety and biomedical detection.

  14. Molecular detection of CTX-M-15-type β-lactamases in Escherichia coli strains from Senegal

    Directory of Open Access Journals (Sweden)

    M.L. Dia

    2016-01-01

    Full Text Available We aimed to detect the extended-spectrum β-lactamases (ESBLs secreted by clinical strains of Escherichia coli at Fann University Hospital in Dakar and to characterize them molecularly. We identified 32 isolates producing ESBLs. The CTX-M-15 gene was the most frequently detected ESBL gene, detected in 90.63% of the isolates studied.

  15. PCR approach for rapid detection of Escherichia coli in tempe using a specific primer

    Directory of Open Access Journals (Sweden)

    Siti Harnina Bintari

    2014-12-01

    Full Text Available Tempe known as a traditional fermented food originated from Indonesia. It has a unique flavour and texture. It also contains high protein and usually serves to substitute meat, fish, or egg as a complement to rice. The manufacture process of Tempe is quite complex and mostly, the traditional process has not employed the hygienic standard. In the process of Tempe making, there are two critical stages of the whole process; i.e. soaking of soybeans and solid state fermentation by Rhizopus sp. During the process, foodborne pathogen bacteria such as Escherichia coli could contaminate the product of Tempe. The bacterial contamination could be revealed through culture dependent methods which is costly, laborious, and time consuming. Therefore, the culture-independent method such as polymerase chain reaction using a specific primer could be applied to detect target microorganism to save time and labour. In this study, thirty-one Tempe samples collected from different manufacturers in Semarang, Central Java, Indonesia were analysed by PCR. In order to obtain the bacterial genomic DNA, a modified Chelex 100-Microwave method was employed. The results of DNA extraction showed that the method was an applicable method. It gave high quantity and quality of DNA; therefore, it could be applied in the PCR reaction. The DNA samples were employed in PCR for detection of Escherichia coli using Ecoli706F/R. It was found that 27 out of 31 samples were detected having Escherichia coli contamination showed by the presence of the amplified product size 706 bp. The application of this method could significantly reduce costs and time of analysis in the laboratory. Further response after E. coli were detected could be employed, including investigation of the critical factors in Tempe manufacturing process which allowed E. coli contamination.

  16. Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytegenes and Bacillus cereus by Oligonucleotide Microarray

    Directory of Open Access Journals (Sweden)

    Meysam Sarshar

    2015-11-01

    Full Text Available Background: Traditional laboratory methods to detect pathogenic bacteria are time consuming and laborious. Therefore, it is essential to use powerful and reliable molecular methods for quick and simultaneous detection of microbial pathogens. Objectives: The current study aimed to evaluate the capability and efficiency of 23S rDNA sequence for rapid and simultaneous detection of four important food-borne pathogens by an oligonucleotide microarray technique. Materials and Methods: The 23S rDNA sequences of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus were obtained from GenBank databases and used to design the oligonucleotide probes and primers by Vector NTI software. Oligonucleotide probes were placed on a nylon membrane and hybridization was performed between probes and 23S rDNA digoxigenin-labeled polymerase chain reaction (PCR products. Hybridization signals were visualized by NBT/BCIP color development. Results: Positive hybridization color was produced for Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus. The oligonucleotide microarray detected all bacterial strains in a single reaction in less than five hours. The sensitivity of the performed microarray assay was 103 cfu/mL for each species of pathogen. No cross reaction was found between the tested bacterial species. Conclusions: The obtained results indicated that amplification of 23S rDNA gene followed by oligonucleotide microarray hybridization is a rapid and reliable method to identify and discriminate foodborne pathogens tested under the study.

  17. FREQUENCY AND DISTRIBUTION OF DIARRHOEAGENIC ESCHERICHIA COLI STRAINS ISOLATED FROM PEDIATRIC PATIENTS WITH DIARRHOEA IN BOSNIA AND HERZEGOVINA

    OpenAIRE

    Dedeić-Ljubović, AmeLa; Hukić, Mirsada; Bekić, DaRia; Zvizdić, AmrA

    2009-01-01

    Diarrhoeal disease is a major cause of illness and death among infants and young children worldwide. Among the Escherichia coli (E. coli) causing intestinal diseases, there are six well-described categories: enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enteroinvasive E. coli (EIEC), entero-pathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and enterotoxigenic E. coli (ETEC).

  18. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isabel; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  19. Detection of Pathogenic Escherichia coli in Samples Collected at an Abattoir in Zaria, Nigeria and at Different Points in the Surrounding Environment

    Science.gov (United States)

    Kabiru, Lawan Mohammed; Bello, Mohammed; Kabir, Junaid; Grande, Laura; Morabito, Stefano

    2015-01-01

    Pathogenic Escherichia coli can be released with the wastes coming from slaughterhouses into the environment, where they can persist. We investigated the presence of diarrheagenic E. coli in specimens taken at an abattoir located in the Zaria region, Nigeria, in samples of water from the river Koreye, where the effluent from the abattoir spills in, and vegetable specimens taken at a nearby farm. All the isolated E. coli were assayed for the production of Shiga toxins (Stx) by using the Ridascreen verotoxin Immunoassay and by PCR amplification of genes associated with the diarrheagenic E. coli. Three strains from the rectal content of two slaughtered animals and a cabbage were positive for the presence of the Stx-coding genes. Additionally we have isolated one Enteroaggregative E. coli (EAggEC) from the abattoir effluent and two Subtilase-producing E. coli from the slaughterhouse’s effluent and a sample of carrots. Our results provide evidence that pathogenic E. coli can contaminate the environment as a result of the discharge into the environment of untreated abattoir effluent, representing a reservoir for STEC and other diarrheagenic E. coli favouring their spread to crops. PMID:25590145

  20. Detection of Pathogenic Escherichia coli in Samples Collected at an Abattoir in Zaria, Nigeria and at Different Points in the Surrounding Environment

    Directory of Open Access Journals (Sweden)

    Lawan Mohammed Kabiru

    2015-01-01

    Full Text Available Pathogenic Escherichia coli can be released with the wastes coming from slaughterhouses into the environment, where they can persist. We investigated the presence of diarrheagenic E. coli in specimens taken at an abattoir located in the Zaria region, Nigeria, in samples of water from the river Koreye, where the effluent from the abattoir spills in, and vegetable specimens taken at a nearby farm. All the isolated E. coli were assayed for the production of Shiga toxins (Stx by using the Ridascreen verotoxin Immunoassay and by PCR amplification of genes associated with the diarrheagenic E. coli. Three strains from the rectal content of two slaughtered animals and a cabbage were positive for the presence of the Stx-coding genes. Additionally we have isolated one Enteroaggregative E. coli (EAggEC from the abattoir effluent and two Subtilase-producing E. coli from the slaughterhouse’s effluent and a sample of carrots. Our results provide evidence that pathogenic E. coli can contaminate the environment as a result of the discharge into the environment of untreated abattoir effluent, representing a reservoir for STEC and other diarrheagenic E. coli favouring their spread to crops.

  1. Detection of pathogenic Escherichia coli in samples collected at an abattoir in Zaria, Nigeria and at different points in the surrounding environment.

    Science.gov (United States)

    Kabiru, Lawan Mohammed; Bello, Mohammed; Kabir, Junaid; Grande, Laura; Morabito, Stefano

    2015-01-13

    Pathogenic Escherichia coli can be released with the wastes coming from slaughterhouses into the environment, where they can persist. We investigated the presence of diarrheagenic E. coli in specimens taken at an abattoir located in the Zaria region, Nigeria, in samples of water from the river Koreye, where the effluent from the abattoir spills in, and vegetable specimens taken at a nearby farm. All the isolated E. coli were assayed for the production of Shiga toxins (Stx) by using the Ridascreen verotoxin Immunoassay and by PCR amplification of genes associated with the diarrheagenic E. coli. Three strains from the rectal content of two slaughtered animals and a cabbage were positive for the presence of the Stx-coding genes. Additionally we have isolated one Enteroaggregative E. coli (EAggEC) from the abattoir effluent and two Subtilase-producing E. coli from the slaughterhouse's effluent and a sample of carrots. Our results provide evidence that pathogenic E. coli can contaminate the environment as a result of the discharge into the environment of untreated abattoir effluent, representing a reservoir for STEC and other diarrheagenic E. coli favouring their spread to crops.

  2. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

    Directory of Open Access Journals (Sweden)

    Jinliang Wang

    2016-03-01

    Full Text Available Shiga toxin-producing Escherichia coli O157:H7 (STEC cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2 that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA; one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  3. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

    Science.gov (United States)

    Wang, Jinliang; Katani, Robab; Li, Lingling; Hegde, Narasimha; Roberts, Elisabeth L; Kapur, Vivek; DebRoy, Chitrita

    2016-03-25

    Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 10⁵ CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  4. Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods : interlaboratory study

    NARCIS (Netherlands)

    Capps, K.L.; McLaughlin, E.M.; Murray, A.W.A.; Aldus, C.F.; Wyatt, G.M.; Peck, M.W.; Amerongen, van A.; Ariens, R.M.C.; Wichers, J.H.; Bayliss, C.L.; Wareing, D.R.A.; Bolton, F.J.

    2004-01-01

    An interlaboratory study was conducted for the validation of 3 methods for the detection of all verotoxin-producing Escherichia coli (VTEC) in foods. The methods were a multi-analyte 1-step lateral flow immunoassay (LFIA) for detection of E. coli O157 and verotoxin (VT); an enzyme-linked

  5. Detection of EnteroinvasiveEscherichia coli by PCR technique in Children with Diarrhea

    Directory of Open Access Journals (Sweden)

    v Aein

    2014-11-01

    Full Text Available Background & aim: EnteroinvasiveEscherichia coliis one of the important agents of invasion to intestinal epithelial cells, damage and cell death which due to dysentery. The aim of this study wastoDetection of EnteroinvasiveEscherichia coli by PCR technique from Children’s Diarrheain yasuj. Methods:This cross-sectional study was performed on 200 stool samples taken from children with diarrhea in Yasuj. After initial identification of E.coli strains by culture and biochemical tests, EIEC gene such as ipaH detected by PCR technicque and antibiotic susceptibility of isolates was evaluated by using disc diffusion (CLSI method. Results: Out of all examined samples, 16(8% EIEC were separated. Antibiotic susceptibility test showed that the most susceptible antibiotic is ciprofloxacin for EIEC and also most resistant antibiotic is ceftizoxime. Conclusion: Results showed that EIEC strains have a moderate prevalence than other studies in our study area. Therefore, for importance of this strain to producing dysentery, hospital-wide surveillance using molecular techniques hase been proposed in other regions of country.

  6. Detection of verotoxin producing Escherichia coli in marine environments of the Caribbean.

    Science.gov (United States)

    Walker, Trisha J; Bachoon, D S; Otero, Ernesto; Ramsubhag, Adesh

    2013-11-15

    The goal of this study was to determine the potential for Enterohemorrhagic Escherichia coli O157:H7 (EHEC) contamination in tropical marine waters. Samples were collected from urban, suburban, and rural sites around the islands of Puerto Rico and The Republic of Trinidad and Tobago. Quantification of E. coli and EHEC was evaluated using MI plates and qPCR. EHEC was detected in six sites in Puerto Rico: West of La Parguera Town, Boquilla, Oro Creek, Fishers Association, Joyuda Lagoon, and Boqueron Wetland Creek and in two rural sites in Trinidad: Balandra Bay and Quinam Bay. Plate count enumeration of E. coli was not a reliable indicator for the presence of EHEC. The sites where EHEC was detected on both islands are used for recreational bathing, water sports and recreational/commercial fisheries and therefore pose a public potential health risk. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. High sensitive and selective Escherichia coli detection using immobilized optical fiber microprobe

    Science.gov (United States)

    Li, Yanpeng; Sun, Qizhen; Luo, Yiyang; Li, Yue; Gong, Andong; Zhang, Haibin; Liu, Deming

    2017-04-01

    We proposed and demonstrated a stable, label-free bacteriophage-based sensor of Escherichia coli using microfiber probe. T4 Bacteriophage was covalently immobilized on microfiber surface and E.coli concentration was investigated using the high accurate spectral interference mechanism. By immersing microfiber sensor into different concentration E.coli solution, the relationship between resonant wavelength shift and E.coli concentration was analyzed in the range of 103-107cfu/ml. The proposed method is capable of reliable detection of E.coli concentration as low as 103cfu/ml with a fast response time about 10minutes, which makes the real-time detection of E.coli move on a giant step. Additionally, the sensor has great potential to be applied in the fields like environment monitoring and food safety.

  8. Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR.

    Science.gov (United States)

    Barak, J D; Sananikone, K; Delwiche, M J

    2005-01-01

    To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing. Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between 10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively. The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water. The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.

  9. Direct detection by PCR of Escherichia coli O157 and enteropathogens in patients with bloody diarrhea.

    Science.gov (United States)

    Takeshi, K; Ikeda, T; Kubo, A; Fujinaga, Y; Makino, S; Oguma, K; Isogai, E; Yoshida, S; Sunagawa, H; Ohyama, T; Kimura, H

    1997-01-01

    Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 10(1) CFUs/0.5 g of stool sample. PCR was completed within 6 hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy-to-read screening method for the detection of Shiga-like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.

  10. Selection of antibiotics in detection procedure of Escherichia coli O157:H7 in vegetables

    Science.gov (United States)

    Hoang, Hoang A.; Nhung, Nguyen T. T.

    2017-09-01

    Detection of Escherichia coli O157:H7 in ready-to-eat fresh vegetables is important since this bacteria is considered as one of the most important pathogens in relation to public health. However, it could be a big challenge for detection of initial low concentrations of E. coli O157:H7 in the samples. In this study, selection of antibiotics that suppress growth of background bacteria to enable detection of E. coli O157:H7 in ready-to-eat fresh vegetables was investigated. Firstly, different combinations of two antibiotics, i.e. novobiocin (N) and vancomycin (V), in BHI broth were conducted. The three antibiotic combinations were preliminary examined their effect on the growth of E. coli O157:H7 and Bacillus spp. in broth based on OD600nm measurement. The combination of both the antibiotics was selected to examine their possibility to support detection of E. coli O157:H7 in vegetables. It was successful when two antibiotics showed their support in detection of E. coli O157:H7 at very low concentration of 2 CFU per one gram of lettuce. Usage of these antibiotics is simple and cheap in the detection procedure and could be applied to other types of ready-to-eat fresh vegetables popular in Vietnam.

  11. Detection of Escherichia coli, Salmonella species, and Vibrio cholerae in tap water and bottled drinking water in Isfahan, Iran.

    Science.gov (United States)

    Momtaz, Hassan; Dehkordi, Farhad Safarpoor; Rahimi, Ebrahim; Asgarifar, Amin

    2013-06-07

    The quality of drinking water has an important role in human infection and disease. This study was aimed at comparing polymerase chain reaction and culture in detecting Escherichia coli, Salmonella species and Vibrio cholera in tape water and bottled drinking water in various seasons in Isfahan province, Iran. A total of 448 water samples from tap water and bottled mineral water were taken over 6 months, from July 2010 to December 2010, and after filtration, samples were examined by culture and polymerase chain reaction methods for detection of Escherichia coli, Salmonella species, and Vibrio cholerae. The culture method showed that 34 (7.58%), 4 (0.89%) and 3 (0.66%) of all 448 water samples were positive for Escherichia coli, Salmonella species, and Vibrio cholera, respectively. The uidA gene from Escherichia coli, IpaB gene from Salmonella species, and epsM gene from Vibrio cholera were detected in 38 (26.38%), 5 (3.47%), and 3 (2.08%) of 144 tap-water samples, respectively. Escherichia coli was detected in 8 (2.63%) of 304 samples of bottled drinking water from 5 companies. The water of southern part of Isfahan and company 5 had the highest prevalence of bacteria. The Escherichia coli water contamination was significantly higher (P waters of southern part and tap waters of central part of Isfahan. This study showed that the polymerase chain reaction assays can be an extremely accurate, fast, safe, sensitive and specific approach to monitor drinking water quality from purification facilities and bottled water companies. Also, our study confirmed the presence of Escherichia coli, Salmonella species, and Vibrio cholerae as water-borne pathogens in tap water and bottled drinking water of Isfahan, Iran. The present study showed the important public health problem in Isfahan, Iran.

  12. Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium

    Science.gov (United States)

    Wu, Wen-he; Li, Min; Wang, Yue; Ouyang, Hou-xian; Wang, Lin; Li, Ci-xiu; Cao, Yu-chen; Meng, Qing-he; Lu, Jian-xin

    2012-11-01

    Herein we reported the development of aptamer-based biosensors (aptasensors) based on label-free aptamers and gold nanoparticles (AuNPs) for detection of Escherichia coli ( E. coli) O157:H7 and Salmonella typhimurium. Target bacteria binding aptamers are adsorbed on the surface of unmodified AuNPs to capture target bacteria, and the detection was accomplished by target bacteria-induced aggregation of the aptasensor which is associated as red-to-purple color change upon high-salt conditions. By employing anti- E. coli O157:H7 aptamer and anti- S. typhimurium aptamer, we developed a convenient and rapid approach that could selectively detect bacteria without specialized instrumentation and pretreatment steps such as cell lysis. The aptasensor could detect as low as 105colony-forming units (CFU)/ml target bacteria within 20 min or less and its specificity was 100%. This novel method has a great potential application in rapid detection of bacteria in the near future.

  13. Identification of Diarrheagenic Escherichia coli Strains from Avian Organic Fertilizers

    Directory of Open Access Journals (Sweden)

    Juan Puño-Sarmiento

    2014-08-01

    Full Text Available The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%, three strains as Shiga toxin-producing (STEC; 4.7%, 10 strains as enteroaggregative (EAEC; 12.5%, but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

  14. Evaluation of Rectoanal Mucosal Swab Sampling for Molecular Detection of Enterohemorrhagic Escherichia coli in Beef Cattle.

    Science.gov (United States)

    Agga, Getahun E; Arthur, Terrance M; Hinkley, Susanne; Bosilevac, Joseph M

    2017-04-01

    Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC), and contaminated beef products are a source of human infections. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven EHEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw ground beef. Sampling a large number of animals for EHEC surveillance or evaluations of EHEC-focused preharvest interventions requires a convenient and robust sampling method. We evaluated the diagnostic performance of rectoanal mucosal swab (RAMS) for the detection of the top seven EHEC serogroups. Paired fecal grab (FG) and RAMS samples were collected from 176 beef cattle and tested using the NeoSEEK Shiga toxin-producing E. coli (STEC) confirmation method. The prevalence of virulence-associated genes (stx 1 , stx 2 , stx 2c , eae, and nleB) was higher in RAMS than in FG samples. The results of the two methods had poor agreement, as indicated by kappa statistics, for the detection of the seven serogroups. When FG and RAMS results were combined for comparison, RAMS was more sensitive than FG for the detection of serogroups O103 (82% versus 39%), O157 (75% versus 67%), and O45 (79% versus 73%) with similar sensitivity for the detection of serogroup O145 (67%). Serogroups O111 and O121 were detected from one and two samples, respectively, by FG and were not detected by RAMS. Serogroup O26 was not detected with either method. RAMS appears to be equivalent or superior to FG sampling for detection of the top seven EHEC serogroups in the feces of beef cattle with the NeoSEEK STEC confirmation test.

  15. Self-assembled monolayers-based immunosensor for detection of Escherichia coli using electrochemical impedance spectroscopy

    International Nuclear Information System (INIS)

    Geng Ping; Zhang Xinai; Meng Weiwei; Wang Qingjiang; Zhang Wen; Jin Litong; Feng Zhen; Wu Zirong

    2008-01-01

    An electrochemical impedance immunosensor for the detection of Escherichia coli was developed by immobilizing anti-E. coli antibodies at an Au electrode. The immobilization of antibodies at the Au electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the self-assembled monolayer (SAM). The surface characteristics of the immunosensor before and after the binding reaction of antibodies with E. coli were characterized by atomic force microscopy (AFM). The immobilization of antibodies and the binding of E. coli cells to the electrode could increase the electro-transfer resistance, which was directly detected by electrochemical impedance spectroscopy (EIS) in the presence of Fe(CN) 6 3- /Fe(CN) 6 4- as a redox probe. A linear relationship between the electron-transfer resistance and the logarithmic value of E. coli concentration was found in the range of E. coli cells from 3.0 x 10 3 to 3.0 x 10 7 cfu mL -1 with the detection limit of 1.0 x 10 3 cfu mL -1 . With preconcentration and pre-enrichment steps, it was possible to detect E. coli concentration as low as 50 cfu/mL in river water samples

  16. Different cellular origins and functions of extracellular proteins from Escherichia coli O157:H7 and O104:H4 as determined by comparative proteomic analysis

    Science.gov (United States)

    Escherichia coli is a diverse species of bacteria, including several pathotypes that cause a variety of diseases in humans. Enterohemorrhagic E. coli (EHEC) and recently emerged shigatoxingenic enteroaggregative E. coli (EAEC) produce Shigatoxins and are major foodborne pathogens that can cause hem...

  17. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

    Science.gov (United States)

    Murinda, Shelton E; Ibekwe, A Mark; Zulkaffly, Syaizul; Cruz, Andrew; Park, Stanley; Razak, Nur; Paudzai, Farah Md; Ab Samad, Liana; Baquir, Khairul; Muthaiyah, Kokilah; Santiago, Brenna; Rusli, Amirul; Balkcom, Sean

    2014-07-01

    Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

  18. Evaluation of Eight Different Cephalosporins for Detection of Cephalosporin Resistance in Salmonella enterica and Escherichia coli

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Hasman, Henrik; Veldman, K

    2010-01-01

    This study evaluates the efficacy of eight different cephalosporins for detection of cephalosporin resistance mediated by extended spectrum beta-lactamases (ESBL) and plasmidic AmpC beta-lactamases in Salmonella and Escherichia coli. A total of 138 E. coli and 86 Salmonella isolates with known beta......-resistant but cephalosporin-susceptible, 56 ESBL isolates and 19 isolates with plasmidic AmpC, as well as 10 ampC hyper-producing E. coli. The minimum inhibitory concentration distributions and zone inhibitions varied with the tested compound. Ampicillin-resistant isolates showed reduced susceptibility to the cephalosporins...... be the recommended substance for monitoring because of some ability in separating ampC hyper-producing E. coli from ESBL and plasmidic AmpC isolates....

  19. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  20. Detecting the Significant Flux Backbone of Escherichia coli metabolism.

    Science.gov (United States)

    Güell, Oriol; Sagués, Francesc; Serrano, M Ángeles

    2017-05-01

    The heterogeneity of computationally predicted reaction fluxes in metabolic networks within a single flux state can be exploited to detect their significant flux backbone. Here, we disclose the backbone of Escherichia coli, and compare it with the backbones of other bacteria. We find that, in general, the core of the backbones is mainly composed of reactions in energy metabolism corresponding to ancient pathways. In E. coli, the synthesis of nucleotides and the metabolism of lipids form smaller cores which rely critically on energy metabolism. Moreover, the consideration of different media leads to the identification of pathways sensitive to environmental changes. The metabolic backbone of an organism is thus useful to trace simultaneously both its evolution and adaptation fingerprints. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  1. Development of a sensor for the detection of Escherichia coli in brackish waters

    Directory of Open Access Journals (Sweden)

    Mancuso Monique

    2016-03-01

    Full Text Available Monitoring of bacterial pathogens is important for marine environmental protection, because the presence of these microorganisms can be a serious risk for human health. For this reason, a portable sensor implemented as an electronic embedded system featuring disposable measurement cells was used to evaluate the ability and sensitivity of detection of Escherichia coli (E. coli as an indicator of fecal pollution in transitional environments and a water sample added with E. coli (102 CFU/mL was assayed. The first result obtained from the laboratory experiment seems promising for the determination of E. coli in environmental samples, though further improvements will be needed for the field application of this sensor in marine and brackish waters.

  2. Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia

    Directory of Open Access Journals (Sweden)

    K. Chishimba

    2016-01-01

    Full Text Available The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL- producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of blaCTX-M, blaSHV, and blaTEM genes. Overall 20.1%, 77/384, (95% CI; 43.2–65.5% of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77; CI: 75.7–92 of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia.

  3. Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia.

    Science.gov (United States)

    Chishimba, K; Hang'ombe, B M; Muzandu, K; Mshana, S E; Matee, M I; Nakajima, C; Suzuki, Y

    2016-01-01

    The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL-) producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of bla CTX-M, bla SHV, and bla TEM genes. Overall 20.1%, 77/384, (95% CI; 43.2-65.5%) of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77; CI: 75.7-92) of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia.

  4. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    M Shanthi

    2012-01-01

    Full Text Available Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR. Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25 and K. pneumoniae (n = 52 were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI guidelines. Minimum inhibitory concentration (MIC to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL. Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test. Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii, DHA (Dhahran Hospital, Saudi Arabia, ACC (Ambler class C, EBC (Amp C origin - Enterobacter cloacae groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

  5. Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by gold nanoparticle optical sensing

    Science.gov (United States)

    Shiga-toxin producing Escherichia coli (STEC) strains (“Big Six” – O26, O45, O103, O111, O121, O145, and O157) represent significant groups of pathogens responsible for foodborne diseases. The objective of this study was to develop a colorimetric optical sensing assay that can simultaneously detect ...

  6. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

    NARCIS (Netherlands)

    Noguera, P.; Posthuma-Trumpie, G.A.; Tuil, van M.; Wal, van der F.J.; Boer, de A.; Moers, A.P.H.A.; Amerongen, van A.

    2011-01-01

    The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay,

  7. Attomole DNA electrochemical sensor for the detection of Escherichia coli O157.

    Science.gov (United States)

    Liao, Wei-Ching; Ho, Ja-An Annie

    2009-04-01

    Enterohemorrhagic Escherichia coli O157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal Ru(NH(3))(6)(3+) was measured using square wave voltammetry, yielding a sigmoidally shaped dose-response curve whose linear portion was over the range from 1 to 10(6) fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 microL of a 0.15 pM solution).

  8. Evaluation of eight different cephalosporins for detection of cephalosporin resistance in Salmonella enterica and Escherichia coli.

    Science.gov (United States)

    Aarestrup, Frank M; Hasman, Henrik; Veldman, Kees; Mevius, Dik

    2010-12-01

    This study evaluates the efficacy of eight different cephalosporins for detection of cephalosporin resistance mediated by extended spectrum beta-lactamases (ESBL) and plasmidic AmpC beta-lactamases in Salmonella and Escherichia coli. A total of 138 E. coli and 86 Salmonella isolates with known beta-lactamase genes were tested for susceptibility toward cefoperazone, cefotaxime, cefpodoxime, cefquinome, ceftazidime, ceftiofur, ceftriaxone, and cefuroxime using minimum inhibitory concentration determinations and disc diffusion. The collection consisted of 84 ampicillin-susceptible, 57 ampicillin-resistant but cephalosporin-susceptible, 56 ESBL isolates and 19 isolates with plasmidic AmpC, as well as 10 ampC hyper-producing E. coli. The minimum inhibitory concentration distributions and zone inhibitions varied with the tested compound. Ampicillin-resistant isolates showed reduced susceptibility to the cephalosporins compared to ampicillin-susceptible isolates. Cefoperazone, cefquinome, and cefuroxime were not useful in detecting isolates with ESBL or plasmidic AmpC. The best substances for detection were cefotaxime, cefpodoxime, and ceftriaxone, whereas ceftazidime and ceftiofur were not as efficient. Ceftriaxone may be the recommended substance for monitoring because of some ability in separating ampC hyper-producing E. coli from ESBL and plasmidic AmpC isolates.

  9. Detection of virulence genes and the phylogenetic groups of Escherichia coli isolated from dogs in Brazil

    Directory of Open Access Journals (Sweden)

    Fernanda Morcatti Coura

    2018-02-01

    Full Text Available ABSTRACT: This study identified the virulence genes, pathovars, and phylogenetic groups of Escherichia coli strains obtained from the feces of dogs with and without diarrhea. Virulence genes and phylogenetic group identification were studied using polymerase chain reaction. Thirty-seven E. coli isolates were positive for at least one virulence factor gene. Twenty-one (57.8% of the positive isolates were isolated from diarrheal feces and sixteen (43.2% were from the feces of non-diarrheic dogs. Enteropathogenic E. coli (EPEC were the most frequently (62.2% detected pathovar in dog feces and were mainly from phylogroup B1 and E. Necrotoxigenic E. coli were detected in 16.2% of the virulence-positive isolates and these contained the cytotoxic necrotizing factor 1 (cnf1 gene and were classified into phylogroups B2 and D. All E. coli strains were negative for the presence of enterotoxigenic E. coli (ETEC enterotoxin genes, but four strains were positive for ETEC-related fimbriae 987P and F18. Two isolates were Shiga toxin-producing E. coli strains and contained the toxin genesStx2 or Stx2e, both from phylogroup B1. Our data showed that EPEC was the most frequent pathovar and B1 and E were the most common phylogroups detected in E. coli isolated from the feces of diarrheic and non-diarrheic dogs.

  10. Detection of molecular changes induced by antibiotics in Escherichia coli using vibrational spectroscopy

    Science.gov (United States)

    Xuan Nguyen, N. T.; Sarter, Samira; Hai Nguyen, N.; Daniel, Philippe

    2017-08-01

    This study aimed to test Raman (400-1800 cm- 1) and Infra-red (1900-500 cm- 1) spectroscopies followed by statistical analysis (principal component analysis) to detect molecular changes induced by antibiotics (ampicillin, cefotaxime - cell wall synthesis inhibitors, tetracycline - protein synthesis inhibitor, ciprofloxacin - DNA synthesis inhibitor) against Escherichia coli TOP10. In case of ampicillin and cefotaxime, a decrease in protein bands in both Raman (1240, 1660 cm- 1), and IR spectra (1230, 1530, 1630 cm- 1), and an increase in carbohydrate bands (1150, 1020 cm- 1) in IR spectra were observed. Tetracycline addition caused an increase in nucleic acid bands (775, 1478, 1578 cm- 1), a sharp decrease in phenylalanine (995 cm- 1) in Raman spectra and the amide I and amide II bands (1630, 1530 cm- 1) in IR spectra, an increase in DNA in both Raman (1083 cm- 1) and IR spectra (1080 cm- 1). Regarding ciprofloxacin, an increase in nucleic acids (775, 1478, 1578 cm- 1) in Raman spectra and in protein bands (1230, 1520, 1630 cm- 1), in DNA (1080 cm- 1) in IR spectra were detected. Clear discrimination of antibiotic-treated samples compared to the control was recorded, showing that Raman and IR spectroscopies, coupled to principal component analysis for data, could be used to detect molecular modifications in bacteria exposed to different classes of antibiotics. These findings contribute to the understanding of the mechanisms of action of antibiotics in bacteria.

  11. Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Zhang Zheng

    2007-12-01

    Full Text Available Abstract Background The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens. Results The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity. In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/μL and 103 cfu/mL per reaction. Conclusion The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

  12. Single walled carbon nanotube-based junction biosensor for detection of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Kara Yamada

    Full Text Available Foodborne pathogen detection using biomolecules and nanomaterials may lead to platforms for rapid and simple electronic biosensing. Integration of single walled carbon nanotubes (SWCNTs and immobilized antibodies into a disposable bio-nano combinatorial junction sensor was fabricated for detection of Escherichia coli K-12. Gold tungsten wires (50 µm diameter coated with polyethylenimine (PEI and SWCNTs were aligned to form a crossbar junction, which was functionalized with streptavidin and biotinylated antibodies to allow for enhanced specificity towards targeted microbes. In this study, changes in electrical current (ΔI after bioaffinity reactions between bacterial cells (E. coli K-12 and antibodies on the SWCNT surface were monitored to evaluate the sensor's performance. The averaged ΔI increased from 33.13 nA to 290.9 nA with the presence of SWCNTs in a 10(8 CFU/mL concentration of E. coli, thus showing an improvement in sensing magnitude. Electrical current measurements demonstrated a linear relationship (R2 = 0.973 between the changes in current and concentrations of bacterial suspension in range of 10(2-10(5 CFU/mL. Current decreased as cell concentrations increased, due to increased bacterial resistance on the bio-nano modified surface. The detection limit of the developed sensor was 10(2 CFU/mL with a detection time of less than 5 min with nanotubes. Therefore, the fabricated disposable junction biosensor with a functionalized SWCNT platform shows potential for high-performance biosensing and application as a detection device for foodborne pathogens.

  13. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

    NARCIS (Netherlands)

    Noguera, P.S.; Posthuma-Trumpie, G.A.; Tuil, Van M.; Wal, van der F.J.; Boer, De A.; Moers, A.P.H.A.; Amerongen, Van A.

    2011-01-01

    The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1,

  14. Detection of Escherichia Coli Bacteria in Wastewater by using Graphene as a Sensing Material

    Science.gov (United States)

    Wibowo, K. M.; Sahdan, M. Z.; Ramli, N. I.; Muslihati, A.; Rosni, N.; Tsen, V. H.; Saim, H.; Ahmad, S. A.; Sari, Y.; Mansor, Z.

    2018-04-01

    Graphene is a family of carbon bonded in hexagonal honeycomb crystalline structure that has many superior properties. It was very suitable to be applied on sensor application due to the superior properties on electrical, physical, and optical. Furthermore, graphene also provide a large detection area since it has 2D structure. In this research, we develop graphene as a nanosensor for detection of Escherichia coli (E. coli) bacteria. The sample E. coli bacteria were cultured from domestic wastewater by using plate culture method and then isolated to get pure single colony. The serial dilution was performed to create different concentration of bacteria. Field emission scanning electron microscope and biochemical test were performed to ensure the sample genuinely target E. coli that defined by the physical size and optical properties. Raman spectroscopy measurements were also performed on the grapheme films, and it was found that the ratio of G peak and D peak intensity changing do to the presence of E. coli. The electrical properties of graphene shows the increasing number of the bacteria 4 to 273 cfu result in decreasing the resistance from 4.371 to 3.903 ohm gradually.

  15. FoodBioTimerAssay: a new microbiological biosensor for detection of Escherichia coli food contamination

    Directory of Open Access Journals (Sweden)

    Francesca Berlutti

    2008-09-01

    Full Text Available

    Background: Prevention of foodborne diseases is a fundamental goal for public health and industries engaged in food preparation and distribution. The correct procedure to ensure an effective prevention of foodborne diseases consists essentially in microbiological monitoring and enumeration of indicator microorganisms of faecal contamination at critical control points along the food producing procedures. Here, we propose a new microbiological biosensor, called FoodBioTimerAssay (FBTA, for rapid and reliable detection of Escherichia coli as indicator of faecal contamination in food and surface samples.

    Methods: A total of 122 samples were analysed using both experimental FBTA and Reference method. FBTA employs FBTM medium and counts bacteria through microbial metabolism measure: the time required for colour switch (red-to-yellow of FBTM, due to E. coli metabolism, is correlated to initial bacterial concentration.

    Results: FBTA results showed an overall agreement percentage with Reference method equal to 97.54%. Discrepancies concerned three samples (1 food and 2 surface samples. Moreover, the time required to perform FBTA method was 3-fold shorter than Reference one.

    Conclusions: FBTA method may be considered a useful tool for detection of E. coli contamination in food and surface samples. Therefore, FBTA method may be successfully employed in risk analysis of foodborne diseases.

  16. Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection.

    Science.gov (United States)

    Sangdee, Aphidech; Natphosuk, Sitakan; Srisathan, Adunwit; Sangdee, Kusavadee

    2013-02-01

    The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 10(0) CFU/ml of genomic DNA and cells of E. coli, respectively.

  17. Electrochemical detection of specific DNA and respiratory activity of Escherichia coli

    International Nuclear Information System (INIS)

    Yamanaka, Keiichiro; Ikeuchi, Tomohiko; Saito, Masato; Nagatani, Naoki; Tamiya, Eiichi

    2012-01-01

    We present two rapid and simplified detection methods for Escherichia coli involving the use of a hand-held potentiostat and a disposable screen-printed carbon electrode. E. coli is one of the indicator organisms used to access for food safety. Commonly, microbiological culture techniques take more than one day to yield results and therefore, a simple, cost-effective, in situ detection system is required for testing food safety. This report describes two complementary techniques for high- and low-sensitivity detection of E. coli. High-sensitivity detection relies upon quantification of DNA amplification by using polymerase chain reaction (PCR), while the simplified, low-sensitivity detection can be obtained through measurement of oxygen consumption due to respiration; importantly, both techniques utilize the same type of electrode. The former entails mixing the PCR mixture with Hoechst, an electro-active DNA intercalator, and then, measuring the oxidation current. Binding of Hoechst molecules to the amplified DNA causes the peak current to decrease because of the slow diffusion of the Hoechst-amplified DNA complex to the electrode surface. The results showed that the oxidation peak current of Hoechst decreased depending on the number of E. coli cells added to the PCR mixture as the template for amplification, and the sensitivity of the method was as low as a single bacterium. Oxygen consumption was detected by direct measurement of the cell-containing culture medium. This method required only 10 μL to be applied on the screen-printed electrode, and the reduction in oxygen current was clearly observed within 30 min when a minimum of 1 × 10 5 cells were present. These results were obtained without purifying the culture, and the samples were applied onto the electrode without any surface modifications. The techniques describes in this report are versatile, because they require the same type of electrode, have simplistic nature, use a hand-held potentiostat, and have

  18. Escherichia coli O104 associated with human diarrhea, South Africa, 2004-2011.

    Science.gov (United States)

    Tau, Nomsa P; Meidany, Parastu; Smith, Anthony M; Sooka, Arvinda; Keddy, Karen H

    2012-08-01

    To determine the origin of >4,000 suspected diarrheagenic Escherichia coli strains isolated during 2004-2011 in South Africa, we identified 7 isolates as serotype O104; 5 as enteroaggregative E. coli O104:H4, and 2 as enteropathogenic E. coli O104:non-H4. Pulsed-field gel electrophoresis showed that these isolates were unrelated to the 2011 E. coli O104:H4 outbreak strain from Germany.

  19. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  20. Evaluation of an enzyme immunoassay for verotoxin detection in Escherichia coli.

    Science.gov (United States)

    Frias, C; Majò, M; Margall, N; Llobet, T; Mirelis, B; Prats, G

    1996-09-01

    Verotoxin-producing Escherichia coli strains (VTEC) cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. Laboratory diagnosis by conventional methods is slow and cumbersome. The results of a new rapid enzyme immunoassay (EIA Premier EHEC) for verotoxin detection both in isolated strains and in clinical samples are presented, and they are compared with cell culture (CC) and polymerase chain reaction (PCR) techniques. Fifty-four strains have been analyzed by both EIA and PCR, and 33 by all three methods. The kit has also been evaluated for experimentally infected stool samples directly and after their enrichment on MacConkey broth. Nineteen, out of the 54 strains, were positive by EIA and 20 by PCR. The results of the 33 strains evaluated by the three techniques were coincident with one exception. The latter was uninterpretable by CC, negative by EIA and positive by PCR. The sensitivity of the kit for experimentally infected stool samples was approximately 5 x 10(7) bacteria/ml in the direct test, and 5 x 10(4) bacteria/ml after broth enrichment. EIA sensitivity and specificity were similar to those of CC and PCR. The diagnostic times were 18h for EIA, 3 days for PCR and 5 days for CC. Sensitivity, rapidity and ease of performance make this technique especially valuable for clinical diagnosis.

  1. Ambient-temperature incubation for the field detection of Escherichia coli in drinking water.

    Science.gov (United States)

    Brown, J; Stauber, C; Murphy, J L; Khan, A; Mu, T; Elliott, M; Sobsey, M D

    2011-04-01

    Escherichia coli is the pre-eminent microbiological indicator used to assess safety of drinking water globally. The cost and equipment requirements for processing samples by standard methods may limit the scale of water quality testing in technologically less developed countries and other resource-limited settings, however. We evaluate here the use of ambient-temperature incubation in detection of E. coli in drinking water samples as a potential cost-saving and convenience measure with applications in regions with high (>25°C) mean ambient temperatures.   This study includes data from three separate water quality assessments: two in Cambodia and one in the Dominican Republic. Field samples of household drinking water were processed in duplicate by membrane filtration (Cambodia), Petrifilm™ (Cambodia) or Colilert® (Dominican Republic) on selective media at both standard incubation temperature (35–37°C) and ambient temperature, using up to three dilutions and three replicates at each dilution. Matched sample sets were well correlated with 80% of samples (n = 1037) within risk-based microbial count strata (E. coli CFU 100 ml−1 counts of 1000), and a pooled coefficient of variation of 17% (95% CI 15–20%) for paired sample sets across all methods.   These results suggest that ambient-temperature incubation of E. coli in at least some settings may yield sufficiently robust data for water safety monitoring where laboratory or incubator access is limited.

  2. Detection and Classification of Live and Dead Escherichia coli by Laser-Induced Breakdown Spectroscopy

    Science.gov (United States)

    Sivakumar, P.; Fernández-Bravo, A.; Taleh, L.; Biddle, J.F.

    2015-01-01

    Abstract A common goal for astrobiology is to detect organic materials that may indicate the presence of life. However, organic materials alone may not be representative of currently living systems. Thus, it would be valuable to have a method with which to determine the health of living materials. Here, we present progress toward this goal by reporting on the application of laser-induced breakdown spectroscopy (LIBS) to study characteristics of live and dead cells using Escherichia coli (E. coli) strain K12 cells as a model organism since its growth and death in the laboratory are well understood. Our goal is to determine whether LIBS, in its femto- and/or nanosecond forms, could ascertain the state of a living organism. E. coli strain K12 cells were grown, collected, and exposed to one of two types of inactivation treatments: autoclaving and sonication. Cells were also kept alive as a control. We found that LIBS yields key information that allows for the discrimination of live and dead E. coli bacteria based on ionic shifts reflective of cell membrane integrity. Key Words: E. coli—Trace elements—Live and dead cells—Laser-induced breakdown spectroscopy—Atomic force microscopy. Astrobiology 15, 144–153. PMID:25683088

  3. Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

    Science.gov (United States)

    Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

    2018-04-01

    In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

  4. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Dept. de Biofisica e Biometria. Lab. de Radio e Fotobiologia]. E-mail: jcmattos@uerj.br

    2008-12-15

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl{sub 2}) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl{sub 2} in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl{sub 2} was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  5. Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method.

    Science.gov (United States)

    Hara-Kudo, Y; Konuma, H; Nakagawa, H; Kumagai, S

    2000-02-01

    We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.

  6. Detection of Escherichia coli in Biofilms from Pipe Samples and Coupons in Drinking Water Distribution Networks▿

    Science.gov (United States)

    Juhna, T.; Birzniece, D.; Larsson, S.; Zulenkovs, D.; Sharipo, A.; Azevedo, N. F.; Ménard-Szczebara, F.; Castagnet, S.; Féliers, C.; Keevil, C. W.

    2007-01-01

    Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of β-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration

  7. Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds.

    Science.gov (United States)

    Wu, F M; Beuchat, L R; Wells, J G; Slutsker, L; Doyle, M P; Swaminathan, B

    2001-12-04

    Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat. We evaluated the effectiveness of various treatments in releasing E. coli O157:H7 from seeds. The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0. 1, 3, 6, or 15 h), soaking temperature (4 or 21 degrees C), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined. Blending or stomaching for 1 or 2 min, and soaking for 1 h or longer at 4 or 21 degrees C, respectively, resulted in maximum release of E. coli O157:H7 from seeds. Soaking seeds at 37 degrees C for 15 h increased cell populations of E. coli O157:H7 by approximately 3.6 log10 CFU/g, likely due to bacterial growth. The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 degrees C, whereas at 37 degrees C, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 degrees C for up to 13 weeks and on sprouts derived from these seeds was compared. E. coli O157:H7 inoculated on seeds at 2.0 log10 CFU/g was detected after storage of seeds for up to 8 weeks at 37 degrees C and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 degrees C and was not isolated from sprouts produced from these seeds. Identifying seed treatment methods that enhance removal of E. coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds. With a combination of optimal conditions for detecting the pathogen, i.e. soaking seeds for 1 h and pummeling seeds for 1 min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E. coli O157

  8. Detection of Shiga Toxin-Producing Escherichia coli from Nonhuman Sources and Strain Typing.

    Science.gov (United States)

    Beutin, Lothar; Fach, Patrick

    2014-06-01

    Shiga toxin-producing Escherichia coli (STEC) strains are commonly found in the intestine of ruminant species of wild and domestic animals. Excretion of STEC with animal feces results in a broad contamination of food and the environment. Humans get infected with STEC through ingestion of contaminated food, by contact with the environment, and from STEC-excreting animals and humans. STEC strains can behave as human pathogens, and some of them, called enterohemorrhagic E. coli (EHEC), may cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Because of the diversity of STEC types, detection strategies for STEC and EHEC are based on the identification of Shiga toxins or the underlying genes. Cultural enrichment of STEC from test samples is needed for identification, and different protocols were developed for this purpose. Multiplex real-time PCR protocols (ISO/CEN TS13136 and USDA/FSIS MLG5B.01) have been developed to specifically identify EHEC by targeting the LEE (locus of enterocyte effacement)-encoded eae gene and genes for EHEC-associated O groups. The employment of more genetic markers (nle and CRISPR) is a future challenge for better identification of EHEC from any kinds of samples. The isolation of STEC or EHEC from a sample is required for confirmation, and different cultivation protocols and media for this purpose have been developed. Most STEC strains present in food, animals, and the environment are eae negative, but some of these strains can cause HC and HUS in humans as well. Phenotypic assays and molecular tools for typing EHEC and STEC strains are used to detect and characterize human pathogenic strains among members of the STEC group.

  9. Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Chen, Minghui; Yu, Zhibiao; Liu, Daofeng; Peng, Tao; Liu, Kun; Wang, Shuying; Xiong, Yonghua; Wei, Hua; Xu, Hengyi; Lai, Weihua

    2015-05-30

    Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14×10(3) CFU mL(-1). The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Phylogenetic typing and molecular detection of virulence factors of avian pathogenic Escherichia coli isolated from colibacillosis cases in Japanese quail

    OpenAIRE

    Alizade, Hesam; Ghanbarpour, Reza; Jajarami, Maziar; Askari, Asma

    2017-01-01

    Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is an economic threat to the poultry industry throughout the world. Some of the virulence genes may enhance the ability of E. coli isolates to grow in the tissues of broilers. The APEC strains are assigned to a few distinct phylogenetic groups. The purpose of the present study was to detect the virulence genes and phylogenetic groups of E. coli isolates from colibacillosis cases in Japanese quail in 2014 in Kerman, Iran. In the...

  11. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  12. Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates.

    Science.gov (United States)

    Paixão, A C; Ferreira, A C; Fontes, M; Themudo, P; Albuquerque, T; Soares, M C; Fevereiro, M; Martins, L; Corrêa de Sá, M I

    2016-07-01

    Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E.coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E.coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival. © 2016 Poultry Science Association Inc.

  13. Detection of Escherichia coli Serogroups O26 and O113 by PCR Amplification of the wzx and wzy Genes

    OpenAIRE

    DebRoy, Chitrita; Roberts, Elisabeth; Kundrat, James; Davis, Michael A.; Briggs, Connie E.; Fratamico, Pina M.

    2004-01-01

    PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple...

  14. Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of the wzx and wzy genes.

    Science.gov (United States)

    DebRoy, Chitrita; Roberts, Elisabeth; Kundrat, James; Davis, Michael A; Briggs, Connie E; Fratamico, Pina M

    2004-03-01

    PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as replacing antigen-based serotyping.

  15. Prevalence and characterization of diarrheagenic Escherichia coli isolated from adults and children in Mangalore, India

    Directory of Open Access Journals (Sweden)

    Veena A Shetty

    2012-01-01

    Full Text Available Background: Diarrheal diseases are a major cause of morbidity and mortality in resource-limited countries. Among the bacterial pathogens, diarrheagenic E. coli (DEC are most frequently implicated in cases of epidemic and endemic diarrhea worldwide. The objective of this study was to determine the prevalence of DEC in stool specimens from patients with acute diarrhea using polymerase chain reaction (PCR. Materials and Methods: Escherichia coli stool samples were collected from 115 hospitalized children and adults with acute diarrhea in Mangalore, a coastal city, in southern India. PCR amplification of eae, bfp, stx, ehx genes were used for detection of enteropathogenic (EPEC and shigatoxigenic E. coli (STEC, lt and st genes were used for enterotoxigenic E. coli (ETEC and astA gene for enteroaggregative E. coli (EAEC. Results: During the 24 month study period, of the 115 stool samples, DEC type was detected in 20 (17.4% using the PCR method. The most prevalent DEC was atypical EPEC accounting for 12 (10.4% cases followed by 4 cases of EAEC (3.4% and 4 of STEC (3.4%. No ETEC strains were isolated from any of the examined stool samples. Conclusion: This study suggests that the atypical EPEC are the newly emerging group among DEC stains in Southern India. Further studies are needed to evaluate the epidemiology and virulence properties of atypical EPEC strains.

  16. The use of a novel nanoLuc-based reporter phage for the detection of Escherichia coli O157:H7

    Science.gov (United States)

    Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food, as they are host-specific and able to differentiate liv...

  17. Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples.

    Science.gov (United States)

    Maheux, Andrée F; Picard, François J; Boissinot, Maurice; Bissonnette, Luc; Paradis, Sonia; Bergeron, Michel G

    2009-07-01

    The analytical performance of 9 different PCR primer sets designed to detect Escherichia coli and Shigella in water has been evaluated in terms of ubiquity, specificity, and analytical detection limit. Of the 9 PCR primer sets tested, only 3 of the 5 primer sets targeting uidA gene and the primer set targeting tuf gene amplified DNA from all E. coli strains tested. However, of those 4 primer sets, only the primer set targeting the tuf gene also amplified DNA from all Shigella strains tested. For the specificity, only the primer sets targeting the uidA gene were 100% specific although the primer sets targeting 16S rRNA, phoE, and tuf genes only amplified Escherichia fergusonii as non-specific target. Finally, the primer set targeting the 16S-ITS-23S gene region, was not specific as it amplified DNA from many other Enterobacteriaceae species. In summary, only the assay targeting the tuf gene detected all E. coli/Shigella strains tested in this study. However, if it becomes important to discriminate between E. coli and E. fergusonii, assays targeting the uidA gene would represent a good choice although none of them were totally ubiquitous to detect of the presence of Shigella strains.

  18. Detection of Escherichia coli O157 and non-O157 Serogroups in ...

    African Journals Online (AJOL)

    Cattle have remained an important primary reservoir of both Escherichia coli O157 and non-O157 serotypes implicated in many diarrhoeal disease outbreaks in animals and humans worldwide. The prevalence of these diarrhoeagenic agents in cattle and environmental sources has not been ascertained in Northeast ...

  19. Strategy for Accurate Detection of Escherichia Coli O157:H7 in Ground Pork Using a Lateral Flow Immunoassay.

    Science.gov (United States)

    Cheng, Song; Chen, Ming-Hui; Zhang, Gang-Gang; Yu, Zhi-Biao; Liu, Dao-Feng; Xiong, Yong-Hua; Wei, Hua; Lai, Wei-Hua

    2017-04-02

    Escherichia coli O157:H7 is known to cause serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. A gold nanoparticle lateral flow immunoassay (Au-LFIA) was used to detect Escherichia coli O157:H7 in ground pork samples. False-positive results were detected using Au-LFIA; a Citrobacter freundii strain was isolated from the ground pork samples and identified by using CHROmagar TM plates, API 20E, and 16S RNA sequencing. Since C. freundii showed cross-reactivity with E. coli O157:H7 when Au-LFIA test strips were used, a novel method combining modified enrichment with a lateral flow immunoassay for accurate and convenient detection of E. coli O157:H7 in ground pork was developed in this study to minimize these false positives. MacConkey broth was optimized for E. coli O157:H7 enrichment and C. freundii inhibition by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime. Using the proposed modified enrichment procedure, the false-positive rate of ground pork samples spiked with 100 CFU/g C. freundii decreased to 5%.

  20. Strategy for Accurate Detection of Escherichia Coli O157:H7 in Ground Pork Using a Lateral Flow Immunoassay

    Directory of Open Access Journals (Sweden)

    Song Cheng

    2017-04-01

    Full Text Available Escherichia coli O157:H7 is known to cause serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. A gold nanoparticle lateral flow immunoassay (Au-LFIA was used to detect Escherichia coli O157:H7 in ground pork samples. False-positive results were detected using Au-LFIA; a Citrobacter freundii strain was isolated from the ground pork samples and identified by using CHROmagarTM plates, API 20E, and 16S RNA sequencing. Since C. freundii showed cross-reactivity with E. coli O157:H7 when Au-LFIA test strips were used, a novel method combining modified enrichment with a lateral flow immunoassay for accurate and convenient detection of E. coli O157:H7 in ground pork was developed in this study to minimize these false positives. MacConkey broth was optimized for E. coli O157:H7 enrichment and C. freundii inhibition by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime. Using the proposed modified enrichment procedure, the false-positive rate of ground pork samples spiked with 100 CFU/g C. freundii decreased to 5%.

  1. Dual priming oligonucleotide (DPO)-based multiplex PCR assay for specific detection of four diarrhoeagenic Escherichia coli in food.

    Science.gov (United States)

    Xu, Y-G; Liu, Z-M; Guan, X-T; Cui, L-C; Li, S-L

    2015-08-01

    In this study, a dual priming oligonucleotide (DPO)-based multiplex PCR assay was developed for the specific detection of four foodborne diarrhoeagenic Escherichia coli (DEC) in food, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) O157:H7 and enteroinvasive E. coli (EIEC). Species-specific DPO primers were designed based on rfbE, LT, ipaH and bfpA genes for EHEC O157:H7, ETEC, EIEC and EPEC respectively. Our optimized DPO-based multiplex PCR assay was able to simultaneously detect these DEC from pure cultures, spiked food or environmental sample with an analytical detection limit of E. coli in food. © 2015 The Society for Applied Microbiology.

  2. Prevalence of diarrheagenic Escherichia coli virulence genes in the feces of slaughtered cattle, chickens, and pigs in Burkina Faso

    Science.gov (United States)

    Kagambèga, Assèta; Martikainen, Outi; Siitonen, Anja; Traoré, Alfred S; Barro, Nicolas; Haukka, Kaisa

    2012-01-01

    We investigated the prevalence of the virulence genes specific for five major pathogroups of diarrheagenic Escherichia coli (DEC) in primary cultures from feces of animals slaughtered for human consumption in Burkina Faso. For the study, 704 feces samples were collected from cattle (n = 304), chickens (n = 350), and pigs (n = 50) during carcass processing. The presence of the virulence-associated genes in the mixed bacterial cultures was assessed using 16-plex polymerase chain reaction (PCR). Virulence genes indicating presence of DEC were detected in 48% of the cattle, 48% of the chicken, and 68% of the pig feces samples. Virulence genes specific for different DECs were detected in the following percentages of the cattle, chicken, and pig feces samples: Shiga toxin-producing E. coli (STEC) in 37%, 6%, and 30%; enteropathogenic E. coli (EPEC) in 8%, 37%, and 32%; enterotoxigenic E. coli (ETEC) in 4%, 5%, and 18%; and enteroaggregative E. coli (EAEC) in 7%, 6%, and 32%. Enteroinvasive E. coli (EIEC) virulence genes were detected in 1% of chicken feces samples only. The study was the first of its kind in Burkina Faso and revealed the common occurrence of the diarrheal virulence genes in feces of food animals. This indicates that food animals are reservoirs of DEC that may contaminate meat because of the defective slaughter and storage conditions and pose a health risk to the consumers in Burkina Faso. PMID:23170227

  3. Selective detection of Escherichia coli by imaging of the light intensity transmitted through an optical disk

    Science.gov (United States)

    Shiramizu, Hideyuki; Kuroda, Chiaki; Ohki, Yoshimichi; Shima, Takayuki; Wang, Xiaomin; Fujimaki, Makoto

    2018-03-01

    We have developed an optical disk system for imaging transmitted light from Escherichia coli dispersed on an optical disk. When E. coli was stained using Bismarck brown, the transmittance was found to decrease in images obtained at λ = 405 nm. The results indicate that transmittance imaging is suitable for finding the difference in light intensity between stained and unstained E. coli, whereas the reflectance images were scarcely changed by staining. Therefore, E. coli can be selectively discriminated from abiotic contaminants using transmittance imaging.

  4. Detection of toxin genes in Escherichia coli isolated from normal dogs and dogs with diarrhea.

    OpenAIRE

    Hammermueller, J; Kruth, S; Prescott, J; Gyles, C

    1995-01-01

    The etiology of acute, nonviral diarrhea in dogs is poorly understood. Enterotoxigenic and verotoxigenic Escherichia coli are causal agents of diarrhea in humans, pigs, and cattle, but the association of these toxigenic E. coli with diarrhea in dogs has not been explored to a significant extent. In this study, DNA hybridization and PCR amplification were used to identify the frequency with which the genes for E. coli enterotoxins (STap, STb, and LTI) and verotoxins (VT1 and VT2) occur in asso...

  5. [Epidemiological characteristics of diarrheagenicEscherichia coliamong diarrhea outpatients in China, 2012-2015].

    Science.gov (United States)

    Zhang, Z K; Lai, S J; Yu, J X; Yang, W Q; Wang, X; Jing, H Q; Li, Z J; Yang, W Z

    2017-04-10

    Objective: To understand the epidemiological characteristics of diarrheagenic Escherichia (E.) coli (DEC) among diarrhea outpatients in China. Methods: Diarrhea surveillance program was conducted in outpatient and emergency departments from 170 hospitals that under the sentinel programs in 27 provinces, from 2012-2015. Clinical and epidemiological data regarding diarrhea patients were collected, with fecal specimens sampled and tested for DEC in 92 network-connected laboratories. Results: Among all the 46 721 diarrhea cases, 7.7 % of them appeared DEC positive in those with geographic heterogeneity. In 2 982 cases (6.4 % ) with available data on PCR subtypes of DEC, enteroaggregative E. coli (EAEC, 1 205 cases, 40.4 % ) appeared the most commonly seen pathogens, followed by enteropathogenic E. coli (EPEC, 815 cases, 27.3 % ), and enterotoxigenic E.coli (ETEC, 653 cases, 21.9 % ). The highest positive rate of DEC was observed in outpatients of 25-34 years old (10.1 % ), living in the warm temperate zones (11.1 % ), and with mucous-like stool (9.4 % ). The positive rate of DEC showed a strong seasonal pattern, with peaks in summer, for all the subtypes. Conclusions: DEC seemed easy to be detected among diarrhea outpatients in China, with EAEC, EPEC and ETEC the most commonly identified subtypes. Epidemiological characteristics regarding the heterogeneities of DEC appeared different, in regions, age groups and seasons. Long-term surveillance programs should be strengthened to better understand the epidemiology of DEC, in China.

  6. A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei [College of Food Science and Engineering, Ocean University of China, Qingdao 266003 (China); Zhao, Shiming [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Mao, Yiping [Yueyang Institute for Food and Drug Control, Yueyang 430198 (China); Fang, Zhiyuan [Affiliated Tumor Hospital of Guangzhou Medical University, Guangzhou 510095 (China); Lu, Xuewen [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China); Zeng, Lingwen, E-mail: zeng6@yahoo.com [Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China)

    2015-02-25

    Highlights: • Limit of detection as low as 10 CFU mL{sup −1}Escherichia coli O157:H7. • No need of antibodies and substituted with aptamers. • Isothermal strand displacement amplification for signal amplification. • Results observed by the naked eye. • Great potential application in the area of food control. - Abstract: Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

  7. Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; Denayer, Sarah; Botteldoorn, Nadine; Dierick, Katelijne; De Keersmaecker, Sigrid C J; Roosens, Nancy H

    2018-04-01

    A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

  8. [A new method for the detection of coliforms and Escherichia coli in water intended for human consumption.].

    Science.gov (United States)

    Bonadonna, Lucia; Cataldo, Claudia; Chiaretti, Gianluca; Coccia, Annamaria; Semproni, Maurizio

    2005-01-01

    The ISO reference method, defined by the European Drinking Water Directive for the enumeration of total coliforms and Escherichia coli in water intended for human consumption, has various limitations, especially related to discrepancies observed with the new taxonomic classification of the coliform group. A study was therefore performed to compare the above reference method with another rapid method, the DST/Colilert, and to evaluate the phenotypical characteristics of isolated microrganisms. The ISO reference method failed to detect a significant proportion of coliforms and E. coli in water and furthermore, it enumerated microrganisms belonging to other groups. The DST/Colilert method was found instead to be a suitable alternative method for the detection of bacterial indicators.

  9. Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes.

    Science.gov (United States)

    Schiebel, Juliane; Böhm, Alexander; Nitschke, Jörg; Burdukiewicz, Michał; Weinreich, Jörg; Ali, Aamir; Roggenbuck, Dirk; Rödiger, Stefan; Schierack, Peter

    2017-12-15

    Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli ). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC. IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device

  10. Colonial morphology of Escherichia coli: impact of detection in clinical specimens

    Directory of Open Access Journals (Sweden)

    Lucia Barcella

    2016-06-01

    Full Text Available As part of bacteriological identification, the macroscopic observation of the colonies on a culture medium is a critical step as it allows the clinical microbiologist to conduct a primary screening of microorganisms and in some cases even to identify them. This study shows how the macroscopic observation of the colonies, in particular of the colonies of Escherichia coli isolated on MacConkey agar from various clinical specimens, allowed to identify 5 classes of morphological variants of the colonies or colonial morphology, thanks to the knowledge and the experience gained by the clinical microbiologist.

  11. A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

    Science.gov (United States)

    Balakrishnan, Baskar; Barizuddin, Syed; Wuliji, Tumen; El-Dweik, Majed

    2016-08-16

    Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples.

    Science.gov (United States)

    Yuan, Yanfang; Xu, Wentao; Zhai, Zhifang; Shi, Hui; Luo, Yunbo; Chen, Zhuojun; Huang, Kunlun

    2009-10-01

    Escherichia coli, Listeria monocytogenes, and Salmonella spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for E. coli O157, L. monocytogenes, and Salmonella spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.

  13. [Occurrence of Shiga toxigenic Escherichia coli strains in pigs and cattle at slaughterhouses in the Czech Republic in 2013].

    Science.gov (United States)

    Koláčková, Ivana; Házová, Klára; Skočková, Alena; Karpíšková, Renáta

    2014-06-01

    This study was performed in cooperation with the State Veterinary Administration (SVA) in order to monitor the occurrence of Shiga toxin-producing Escherichia coli isolates in swabs from the carcasses of pigs and cattle at slaughterhouses. From June to August 2013, SVA staff took 168 swabs from cattle and 318 from pigs at 157 different slaughters in the Czech Republic. Basic processing of the samples was carried out in the State Veterinary Institutes (SVIs) in Prague, Jihlava and Olomouc according to the methodical process coordinated by the National reference laboratory (NRL) for Escherichia coli (Czech Ministry of Agriculture). The procedure was based on the guideline ISO TS 13136. Out of the 486 swabs, twenty-two positive samples were detected. There were a total of 22 isolates of Shiga toxigenic E. coli (STEC) and 1 strain with the characteristic of enterohemorrhagic E. coli (EHEC). Genes typical for enteroaggregative E. coli (EAggEC) were not found in any of the isolates. Most STEC strains originated from pigs. The stx1 gene was detected twice (stx1a, stx1d) and the stx2 gene 13 times (12 times stx2e, once stx2a). Seven STEC isolates were detected from samples of cattle origin. One strain was stx1 (stx1a) -positive, the stx2 gene was found 6 times (4 stx2e, 1 stx2a and 1 stx2c). One isolate carried simultaneously both stx1a and stx2a. Each of the serogroups O91, O113 and O146 described as etiological agents of severe disease in humans were detected only once. None of these strains harbored additional virulence factors typical for strains causing serious illness. RESULTS of this study show the overall prevalence of Shiga toxigenic E. coli of 4.5 % and 0.2 % of enterohemorrhagic strains in the studied samples. Raw meat originating from local farms does not currently represent an important source of STEC for humans.

  14. PCR detection and serotyping of enterotoxigenic and shigatoxigenic Escherichia coli isolates obtained from chicken meat in Mumbai, India

    Directory of Open Access Journals (Sweden)

    R. J. Zende,

    2013-08-01

    Full Text Available Aim: Present study was undertaken to find out the frequency of few virulent genes and prevalence of related strains of Escherichia coli isolated from chicken meat obtained from chicken retail shops by Polymerase Chain Reaction (PCR.Materials and Methods: 66 samples of freshly slaughtered chicken meat were collected from 22 identified retail shops located at Mumbai city, randomly. Processed meat samples were cultured in EMB agar and presumptive colonies were confirmed by various biochemical tests. PCR method was accustomed for identification of the genes coding for heat-stable enterotoxin a (STa, heat labile enterotoxin (LT, shiga-like toxins 1 and 2 (SLT1 and SLT2. E. coli isolates were sent to National Salmonella and Escherichia Centre, CRI, Kasauli, HP, India for serotyping.Results: 11 (16.67% E. coli strains were isolated from 66 chicken meat samples. 3 (27.27% out of 11 harbored the gene for SLT2, and 2 (18.18% for STa. None of the strain contains SLT1 and LT genes. Serotypes detected were rough, O2, O20, O22, O102 each for one isolate and 6 isolates were untypable (UT.Conclusion: The results concluded that chicken meat samples analysed harbored genes for shiga like toxins and enterotoxins and different serotypes of E. coli. These findings indicating that regular monitoring of chicken meat is essential for this pathogen to prevent potential public health problems.

  15. Infection strategies of enteric pathogenic Escherichia coli.

    Science.gov (United States)

    Clements, Abigail; Young, Joanna C; Constantinou, Nicholas; Frankel, Gad

    2012-01-01

    Enteric Escherichia coli (E. coli) are both natural flora of humans and important pathogens causing significant morbidity and mortality worldwide. Traditionally enteric E. coli have been divided into 6 pathotypes, with further pathotypes often proposed. In this review we suggest expansion of the enteric E. coli into 8 pathotypes to include the emerging pathotypes of adherent invasive E. coli (AIEC) and Shiga-toxin producing enteroaggregative E. coli (STEAEC). The molecular mechanisms that allow enteric E. coli to colonize and cause disease in the human host are examined and for two of the pathotypes that express a type 3 secretion system (T3SS) we discuss the complex interplay between translocated effectors and manipulation of host cell signaling pathways that occurs during infection.

  16. [Virulence mechanisms of enteropathogenic Escherichia coli].

    Science.gov (United States)

    Farfán-García, Ana Elvira; Ariza-Rojas, Sandra Catherine; Vargas-Cárdenas, Fabiola Andrea; Vargas-Remolina, Lizeth Viviana

    2016-08-01

    Acute diarrheal disease (ADD) is a global public health problem, especially in developing countries and is one of the causes of mortality in children under five. ADD etiologic agents include viruses, bacteria and parasites in that order. Escherichia coli bacteria it is classified as a major diarrheagenic agent and transmitted by consuming contaminated water or undercooked foods. This review compiled updates on information virulence factors and pathogenic mechanisms involved in adhesion and colonization of seven pathotypes of E. coli called enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), shigatoxigenic E. coli (STEC), enteroaggregative E. coli (EAEC) and diffusely-adherent E. coli (DAEC). A final pathotype, adherent-invasive E. coli (AIEC) associated with Crohn's disease was also reviewed. The diarrheagenic pathotypes of E. coli affect different population groups and knowledge of the molecular mechanisms involved in the interaction with the human is important to guide research towards the development of vaccines and new tools for diagnosis and control.

  17. Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification.

    Science.gov (United States)

    Zhao, Xihong; Wang, Jun; Forghani, Fereidoun; Park, Joong-Hyun; Park, Myoung-Su; Seo, Kun-Ho; Oh, Deog-Hwan

    2013-12-01

    Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by 3.0 μg/ml PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.

  18. Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth.

    Science.gov (United States)

    Jasson, Vicky; Sampers, Imca; Botteldoorn, Nadine; López-Gálvez, Francisco; Baert, Leen; Denayer, Sarah; Rajkovic, Andreja; Habib, Ihab; De Zutter, Lieven; Debevere, Johan; Uyttendaele, Mieke

    2009-11-15

    A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.

  19. PCR for the Specific Detection of an Escherichia coli O157:H7 Laboratory Control Strain.

    Science.gov (United States)

    Knowles, Michael; Lambert, Dominic; Huszczynski, George; Gauthier, Martine; Blais, Burton W

    2015-09-01

    Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.

  20. Antimicrobial susceptibility and molecular detection of chloramphenicol and florfenicol resistance among Escherichia coli isolates from diseased chickens

    Science.gov (United States)

    Li, Xin-Sheng; Wang, Gui-Qin; Cui, Bao-An; Zhang, Su-Mei; Shen, Jian-Zhong

    2007-01-01

    Seventy Escherichia coli isolates recovered from diseased chickens diagnosed with colibacillosis in Henan Province, China, between 2004 and 2005 were characterized for antimicrobial susceptibility profiles via a broth doubling dilution method. Overall, the isolates displayed resistance to trimethoprim-sulfamethoxazole (100%), oxytetracycline (100%), ampicillin (83%), enrofloxacin (83%), and ciprofloxacin (81%), respectively. Among the phenicols, resistance was approximately 79% and 29% for chloramphenicol and florfenicol, respectively. Molecular detection revealed that the incidence rates of the floR, cmlA, cat1, cat2 and cat3 were 29, 31, 16, 13, and 0%, respectively. Additionally, 10% of the isolates were positive for both floR and cmlA. As these antimicrobial agents may potentially induce cross-resistance between animal and human bacterial pathogens, their prudent use in veterinary medicine is highly recommended. PMID:17679770

  1. First environmental sample containing plasmid-mediated colistin-resistant ESBL-producing Escherichia coli detected in Norway.

    Science.gov (United States)

    Jørgensen, Silje Bakken; Søraas, Arne; Arnesen, Lotte Stenfors; Leegaard, Truls; Sundsfjord, Arnfinn; Jenum, Pål A

    2017-09-01

    We hereby report the detection of the plasmid borne mcr-1 gene conferring colistin resistance in an extended-spectrum β-lactamase (ESBL) producing Escherichia coli ST10 strain retrieved from seawater at a public beach in Norway. The sample was collected in September 2010 and was investigated by whole-genome sequencing in 2016. This report illustrates that E. coli strains carrying plasmid-mediated colistin resistance genes have also reached areas where this drug is hardly used at all. Surveillance of colistin resistance in environmental, veterinary, and human strains is warranted also in countries where colistin resistance is rare in clinical settings. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  2. Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Possan, A.L. [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Menti, C. [Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Beltrami, M. [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Santos, A.D. [Instituto de Física, Universidade de São Paulo, São Paulo, SP (Brazil); Roesch-Ely, M. [Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Missell, F.P., E-mail: fmissell@yahoo.com [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil)

    2016-01-01

    Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembled monolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift. - Highlights: • Magnetoelastic biosensors to capture Escherichia coli were produced. • Surface roughness of biosensors was varied in the range R{sub a} = 0.3–0.52 μm. • Rough surfaces show faster response, polished surfaces have larger frequency shift.

  3. On-line detection of Escherichia coli intrusion in a pilot-scale drinking water distribution system.

    Science.gov (United States)

    Ikonen, Jenni; Pitkänen, Tarja; Kosse, Pascal; Ciszek, Robert; Kolehmainen, Mikko; Miettinen, Ilkka T

    2017-08-01

    Improvements in microbial drinking water quality monitoring are needed for the better control of drinking water distribution systems and for public health protection. Conventional water quality monitoring programmes are not always able to detect a microbial contamination of drinking water. In the drinking water production chain, in addition to the vulnerability of source waters, the distribution networks are prone to contamination. In this study, a pilot-scale drinking-water distribution network with an on-line monitoring system was utilized for detecting bacterial intrusion. During the experimental Escherichia coli intrusions, the contaminant was measured by applying a set of on-line sensors for electric conductivity (EC), pH, temperature (T), turbidity, UV-absorbance at 254 nm (UVAS SC) and with a device for particle counting. Monitored parameters were compared with the measured E. coli counts using the integral calculations of the detected peaks. EC measurement gave the strongest signal compared with the measured baseline during the E. coli intrusion. Integral calculations showed that the peaks in the EC, pH, T, turbidity and UVAS SC data were detected corresponding to the time predicted. However, the pH and temperature peaks detected were barely above the measured baseline and could easily be mixed with the background noise. The results indicate that on-line monitoring can be utilized for the rapid detection of microbial contaminants in the drinking water distribution system although the peak interpretation has to be performed carefully to avoid being mixed up with normal variations in the measurement data. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015

    DEFF Research Database (Denmark)

    Hasman, H.; Hammerum, A. M.; Hansen, F.

    2015-01-01

    The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131...

  5. Phenotypic and molecular detection of BLACTX-M gene extended-spectrum beta-lactamases in escherichia coli and klebsiella pneumoniae of north sumatera isolates

    Science.gov (United States)

    Hasibuan, Mirzan; Suryanto, Dwi; Lia Kusumawati, R.

    2018-03-01

    The application of antibiotics expanded-spectrum third-generation cephalosporin for the treatment of infectious diseases in hospitals is known contribute to increasing resistance due to the presence of the blaCTX-M gene in the bacteria producing ESBLs. This study was aimed to detect ESBLs, isolate phenotype and blaCTX-M genes on Escherichia coli and Klebsiella pneumoniae collected from H. Adam Malik Central Hospital. Phenotypes of the bacterial were detection using Vitek two compact, while the blaCTX-M genes were detection using polymerase chain reaction technique. The results showed that 85 (100%) isolates were ESBLs consisted of 41(48%) of Escherichia coli, and 44 (52%) of Klebsiella pneumoniae, respectively. blaCTX-M genes were detection in 62 (72.94%) of the isolates which 31 (36.47%) were Escherichia coli, and 31 (36.47%) of the isolates were Klebsiella pneumoniae, respectively. This study indicates the high prevalence of blaCTX-M genes in Escherichia coli and Klebsiella pneumoniea causing bacterial antibiotic resistance.

  6. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

    Science.gov (United States)

    Kuwayama, Masaru; Shigemoto, Naoki; Oohara, Sachiko; Tanizawa, Yukie; Yamada, Hiroko; Takeda, Yoshihiro; Matsuo, Takeshi; Fukuda, Shinji

    2011-07-01

    We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Detection and characterization of Escherichia coli O157:H7 from feral pigeon in Qom province, Iran

    Directory of Open Access Journals (Sweden)

    Hossein Esameili

    2015-02-01

    Full Text Available Objective: To detect and characterize Escherichia coli (E. coli O157:H7 in feral pigeons in Qom province, Iran. Methods: In this survey, 290 cloacal samples were obtained from trapped feral pigeons in Qom province. Microbiological, biochemical and serological examinations were done to detect the E. coli O157:H7. Isolates were subjected to multiplex polymerase chain reaction for the detection of stx1, stx2, eaeA and hlyA genes. Results: Four samples (1.38% were positive for E. coli O157:H7 by using O157 and H7 antisera and only one E. coli O157:H7 strain isolated showed the presence of stx1, stx2, eaeA and hlyA genes. Conclusions: The results of present survey revealed that feral pigeons in Qom province had the potential to be a reservoir of E. coli O157:H7. The low prevalence of E. coli O157:H7 can be attributed to sampling each pigeon just once and fecal culture limits, and true prevalence of the E. coli O157:H7 might be higher than our findings.

  8. Occurrence of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae isolates.

    Directory of Open Access Journals (Sweden)

    Peng Wang

    Full Text Available Adequate detection of the production of carbapenemase in Enterobacteriaceae isolates is crucial for infection control measures and the appropriate choice of antimicrobial therapy. In this study, we investigated the frequency of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae clinical isolates by the modified Hodge test (MHT. Three hundred and one E. coli and K. pneumoniae clinical isolates were investigated. All produced extended spectrum β-lactamases (ESBLs but were susceptible to carbapenems. Antimicrobial susceptibility testing was performed by the disk diffusion and agar dilution methods. The MHT was performed using the standard inoculum of test organisms recommended by the CLSI. Genes that encoded ESBLs and carbapenemases were identified by PCR and DNA sequencing. Among the 301 clinical isolates, none of the isolates conformed to the criteria for carbapenemase screening recommended by the CLSI. The susceptibility rates for imipenem, meropenem, and ertapenem all were 100.0%, 100.0%, and 100.0%, respectively. Of the 301 E. coli and K. pneumoniae isolates, none produced carbapenemase. The MHT gave a positive result for 3.3% (10/301 of the isolates. False positive results can occur when the MHT is used to detect carbapenemase in ESBL-producing isolates and clinical laboratories must be aware of this fact.

  9. Prevalence and Antibiogram Profiling of Escherichia coli Pathotypes Isolated from the Kat River and the Fort Beaufort Abstraction Water

    Directory of Open Access Journals (Sweden)

    Nolonwabo Nontongana

    2014-08-01

    Full Text Available Escherichia coli is a widespread bacterium encompassing a variety of strains, ranging from highly pathogenic strains, causing worldwide outbreaks of severe diseases to avirulent, well characterized safe laboratory strains. This study evaluated the prevalence and antibiogram profiles of E. coli pathotypes isolated from the Kat River and Fort Beaufort abstraction water. A total of 171 out of 278 confirmed E. coli isolates were positive for at least one pathogenic determinant and these included enteropathogenic E. coli (6%, enterotoxigenic E. coli (47%, uropathogenic E. coli (2%, neonatal meningitis E. coli (5%, diffusely adherent E. coli (1% and enterohaemorrhagic E. coli (1%. Interestingly, enteroinvasive and enteroaggregative E. coli were not detected. The phenotypic antibiogram profiles of the isolates revealed that all were resistant to penicillin G, while 98% and 38% of the pathotypes were resistant to ampicillin and trimethoprim-sulphamethoxazole, respectively. About 8% of the isolates were resistant to streptomycin. More than half of the isolates exhibited multiple antibiotic resistance with 44% being resistant to three antibiotics and 8% resistant to four antibiotics. We conclude that the Kat River is a reservoir of potentially virulent antibiotic resistant E. coli strains that can cause serious health risks to humans who drink raw water from this river, or in the case that consumption of treated drinking water coincides with failed drinking water processes.

  10. Method of Detecting Coliform Bacteria and Escherichia Coli Bacteria from Reflected Light

    Science.gov (United States)

    Vincent, Robert (Inventor)

    2013-01-01

    The present invention relates to a method of detecting coliform bacteria in water from reflected light and a method of detecting Eschericha Coli bacteria in water from reflected light, and also includes devices for the measurement, calculation and transmission of data relating to that method.

  11. Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples.

    Science.gov (United States)

    Song, Chunmei; Liu, Jinxin; Li, Jianwu; Liu, Qing

    2016-11-15

    A pattern of signal amplification lateral flow immunoassay (LFIA) for pathogen detection, which used fluorescein isothiocyanate (FITC) labeled antigen and antibody for dual FITC-LFIA was developed. Escherichia coli O157:H7 (E.coli O157:H7) was selected as the model analyte. In the signal amplification LFIA method, FITC was mixed with sample culture medium, with the presence of E.coli O157:H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation, creating a fluorescent antigen probe. This antigen probe was added to LFIA, which already contained E.coli O157:H7 monoclonal antibodies-FITC (McAb-E.coli O157:H7-FITC) dispersed in the conjugate pad. Another E.coli O157:H7 McAb was the test line, and goat anti-mouse IgG antibody was the control line in nitrocellulose (NC) membrane. The visual limit of detection (LOD) of the strip for qualitative detection was 10(5) CFU/mL while the LOD for semi-quantitative detection could down to 10(4) CFU/mL by using scanning reader. Signal amplification LFIA was perfectly applied to the detection of food samples with E.coli O157:H7. The LOD was substantially improved to 1 CFU/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 8 and 8h respectively. The results of this method was more sensitive by 10-fold than the conventional colloidal gold (CG) based strips and comparable to the traditional ELISA. This simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring food-borne pathogens in food-safety testing. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Genetic relationship of diarrheagenic Escherichia coli pathotypes among the enteropathogenic Escherichia coli O serogroup

    Directory of Open Access Journals (Sweden)

    Silvia Y Bando

    2007-03-01

    Full Text Available The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC and enterohemorrhagic E. coli (EHEC. These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.

  13. Detection and Molecular Characterization of Sorbitol Negative Shiga Toxigenic Escherichia Coli in Chicken from Northwest of Iran

    Directory of Open Access Journals (Sweden)

    Aram Mokarizadeh

    2011-09-01

    Full Text Available AbstractShiga toxin-producing Escherichia coli (STEC are food-borne pathogens primarily associated with the consumption of contaminated ground beef and are an important food safety concern worldwide. STEC has been found to produce a family of related cytotoxins known as Shiga toxins (Stxs. Shiga toxins have been classified into two major classes, Stx1 and Stx2. A single strains of STEC can produce Stx1, Stx2 (or its variants or both. The aims of this project were to determine the prevalence and molecular characteristics of STEC isolates from chicken flocks in Northwest of Iran. A total of 350 fecal samples from 28 broiler farms were screened for the presence of STEC by conventional culture methods and polymerase chain reaction (PCR. All samples were initially subjected to phenotypical analysis using the Sorbitol MacConkey agar plate for the detection of the sorbitol negative E. coli, and then for genotypic analysis, by multiplex PCR for detection of stx1and stx2 genes. STEC were isolated from 14 (4 % chicken fecal samples. To our knowledge, this is the first report of isolation of STEC from poultry in Iran. To conclude, this work revealed the presence STEC strains harboring stx1 and stx2 gene in healthy chicken fecal samples in Northwest of Iran suggesting they can play as an important potential source of contamination for people working on broiler farms or are in contact with chicken carcasses at meat processing plants.

  14. Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction.

    Science.gov (United States)

    Asano, Ryoki; Kubori, Kiyohide; Ozutsumi, Yuhei; Yamamoto, Nozomi; Otrawa, Kenichi; Nakai, Yutaka

    2011-01-01

    Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.

  15. Optical Detection of Paraoxon Using Single-Walled Carbon Nanotube Films with Attached Organophosphorus Hydrolase-Expressed Escherichia coli

    Directory of Open Access Journals (Sweden)

    Intae Kim

    2015-05-01

    Full Text Available In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

  16. Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs

    DEFF Research Database (Denmark)

    Jurado-Rabadan, Sonia; de la Fuente, Ricardo; Ruiz-Santa-Quiteria, Jose A.

    2014-01-01

    from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. Results: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene......Background: In Escherichia coli the genes involved in the acquisition of tetracycline resistance are mainly tet(A) and tet(B). In addition, tet(M) is the most common tetracycline resistance determinant in enterococci and it is associated with conjugative transposons and plasmids. Although tet......(M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates...

  17. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Science.gov (United States)

    Gutiérrez-Del-Río, Ignacio; Marín, Laura; Fernández, Javier; Álvarez San Millán, María; Ferrero, Francisco Javier; Valledor, Marta; Campo, Juan Carlos; Cobián, Natalia; Méndez, Ignacio; Lombó, Felipe

    2018-01-01

    Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins) failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking water.

  18. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Directory of Open Access Journals (Sweden)

    Ignacio Gutiérrez-Del-Río

    Full Text Available Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking

  19. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli.

    Science.gov (United States)

    Joensen, Katrine Grimstrup; Scheutz, Flemming; Lund, Ole; Hasman, Henrik; Kaas, Rolf S; Nielsen, Eva M; Aarestrup, Frank M

    2014-05-01

    Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

  20. Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia.

    Science.gov (United States)

    Bagus Wasito, Eddy; Shigemura, Katsumi; Osawa, Kayo; Fardah, Alpha; Kanaida, Akiho; Raharjo, Dadik; Kuntaman, K; Hadi, Usman; Harijono, Sugeng; Marto Sudarmo, Subijanto; Nakamura, Tatsuya; Shibayama, Keigo; Fujisawa, Masato; Shirakawa, Toshiro

    2017-07-24

    The purpose of this study was to investigate extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilities, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p < 0.0001) and the quinolones nalidixic acid (p=0.004) and ciprofloxacin (p < 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.

  1. MOLECULAR DETECTION OF ANTIBIOTIC-RESISTANCE DETERMINANTS IN ESCHERICHIA COLI ISOLATED FROM THE ENDANGERED AUSTRALIAN SEA LION (NEOPHOCA CINEREA).

    Science.gov (United States)

    Delport, Tiffany C; Harcourt, Robert G; Beaumont, Linda J; Webster, Koa N; Power, Michelle L

    2015-07-01

    Greater interaction between humans and wildlife populations poses significant risks of anthropogenic impact to natural ecosystems, especially in the marine environment. Understanding the spread of microorganisms at the marine interface is therefore important if we are to mitigate adverse effects on marine wildlife. We investigated the establishment of Escherichia coli in the endangered Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and captive sea lion populations. Fecal samples were collected from wild colonies March 2009-September 2010 and from captive individuals March 2011-May 2013. Using molecular screening, we assigned a phylotype to E. coli isolates and determined the presence of integrons, mobile genetic elements that capture gene cassettes conferring resistance to antimicrobial agents common in fecal coliforms. Group B2 was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, B1, and D also identified. Integrons were not observed in E. coli (n = 21) isolated from wild sea lions, but were identified in E. coli from captive animals (n = 16), from which class I integrases were detected in eight isolates. Sequencing of gene cassette arrays identified genes conferring resistance to streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II integrases were not detected in the E. coli isolates. The frequent detection in captive sea lions of E. coli with resistance genes commonly identified in human clinical cases suggests that conditions experienced in captivity may contribute to establishment. Identification of antibiotic resistance in the microbiota of Australian sea lions provides crucial information for disease management. Our data will inform conservation management strategies and provide a mechanism to monitor microorganism dissemination to sensitive pinniped populations.

  2. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

    Science.gov (United States)

    March, S B; Ratnam, S

    1986-05-01

    Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.

  3. Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR.

    Science.gov (United States)

    Liu, Yarui; Mustapha, Azlin

    2014-01-17

    Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water.

    Science.gov (United States)

    Namura, Mariko; Hijikata, Tomonori; Miyanaga, Kazuhiko; Tanji, Yasunori

    2008-01-01

    Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.

  5. Molecular Analysis of Cytolysin A (ClyA) in Pathogenic Escherichia coli Strains

    OpenAIRE

    Ludwig, Albrecht; von Rhein, Christine; Bauer, Susanne; Hüttinger, Christian; Goebel, Werner

    2004-01-01

    Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E. coli K-12. In this study we examined various clinical E. coli isolates with regard to the presence and integrity of clyA. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E. coli (STEC) strains, all 7 tested enteroinvasive E. coli (EIEC) strains, 6 of 8 enteroaggregative E. coli (EAEC) strains, and 4 of 7 tested e...

  6. Rapid Detection Of Escherichia coli Enterohemorragic (EHEC) Bacteria by PCR (Polymerase Chain Reaction) methods

    International Nuclear Information System (INIS)

    Sudrajat, Dadang; R, Maria Lina; Suhadi, F.

    2000-01-01

    A polymerase Chain Reaction (PCR) assay for detect presence of enterohemmoragic Eschericha coli O157:H7 was carried out. DNA was extracted from bacterial cells with CTBA-phenol-chloroform and precipitated with isopropanol. To test sensitivity of PCR amplifies reaction, serial dilutions of E. coli DNA solution were prepared bwtween 1 mu g-1 ng/mu l. A single pair oligonucleotide primer SLTI-F and SLTI-R derived from shiga-like-toxin genes was used in amplification method. The results shows that 1 ng/mu l of E. coli DNA could be detected using the primers SLTI-F and SLTI-R with the position of 140 bp DNA fragment

  7. Comparison of detection methods for extended-spectrum beta-lactamases in Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Ewelina Kałużna

    2014-06-01

    Full Text Available Introduction: Detection of extended-spectrum beta-lactamases (ESBLs could be a major challenge for microbiologists – the difficulties arise mainly from the phenotypic differences among strains.Materials and Methods: Evaluation of ESBLs was performed on 42 strains of E. coli by: 1 DDST on MHA, 2 DDST on MHA with cloxacillin, 3 CT on MHA, according to CLSI, 4 CT on MHA with cloxacillin, 5 Etest ESBL (AB Biodisk, 6 CHROMagarTM ESBL (GRASO, 7 ChromID® ESBL (bioMérieux, and 8 automatic system VITEK2 ESBL test (bioMérieux.Result: Positive results were obtained for 20 strains using method 1, for 18 strains using method 2, 17 by method 3, 14 by method 4, 11 by method 5, 39 by method 6, 40 by method 7, and 15 by method 8. Using Etest ESBL 6.0 non-determinable results were obtained. The most consistent results were obtained when comparing the results of method 3 with results of method 2 (97.6%, and comparing the results obtained using methods 3 and 8 (95.2%.Conclusions: Based on our study we conclude that the chromogenic media can only be used as a screening method for the detection of ESBLs in E. coli rods. Etest is less useful compared to other phenotype methods, due to the impossibility of obtaining results for all the tested strains. Adding cloxacillin to MHA does not increase the frequency of detection of ESBLs in E. coli strains. DDST seems to be the most reliable among phenotypic methods for the detection of ESBLs in E. coli rods.

  8. Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry

    Directory of Open Access Journals (Sweden)

    Bradley J. Hernlem

    2014-05-01

    Full Text Available Protozoa are known to harbor bacterial pathogens, alter their survival in the environment and make them hypervirulent. Rapid non-culture based detection methods are required to determine the environmental survival and transport of enteric pathogens from point sources such as dairies and feedlots to food crops grown in proximity. Grazing studies were performed on a soil isolate of Tetrahymena fed green fluorescent protein (GFP expressing Escherichia coli O157:H7 to determine the suitability of the use of such fluorescent prey bacteria to locate and sort bacterivorous protozoa by flow cytometry. In order to overcome autofluorescence of the target organism and to clearly discern Tetrahymena with ingested prey versus those without, a ratio of prey to host of at least 100:1 was determined to be preferable. Under these conditions, we successfully sorted the two populations using short 5 to 45 min exposures of the prey and verified the internalization of E. coli O157:H7 cells in protozoa by confocal microscopy. This technique can be easily adopted for environmental monitoring of rates of enteric pathogen destruction versus protection in protozoa.

  9. Detection of virulence factors and antimicrobial resistance patterns in shiga toxin-producing Escherichia coli isolates from sheep

    Directory of Open Access Journals (Sweden)

    Marcos R.A. Ferreira

    2015-09-01

    Full Text Available Abstract: In order to detect virulence factors in Shiga toxin-producing Escherichia coli (STEC isolates and investigate the antimicrobial resistance profile, rectal swabs were collected from healthy sheep of the races Santa Inês and Dorper. Of the 115 E. coli isolates obtained, 78.3% (90/115 were characterized as STEC, of which 52.2% (47/90 carried stx1 gene, 33.3% (30/90 stx2 and 14.5% (13/90 both genes. In search of virulence factors, 47.7% and 32.2% of the isolates carried the genes saa and cnf1. According to the analysis of the antimicrobial resistance profile, 83.3% (75/90 were resistant to at least one of the antibiotics tested. In phylogenetic classification grouped 24.4% (22/90 in group D (pathogenic, 32.2% (29/90 in group B1 (commensal and 43.3% (39/90 in group A (commensal. The presence of several virulence factors as well as the high number of multiresistant isolates found in this study support the statement that sheep are potential carriers of pathogens threatening public health.

  10. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-09-01

    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  11. DETECTION OF THERMAL SUBLETHAL INJURY IN ESCHERICHIA COLI VIA THE SELECTIVE MEDIUM PLATING TECHNIQUE: MECHANISMS AND IMPROVEMENTS

    Directory of Open Access Journals (Sweden)

    Laura Espina

    2016-08-01

    Full Text Available In food preservation, the selective medium plating technique (SMPT is commonly used in order to detect and quantify the amount of sublethally injured cells in their bacterial cytoplasmic membranes after inimical treatments. From an applicative point of view, this information is of use in the synergistic combination of different preservation technologies, so that cells that are sublethally injured after one or more processes can end up being entirely inactivated by other hurdle(s. However, little work has been done to explain the reasons for the inability of sublethally injured cells to outgrow in selective agar media (containing the osmolyte NaCl as a selective agent, whereas they are able to grow in non-selective agar media. This research could contribute to explain this technique’s limits. In the present paper, the performance of SMPT on Escherichia coli cells after heat treatments is explored by applying different selective agents in the recovery media, using several mutants lacking factors involved in osmoregulation, and also by examining the integrity of the cytoplasmic membrane. In view of the results, the possibility of a specific toxic effect of Na+ as the main mechanism under SMPT is discarded, and the same level of sublethal injury is detected using KCl instead of NaCl. The synthesis of the osmoprotectant trehalose determined the maximum osmotolerance of intact cells to the selective agents, but was not crucial in the quantification of sublethal injury. Moreover, the extent of sublethal injury detected via SMPT was directly correlated with the physical loss of integrity of the cell membrane as measured with the propidium iodide-exclusion technique when that dye was added before thermal treatments. The present work confirms the adequacy of SMPT as a tool for detecting the occurrence and quantity of sublethally injured cells and thus, for efficiently designing combined preservation treatments. Additionally, we propose the combination

  12. Evaluation of real time PCR assays for the detection and enumeration of enterohemorrhagic Escherichia coli directly from cattle feces.

    Science.gov (United States)

    Luedtke, Brandon E; Bono, James L; Bosilevac, Joseph M

    2014-10-01

    Shiga toxin-producing Escherichia coli are a growing concern in the area of food safety, and the United States Department of Agriculture Food Safety and Inspection Service has identified the serotypes O26, O45, O103, O111, O121, O145, and O157 as adulterants in certain types of raw beef. The most relevant to human disease are the enterohemorrhagic E. coli (EHEC) strains that possess intimin (eae), Shiga toxin 1 and/or 2 (stx1-2), and in most cases the conserved pO157 or pO157 like virulence plasmid. Contamination of raw beef with EHEC is likely to occur via the transfer of cattle feces on hides to the carcass. To detect EHEC directly from cattle feces, we evaluated the utility of a multiplex real time PCR assay that targets the EHEC associated gene target ecf1 in combination with eae and stx1-2. Our assay had an increased sensitivity and provided a reliable limit of detection (LOD) of 1.25×10(3)colony-forming unitspermL (CFUs/mL) in an EHEC spiked fecal background. In addition, we evaluated the use of a duplex qPCR assay using ecf1 for the enumeration of total EHEC directly from cattle feces. The reliable limit of quantification (LOQ) was determined to be 1.25×10(3)CFUs/mL. Our assay requires minimal sample processing and provides LOD and LOQ of EHEC directly from cattle feces that are the lowest reported. The application of this assay towards the identification of cattle shedding EHEC at a level above 1.25×10(3)CFUs/mL could be a first line of defense in identifying cattle shedding these pathogens. Published by Elsevier B.V.

  13. Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives.

    Science.gov (United States)

    Shemer, Benjamin; Yagur-Kroll, Sharon; Hazan, Carina; Belkin, Shimshon

    2018-02-15

    DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in E. coli , have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells. IMPORTANCE The information provided in this article sheds new light both on the microbial biodegradability of nitroaromatic compounds and on the metabolic capabilities of E. coli By doing so, it also clarifies the pathway leading to the previously unexplained induction of the E. coli yqjF gene by 2,4-dinitrotoluene, an impurity that accompanies 2,4,6-trinitrotoluene (TNT)-based explosives. Our improved understanding of these processes will serve to molecularly enhance the performance of a previously described microbial bioreporter of buried landmines and other explosive devices, in which the yqjF gene promoter serves as the sensing element. Copyright © 2018 American Society for Microbiology.

  14. Genetic detection of extended-spectrum beta-lactamase-containing Escherichia coli isolates from birds of prey from Serra da Estrela Natural Reserve in Portugal.

    Science.gov (United States)

    Pinto, Luís; Radhouani, Hajer; Coelho, Céline; Martins da Costa, Paulo; Simões, Roméo; Brandão, Ricardo M L; Torres, Carmen; Igrejas, Gilberto; Poeta, Patrícia

    2010-06-01

    Extended-spectrum beta-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following beta-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

  15. Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal▿

    Science.gov (United States)

    Pinto, Luís; Radhouani, Hajer; Coelho, Céline; Martins da Costa, Paulo; Simões, Roméo; Brandão, Ricardo M. L.; Torres, Carmen; Igrejas, Gilberto; Poeta, Patrícia

    2010-01-01

    Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1). PMID:20418435

  16. Evaluation of BBL CHROMagar O157 versus sorbitol-MacConkey medium for routine detection of Escherichia coli O157 in a centralized regional clinical microbiology laboratory.

    Science.gov (United States)

    Church, D L; Emshey, D; Semeniuk, H; Lloyd, T; Pitout, J D

    2007-09-01

    The performance of BBL CHROMagar O157 (CHROM) versus that of sorbitol-MacConkey (SMAC) media for detection of Escherichia coli O157 was determined for a 3-month period. Results for 27/3,116 (0.9%) stool cultures were positive. CHROM had a higher sensitivity (96.30%) and negative predictive value (100%) and a better diagnostic efficiency than SMAC. Labor and material costs decreased when CHROM was used.

  17. Prevalence of virulence genes associated with pathogenic Escherichia coli strains isolated from domestically harvested rainwater during low- and high-rainfall periods.

    Science.gov (United States)

    Dobrowsky, P H; van Deventer, A; De Kwaadsteniet, M; Ndlovu, T; Khan, S; Cloete, T E; Khan, W

    2014-03-01

    The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.

  18. The effect of Enterococcus faecium M74 feed additive on the extended-spectrum beta-lactamases/AmpC-positive Escherichia coli faecal counts in pre-weaned dairy calves

    Directory of Open Access Journals (Sweden)

    Jana Šmídková

    2017-01-01

    Full Text Available The increasing occurrence of extended-spectrum beta-lactamases and/or AmpC-positive Escherichia coli among different species of food producing animals poses a threat to public health. The animal gut plays a key role in the development of antibiotic resistant bacteria, allowing the selection, multiplication and subsequent contamination of the farm environment. However, applicable procedures for reducing such bacteria on farms are currently unavailable. The present study was aimed to determine whether a probiotic administration to new-born dairy calves would reduce faecal shedding of extended-spectrum beta-lactamases and/or AmpC-positive Escherichia coli during the pre-weaning period. Ten randomly assigned new-born Holstein calves on a dairy farm with recent evidence of high occurrence of AmpC-positive Escherichia coli among calves were treated by a probiotic mix within 12 h after birth. Nine control calves were not treated. Faecal samples were collected from each calf daily on days 2 through 5, and then on days 7, 10, and 14. The faecal samples were cultured, and the mean numbers of cefotaxime-resistant Escherichia coli and confirmed enteroaggregative Escherichia coli were compared between the two groups. Results suggested that the Enterococcus faecium probiotic treatment (Enterococcus faecium M74, NCIMB 11181 of new-born calves did not reduce the enteroaggregative Escherichia coli counts in their faeces. There was no significant difference in the shedding of enteroaggregative Escherichia coli between the probiotic-treated and control calves throughout the two-week study period.

  19. Comparison of 4 label-based immunochromatographic assays for the detection of Escherichia coli O157:H7 in milk.

    Science.gov (United States)

    Luo, Kai; Hu, Liming; Guo, Qi; Wu, Chenghui; Wu, Songsong; Liu, Daofeng; Xiong, Yonghua; Lai, Weihua

    2017-07-01

    Immunochromatographic assays (ICA) are widely used to detect pathogens. In this study, we used traditional gold nanoparticles (GNP), quantum dots (QD), fluorescent nanoparticles (FNP), and europium (Eu) (III) chelate nanoparticles (EuNP) as ICA labels. We first compared the ability of the 4 ICA test strips to quantitatively detect Escherichia coli O157:H7 in milk. We then optimized various parameters influencing the ICA. The sensitivity to E. coli O157:H7 of the GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA was 2.5 × 10 4 , 5 × 10 3 , 1.0 × 10 3 , and 5.0 × 10 2 cfu mL -1 , respectively. The EuNP-ICA exhibited the highest sensitivity. The amounts of monoclonal antibodies (mAb) per GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA test strip were 0.16, 0.37, 0.04, and 0.10 μg, respectively. The corresponding coefficients of variation were 7.4 to 15.8%, 10.4 to 18.6%, 2.7 to 7.8%, and 6.9 to 10.5%, respectively. The FNP-ICA required the least mAb per test strip and had the best coefficient of variation. The linear ranges of GNP-ICA, QD-ICA, FNP-ICA, and EuNP-ICA were 1.0 × 10 4 to 1.0 × 10 6 , 2.5 × 10 3 to 1.0 × 10 6 , 2.5 × 10 2 to 2.5 × 10 5 , and 2.5 × 10 2 to 2.5 × 10 5 cfu mL -1 , respectively. The FNP-ICA and EuNP-ICA had wider linear ranges than GNP-ICA and QD-ICA. Additionally, the FNP-ICA and EuNP-ICA showed better tolerance than GNP-ICA and QD-ICA in the milk samples. The FNP-ICA and EuNP-ICA showed remarkable potential for detection of pathogens in milk. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  20. Detection of plasmid-borne extended-spectrum β-lactamase (ESBL) genes in Escherichia coli isolates from bovine mastitis.

    Science.gov (United States)

    Freitag, Christin; Michael, Geovana Brenner; Kadlec, Kristina; Hassel, Melanie; Schwarz, Stefan

    2017-02-01

    Extended-spectrum β-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009-2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n=16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n=3), ST117 (n=2), ST361 (n=1), ST362 (n=1), ST540 (n=1), ST1431 (n=2), ST1508 (n=1), and the novel ST5447 (n=1)] and the phylogenetic groups A (n=6), B1 (n=2), B2 (n=1) and D (n=3). ESBL genes bla CTX-M-1 (n=5), bla CTX-M-2 (n=2), bla CTX-M-14 and bla CTX-M-15 (n=4) were found on conjugative plasmids (35-225kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2+P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to β-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of β-lactam antibiotics. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Detection and characterization of extended-spectrum β-lactamase-producing Escherichia coli from chicken production chains in Nigeria.

    Science.gov (United States)

    Ojo, Olufemi E; Schwarz, Stefan; Michael, Geovana B

    2016-10-15

    A total of 405 Escherichia coli from the chicken production chains in Nigeria were investigated for ESBL-production and 4 isolates were identified as ESBL producers. They were characterized by XbaI-PFGE, multilocus sequence typing (MLST), phylotyping, sequencing of porin and regulatory genes and of the regulatory region of chromosomal ampC genes. Transformed ESBL gene-carrying plasmids were characterized by S1-nuclease, replicon typing, conjugation, digestion and PCRs for detection of the genetic environment of ESBL genes. Susceptibility testing, PCRs for the resistance genes, integrons, and the DNA microarray were performed with both, the original isolates and the transformants. All ESBL-producing isolates harboured bla CTX-M-15 genes located on non-conjugative plasmids (120-155kb). Three isolates with closely related/indistinguishable XbaI-patterns belonged to phylogroup A, and MLST sequence type ST10 and the fourth to phylogroup D and ST405. Resistance to aminoglycosides, sulfonamides/trimethoprim, quinolones, and tetracyclines were seen in all isolates. Incompatibility group IncFIB bla CTX-M-15 -carrying plasmids were detected in the three related isolates which carried also a class 1 integron (aadA2-orfF-dfrA12) and the resistance genes bla OXA-1 , bla TEM-1 , aac(3')-IIa, aac(6')-Ib-cr, sul1, sul2, and tet(A). The IncFIA-IncFIB-IncI1 bla CTX-M-15 -carrying plasmid harboured additionally the resistance genes aac(3')-IIa and tet(B). The bla CTX-M-15 genes were associated with ISEcp1 and Δorf477. ESBL-producing isolates showed elevated MICs to cefoxitin (16-64mg/L) and ertapenem MICs (0.5-2.0mg/L) mainly due to alterations in the porin genes. The virulence genes astA and prfB were detected. Although a low prevalence of ESBL-producing isolates was found, co-located resistance genes on the ESBL gene-carrying plasmids may facilitate the dissemination of them. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals.

    Science.gov (United States)

    Abraham, Sam; Jordan, David; Wong, Hui S; Johnson, James R; Toleman, Mark A; Wakeham, David L; Gordon, David M; Turnidge, John D; Mollinger, Joanne L; Gibson, Justine S; Trott, Darren J

    2015-12-01

    This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n=324) from Australian food-producing animals [cattle (n=169), porcine (n=114), poultry (n=32) and sheep (n=9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0-88.6%), ampicillin (9.4-71.1%), trimethoprim/sulfamethoxazole (11.1-67.5%) and streptomycin (21.9-69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of bla CMY-2 . This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages. Copyright © 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights

  3. Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads

    Science.gov (United States)

    Luciani, Mirella; Di Febo, Tiziana; Zilli, Katiuscia; Di Giannatale, Elisabetta; Armillotta, Gisella; Manna, Laura; Minelli, Fabio; Tittarelli, Manuela; Caprioli, Alfredo

    2016-01-01

    Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 103 E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 102 E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 101 E. coli O104:H4 initial load). The specificity was 100%. PMID:27379071

  4. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    Science.gov (United States)

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

  5. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

    Science.gov (United States)

    Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

  6. Detection of Shiga-like toxin producing Escherichia coli from raw milk cheeses produced in Wallonia [Belgium].

    Directory of Open Access Journals (Sweden)

    El-Lioui M.

    1999-01-01

    Full Text Available Shiga-like toxin Escherichia coli (STEC implicated in aqueous diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome, has become a serious health problem in various countries. In Belgium, all cases are sporadic and no outbreak has been detected so far. Cattle are thought to be a reservoir for E. coli O157:H7, and many foodborne diseases have been associated with the consumption of minced beef, beefburgers and raw milk. Recently, foodborne outbreaks were concerned with different unusual foods such as acidic products. Although some data suggest that STEC are not prevalent within dairy products, the aim of this work was to assess the prevalence of E. coli O157 and non-O157 STEC in raw milk cheeses produced in the southern part of Belgium (Wallonia. For this purpose, 153 frozen samples of soft and semi-soft cheeses made with raw cow, ewe and goat milk were analysed for the presence of E. coli O157 and STEC. By using a dynabeads immunomagnetic separation technique (Dynabeads anti-E. coli O157, Dynal followed by streaking onto sorbitol MacConckey agar, no sample was found contaminated by E. coli O157 serotype. By using polymerase chain reaction achieved from a loopful of confluent bacterial material growing onto MacConckey agar, the use of consensus primers detected stx genes in 11.1/ of the samples but Shiga-like toxin producing strains could be isolated only in five of them (3.3/. The isolation rate seems to be optimum for samples with a thermotolerant coliform count arround or below 102 cfu per g. The five Shiga-like toxin isolates were identified as belonging to the species Hafnia alvei or Enterobacter amnigenius without any accessory virulence factors needed to cause illness. Nevertheless, because of the ability of STEC to survive adverse conditions and the possibility for commensal non-pathogenic enteric bacteria to become pathogenic, raw milk cheeses are to be considered at risk for foodborne STEC contamination.

  7. 3M™ Molecular detection system versus MALDI-TOF mass spectrometry and molecular techniques for the identification of Escherichia coli 0157:H7, Salmonella spp. &Listeria spp.

    Science.gov (United States)

    Loff, Marché; Mare, Louise; de Kwaadsteniet, Michele; Khan, Wesaal

    2014-06-01

    The aim of this study was to compare standard selective plating, conventional PCR (16S rRNA and species specific primers), MALDI-TOF MS and the 3M™ Molecular Detection System for the routine detection of the pathogens Listeria, Salmonella and Escherichia coli 0157:H7 in wastewater and river water samples. MALDI-TOF MS was able to positively identify 20/21 (95%) of the E. coli isolates obtained at genus and species level, while 16S rRNA sequencing only correctly identified 6/21 (28%) as E. coli strains. None of the presumptive positive Listeria spp. and Salmonella spp. isolates obtained by culturing on selective media were positively identified by MALDI-TOF and 16S rRNA analysis. The species-specific E. coli 0157:H7 PCR described in this present study, was not able to detect any E. coli 0157:H7 strains in the wastewater and river water samples analysed. However, E. coli strains, Listeria spp., L. monocytogenes and Salmonella spp. were detected using species specific PCR. Escherichia coli 0157:H7, Listeria spp. and Salmonella spp. were also sporadically detected throughout the sampling period in the wastewater and river water samples analysed by the 3M™ Molecular Detection System. MALDI-TOF MS, which is a simple, accurate and cost-effective detection method, efficiently identified the culturable organisms, while in the current study both species specific PCR (Listeria spp. and Salmonella spp.) and 3M™ Molecular Detection System could be utilised for the direct routine analysis of pathogens in water sources. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Detection, Virulence Gene Assessment and Antibiotic Resistance Pattern of O157 Enterohemorrhagic Escherichia coli in Tabriz, Iran.

    Science.gov (United States)

    Akhi, Mohammad Taghi; Ostadgavahi, Ali Toloue; Ghotaslou, Reza; Asgharzadeh, Mohammad; Pirzadeh, Tahereh; Sorayaei Sowmesarayi, Vida; Memar, Mohammad Yousef

    2015-11-01

    Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen and infection with this organism causes illnesses such as bloody diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Considering the lack of any information about the prevalence rate and the antibiotic resistance pattern of O157:H7 serotype in Tabriz, finding answers to the above mentioned subjects was among the goals of this study. Two hundred E. coli strains from diarrheal or non-diarrheal stools of outpatients and hospitalized cases in Tabriz Imam Reza hospital were isolated between September and December 2014 using MacConkey agar and standard biochemical tests and then cultured on sorbitol MacConkey agar. The sorbitol-negative isolates were confirmed as the O157 serotype using O157 antisera. A multiplex polymerase chain reaction (PCR) method was used for the detection of stx-1, stx-2, eae, and mdh genes and the antibiotic resistance pattern of these isolates was determined using Kirby-Bauer method and clinical and laboratory standards institute (CLSI) standards. Of the isolates 11 (5.5%) were sorbitol-negative, which were later analyzed by multiplex PCR and the results revealed that 2 (18.18%) isolates contained the stx-1 gene, 10 (90.91%) contained the stx-2 gene, and 5 (45.45%) contained the eae gene. The stx-2 and eae genes were the most commonly encountered virulence factors. All or most of the isolates were susceptible to ceftazidime (100%), gentamicin (100%), ciprofloxacin (100%), nalidixic acid (90.9%), trimetoprim sulfamethoxazole (90.9%), chloramphenicol (90.9%), ampicillin (81.8%), and cephalothin (72.7%). On the contrary, moderate susceptibility of the isolates to doxycycline (54.5%) was observed. Due to the low frequency of STEC O157 and the high susceptibility rates of the isolates to the tested antibiotics in this study, STEC O157 has not become a major problem in Tabriz yet, but comprehensive microbiological surveillance programs that provide early warning and limit

  9. Evaluation of immunomagnetic separation and PCR for the detection of Escherichia coli O157 in animal feces and meats

    NARCIS (Netherlands)

    Islam, M.A.; Heuvelink, A.E.; Talukder, K.A.; Zwietering, M.H.; Boer, de E.

    2006-01-01

    Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)-based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is

  10. Evaluation of an inexpensive growth medium for direct detection of Escherichia coli in temperate and sub-tropical waters

    CSIR Research Space (South Africa)

    Bain, RES

    2015-10-01

    Full Text Available prototrophic and auxotrophic variants of Escherichia coli and other Enterobacteriaceae commonly implicated in urinary tract infections. Diag- nostic Microbiology and Infectious Disease 22: 261–266. PMID: 8565414 38. McDaniels AE, Rice EW, Reyes AL, Johnson CH...

  11. Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry

    NARCIS (Netherlands)

    Dierikx, C.M.; Essen-Zandbergen, van A.; Veldman, K.T.; Smith, H.E.; Mevius, D.J.

    2010-01-01

    To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006

  12. Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark

    DEFF Research Database (Denmark)

    Hammerum, Anette Marie; Sandvag, Dorthe; Andersen, Sigrid R.

    2006-01-01

    The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n = 35), 20% (n = 38) and 26% (n = 161) of the E. coli...... isolates obtained from humans, pork and pigs, respectively, were resistant to sulphonamide. All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, sul3 and intl1 genes by PCR. The sul1 gene was detected in 40% (n = 14), 29% (n = 11) and 55% (n = 88...

  13. High prevalence of diarrheagenic Escherichia coli carrying toxin-encoding genes isolated from children and adults in southeastern Brazil.

    Science.gov (United States)

    Spano, Liliana Cruz; da Cunha, Keyla Fonseca; Monfardini, Mariane Vedovatti; de Cássia Bergamaschi Fonseca, Rita; Scaletsky, Isabel Christina Affonso

    2017-12-18

    Diarrheagenic Escherichia coli (DEC) are important bacterial causes of childhood diarrhea in Brazil, but its impact in adults is unknown. This study aimed at investigating DEC among children and adults living in endemic areas. A total of 327 stools specimens were collected from children (n = 141) and adults (n = 186) with diarrhea attending health centers. Diarrheagenic E. coli (DEC) were identified by their virulence genes (multiplex polymerase chain reaction) and HEp-2 cell adherence patterns. DEC were detected in 56 (40%) children and 74 (39%) adults; enteroaggregative E. coli (EAEC) (23%) was the most prevalent pathotype, followed by diffusely adherent E. coli (DAEC) (13%), and occurred at similar frequencies in both diarrheal groups. Atypical enteropathogenic E. coli (aEPEC) strains were recovered more frequently from children (6%) than from adults (1%). Twenty-six percent of the EAEC were classified as typical EAEC possessing aggR gene, and carried the aap gene. EAEC strains carrying aggR-aap-aatA genes were significantly more frequent among children than adults (p < 0.05). DAEC strains possessing Afa/Dr. genes were detected from children (10%) and adults (6%). EAEC and DAEC strains harboring genes for the EAST1 (astA), Pet, Pic, and Sat toxins were common in both diarrheal groups. The astA and the porcine AE/associated adhesin (paa) genes were found in most of aEPEC strains. High levels of resistance to antimicrobial drugs were found among DAEC and aEPEC isolates. The results show a high proportion of EAEC and DAEC carrying toxin-encoding genes among adults with diarrhea.

  14. The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O 157.

    Science.gov (United States)

    Kleanthous, H; Fry, N K; Smith, H R; Gross, R J; Rowe, B

    1988-10-01

    Using DNA probes specific for the genes encoding Vero cytotoxins 1 and 2 in hybridization experiments on faecal samples, Vero cytotoxin-producing Escherichia coli (VTEC) of serogroup O 157 were detected in 21 of 63 cases of haemorrhagic colitis, 9 of 31 cases of non-bloody diarrhoea and 14 of 68 cases of haemolytic uraemic syndrome. Compared with these results sorbitol-MacConkey agar in conjunction with a specific O 157 antiserum gave a sensitivity of 62% in haemorrhagic colitis, 56% in non-bloody diarrhoea and 57% in haemolytic uraemic syndrome. The specificity of this method was 100% in all three groups. This demonstrates that sorbitol-MacConkey agar is a useful screening method for the detection of VTEC of serogroup O 157 when used in conjunction with a specific homologous antiserum. However, this method does not detect VTEC belonging to other serogroups and such strains were found, particularly in cases of haemolytic uraemic syndrome.

  15. Fast detection of both O157 and non-O157 shiga-toxin producing Escherichia coli by real-time optical immunoassay.

    Science.gov (United States)

    Mondani, L; Delannoy, S; Mathey, R; Piat, F; Mercey, T; Slimani, S; Fach, P; Livache, T; Roupioz, Y

    2016-01-01

    Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact. This article presents a simple-to-operate immunoassay for the specific detection of Shiga-toxin producing Escherichia coli (STEC). This approach consists in the on-chip assay detection of viable cells on a specifically designed antibody microarray. By skipping any enrichment step and avoiding the use of labelling agent, this approach based on the Surface Plasmon Resonance imaging of the microarrays turns out to be much faster and more cost effective by comparison with standardized methods. © 2015 The Society for Applied Microbiology.

  16. Characterization of Diarrheagenic Escherichia coli Isolated in Organic Waste Products (Cattle Fecal Matter, Manure and, Slurry) from Cattle's Markets in Ouagadougou, Burkina Faso.

    Science.gov (United States)

    Bako, Evariste; Kagambèga, Assèta; Traore, Kuan Abdoulaye; Bagre, Touwendsida Serge; Ibrahim, Hadiza Bawa; Bouda, Soutongnooma Caroline; Bonkoungou, Isidore Juste Ouindgueta; Kaboré, Saidou; Zongo, Cheikna; Traore, Alfred Sababenejo; Barro, Nicolas

    2017-09-22

    Cattle farming can promote diarrheal disease transmission through waste, effluents or cattle fecal matter. The study aims to characterize the diarrheagenic Escherichia coli (DEC) isolated from cattle feces, manure in the composting process and slurry, collected from four cattle markets in Ouagadougou. A total of 585 samples (340 cattle feces, 200 slurries and 45 manures in the composting process) were collected from the four cattle markets between May 2015 and May 2016. A multiplex Polymerase Chain Reaction (PCR), namely 16-plex PCR, was used to screen simultaneously the virulence genes specific for shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC). DEC was detected in 10.76% of samples. ETEC was the most prevalent (9.91%). STEC and EAEC have been observed with the same rate (0.51%). ETEC were detected in 12.64% of cattle feces, in 6.66% of manure in the composting process and in 5% of slurry. STEC were detected in 0.58% of cattle feces and in 2.22% of manure in the composting process. EAEC was detected only in 1% of slurry and in 2.22% of manure in the composting process. ETEC strains were identified based on estIa gene and/or estIb gene and/or elt gene amplification. Of the 58 ETEC, 10.34% contained astA , 17.24% contained elt , 3.44% contained estIa and 79.31% contained estIb . The two positive EAEC strains contained only the aggR gene, and the third was positive only for the pic gene. The results show that effluent from cattle markets could contribute to the spreading of DEC in the environment in Burkina Faso.

  17. Characterization of Diarrheagenic Escherichia coli Isolated in Organic Waste Products (Cattle Fecal Matter, Manure and, Slurry from Cattle’s Markets in Ouagadougou, Burkina Faso

    Directory of Open Access Journals (Sweden)

    Evariste Bako

    2017-09-01

    Full Text Available Cattle farming can promote diarrheal disease transmission through waste, effluents or cattle fecal matter. The study aims to characterize the diarrheagenic Escherichia coli (DEC isolated from cattle feces, manure in the composting process and slurry, collected from four cattle markets in Ouagadougou. A total of 585 samples (340 cattle feces, 200 slurries and 45 manures in the composting process were collected from the four cattle markets between May 2015 and May 2016. A multiplex Polymerase Chain Reaction (PCR, namely 16-plex PCR, was used to screen simultaneously the virulence genes specific for shiga toxin-producing E. coli (STEC, enteropathogenic E. coli (EPEC, enterotoxigenic E. coli (ETEC, enteroinvasive E. coli (EIEC and enteroaggregative E. coli (EAEC. DEC was detected in 10.76% of samples. ETEC was the most prevalent (9.91%. STEC and EAEC have been observed with the same rate (0.51%. ETEC were detected in 12.64% of cattle feces, in 6.66% of manure in the composting process and in 5% of slurry. STEC were detected in 0.58% of cattle feces and in 2.22% of manure in the composting process. EAEC was detected only in 1% of slurry and in 2.22% of manure in the composting process. ETEC strains were identified based on estIa gene and/or estIb gene and/or elt gene amplification. Of the 58 ETEC, 10.34% contained astA, 17.24% contained elt, 3.44% contained estIa and 79.31% contained estIb. The two positive EAEC strains contained only the aggR gene, and the third was positive only for the pic gene. The results show that effluent from cattle markets could contribute to the spreading of DEC in the environment in Burkina Faso.

  18. Detection of Escherichia coli O157:H7 and Salmonella in ground beef by a bead-free quantum dot-facilitated isolation method.

    Science.gov (United States)

    Wang, Luxin; Wu, Chung-Shieh; Fan, Xudong; Mustapha, Azlin

    2012-05-01

    The aims of this study were to introduce a new immunological bead-free cell detection method using quantum dots (QDs) as reporter markers for foodborne pathogen detection. QDs are nanosized particles with long-term photostability, high quantum yield, broad absorption spectra, and narrow, symmetric emission and high signal-to-noise ratio. The chemical compound [(1-ethyl-3-3-dimethylaminopropyl) carbodiimide hydrochloride] (EDC) and protein A were used as crosslinkers for manufacturing QD-antibody conjugates. To minimize the inhibition of QD fluorescence by the magnetic beads, the beads were removed after the primary pathogen isolation and before fluorescence measurement. Detection signals were increased four-fold after employing the bead-free isolation method. With a 24-h enrichment, the bead-free QD-facilitated detection method was able to detect 10 CFU/g Escherichia coli O157:H7 and Salmonella from artificially contaminated ground beef. To our knowledge, this detection method is the first research that combined a new EDC-protein A QD-labeling technique and bead-free fluorescence measurement to detect E. coli O157:H7 and Salmonella in ground beef. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions

    International Nuclear Information System (INIS)

    Cupples, C.G.; Miller, J.H.

    1989-01-01

    We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains

  20. Label-Free 3D Ag Nanoflower-Based Electrochemical Immunosensor for the Detection of Escherichia coli O157:H7 Pathogens

    Science.gov (United States)

    Huang, He; Liu, Minghuan; Wang, Xiangsheng; Zhang, Wenjie; Yang, Da-Peng; Cui, Lianhua; Wang, Xiansong

    2016-11-01

    It is highly desirable to develop a rapid and simple method to detect pathogens. Combining nanomaterials with electrochemical techniques is an efficient way for pathogen detection. Herein, a novel 3D Ag nanoflower was prepared via a biomineralization method by using bovine serum albumin (BSA) as a template. It was adopted as a sensing interface to construct an electrochemical bacteria immunosensor for the rapid detection of foodborne pathogens Escherichia coli ( E. coli) O157:H7. Bacterial antibody was immobilized onto the surface of Ag nanoflowers through covalent conjugation. Electrochemical impedance spectroscopy (EIS) was used to detect and validate the resistance changes, where [Fe(CN)6]3-/4- acted as the redox probe. A linear relation between R et and E. coli concentration was obtained in the E. coli concentration range of 3.0 × 102-3.0 × 108 cfu mL-1. The as-prepared biosensor gave rise to an obvious response to E. coli but had no distinct response to Cronobacter sakazakii, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus albus, Lactobacillus easei, and Shigella flexneri, revealing a high selectivity for the detection of the pathogens down to 100 cfu mL-1 in a short time. We believe that this BSA-conjugated 3D Ag nanoflowers could be used as a powerful interface material with good conductivity and biocompatibility for improving pathogen detection and treatment in the field of medicine, environment, and food safety.

  1. Simultaneous Detection of Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes at a Very Low Level Using Simultaneous Enrichment Broth and Multichannel SPR Biosensor.

    Science.gov (United States)

    Zhang, Xiaoguang; Tsuji, Sachiko; Kitaoka, Hayato; Kobayashi, Hiroshi; Tamai, Mitsuru; Honjoh, Ken-Ichi; Miyamoto, Takahisa

    2017-10-01

    Detection of foodborne pathogens at very low levels is still a challenge. A custom-built multichannel surface plasmon resonance (SPR) biosensor and simultaneous enrichment broth (SEB) were used to develop a simultaneous detection method for 3 important foodborne pathogens, Escherichia coli O157:H7 (O157:H7), Salmonella enteritidis, and Listeria monocytogenes, at a very low level. These 3 foodborne pathogens at a very low level (14, 6, and 28 CFU/25 g (mL) for O157:H7, S. enteritidis, and L. monocytogenes, respectively) were inoculated in SEB and incubated at 37 ˚C for 24 h. Sample prepared from the simultaneous enrichment culture was analyzed using the multichannel SPR biosensor and sensor chip immobilized with polyclonal antibodies specific to each of the target pathogens. O157:H7, S. enteritidis, and L. monocytogenes in chicken were detected simultaneously at an inoculum dose of 14, 6, and 28 CFU/25 g, respectively. Our method using a custom-built multichannel SPR biosensor and enrichment in SEB is expected as a rapid and simultaneous detection method for low levels of O157:H7, S. enteritidis, and L. monocytogenes in food. Our method is expected as a rapid and simultaneous detection method for pathogens at very low levels. It has great potential for safety control of food and microbiological detection applications. © 2017 Institute of Food Technologists®.

  2. An Investigation of beta-lactam antibiotics resistance in Escherichia coli isolates and molecular detection of Escherichia coli O157:H7 in cage birds from Shahrekord, Iran

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    Hossein Tahmasby

    2014-04-01

    Full Text Available Introduction: Cage birds can harbor human pathogens and contribute to the transmission and spread of drug resistant infectious agents to human. Since many people are interested in keeping cage birds, present study was conducted in cage birds from Shahrekord to investigate the beta-lactam antibiotics resistant E. coli and molecular detection of E. coli O157:H7 that is responsible for outbreaks of human intestinal diseases and fatal haemolytic-uraemic syndrome worldwide. Materials and methods: Altogether 256 samples of cage birds (lovebirds, quails, nightingales, parrots, mynahs, goldfinchs, finches, kingbirds, peacocks, and pheasants faeces were collected with sterile cotton swabs from different areas of Shahrekord, Iran. Swabs were placed directly into Tryptone Soya Broth (TSB. In the laboratory, samples were streaked onto MacConkey agar and also Sorbitol MacConkey agar as selective plating media. Then, antibiogram tests were performed using disc diffusion method. Suspected colonies to E. coli O157:H7 were tested by polymerase chain reaction (PCR. Results: E. coli was isolated from 31 (12.1% out of 256 the samples. Resistance of isolates to Imipenem, Cefotaxime, Cefixime, Cefalexin, Amoxicillin, Penicillin G and Oxacillin was 0, 3.2, 16.1, 90.3, 100, 100 and 100% respectively. E. coli O157:H7 was not found in any samples. Discussion and conclusion: Although cage birds were not sourcee or carriers of E. coli O157:H7 in the studied region, they harbored beta-lactam antibiotics resistant E. coli and could be an important component of drug-resistant infections transmission from cage-birds to human, especially to kids and can pose a potential risk to human health. For this reason, it is recommended to make pet birds owners and general public aware of potential dangers of cage bird keeping.

  3. Seroprevalence and molecular epidemiology of EAST1 gene-carrying Escherichia coli from diarrheal patients and raw meats.

    Science.gov (United States)

    Sukkua, Kannika; Manothong, Somruthai; Sukhumungoon, Pharanai

    2017-03-31

    Several Escherichia coli pathotypes have been reported in Thailand; however, information on enteroaggregative heat-stable enterotoxin 1 (EAST1)-carrying E. coli (EAST1-EC) is insufficient. Previous reports show that consumption of raw meats causes diarrheagenic E. coli infections. In this study, we investigated the seroprevalence and genetic relationship of EAST1-EC from clinical and raw meat samples. Diarrheal patients and raw meat samples were investigated for the presence of EAST1-EC by performing polymerase chain reaction (PCR) to detect astA. Serotyping, antimicrobial susceptibility tests, and PCR-based phylogenetic group assay were performed. Molecular epidemiology of E. coli strains from clinical and raw meat samples was determined using repetitive element-PCR typing, BOX-PCR, and ERIC2-PCR. Results showed that 11.2% (17/152) of clinical samples and 53.3% (16/30) of raw meat samples had EAST1-EC. In all, 24 and 36 EAST1-EC strains were successfully isolated from 17 clinical and 16 raw meat samples, respectively. These strains had astA but did not possess the indicative genes of other E. coli pathotypes and were therefore classified as EAST1-EC. Most of these strains were multidrug resistant and were classified into nine serogroups. Molecular genotyping showed identical DNA fingerprint among EAST1-EC serotype O15 strains from clinical and raw chicken samples, suggesting that they were derived from the same bacterial clone. Our results indicated a high prevalence of multidrug-resistant EAST1-EC strains in clinical and environmental samples in Thailand belonging to nine serogroups. Moreover, the study highlighted the close association between infections caused by EAST1-EC serotype O15 and raw meat consumption.

  4. Fiber-Optic Biosensor Employing Alexa-Fluor Conjugated Antibody for Detection of Escherichia coli O157:H7 from Ground Beef in Four Hours

    Science.gov (United States)

    Geng, Tao; Uknalis, Joe; Tu, Su-I; Bhunia, Arun K.

    2006-01-01

    Fiber optic biosensor has a great potential to meet the need for rapid, sensitive, and real-time microbial detection systems. We developed an antibody-based fiber-optic biosensor to rapidly detect low levels of Escherichia coli O157:H7 cells in ground beef. The principle of the sensor is a sandwich immunoassay using an antibody which is specific for E. coli O157:H7. A polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction that served as a capture antibody. An Alexa Fluor 647 dye-labeled antibody to E. coli O157:H7 was used to detect cells and generate a specific fluorescent signal, which was acquired by launching a 635 nm laser-light from an Analyte-2000. Fluorescent molecules within several hundred nanometers of the fiber were excited by an evanescent wave, and a portion of the emission light from fluorescent dye transmitted by the fiber and collected by a photodetector at wavelengths of 670 to 710 nm quantitatively. This immunosensor was specific for E. coli O157:H7 compared with multiple other foodborne bacteria. In addition, the biosensor was able to detect as low as 103 CFU/ml pure cultured E. coli O157:H7 cells grown in culture broth. Artificially inoculated E. coli O157:H7 at concentration of 1 CFU/ml in ground beef could be detected by this method after only 4 hours of enrichment.

  5. Association of untypeable enteropathogenic Escherichia coli (EPEC) strains with persistent diarrhea in children from the region of lower Silesia in Poland.

    Science.gov (United States)

    Duda-Madej, Anna; Gościniak, Grazyna; Andrzejewska, Barbara; Duda, Anna Krystyna; Sobieszczańska, Beata

    2013-01-01

    Enteropathogenic Escherichia coli strains (EPEC) carrying the eae gene encoding intimin are divided into typical strains producing bundle forming pili, encoded by the bfpA gene, and atypical strains lacking the gene. In the study typical and atypical EPEC that did not agglutinated with EPEC polyvalent antisera but carrying virulence factors characteristic to other pathogenic E. coli i.e. diffusely adhering and enteroaggregative E. coli were isolated from 24 (43.6%) of 55 children > 10 years old with persistent diarrhea. These results indicated that non-typeable typical and atypical EPEC can contribute to chronic intestinal infections in teenagers.

  6. A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Qi H

    2011-11-01

    Full Text Available Hui Qi1, Zhen Zhong1, Han-Xin Zhou1, Chun-Yan Deng1, Hai Zhu2, Jin-Feng Li2, Xi-Li Wang2, Fu-Rong Li1,31Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People's Hospital, Jinan University, 2Shenzhen Bioeasy Biotechnologies Co, Ltd, 3Shenzhen Institute of Gerontology, Shenzhen, People's Republic of ChinaBackground: Escherichia coli O157:H7 (E. coli O157:H7 is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary.Methods: In the present study, immunomagnetic nanoparticles (IMPs were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA and used to establish highly sensitive and rapid kits (IMPs+ICA to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy.Results: The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 µg/mg. The sensitivity of ICA and IMPs+ICA was 105 colony-forming units/mL and 103 CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA.Conclusion: The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.Keywords: colloidal gold, immunomagnetic nanoparticles, Escherichia coli O157:H7, immunochromatographic assay

  7. [Hygienic-sanitary quality in abattoirs from Tucuman province, Argentina. Detection, isolation and characterization of Shiga toxin-producing Escherichia coli].

    Science.gov (United States)

    Pérez Terrazzino, Gabriela B; Condorí, Marina S; López Campo, Alejandro; Vega, Silvia; Carbonari, Carolina; Chinen, Isabel; Rivas, Marta; de Castillo, Marta C; Jure, María A

    Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), and the strategies to prevent the transmission of these microorganisms are concentrated in the slaughtering plant. The aim of this study was to evaluate the hygienic-sanitary quality and the frequency of detection of STEC in beef carcasses in abattoirs from Tucuman province. Two hundred and seventy four beef carcass sponges were processed; the count of generic E. coli was marginal in 9 (3,3%) of them. Escherichia coli O157 was isolated in 4 (1,4%) samples; 2 of which were characterized as stx 2c(vh-a) /eae/ehxA whereas the other 2 were non-toxigenic strains. Non-O157 E. coli ONT:H49, stx 2a /ehxA/saa was isolated from 1 sample (0,4%). In this work the quality of the analyzed product indicates that the good practices of manufacture are fulfilled in slaughtering facilities in Tucumán province. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  8. The detection of K88, K99 fimbrial antigen and enterotoxin genes of Escherichia coli isolated from piglets and calves with diarrhoea in Indonesia

    Directory of Open Access Journals (Sweden)

    Supar

    1996-03-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC strains cause diarrhoeal disease in piglets and calves in Indonesia. These strains possess two virulence factors namely attachment and enterotoxin antigens . These factors could be detected phenotypically and genetically. Haemolytic Escherichia coli (E coli isolates possessing K88 fimbrial antigen associated with 0-group 108 and 149. They were positive for K88 gene and demonstrated their ability to produce heat labile enterotoxin (LT and genetically were all positive for LT gene . Seventeen isolates ofE coli K88 which associated with 0-group 149 were positive forSTb gene, other O-serotypes were negative . Ten isolates of Ecoli K88 which associated with 0-group 108 possessed K88, K99, LT and STa genes, but negative for STb gene . However, phenotypically the K99 antigen and STa toxin were not expressed under laboratory conditions, the reason was not well understood . E. coli K99 strains isolated from calves wit h diarrhoea were all associated with 0-group 9 and produced STa toxin when tested by suckling mousse bioassay. The E. coli K99 calf isolates were all hybridized with K99 and STa gene only . It is likely that K99 gene is associated with STa gene . The DNA hybridization technique is more convenience to be used for confirmation diagnosis of colibacillosis, however, not all veterinary laboratories could perform these tests .

  9. Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan--Quantitative Shiga toxin detection from stool during EHEC outbreak.

    Science.gov (United States)

    Yamasaki, Eiki; Watahiki, Masanori; Isobe, Junko; Sata, Tetsutaro; Nair, G Balakrish; Kurazono, Hisao

    2015-10-27

    Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

  10. Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    Reischl, Udo; Youssef, Mohammad T.; Kilwinski, Jochen; Lehn, Norbert; Zhang, Wen Lan; Karch, Helge; Strockbine, Nancy A.

    2002-01-01

    PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains. PMID:12089277

  11. Detección de Escherichia coli O157: H7 en carne picada fresca y hamburguesas congeladas Escherichia coli O157: H7 detection in fresh ground beef and hamburgers

    Directory of Open Access Journals (Sweden)

    M. A. Marzocca

    2006-03-01

    Full Text Available Escherichia coli O157:H7 es un patógeno emergente asociado a enfermedades transmitidas por alimentos. En el año 1982 fue reconocido por primera vez, en los Estados Unidos, como causante de dos brotes de colitis hemorrágica. Hoy se sabe que la mayoría de los casos de síndrome urémico hemolítico son ocasionados por este microorganismo. El objetivo del trabajo fue detectar su presencia en carne picada fresca y hamburguesas congeladas, muestras obtenidas en puntos de venta de nuestra cadena de supermercados en un total de 37 y 43, respectivamente, en el período comprendido entre abril de 2003 y agosto de 2004. Las mismas fueron procesadas utilizando el caldo selectivo para enriquecimiento EC modificado conteniendo novobiocina, seguido de la aplicación de un método de inmunocaptura (TECRA E. COLI O157 IMMUNOCAPTURE TM ECOICM 20, y posterior aislamiento en agar MacConkey sorbitol suplementado con cefixima y telurito de potasio y un medio cromogénico. Las cepas sospechosas fueron caracterizadas a genotípicamente mediante la detección de los genes de virulencia stx1, stx2, eaeA y EHEC-hlyA por PCR e hibridación con sondas genéticas, b fenotípicamente mediante la determinación del serotipo, sensibilidad a los antimicrobianos por el método de difusión de Kirby-Bauer y producción de Stx por ensayo de citotoxicidad específica en células Vero. Se aisló E. coli O157:H7 en una sola muestra de carne picada fresca (2,7% caracterizada como gen eae (+/stx2/EHEC-hlyA.Escherichia coli O157:H7 is an emergent pathogen associated with food transmitted diseases. In 1982, Escherichia coli 0157:H7 was for the first time identified as the cause of two hemorrhagic colitis outbreaks in the United States. It is now well known that most cases of hemolytic uremic syndrome are caused by these bacteria. The objective of this work was to detect the microorganism in fresh ground beef and hamburgers. From April 2003 to August 2004 samples were taken at sale

  12. A new amperometric method for rapid detection of Escherichia coli density using a self-assembled monolayer-based bienzyme biosensor

    International Nuclear Information System (INIS)

    Tang Hui; Zhang Wen; Geng Ping; Wang Qingjiang; Jin Litong; Wu Zirong; Lou Min

    2006-01-01

    A new amperometric method was developed for rapid detection of Escherichia coli (E. coli) density using a bienzyme biosensor. The bienzyme biosensor was fabricated based on the covalent immobilization of laccase and horseradish peroxidase (HRP) at indium tin oxide (ITO) electrode by (3-aminopropyl) triethoxysilane (APTES) monolayer. The bienzyme biosensor showed a high sensitivity in determination of the polyphenolic compounds, which was microbially generated from the salicylic acid (SA) added into the culture medium during the course of E. coli metabolism. Since the amount of polyphenolic compounds depends on E. coli density, the bienzyme biosensor was applied for the rapid and high sensitive detection of E. coli density after the E. coli solution was incubated in culture medium with salicylic acid for 2.5 h at 37 deg. C. By chronoamperometry, the amplified response current was obtained at the bienzyme biosensor, due to the substrate recycling of the polyphenolic compounds driven by bienzyme-catalyzed oxidation and electrochemical reduction. The amplified response current at the biosensor was linear with the E. coli density ranging from 1.6 x 10 3 to 1.0 x 10 7 cells/mL. The bienzyme biosensor could detect the E. coli density with a detection limit of 9.7 x 10 2 cells/mL within 3 h

  13. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles

    Science.gov (United States)

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL-1 in PBS and 6.8 × 102 to 6.8 × 103 CFU mL-1 in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method. PMID:24864164

  14. An accelerated method for the detection of Extended-Spectrum B- Lactamases in urinary isolates of Escherichia Coli and Klebsiella pneumoniae

    International Nuclear Information System (INIS)

    Kader, Abdulrahman A.; Kumar, A.; Krishna, A.; Zaman, M.N.

    2006-01-01

    We prospectively studied an accelerated phenotypic method by incorporating the double disk synergy test in the standard Kirby-Bauer disk diffusion susceptibility testing, to evaluate a protocol for the rapid detection of extended of extended-spectrum B-lactamases (ESBL) in urinary isolates of Escherichia coli (E. coli) and Klebsiella, pneumoniae (K. pneumoniae). All ESBL-positive isolates were confirmed by the standard Clinical Laboratory Standards Institute (CLSI) confirmatory disk diffusion method. Between November 2004 and December 2005, a total of 6988 urine specimens were analyzed of which 776 (11%) showed significant growth. They included E. coli in 577 cases (74%) and K. pneumoniae in 199 (25.6%). Of these, 63 E. coli (8%) and 15 K. pneumoniae (7.5%) were positive for ESBL by the accelerated and CLSI methods. Compared to the standard CLSI method, the accelerated method reduced the ESBL detection time from two days to one day. We conclude that the accelerated ESBL detection technique used by us in this study is a reliable and rapid method for detecting ESBL in urinary isolates of E. coli and K. pneumoniae. (author)

  15. Improved sample preparation for direct quantitative detection of Escherichia coli O157 in soil using qPCR without pre-enrichment.

    Science.gov (United States)

    Highmore, Callum J; Rothwell, Steve D; Keevil, Charles W

    2017-07-01

    The prominence of fresh produce as a vehicle for foodborne pathogens such as enterohaemorrhagic Escherichia coli (EHEC) O157 is rising, where disease cases can cause hospitalization and in some cases death. This rise emphasises the necessity for accurate and sensitive methods for detection of pathogens in soil, potential sources of contamination of fresh produce. The complexity of the soil matrix has previously proven prohibitive to pathogen detection via molecular methods without the use of a culture enrichment step, thereby excluding the detection of viable but non-culturable cells. Here, a sample preparation procedure to facilitate a direct qPCR assay is developed for the detection of E. coli O157 in soil, bypassing culture steps in favour of sample separation through pulsification release and filtration. In sand and peat-based compost, the method is sensitive to 10 CFU g -1 soil. When testing soils from agricultural sites, it was found that several were qPCR positive for E. coli O157 while being culture-negative, with peat-based compost possessing a concentration of 200 tir gene copies per gram. This procedure offers a rapid, quantitative assessment of the potential presence of E. coli O157 in soils which can act as a prescreen of their suitability to grow fresh produce safely. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-Producing Escherichia coli among Indigenous Individuals in Malaysia

    Directory of Open Access Journals (Sweden)

    Cindy Shuan Ju Teh

    2014-01-01

    Full Text Available We have successfully developed a Loop-mediated isothermal amplification (LAMP assay that could specifically detect generic Escherichia coli (E. coli. This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26. The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12. We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.

  17. Validation of low-volume enrichment protocols for detection of Escherichia coli O157 in raw ground beef components, using commercial kits.

    Science.gov (United States)

    Ahmed, Imtiaz; Hughes, Denise; Jenson, Ian; Karalis, Tass

    2009-03-01

    Testing of beef destined for use in ground beef products for the presence of Escherichia coli O157:H7 has become an important cornerstone of control and verification activities within many meat supply chains. Validation of the ability of methods to detect low levels of E. coli O157:H7 is critical to confidence in test systems. Many rapid methods have been validated against standard cultural methods for 25-g samples. In this study, a number of previously validated enrichment broths and commercially available test kits were validated for the detection of low numbers of E. coli O157:H7 in 375-g samples of raw ground beef component matrices using 1 liter of enrichment broth (large-sample:low-volume enrichment protocol). Standard AOAC International methods for 25-g samples in 225 ml of enrichment broth, using the same media, incubation conditions, and test kits, were used as reference methods. No significant differences were detected in the ability of any of the tests to detect low levels of E. coli O157:H7 in samples of raw ground beef components when enriched according to standard or large-sample:low-volume enrichment protocols. The use of large-sample:low-volume enrichment protocols provides cost savings for media and logistical benefits when handling and incubating large numbers of samples.

  18. Electrochemical Genosensor To Detect Pathogenic Bacteria (Escherichia coli O157:H7) As Applied in Real Food Samples (Fresh Beef) To Improve Food Safety and Quality Control.

    Science.gov (United States)

    Abdalhai, Mandour H; Fernandes, António Maximiano; Xia, Xiaofeng; Musa, Abubakr; Ji, Jian; Sun, Xiulan

    2015-05-27

    The electrochemical genosensor is one of the most promising methods for the rapid and reliable detection of pathogenic bacteria. In a previous work, we performed an efficient electrochemical genosensor detection of Staphylococcus aureus by using lead sulfide nanoparticles (PbSNPs). As a continuation of this study, in the present work, the electrochemical genosensor was used to detect Escherichia coli O157:H7. The primer and probes were designed using NCBI database and Sigma-Aldrich primer and probe software. The capture and signalizing probes were modified by thiol (SH) and amine (NH2), respectively. Then, the signalizing probe was connected using cadmium sulfide nanoparticles (CdSNPs), which showed well-defined peaks after electrochemical detection. The genosensor was prepared by immobilization of complementary DNA on the gold electrode surface, which hybridizes with a specific fragment gene from pathogenic to make a sandwich structure. The conductivity and sensitivity of the sensor were increased by using multiwalled carbon nanotubes (MWCNT) that had been modified using chitosan deposited as a thin layer on the glass carbon electrode (GCE) surface, followed by a deposit of bismuth. The peak currents of E. coli O157:H7 correlated in a linear fashion with the concentration of tDNA. The detection limit was 1.97 × 10(-14) M, and the correlation coefficient was 0.989. A poorly defined current response was observed as the negative control and baseline. Our results showed high sensitivity and selectivity of the electrochemical DNA biosensor to the pathogenic bacteria E. coli O157:H7. The biosensor was also used to evaluate the detection of pathogen in real beef samples contaminated artificially. Compared with other electrochemical DNA biosensors, we conclude that this genosensor provides for very efficient detection of pathogenic bacteria. Therefore, this method may have potential application in food safety and related fields.

  19. A low cost technique for synthesis of gold nanoparticles using microwave heating and its application in signal amplification for detecting Escherichia Coli O157:H7 bacteria

    Science.gov (United States)

    Thanh Ngo, Vo Ke; Giang Nguyen, Dang; Phat Huynh, Trong; Lam, Quang Vinh

    2016-09-01

    In the present work a low cost technique for preparation of gold nanoparticles (AuNPs) using microwave heating was developed. The effect of different elements (precursor reagents, irradiation time, and microwave radiation power) on the final morphology of AuNPs obtained through the microwave assisted technique has been investigated. The characterization of the samples has been carried out by transmission electron microscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy, and powder x-ray diffraction. The results showed that to some extent the above-mentioned characterizations influenced the size of synthetized nanoparticles and application of microwave heating has many advantages such as low cost, rapid preparation and highly uniform particles. As an application in quartz crystal microbalance (QCM) immunosensor, AuNPs are conjugated with the Escherichia coli (E.coli) O157:H7 antibodies for signal amplification to detect E.coli O157:H7 bacteria residual in QCM system.

  20. Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

    Science.gov (United States)

    Giowanella, Melissa; Bozza, Angela; do Rocio Dalzoto, Patricia; Dionísio, Jair Alves; Andraus, Sumaia; Guimarães, Edson Luiz Gomes; Pimentel, Ida Chapaval

    2015-11-01

    Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 μg, streptomycin 10 μg, and ceftazidime 30 μg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

  1. Detection of Healthcare-Related Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Transmission Events Using Combined Genetic and Phenotypic Epidemiology.

    Directory of Open Access Journals (Sweden)

    Anne F Voor In 't Holt

    Full Text Available Since the year 2000 there has been a sharp increase in the prevalence of healthcare-related infections caused by extended-spectrum beta-lactamase (ESBL-producing Escherichia coli. However, the high community prevalence of ESBL-producing E. coli isolates means that many E. coli typing techniques may not be suitable for detecting E. coli transmission events. Therefore, we investigated if High-throughput MultiLocus Sequence Typing (HiMLST and/or Raman spectroscopy were suitable techniques for detecting recent E. coli transmission events.This study was conducted from January until December 2010 at Erasmus University Medical Center, Rotterdam, the Netherlands. Isolates were typed using HiMLST and Raman spectroscopy. A genetic cluster was defined as two or more patients carrying identical isolates. We used predefined definitions for epidemiological relatedness to assess healthcare-related transmission.We included 194 patients; strains of 112 patients were typed using HiMLST and strains of 194 patients were typed using Raman spectroscopy. Raman spectroscopy identified 16 clusters while HiMLST identified 10 clusters. However, no healthcare-related transmission events were detected. When combining data from both typing techniques, we identified eight clusters (n = 34 patients, as well as 78 patients with a non-cluster isolate. However, we could not detect any healthcare-related transmission in these 8 clusters.Although clusters were genetically detected using HiMLST and Raman spectroscopy, no definite epidemiological relationships could be demonstrated which makes the possibility of healthcare-related transmission events highly unlikely. Our results suggest that typing of ESBL-producing E. coli using HiMLST and/or Raman spectroscopy is not helpful in detecting E. coli healthcare-related transmission events.

  2. Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker.

    Science.gov (United States)

    Li, Baoguang; Chen, Jin-Qiang

    2012-08-01

    The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.

  3. Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

    Science.gov (United States)

    Chen, Jin-Qiang

    2012-01-01

    The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 107 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources. PMID:22635992

  4. Sensitive detection of viable Escherichia coli O157:H7 from foods using a luciferase-reporter phage phiV10lux.

    Science.gov (United States)

    Kim, Jinwoo; Kim, Minsik; Kim, Seongmi; Ryu, Sangryeol

    2017-08-02

    Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen. The luxCDABE operon was introduced into the phiV10 genome and allowed the engineered phage phiV10lux to generate bioluminescence proportional to the number of viable E. coli O157:H7 cells without any substrate addition. The phage phiV10lux was able to detect at least 1CFU/ml of E. coli O157:H7 in a pure culture within 40min after 5h of pre-incubation. In artificially contaminated romaine lettuce, apple juice (pH3.51), and ground beef, the reporter phage could detect approximately 10CFU/cm 2 , 13CFU/ml, and 17CFU/g of E. coli O157:H7, respectively. Taken together, the constructed reporter phage phiV10lux could be applied as a powerful tool for rapid and sensitive detection of live E. coli O157:H7 in foods. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Molecular detection of virulence genes and multi-drug resistance patterns in Escherichia coli (STEC) in clinical bovine mastitis: Alborz province, Iran.

    Science.gov (United States)

    Tavakoli, M; Pourtaghi, H

    2017-01-01

    The aim of this study was to identify virulence genes and antimicrobial resistance of Escherichia coli isolated from bovine clinical mastitis in dairy herds in Iran. Sampling was done from 86 inflamed quarters of dairy cows in 8 commercial farms of Alborz province, Iran in summer 2015. Shiga toxin-producing E. coli (STEC) virulence genes were detected by multiplex PCR and multi-drug resistance profiles were confirmed using disk diffusion method. Among 60 E. coli isolated from examined samples, 13 (21.6%) of them were STEC. The results of PCR assay showed that eaeA gene was carried by 4 (30.8%) of STEC isolates. Although stx1 in combination with eaeA gene was detected from 7 (53.8%) of STEC isolates, stx1 and stx2 genes were detected from only 1 (7.7%) of the examined samples. The result of the disk diffusion method showed that all E. coli isolates were resistant to penicillin, tylosin, oxytetracycline, erythromycin, ampicillin, streptomycin and neomycin. However all isolates were susceptible to enrofloxacin. Therefore, according to the results establishing a regular monitoring system for identification of cases with clinical mastitis and conducting antibiotic sensitivity tests are recommended.

  6. Detection of pap, sfa, afa, foc, and fim Adhesin-Encoding Operons in Uropathogenic Escherichia coli Isolates Collected From Patients With Urinary Tract Infection.

    Science.gov (United States)

    Rahdar, Masoud; Rashki, Ahmad; Miri, Hamid Reza; Rashki Ghalehnoo, Mehdi

    2015-08-01

    Uropathogenic Escherichia coli (UPEC) with its virulence factors is the most prevalent cause of urinary tract infection (UTI). This study aimed to determine the occurrence of fim, pap, sfa, and afa genes among 100 UPEC isolates collected from patients diagnosed with UTI. A total of 100 UPEC isolates were obtained from urine samples of patients with UTI. The prevalence of 5 virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc) and afimbrial adhesins (afa) were determined through PCR method. We also investigated the phylogenetic background of all isolates. In addition, the distribution of adhesin-encoding operons between the phylogroups was assessed. The prevalence of genes encoding for fimbrial adhesive systems was 95% for fim, 57% for pap, 16% for foc, and 81% for sfa. The operons encoding for afa afimbrial adhesins were identified in 12% of isolates. The various combinations of detected genes were designated as virulence patterns. The fim gene, which occurred in strains from all phylogenetic groups (A, B1, B2, and D) was evaluated and no significant differences were found among these groups. Conversely, significant differences were observed in relation to pap, afa, foc, and sfa operons. These results indicate that the PCR method is a powerful genotypic assay for the detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as epidemiological studies.

  7. Development of a serogroup-specific multiplex PCR assay to detect a set of Escherichia coli serogroups based on the identification of their O-antigen gene clusters.

    Science.gov (United States)

    Wang, Quan; Ruan, Xiaojuan; Wei, Dongmei; Hu, Zhidong; Wu, Lixia; Yu, Ting; Feng, Lu; Wang, Lei

    2010-10-01

    The Escherichia coli serogroups O115, O126, O137, O158, O165, and O173 are pathogenic strains associated with diarrhea. Molecular approaches such as PCR have been proven to be rapid, inexpensive, and accurate. The sequences of the O-antigen-processing genes wzx and wzy are specific for different O antigens and are generally used as the target genes for the detection and identification of E. coli strains belonging to different O serogroups. In this report, the O-antigen gene clusters of these 6 O serogroups were sequenced, and genes were identified on the basis of homology. By screening these sequences against all 186 E. coli and Shigella strains, we found that the sequences of the wzx and wzy genes were serogroup-specific, and 2 specific primer pairs for each serogroup were screened out. A multiplex PCR assay targeting all 6 serogroups was developed. Twenty-nine strains were used to validate the specificity of the assay. The detection sensitivity was 1ng genomic DNA. As the assay was shown to be accurate and sensitive, it can be used for the identification and detection of strains belonging to these serogroups in stool and other environmental samples after being isolated by culture. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  8. Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples.

    Science.gov (United States)

    Park, Si Hong; Hanning, Irene; Jarquin, Robin; Moore, Philip; Donoghue, Dan J; Donoghue, Ann M; Ricke, Steven C

    2011-03-01

    Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens.

    Science.gov (United States)

    Faron, Matthew L; Ledeboer, Nathan A; Connolly, Jessica; Granato, Paul A; Alkins, Brenda R; Dien Bard, Jennifer; Daly, Judy A; Young, Stephen; Buchan, Blake W

    2017-02-01

    The Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producing Escherichia coli (STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx 1 and stx 2 ), as well as the E. coli serotype O:157-specific marker rfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection of stx 1 and stx 2 and 95.7% sensitive and 99.3% specific for detection of E. coli serotype O:157. All specimens with false-positive results were found to contain stx 1 or stx 2 or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negative stx 1 or stx 2 results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive for stx by an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative for stx 1 and stx 2 following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens. Copyright © 2017 American Society for Microbiology.

  10. Paper-based magnetic nanoparticle-peptide probe for rapid and quantitative colorimetric detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Suaifan, Ghadeer A R Y; Alhogail, Sahar; Zourob, Mohammed

    2017-06-15

    There is a critical and urgent demand for a simple, rapid and specific qualitative and quantitative colorimetric biosensor for the detection of the food contaminant Escherichia coli O157:H7 (E. coli O157:H7) in complex food products due to the recent outbreaks of food-borne diseases. Traditional detection techniques are time-consuming, require expensive instrumentation and are labour-intensive. To overcome these limitations, a novel, ultra-rapid visual biosensor was developed based on the ability of E. coli O157:H7 proteases to change the optical response of a surface-modified, magnetic nanoparticle-specific (MNP-specific) peptide probe. Upon proteolysis, a gradual increase in the golden color of the sensor surface was visually observed. The intensification of color was correlated with the E. coli O157:H7 concentration. The color change resulting from the dissociation of the self-assembled monolayer (SAM) was detected by the naked eye and analysed using an image analysis software (ImageJ) for the purpose of quantitative detection. This biosensor demonstrated high sensitivity and applicability, with lower limits of detection of 12CFUmL -1 in broth samples and 30-300CFUmL -1 in spiked complex food matrices. In conclusion, this approach permits the use of a disposable biosensor chip that can be mass-produced at low cost and can be used not only by food manufacturers but also by regulatory agencies for better control of potential health risks associated with the consumption of contaminated foods. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

    Science.gov (United States)

    Timmons, Chris; Dobhal, Shefali; Fletcher, Jacqueline; Ma, Li Maria

    2013-04-01

    Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

  12. Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.

    Science.gov (United States)

    Miszczycha, Stéphane D; Ganet, Sarah; Duniere, Lysiane; Rozand, Christine; Loukiadis, Estelle; Thevenot-Sergentet, Delphine

    2012-08-01

    Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

  13. Detection and coexistence of six categories of resistance genes in Escherichia coli strains from chickens in Anhui Province, China

    Directory of Open Access Journals (Sweden)

    Lin Li

    2015-12-01

    Full Text Available The aim of this study was to characterise the prevalence of class 1 integrons and gene cassettes, tetracycline-resistance genes, phenicol-resistance genes, 16S rRNA methylase genes, extended-spectrum β-lactamase genes and plasmid-mediated fluoroquinolone resistance determinants in 184 Escherichia coli isolates from chickens in Anhui Province, China. Susceptibility to 15 antimicrobials was determined using broth micro-dilution. Polymerase chain reaction and DNA sequencing were used to characterise the molecular basis of the antibiotic resistance. High rates of antimicrobial resistance were observed; 131 out of the 184 (72.3% isolates were resistant to at least six antimicrobial agents. The prevalences of class 1 integrons, tetracycline-resistance genes, phenicol-resistance genes, 16S rRNA methylase genes, extended-spectrum β-lactamase genes and plasmid-mediated fluoroquinolone resistance determinants were 49.5, 17.4, 15.8, 0.5, 57.6 and 46.2%, respectively. In 82 isolates, 48 different kinds of coexistence of the different genes were identified. Statistical (χ2 analysis showed that the resistance to amoxicillin, doxycycline, florfenicol, ofloxacin and gentamicin had significant differences (P<0.01 or 0.01

  14. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    International Nuclear Information System (INIS)

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  15. Simple sensitive rapid detection of Escherichia coli O157:H7 in food samples by label-free immunofluorescence strip sensor.

    Science.gov (United States)

    Song, Chunmei; Li, Jianwu; Liu, Jinxin; Liu, Qing

    2016-08-15

    A simple, one-step, rapid method to detect Escherichia coli O157: H7 (E. coli O157: H7) using a label-free immunofluorescence strip sensor is presented. Fluorescein isothiocyanate (FITC) was added to the sample culture medium to prepare the fluorescent probe for the label-free strip sensor. With the presence of E. coli O157: H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation and maintain good affinity to the monoclonal antibodies (McAb) against E. coli O157: H7. The direct-type immunofluorescence strip sensor was based on the binding between fluorescent bacteria and the unlabeled McAb immobilized at the test line in nitrocellulose membrane (NC membrane) reaction zone. The visual limit of detection (LOD) of the strip for qualitative detection was 10(6)cells/mL while the LOD for semi-quantitative detection could go down to 10(5)cells/mL by using scanning reader. The LOD was substantially improved to 1cells/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 10 and 8h respectively, which was competitive to some current rapid E. coli O157: H7 detection methods. Besides the obvious advantages, including reduced detection time and operation procedures, the results of this method meet the various detection requirements for E. coli O157: H7 and are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich gold-labeled strips. This is the first report of semi-quantitative immunofluorescence strip for directly detecting foodborne pathogen using only one unlabeled antibody. All detections could be achieved in less than 5min. In addition, this simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring foodborne pathogens in food-safety testing. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application.

    Science.gov (United States)

    Chen, Qingliang; Shi, Xiaolu; Li, Yinghui; Jiang, Yixiang; Lin, Yiman; Qiu, Yaqun; Li, Qingge; Hu, Qinghua

    2014-07-15

    Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC. In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5'end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real-time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR. The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains. The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of

  17. Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

    Science.gov (United States)

    Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

    2014-06-01

    Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

  18. Predictive models for Escherichia coli concentrations at inland lake beaches and relationship of model variables to pathogen detection.

    Science.gov (United States)

    Francy, Donna S; Stelzer, Erin A; Duris, Joseph W; Brady, Amie M G; Harrison, John H; Johnson, Heather E; Ware, Michael W

    2013-03-01

    Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous day's E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium, adenovirus, eaeA (E. coli), ipaH (Shigella), and spvC (Salmonella) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.

  19. Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

    Directory of Open Access Journals (Sweden)

    Letícia B. Rocha

    2013-12-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC produce heat-labile (LT and/or heat-stable enterotoxins (ST. Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.

  20. EXPRESSION OF RECOMBINANT ANTIBODY FRAGMENT, ANTI BNP-SCFV ON THE PERIPLASM OF Escherichia coli FOR THE DETECTION OF HEART FAILURE

    Directory of Open Access Journals (Sweden)

    Shabarni Gaffar

    2017-05-01

    Full Text Available Basic natriuretic peptide (BNP is a polypeptide hormone consist of 32 amino acids that secreted by the heart ventricle to respond the excessive stretching of heart muscle cells. BNP can be used as prognostic marker for patients with heart failure. The presence of BNP in blood can be detected by BNP antibody, which is anti BNP-single chain variable fragment (Anti BNP-SCFV. The antibody is a combination of polypeptides between varying region on the heavy chain (VH and the light chain (VL of immunoglobulin. Anti BNP-SCFV will bind to BNP through the antigen-antibody interaction. Concentration of BNP in a patient’s blood can be detected through the interaction of BNP with Anti BNP-SCFV using immunosensor method. Production of recombinant Anti BNP-SCFV in Escherichia coli as host is reported in the present study. Anti BNP-SCFV was expressed in fusion form with OmpC signal peptide that direct the protein to a periplasmic space. Expression was performed under RhaBad promoter as control using L-rhamnose as inducer. SDS-PAGE characterization showed consistent band at 28 kDa, which was assumed as Anti BNP-SCFV. The optimum expression was found at four hours after induction with 4 mM inducer. Anti BNP-SCFV was secreted from the cell as characterized by the presence of the protein on periplasmic membrane and extracellular fraction.

  1. Microfluidic Integration of a Cloth-Based Hybridization Array System (CHAS) for Rapid, Colorimetric Detection of Enterohemorrhagic Escherichia coli (EHEC) Using an Articulated, Centrifugal Platform.

    Science.gov (United States)

    Geissler, Matthias; Clime, Liviu; Hoa, Xuyen D; Morton, Keith J; Hébert, Harold; Poncelet, Lucas; Mounier, Maxence; Deschênes, Mylène; Gauthier, Martine E; Huszczynski, George; Corneau, Nathalie; Blais, Burton W; Veres, Teodor

    2015-10-20

    We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rfbO157, fliCH7, vt1, and vt2 genes.

  2. Application of multiplex PCR assay based on uidR and fliCH7 genes for detection of Escherichia coli O157:H7 in milk.

    Science.gov (United States)

    Kumar, Ashwani; Grover, Sunita; Kumar Batish, Virender

    2013-01-01

    A highly sensitive and specific multiplex PCR assay has been developed to detect the presence of Escherichia coli O157:H7 from naturally contaminated raw milk samples within 10 h. The primers explored in the assay were targeted against the uidR gene specific for all types of E. coli and the fliCH7 gene specific for the h7 flagellar antigen of E. coli O157:H7. The multiplex PCR assay developed was found to be highly specific as it produced PCR products of 152 bp (E. coli specific) and 625 bp (E. coli O157:H7 specific). The assay was tested for its specificity against different serotypes of E. coli as well as other pathogenic strains like Salmonella, Shigella, Klebsiella, Enterobacter, Staphylococcus aureus, Lactobacillus and Lactococcus etc. When this multiplex PCR assay was directly applied to 24 raw milk samples collected from different sources, E. coli O157:H7 could be detected in one of the milk samples without 4 h enrichment in CT-SMAC broth and three samples after 4 h enrichment in CT-SMAC broth. However, all the pasteurized milk samples gave a negative signal for this organism.

  3. Detection of Class I and II integrons for the assessment of antibiotic and multidrug resistance among Escherichia coli isolates from agricultural irrigation waters in Bulacan, Philippines.

    Science.gov (United States)

    Paraoan, Cielo Emar M; Rivera, Windell L; Vital, Pierangeli G

    2017-05-04

    Contaminated irrigation water may greatly affect not only the quality of produce but also the people exposed to it. In this study, agricultural irrigation waters in Bulacan, Philippines were assessed and found to be contaminated with Escherichia coli (E. coli) ranging from 0.58 to 4.51 log 10 CFU/mL. A total of 79 isolates of E. coli were confirmed through polymerase chain reaction (PCR) amplifying the uidA gene and were tested for phenotypic resistance using 10 antimicrobials through the Kirby-Bauer disc diffusion method. Forty-six isolates (58.22%) were noted to be multidrug resistant (MDR) with high resistance rate to cephalothin, tetracycline, streptomycin, ampicillin, trimethoprim, nalidixic acid, and chloramphenicol. Moreover, this study also examined the prevalence of Class I and II integrons accounting to 67.39% and 17.39%, respectively, of the MDR E. coli strains using multiplex PCR. The results imply that the agricultural water used in Bulacan is contaminated with the fecal material of man or other animals present in the area, and the presence of MDR bacteria, which pose a potential threat to individuals in these areas, is alarming. In addition, detection of integrons could be a good marker for the identification of MDR isolates. Lastly, this study could develop strategies for the proper management of farming sites leading to the detection of food-borne pathogens and prevention of infectious diseases.

  4. Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase.

    Science.gov (United States)

    Alonso, C A; González-Barrio, D; Tenorio, Carmen; Ruiz-Fons, F; Torres, C

    2016-04-01

    Eighty-nine Escherichia coli isolates recovered from faeces of red deer and small mammals, cohabiting the same area, were analyzed to determine the prevalence and mechanisms of antimicrobial resistance and molecular typing. Antimicrobial resistance was detected in 6.7% of isolates, with resistances to tetracycline and quinolones being the most common. An E. coli strain carrying blaCTX-M-1 as well as other antibiotic resistant genes included in an unusual class 1 integron (Intl1-dfrA16-blaPSE-1-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26) was isolated from a deer. The blaCTX-M-1 gene was transferred by conjugation and transconjugants also acquired an IncN plasmid. This strain was typed as ST224, which seems to be well adapted to both clinical and environmental settings. The phylogenetic distribution of the 89 strains varied depending on the animal host. This work reveals low antimicrobial resistance levels among faecal E. coli from wild mammals, which reflects a lower selective pressure affecting these bacteria, compared to livestock. However, it is remarkable the detection of a multi-resistant ESBL-E. coli with an integron carrying clinically relevant antibiotic-resistance genes, which can contribute to the dissemination of resistance determinants among different ecosystems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2015-12-23

    Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10 CFU/25 g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325 g of food samples were spiked with 10 CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples. Copyright © 2015. Published by Elsevier B.V.

  6. Discrimination of enterohemorrhagic Escherichia coli (EHEC) from non-EHEC strains based on detection of various combinations of type III effector genes.

    Science.gov (United States)

    Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

    2013-10-01

    Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains.

  7. A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces.

    Science.gov (United States)

    Stratakos, A Ch; Linton, M; Millington, S; Grant, I R

    2017-03-01

    To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC). Primer sets were selected to amplify the phoA gene (all E. coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared with conventional culture. The limits of detection 50% (LOD 50 ) of the multiplex LAMP assay were 2·8 (95% CI 2·4-3·3), 3·2 (95% CI 2·5-3·9) and 2·8-3·2 (95% CI 2·1-3·5) log CFU per g for the phoA, stx1 and stx2 genes, respectively. When validated by testing retail beef and bovine faeces samples, good correlation between E. coli counts indicated by the LAMP assay and culture was observed; however, false-negative LAMP assay results were obtained for 12·5-14·7% of samples. A rapid, multiplex LAMP assay for direct quantification of E. coli and specific detection of VTEC in beef and faeces was successfully developed. Further optimisation of the assay would be needed to improve detection sensitivity. The multiplex LAMP assay represents a rapid alternative to culture for monitoring E. coli levels on beef for hygiene monitoring purposes, and, potentially, a method for detection of VTEC in beef and faeces. © 2016 The Society for Applied Microbiology.

  8. Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic Escherichia coli

    Science.gov (United States)

    Reischl, Udo; Youssef, Mohammad T.; Wolf, Hans; Hyytia-Trees, Eija; Strockbine, Nancy A.

    2004-01-01

    To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC. PMID:15364995

  9. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    Science.gov (United States)

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A Laboratory-Developed TaqMan Array Card for Simultaneous Detection of 19 Enteropathogens

    Science.gov (United States)

    Liu, Jie; Gratz, Jean; Amour, Caroline; Kibiki, Gibson; Becker, Stephen; Janaki, Lalitha; Verweij, Jaco J.; Taniuchi, Mami; Sobuz, Shihab U.; Haque, Rashidul; Haverstick, Doris M.

    2013-01-01

    The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and

  11. Detection of Escherichia coli in ready-to-eat fresh vegetables using broad-host-range recombinant phages.

    Science.gov (United States)

    Hoang, Hoang A; Quy, Nguyen T C; Chi, Nguyen V T

    2018-01-16

    To construct a simple method to detect E. coli in ready-to-eat fresh vegetables using broad-host-range recombinant phages. Firstly, a gene encoding Cytochrome c Peroxidase (CCP) chromogenic enzyme was inserted into genomes of wild-type phages IP008 and IP052 to produce recombinant phages IP008BK and IP052BK. They were then used in the production of the chromogenic enzyme (CCP) through infection into E. coli. The method was then examined in the colorimetric detection of E. coli K12 in broth, and its appearance was confirmed by a significant change in absorbance after a few min of the enzyme assay. Secondly, the protocol using the recombinant phages for detection of E. coli in vegetables, i.e. lettuce and mustard greens was investigated. A low E. coli concentration as 4 CFU g -1 vegetable was detected within 16.5 h that is of a shorter duration than agar-plate methods and in some commonly known phage-based methods. Existence of E. coli as a fecal contamination indicator in two types of ready-to-eat fresh vegetables, i.e. lettuce and mustard greens, can be identified by the broad-host-range recombinant phages. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. Detection of qnr, aac(6')-Ib-cr and qepA genes in Escherichia coli isolated from cooked meat products in Henan, China.

    Science.gov (United States)

    Jiang, Xiaobing; Yu, Tao; Wu, Nan; Meng, Hecheng; Shi, Lei

    2014-09-18

    Antimicrobial resistance in Escherichia coli has increased in recent years in China. Antimicrobial resistant isolates and resistance genes of E. coli can be transferred to humans through the food chain and this presents a public health risk. However, few studies have investigated the prevalence of antimicrobial resistance-encoding genes in E. coli isolated from food samples in China. The aim of this study was to investigate the presence of quinolone resistance genes (QRGs) and extended-spectrum β-lactamases (ESBLs) in E. coli isolated from cooked meat products in Henan, China. A total of 75 E. coli isolates (12.1%) were detected from 620 samples. High rates of resistance to the following drugs were observed: tetracycline (56.0%), trimethoprim/sulfamethoxazole (41.3%), streptomycin (29.3%), ampicillin (26.7%) and nalidixic acid (14.7%). Of the 75 isolates, QRGs were present in 10 isolates (13.3%), with qnr and aac(6')-Ib-cr detected alone or in combination in five (6.7%) and eight isolates (10.7%). The qnr genes detected in this study included qnrS (n=3) and qnrA (n=2). The qepA gene was absent among these isolates. Three types of β-lactamase genes were identified in the five ESBL-producing E. coli isolates: blaCTX-M-1, blaCTX-M-9, and blaTEM-1. The qnrS gene was found to be co-transferred with blaCTX-M-1 and blaTEM-1 in one isolate. Our data suggest that cooked meat products may act as reservoirs for multi-resistant bacteria and facilitate the dissemination of antimicrobial resistance genes. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. First Detection of CTX-M-1 in Extended-Spectrum β-Lactamase-Producing Escherichia coli in Seafood from Tunisia.

    Science.gov (United States)

    Said, Leila Ben; Hamdaoui, Mouna; Jouini, Ahlem; Boudabous, Abdellatif; Slama, Karim Ben; Torres, Carmen; Klibi, Naouel

    2017-10-17

    The purpose of this study was to determine the carriage rate of Escherichia coli isolates in seafood, to analyze the phenotype and genotype of antimicrobial resistance in the recovered isolates, and to characterize extended-spectrum β-lactamase (ESBL) E. coli producers. E. coli isolates were recovered from 24 (34.3%) of the 70 seafood samples analyzed, and one isolate per sample was further characterized. Antibiotic resistance was determined by the disk diffusion method in the 24 isolates, with the following results (number of resistant isolates): tetracycline (8), streptomycin (7), ampicillin (6), trimethoprim-sulfamethoxazole (4), chloramphenicol (4), ciprofloxacin (3), cefotaxime (2), and ceftazidime (2). Six isolates showed a multiresistant phenotype (including at least three families of antibiotics). Among tetracycline-resistant E. coli isolates, tet(A) was detected in five isolates and tet(B) in two isolates. The qnr(A) or aac(6')-1b-cr genes were detected in two ciprofloxacin-resistant E. coli isolates, and the sul2 gene in two trimethoprim-sulfamethoxazole-resistant isolates. ESBL-containing E. coli isolates, carrying the bla CTX-M-1 gene, were detected in 2 of the 70 seafood samples, obtained from gilt-head bream aquaculture. The ESBL isolates were typed phylogenetically and by multilocus sequence typing, and they were ascribed to lineage ST48/A and to the new ST3497/B1; these isolates carried the fimA, aer, and papGIII virulence genes. One of the ESBL-producing E. coli isolates carried an unusual class 1 integron (with the array dfr32-ereA-aadA1). Seafood could be a source of multiresistant E. coli isolates for the aquatic environment, and these could enter the food chain.

  14. Comparative evaluation of practical functionality of rapid test format kits for detection of Escherichia coli O157:H7 on lettuce and leafy greens.

    Science.gov (United States)

    D'Lima, C B; Suslow, T V

    2009-12-01

    Multistate outbreaks of Escherichia coli O157:H7 infection in 2005 and 2006 associated with fresh and especially minimally processed produce greatly escalated the application of rapid pathogen detection systems to safety management in this food category. Pathogen testing was rapidly integrated into preharvest qualification for field lots, incoming raw produce, or final product. The raw produce and final product were incorporated into test-and-hold programs, typically within a 10-h time frame. To enhance consumer safety and provide guidance for the industry, an assessment of selected kits in comparison to a culture-based method was undertaken. Four primary kits were compared: the Neogen Reveal, SDI RapidChek, BioControl GDS O157, and Qualicon BAX O157 MP. Nine different leafy greens were freshly harvested and inoculated with a five-isolate mixture of E. coli O157:H7 at 10 CFU/25 g of sample, and cultures were enriched following the specified protocol. The PCR method was most consistent for identifying the presence of the inoculated pathogen in the shortest period of time. For the red-pigmented leafy vegetables red butter lettuce, curly endive, red lettuce, and lollo rosa, 13, 38, 88, and 100% false-negative results, respectively, were obtained with the immunoassays, but PCR detection was minimally affected. Immunoassays were negatively affected by delays in achieving critical threshold populations during the allowed enrichment period. Leafy green type, temperature abuse, and preharvest environment were unlikely to affect the results of PCR-based kits. Findings strongly suggest that product testing systems using 8-h detection cutoffs may give false-negative results. These issues become very important in high-throughput testing and retest protocols for presumptive pathogen-positive lots of produce.

  15. Development of a one-step PCR assay with nine primer pairs for the detection of five diarrheagenic Escherichia coli types.

    Science.gov (United States)

    Oh, Kyung-Hwan; Kim, Soo-Bok; Park, Mi-Sun; Cho, Seung-Hak

    2014-06-28

    Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 10(1) CFU/ml for EHEC, 5 × 10(3) CFU/ml for ETEC expressing lt and sth, 5 × 10(4) CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 10(4) CFU/ml for EAEC, and 5 × 10(2) CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

  16. Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis.

    Directory of Open Access Journals (Sweden)

    Dafni-Maria Kagkli

    Full Text Available Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2 producing E. coli (VTEC or STEC respectively have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157 most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae in the case of VTEC, or aggregative protein (aggR, in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.

  17. Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples.

    Science.gov (United States)

    Stefan, A; Scaramagli, S; Bergami, R; Mazzini, C; Barbanera, M; Perelle, S; Fach, P

    2007-03-01

    This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157 for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

  18. Detection of Extended-Spectrum β-Lactamase and Plasmid-Mediated Quinolone Resistance Determinants in Escherichia coli Isolates from Retail Meat in China.

    Science.gov (United States)

    Yu, Tao; Jiang, Xiaobing; Fu, Kaifei; Liu, Biyun; Xu, Dong; Ji, Shengdong; Zhou, Lijun

    2015-05-01

    The aim of this study was to investigate the presence of extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli isolated from retail meat samples in Henan Province, China. E. coli isolates were detected in 179 of 645 (27.7%) retail meat samples. Resistance of these isolates to antimicrobials was commonly observed, with 78.2% of isolates resistant to streptomycin, 74.3% resistant to tetracycline and 54.2% resistant to trimethoprim/sulfamethoxazole. Of the 179 isolates, 30 (16.7%) expressed ESBL, with blaTEM-1 (n = 17) and bla(CTX-M-14) (n = 9) most commonly mediating the ESBL phenotype. PMQR genes were present in 14 isolates (7.8%), with qnr and aac(6')-Ib-cr detected alone or in combination in nine (5.0%) and seven isolates (3.9%), respectively. The qnr genes detected included qnrS1 (n = 5), qnrA1 (n = 3), and qnrB4 (n = 1). The qepA gene was absent among these isolates. CTX-M-14 was the most prevalent ESBL type among the PMQR-positive isolates. The qnr and aac(6')-Ib-cr genes were found to co-reside and be co-transferred with blaCTX-M-14 or blaTEM-1 in five isolates. Our data suggest that retail meat may act as a reservoir for multi-resistant E. coli and may facilitate the dissemination of resistance genes. © 2015 Institute of Food Technologists®

  19. Escherichia coli

    Science.gov (United States)

    Qian, Cunzhong; Hou, Jiafa

    2017-10-01

    The present study aimed to investigate whether Escherichia coli virulence affects the roles of sex hormone receptors in female dogs with simulated pyometra. A total of 33 healthy, nulliparous, crossbred female dogs were divided into four groups, with 10 dogs in each of the three experimental groups and 3 dogs in the control group. Estradiol was administrated to female dogs in group 1 continuously at 0.6-4.8 mg/kg twice daily for 12 days (the dose doubled every three days), followed by intramuscular injection of 0.2-1.8 mg/kg progesterone. The progesterone was administrated with an initial dose of 0.2 µg/kg and increased 0.2 mg/kg every three days, twice daily until the maximum of 1.8 mg/kg for 24 days and maintained at 1.8 mg/kg for 19 days. Progesterone only was administrated at 1.8 mg/kg in group 2 (twice daily) for 55 continuous days and only estradiol was administered with an initial dose of 0.6 µg/kg (dose doubled every 3 days for 12 days) in group 3 twice daily and maintained at 4.8 mg/kg for the following 43 days. A strongly virulent E. coli strain, nau-b, and a weakly virulent strain, nau-i, were screened. On the 12th day of diestrus, 5 female dogs in each of the experimental groups were inoculated with E. coli nau-i strain, while the other five in each group were inoculated with nau-b strain. Histopathological changes of uterine tissues were microscopically observed 50 days after E. coli inoculation and hormone receptor expression levels were detected by quantitative polymerase chain reaction. Simulated pyometra was observed in dogs administrated with progesterone alone or progesterone combined with estradiol. The clinical symptoms and histopathological observation demonstrated that inoculation with strongly virulent E. coli strain, nau-b, caused earlier onset of pyometra symptoms and more severe pyometra symptoms compared with the weakly virulent E. coli strain, nau-i. Furthermore, estrogen and progesterone receptor levels in dogs with pyometra

  20. Escherichia Coli

    Science.gov (United States)

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  1. Optimization of Synthesis and Modification of ZnSe/ZnS Quantum Dots for Fluorescence Detection ofEscherichia coil.

    Science.gov (United States)

    Wu, Pian; Huang, Ruixue; Li, Guiyin; He, Yayuan; Chen, Cuimei; Xiao, Wen; Ding, Ping

    2018-05-01

    This study prepared an innovative 3-mercaptopropionic acid modified ZnSe/ZnS core/shell quantum dots (MPA-ZnSe/ZnS QDs), and established a rapid fluorescence method to detect the E. coli cells count by using MPA-ZnSe/ZnS QDs as fluorescence probe. The formulation variables and process were optimized using the response surface methodology (RSM). Fluorescence microscopy was used to obtain fluorescence microscope images of MPA-ZnSe/ZnS QDs that bind to bacteria. The fluorescence peak intensity increases with increasing cells count in the range of 101-108 CFU/mL. Compared with the traditional based on fluorescent detection methods, this method is more convenient and useful in the bacterial count determination.

  2. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli.

    OpenAIRE

    Joensen, Katrine Grimstrup; Scheutz, Flemming; Lund, Ole; Hasman, Henrik; Kaas, Rolf Sommer; Nielsen, Eva M.; Aarestrup, Frank Møller

    2014-01-01

    Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-prod...

  3. Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

    OpenAIRE

    Joensen, Katrine Grimstrup; Scheutz, Flemming; Lund, Ole; Hasman, Henrik; Kaas, Rolf S.; Nielsen, Eva M.; Aarestrup, Frank M.

    2014-01-01

    Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-prod...

  4. Detection of verotoxin (Shiga-like toxin-producing and eae harboring Escherichia coli in some wild captive and domestic Equidae and Canidae

    Directory of Open Access Journals (Sweden)

    Koochakzadeh, A.

    2014-11-01

    Full Text Available The aim of this study was to investigate the prevalence of STEC and EPEC strains and E. coli O157 serogroup in some Equidae and Canidae. The fecal samples of 79 animals from 6 different species were evaluated for presence of these strains. All the Isolates were tested for virulence genes using multiplex-PCR. Non-sorbitol fermenting (NSF Escherichia coli isolates and positive strains for virulence factors were subjected to serogroupe specific PCR for rfb O157 gene. None of the STEC, EPEC and NFS strains in this study belonged to O157 serogroupe. While 36.64% of animals carried strains positive for one or more of the virulence factors tested, and 18.9% of animals harbored STEC strains (stx1, stx2 was not detected in this study. eae and Ehly positive strains were found in 3.79% and 22.7% of animals respectively. In conclusion, these species can act as a reservoir for EPEC and STEC strains. Also, since the study was conducted in some parts of Iran, a more accurate conclusion needs more distributed sampling. To our knowledge this is the first study which reports the faecal shedding of STEC and EPEC from wild captive Canidae and Equidae in Iran.

  5. Addition of Rifampicin to Bolton Broth to Inhibit Extended-Spectrum β-Lactamase-Producing Escherichia coli for the Detection of Campylobacter.

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Young-Ji; Kim, Young-Jo; Jung, Ji Young; Bae, Dongryeoul; Khan, Saeed; Seo, Kun-Ho; Sung, Kidon

    2017-07-01

    Exponential growth of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL-producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL-producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim-resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%). © 2017 Institute of Food Technologists®.

  6. Antimicrobial susceptibility testing of Escherichia coli strains isolated from urinary tract infections to fluoroquinolones and detection of gyrA mutations in resistant strains

    Directory of Open Access Journals (Sweden)

    Akbari-Nakhjavani F.

    2007-05-01

    Full Text Available Widespread uses of fluoroquinolones have resulted in increasing incidences of resistance against these agents all over the world. The aim of this study was to assess, susceptibility of Escherichia coli strains from patients with Urinary Tract Infection against common fluoroquinolones and detection of mutations in the gyrA gene. Antimicrobial susceptibility testing of 164 E.coli isolates from patients with UTI, was evaluated by disk agar diffusion (DAD and MIC methods. Polymerase chain reaction of E.coli strains were performed by amplification of Quinolone Resistance Determining Region (QRDR of gyrA gene. PCR products were tested by Conformational Sensitive Gel Electrophoresis (CSGE and those with hetrodublexes were selected and examined by DNA sequencing. According to disc agar diffusion, 49.3% were resistant to nalidixic acid, 41.4% to norfloxacin, 44.5% to ofloxacin and 40.2 % to ciprofloxacin. By Minimal Inhibitory Concentration (MIC testing a high-level of resistance (42.1% to ciprofloxacin was observed. Mutations in codons 83 and 87 in all 81 isolates were positive by CSGE method.

  7. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Characteristics of Clusters of Salmonella and Escherichia coli O157 Detected by Pulsed-Field Gel Electrophoresis that Predict Identification of Outbreaks.

    Science.gov (United States)

    Jones, Timothy F; Sashti, Nupur; Ingram, Amanda; Phan, Quyen; Booth, Hillary; Rounds, Joshua; Nicholson, Cyndy S; Cosgrove, Shaun; Crocker, Kia; Gould, L Hannah

    2016-12-01

    Molecular subtyping of pathogens is critical for foodborne disease outbreak detection and investigation. Many clusters initially identified by pulsed-field gel electrophoresis (PFGE) are not confirmed as point-source outbreaks. We evaluated characteristics of clusters that can help prioritize investigations to maximize effective use of limited resources. A multiagency collaboration (FoodNet) collected data on Salmonella and Escherichia coli O157 clusters for 3 years. Cluster size, timing, extent, and nature of epidemiologic investigations were analyzed to determine associations with whether the cluster was identified as a confirmed outbreak. During the 3-year study period, 948 PFGE clusters were identified; 849 (90%) were Salmonella and 99 (10%) were E. coli O157. Of those, 192 (20%) were ultimately identified as outbreaks (154 [18%] of Salmonella and 38 [38%] of E. coli O157 clusters). Successful investigation was significantly associated with larger cluster size, more rapid submission of isolates (e.g., for Salmonella, 6 days for outbreaks vs. 8 days for nonoutbreaks) and PFGE result reporting to investigators (16 days vs. 29 days, respectively), and performance of analytic studies (completed in 33% of Salmonella outbreaks vs. 1% of nonoutbreaks) and environmental investigations (40% and 1%, respectively). Intervals between first and second cases in a cluster did not differ significantly between outbreaks and nonoutbreaks. Molecular subtyping of pathogens is a rapidly advancing technology, and successfully identifying outbreaks will vary by pathogen and methods used. Understanding criteria for successfully investigating outbreaks is critical for efficiently using limited resources.

  9. Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment.

    Science.gov (United States)

    Lee, Jung-Lim; Levin, Robert E

    2011-05-01

    A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10(2) CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal's ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10(0) to 1.0 × 10(4) CFU/g by using Rti-PCR. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state.

    Science.gov (United States)

    Yan, Muxia; Xu, Ling; Jiang, Hua; Zhou, Zhenwen; Zhou, Shishui; Zhang, Li

    2017-04-01

    In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Detection of Enterohemorrhagic Escherichia coli Related Genes in E. coli Strains Belonging to B2 Phylogroup Isolated from Urinary Tract Infections in Combination with Antimicrobial Resistance Phenotypes

    Directory of Open Access Journals (Sweden)

    Hamid Staji

    2017-07-01

    Full Text Available Background:  This study was conducted to detect the prevalence of EHEC virulence genes and antimicrobial resistance profile of Escherichia coli strains belonging to B2 phylogroup implicated in Urinary tract infections in Semnan, Iran.Methods:   From 240 urine samples 160 E. coli strains were isolated, biochemically. Then, E. coli isolates were examined by Multiplex-PCR for phylogenetic typing and detection of virulence genes (hly, stx1, stx2, eae associated with Enterohemorrhagic E. coli. Finally, Antimicrobial resistance of E. coli isolates were characterized using Disk Diffusion method.  Results:  From 160 E. coli isolates, 75 strains (47% were assigned to B2 phylogenetic group and prevalence of virulence genes were as follow: hly (21.3%, stx1 (16%, stx2 (10.6% and eae (6.7%, subsequently.  Phenotypic antimicrobial resistance of B2 isolates showed that all isolates were sensitive to Meropenem and Furazolidone and then highest frequency of resistance was observed to Streptomycin, Oxytetracycline, Neomycin, Nalidixic acid and Ampicillin (98.7% to 49.3%. Also low resistance prevalence was observed in case of Ceftizoxime, Lincospectin, Imipenem, Chloramphenicol and flurefenicole (16% to 1.3%.Conclusion:   The data suggest a high prevalence of antibiotic resistance in UPEC strains belonging to B2 phylogroup even for the antimicrobials using in pet and farm animals and their potential to cause EHEC specific clinical symptoms which may represent a serious health risk since these strains can be transmitted to GI tract and act as a reservoir for other uropathogenic E. coli and commensal strains.

  12. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures.

    Science.gov (United States)

    Stapp, J R; Jelacic, S; Yea, Y L; Klein, E J; Fischer, M; Clausen, C R; Qin, X; Swerdlow, D L; Tarr, P I

    2000-09-01

    We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.

  14. A disposable electrochemical immunosensor arrays using 4-channel screen-printed carbon electrode for simultaneous detection of Escherichia coli O157:H7 and Enterobacter sakazakii

    International Nuclear Information System (INIS)

    Dou, Wenchao; Tang, Weilu; Zhao, Guangying

    2013-01-01

    An electrochemical immunosensor for Escherichia coli O157:H7 (E. coli O157:H7) and Enterobacter sakazakii (E. sakazakii) detection using carbon screen-printed low-density arrays is reported. The sensors were fabricated based on screen-printed carbon arrays containing four carbon working electrode, an integrated carbon counter electrodes and an integrated Ag/AgCl reference electrode. Multi-walled carbon nanotubes (MWCNTs)/sodium alginate (SA)/carboxymethyl chitosan (CMC) composite films were coated on all the working electrodes to enhance the sensitization of the electrode. Horseradish peroxidases (HRP) labeled antibodies of two bacteria were immobilize on different working electrode of the same screen-printed electrode respectively. The immobilization of MWCNTs, HRP labeled antibodies onto the screen-printed carbon electrodes was examined using atom force microscopy (AFM) and cyclic voltammetry (CV). The analytical performance of proposed immunosensor arrays toward E. sakazakii and E. coli O157:H7 was investigated by AFM and CV. Under optimal conditions, the linear range of E. sakazakii and E. coli O157:H7 were from 10 4 to 10 10 cfu/ml, with a detection limit of 4.57 × 10 3 cfu/ml (S/N = 3) and 3.27 × 10 3 cfu/ml (S/N = 3), respectively. The specificity, reproducibility, stability and accuracy of the proposed immunosensor arrays were also evaluated. Two antibodies modified work electrodes were tested and compared in terms of sensitivity and ability to recognize different pathogenic biological species

  15. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests.

    Science.gov (United States)

    Wang, Rong; Schmidt, John W; Arthur, Terrance M; Bosilevac, Joseph M

    2013-04-01

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS(®) and DuPont Qualicon BAX(®)-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C(T)) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C(T) values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10(6) cells/sample or higher, which are levels not typically seen in ground beef. Published by Elsevier Ltd.

  16. Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant Escherichia coli and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant E. coli Isolates

    OpenAIRE

    Wu, Beibei; Xia, Chun; Du, Xiangdang; Cao, Xingyuan; Shen, Jianzhong

    2006-01-01

    To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromato...

  17. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

    Science.gov (United States)

    Li, Baoguang; Liu, Huanli; Wang, Weimin

    2017-11-09

    Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

  18. Discrimination of Enterohemorrhagic Escherichia coli (EHEC) from Non-EHEC Strains Based on Detection of Various Combinations of Type III Effector Genes

    Science.gov (United States)

    Delannoy, Sabine; Beutin, Lothar

    2013-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains. PMID:23884997

  19. Loop-mediated isothermal amplification assay targeting the blaCTX-M9 gene for detection of extended spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

    Science.gov (United States)

    Thirapanmethee, Krit; Pothisamutyothin, Kanokporn; Nathisuwan, Surakit; Chomnawang, Mullika T; Wiwat, Chanpen

    2014-12-01

    Extended-spectrum β-lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β-lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL-producing bacteria is necessary for identification, prevention and treatment. Loop-mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL-producing bacteria by a LAMP technique. ESBLs-producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double-disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla(CTX-M9) gene was designed based on bla(CTX-M9) from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder-like patterns of band sizes from 226 bp of the bla(CTX-M9) DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla(CTX-M9) was greater than that of the PCR method by at least 10,000-fold. These results showed that the LAMP primers specifically amplified only the bla(CTX-M9) gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  20. Detection of acrA, acrB, aac(6')-Ib-cr, and qepA genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae.

    Science.gov (United States)

    Heidary, Mohsen; Bahramian, Aghil; Hashemi, Ali; Goudarzi, Mehdi; Omrani, Vahid Fallah; Eslami, Gita; Goudarzi, Hossein

    2017-03-01

    The distribution of drug resistance among clinical isolates of Escherichia coli and Klebsiella pneumoniae has limited the therapeutic options. The aim of this study was to report the prevalence of quinolone resistance genes among E. coli and K. pneumoniae clinical strains isolated from three educational hospitals of Tehran, Iran. A total of 100 strains of E. coli from Labbafinejad and Taleghani Hospitals and 100 strains of K. pneumoniae from Mofid Children and Taleghani Hospitals were collected between January 2013 and May 2014. Antimicrobial susceptibility tests were done by disk diffusion method based on Clinical and Laboratory Standards Institute guidelines. Detection of qepA, aac(6')-Ib-cr, acrA, and acrB genes was done by polymerase chain reaction (PCR). In this study, fosfomycin and imipenem against E. coli and fosfomycin and tigecycline against K. pneumoniae had the best effect in antimicrobial susceptibility tests. PCR assay using specific primers demonstrated that the prevalence of qepA, aac(6')-Ib-cr, acrA, and acrB genes among the 100 E. coli isolates was 0 (0%), 87 (87%), 92 (92%), and 84 (84%), respectively. The prevalence of qepA, aac(6')-Ib-cr, acrA, and acrB genes among the 100 K. pneumoniae isolates was 4 (4%), 85 (85%), 94 (94%), and 87 (87%), respectively. The distribution of qepA, aac(6')-Ib-cr, acrA, and acrB resistance determinants in E. coli and K. pneumoniae is a great concern. Therefore, infection control and prevention of spread of drug-resistant bacteria need careful management of medication and identification of resistant isolates.

  1. Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

    Science.gov (United States)

    Delannoy, Sabine; Chaves, Byron D.; Ison, Sarah A.; Webb, Hattie E.; Beutin, Lothar; Delaval, José; Billet, Isabelle; Fach, Patrick

    2016-01-01

    Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013. PMID:26834723

  2. Development of a multiplex PCR assay for detection of Shiga toxin-producing Escherichia coli, enterohemorrhagic E. coli, and enteropathogenic E. coli strains.

    Science.gov (United States)

    Botkin, Douglas J; Galli, Lucía; Sankarapani, Vinoth; Soler, Michael; Rivas, Marta; Torres, Alfredo G

    2012-01-01

    Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain's respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 10(4) CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms.

  3. Revisiting the STEC testing approach: using espK and espV to make enterohemorrhagic Escherichia coli (EHEC detection more reliable in beef

    Directory of Open Access Journals (Sweden)

    Sabine eDelannoy

    2016-01-01

    Full Text Available Current methods for screening Enterohemorrhagic Escherichia coli (EHEC O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of ‘false positive’ results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1,739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50% of these tested negative for espK, espV, ureD, and Z2098, but twelve out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority opinion on pathogenic STEC published in 2013.

  4. Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes inEscherichia coliIsolated from Chicken Feces and Houseflies after Apramycin Administration.

    Science.gov (United States)

    Zhang, Anyun; Li, Yunxia; Guan, Zhongbin; Tuo, Hongmei; Liu, Dan; Yang, Yanxian; Xu, Changwen; Lei, Changwei; Wang, Hongning

    2018-01-01

    The aim of this study was to evaluate the influence of apramycin administration on the development of antibiotic resistance in Escherichia coli ( E. coli ) strains isolated from chicken feces and houseflies under field conditions. Chickens in the medicated group ( n = 25,000) were given successive prophylactic doses (0.5 mg/l) of apramycin in their drinking water from Days 1 to 5, while no antibiotics were added to the un-medicated groups drinking water ( n = 25,000). Over 40 days, a total of 1170 E. coli strains were isolated from fecal samples obtained from medicated and un-medicated chickens and houseflies from the same chicken farm. Apramycin MIC90 values for E. coli strains obtained from the medicated group increased 32-128 times from Days 2 to 6 (256-1024 μg/ml) when compared to those on Day 0 (8 μg/ml). Strains isolated from un-medicated chickens and houseflies had consistently low MIC90 values (8-16 μg/ml) during the first week, but showed a dramatic increase from Days 8 to 10 (128-1024 μg/ml). The apramycin resistance gene aac(3)-IV was detected in E. coli strains from medicated ( n = 71), un-medicated ( n = 32), and housefly groups ( n = 42). All strains positive for aac(3)-IV were classified into 12 pulsed-field gel electrophoresis (PFGE) types. PFGE types A, E, and G were the predominant types in both the medicated and housefly groups, suggesting houseflies play an important role in spreading E. coli -resistant strains. Taken together, our study revealed that apramycin administration could facilitate the occurrence of apramycin-resistant E. coli and the apramycin resistance gene acc(3)-IV . In turn, these strains could be transmitted by houseflies, thus increasing the potential risk of spreading multi-drug-resistant E. coli to the public.

  5. Detection by hyperspectral imaging of shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 on rainbow agar.

    Science.gov (United States)

    Windham, William R; Yoon, Seung-Chul; Ladely, Scott R; Haley, Jennifer A; Heitschmidt, Jerry W; Lawrence, Kurt C; Park, Bosoon; Narrang, Neelam; Cray, William C

    2013-07-01

    The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due to the lack of suitable agar media. The lack of distinct phenotypic color variation among non-O157serogroups cultured on chromogenic agar poses a challenge in selecting colonies for confirmation. In this study, visible and near-infrared hyperspectral imaging and chemometrics were used to detect and classify non-O157 STEC serogroups grown on Rainbow agar O157. The method was first developed by building spectral libraries for each serogroup obtained from ground-truth regions of interest representing the true identity of each pixel and thus each pure culture colony in the hyperspectral agar-plate image. The spectral library for the pure-culture non-O157 STEC consisted of 2,171 colonies, with spectra derived from 124,347 of pixels. The classification models for each serogroup were developed with a k nearest-neighbor classifier. The overall classification training accuracy at the colony level was 99%. The classifier was validated with ground beef enrichments artificially inoculated with 10, 50, and 100 CFU/ml STEC. The validation ground-truth regions of interest of the STEC target colonies consisted of 606 colonies, with 3,030 pixels of spectra. The overall classification accuracy was 98%. The average specificity of the method was 98% due to the low false-positive rate of 1.2%. The sensitivity ranged from 78 to 100% due to the false-negative rates of 22, 7, and 8% for O145, O45, and O26, respectively. This study showed the potential of visible and near-infrared hyperspectral imaging for detecting and classifying colonies of the six non-O157 STEC serogroups. The technique needs to be validated with bacterial cultures directly extracted from meat products and positive

  6. Structural and functional studies of Escherichia coli aggregative adherence fimbriae (AAF/V) reveal a deficiency in extracellular matrix binding.

    Science.gov (United States)

    Jønsson, Rie; Liu, Bing; Struve, Carsten; Yang, Yi; Jørgensen, René; Xu, Yingqi; Jenssen, Håvard; Krogfelt, Karen A; Matthews, Steve

    2017-03-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. The pathogenesis of different EAEC stains is complicated, however, the early essential step begins with attachment of EAEC to intestinal mucosa via aggregative adherence fimbriae (AAFs). Currently, five different variants have been identified, which all share a degree of similarity in the gene organization of their operons and sequences. Here, we report the solution structure of Agg5A from the AAF/V variant. While preserving the major structural features shared by all AAF members, only Agg5A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development.

    Science.gov (United States)

    Yang, Shih-Chun; Lin, Chih-Hung; Aljuffali, Ibrahim A; Fang, Jia-You

    2017-08-01

    Food contamination by pathogenic microorganisms has been a serious public health problem and a cause of huge economic losses worldwide. Foodborne pathogenic Escherichia coli (E. coli) contamination, such as that with E. coli O157 and O104, is very common, even in developed countries. Bacterial contamination may occur during any of the steps in the farm-to-table continuum from environmental, animal, or human sources and cause foodborne illness. To understand the causes of the foodborne outbreaks by E. coli and food-contamination prevention measures, we collected and investigated the past 10 years' worldwide reports of foodborne E. coli contamination cases. In the first half of this review article, we introduce the infection and symptoms of five major foodborne diarrheagenic E. coli pathotypes: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli/enterohemorrhagic E. coli (STEC/EHEC), Shigella/enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and enterotoxigenic E. coli (ETEC). In the second half of this review article, we introduce the foodborne outbreak cases caused by E. coli in natural foods and food products. Finally, we discuss current developments that can be applied to control and prevent bacterial food contamination.

  8. Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

    Science.gov (United States)

    Huang, Zhen; Cui, Xi; Xie, Quan-Yuan; Liu, Dao-Feng; Lai, Wei-Hua

    2016-12-01

    Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

  10. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  11. Identification and antimicrobial resistance prevalence of pathogenic Escherichia coli strains from treated wastewater effluents in Eastern Cape, South Africa.

    Science.gov (United States)

    Adefisoye, Martins A; Okoh, Anthony I

    2016-02-01

    Antimicrobial resistance (AMR) is a global problem impeding the effective prevention/treatment of an ever-growing array of infections caused by pathogens; a huge challenge threatening the achievements of modern medicine. In this paper, we report the occurrence of multidrug resistance (MDR) in Escherichia coli strains isolated from discharged final effluents of two wastewater treatment facilities in the Eastern Cape Province of South Africa. Standard disk diffusion method was employed to determine the antibiotic susceptibility profile of 223 polymerase chain reaction (PCR)-confirmed E. coli isolates against 17 common antibiotics in human therapy and veterinary medicine. Seven virulence associated and fourteen antibiotic resistance genes were also evaluated by molecular methods. Molecular characterization revealed five pathotypes of E. coli in the following proportions: enterotoxigenic ETEC (1.4%), enteropathogenic EPEC (7.6%), enteroaggregative EAEC (7.6%), neonatal meningitis (NMEC) (14.8%), uropathogenic (41.7%), and others (26.9%). Isolates showed varying (1.7-70.6%) degrees of resistance to 15 of the test antibiotics. Multidrug resistance was exhibited by 32.7% of the isolates, with the commonest multiple antibiotic-resistant phenotype (MARP) being AP-T-CFX (12 isolates), while multiple antibiotic-resistant indices (MARI) estimated are 0.23 (Site 1) and 0.24 (Site 2). Associated antibiotic resistance genes detected in the isolates include: strA (88.2%), aadA (52.9%), cat I (15%), cmlA1 (4.6%), blaTEM (56.4%), tetA (30.4%), tetB (28.4%), tetC (42.2%), tetD (50%), tetK (11.8%), and tetM (68.6%). We conclude that municipal wastewater effluents are important reservoirs for the dissemination of potentially pathogenic E. coli (and possibly other pathogens) and antibiotic resistance genes in the aquatic milieu of the Eastern Cape and a risk to public health. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.

    Science.gov (United States)

    Kouguchi, Yoshihiro; Fujiwara, Takako; Teramoto, Miki; Kuramoto, Mika

    2010-08-01

    We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy. Copyright 2010 Elsevier Ltd. All rights reserved.

  13. Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos Detection of virulence factors in Escherichia coli and analysis of Salmonella spp. in psittacines

    Directory of Open Access Journals (Sweden)

    Isadora M. de O. Corrêa

    2013-02-01

    Full Text Available A flora entérica dos psitacídeos é composta principalmente por bactérias Gram positivas. Bactérias Gram negativas, como Escherichia coli e Salmonella spp., apresentam elevado potencial patogênico, sendo consideradas indicativo de problemas de manejo, que poderão culminar em manifestação de doenças em decorrência de fatores estressantes, dietas deficientes e superlotação, combinados com alta carga bacteriana no ambiente. O objetivo deste trabalho foi avaliar a presença de Salmonella spp., Escherichia coli e os fatores de virulência dos genes iss e iutA dos isolados de E. coli. Analisou-se um total de 44 amostras provenientes de psitacídeos criados em cativeiro, sendo estas 15 fragmentos de órgãos de aves submetidas a exame de necropsia e também 29 amostras de swabs de cloaca e inglúvio de papagaios-charão (Amazona pretrei criados em cativeiro. Nenhuma amostra foi positiva para Salmonella spp. Nas amostras de E. coli detectou-se ambos os fatores de virulência pesquisados.The enteric flora of psittacines is mainly composed of Gram positive bacteria. Gram negative bacteria, like Escherichia coli and Salmonella spp., have a high pathogenic potential and can be considerate as an indicative of management problems that may culminate in disease manifestation due to stress factors, poor diets and overcrowding, in combination with a high bacterial load on the environment. The objective of this study was evaluated the presence of Salmonella spp., Escherichia coli and the virulence genes iss and iutA from E. coli isolates. Forty-four samples were analyzed from psittacines living in captivity, which fifteen samples were from organs fragments of necropsied birds, and twenty-nine were from cloacal and crop swabs of red-spectacled parrots (Amazona pretrei keeping in captivity. No samples were positive for Salmonella spp. In the samples in which E. coli was detected, both virulence factors (genes iss and iutA were present.

  14. Detection and Characterization of the Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM Isolates from Humans and Cattle ▿

    OpenAIRE

    Bielaszewska, Martina; Prager, Rita; Vandivinit, Liz; Müsken, Anne; Mellmann, Alexander; Holt, Nicholas J.; Tarr, Phillip I.; Karch, Helge; Zhang, Wenlan

    2008-01-01

    The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide seq...

  15. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

    Science.gov (United States)

    Wasilenko, Jamie L; Fratamico, Pina M; Narang, Neelam; Tillman, Glenn E; Ladely, Scott; Simmons, Mustafa; Cray, William C

    2012-11-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

  16. Detection of an Escherichia coli Sequence Type 167 Strain with Two Tandem Copies of blaNDM-1 in the Chromosome.

    Science.gov (United States)

    Shen, Ping; Yi, Maoli; Fu, Ying; Ruan, Zhi; Du, Xiaoxing; Yu, Yunsong; Xie, Xinyou

    2017-01-01

    New Delhi metallo-β-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the bla NDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the bla NDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of bla NDM-1 , the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same bla NDM-1 gene-surrounding structure as Y5. The bla NDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-bla NDM-1 -bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the bla NDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated bla NDM-1 , which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor bla NDM-1 . Copyright © 2016 American Society for Microbiology.

  17. OCORRÊNCIA DE Escherichia coli O157: H7 EM PRODUTOS CÁRNEOS E SENSIBILIDADE DOS MÉTODOS DE DETECÇÃO OCCURRENCE OF Escherichia coli O157:H7 IN MEAT PRODUCTS AND SENSIBILITY OF THE DETECTION METHODS

    Directory of Open Access Journals (Sweden)

    Neusely da SILVA

    2001-08-01

    Full Text Available Foi verificada a ocorrência de Escherichia coli O157:H7 em 340 amostras de produtos cárneos e ambiente industrial, provenientes de frigoríficos do Sul e Sudeste do Brasil, no período de abril/98 a abril/99. A presença de E.coli O157:H7 não foi detectada em nenhuma das amostras analisadas e os resultados da avaliação da sensibilidade dos métodos de detecção evidenciaram que tanto o método cultural quanto o imunoensaio da Neogem foram capazes de detectar a presença de E.coli O157:H7 em cultura pura em concentrações iniciais de menos de 0,5Log UFC/mL do caldo de enriquecimento.The occurrence of E.coli O157:H7 was evaluated in 340 samples of meat products and industrial environment of meat manufacturers from the South and Southeast regions of Brazil, from April, 1998 to April, 1999. Pathogen was not detected in any of the samples analysed, and the evaluation of the sensibility of the studied detection methods showed that both, culture and immuno-assay methods detected E.coli O157:H7 in pure culture in initial population levels of 0.5Log CFU/mL of enrichment broth.

  18. Improved detection of extended spectrum beta-lactamase (ESBL-producing Escherichia coli in input and output samples of German biogas plants by a selective pre-enrichment procedure.

    Directory of Open Access Journals (Sweden)

    Thorsten Schauss

    Full Text Available The presence of extended-spectrum beta-lactamase (ESBL-producing Escherichia coli was investigated in input (manure from livestock husbandry and output samples of six German biogas plants in 2012 (one sampling per biogas plant and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%, few SHV-type beta lactamase (6%. Sixty-four blaCTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85% and 9 (13%, and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71% and B1 (27%, only one to group D (2%. Genomic fingerprinting and multilocus sequence typing (MLST showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E

  19. Improved Detection of Extended Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Input and Output Samples of German Biogas Plants by a Selective Pre-Enrichment Procedure

    Science.gov (United States)

    Schauss, Thorsten; Glaeser, Stefanie P.; Gütschow, Alexandra; Dott, Wolfgang; Kämpfer, Peter

    2015-01-01

    The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli was investigated in input (manure from livestock husbandry) and output samples of six German biogas plants in 2012 (one sampling per biogas plant) and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four bla CTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85%) and 9 (13%), and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71%) and B1 (27%), only one to group D (2%). Genomic fingerprinting and multilocus sequence typing (MLST) showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT) genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E. coli in

  20. Development of a rapid capture-cum-detection method for Escherichia coli O157 from apple juice comprising nano-immunomagnetic separation in tandem with surface enhanced Raman scattering.

    Science.gov (United States)

    Najafi, Roya; Mukherjee, Shubhasish; Hudson, Jim; Sharma, Anup; Banerjee, Pratik

    2014-10-17

    A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite-gold (Fe3O4/Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84-94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (ptarget pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe3O4/Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10(2)CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Escherichia coli

    Science.gov (United States)

    Nyanga, Peter Lokamar; Onyuka, Jackson; Webale, Mark Kilongosi; Were, Tom; Budambula, Valentine

    2017-01-01

    In the present study, we investigated the prevalence of E. coli pathotypes and Shigella sero-groups and their antimicrobial profiles among diarrheic children in Nairobi city, Kenya. Although diarrheagenic E. coli pathotypes and Shigella sero-groups are leading causes of diarrhea in children under five years in developing countries, their distribution and antimicrobial resistance vary from place to place and over time in a given region. In a cross-sectional study, we enrolled diarrheic children (n=354) under five years seeking treatment at Mbagathi Hospital, Nairobi city, Kenya,. Stool samples were collected from all children for bacterial culture. Bacterial isolation and identification was performed by conventional microbiological methods. Polymerase chain amplification was used to detect aspU, aggR, andpcvd432 for EAEC, est and elt for ETEC, eae for EPEC, stx for EHEC, and ipaH for EIEC and Shigella species. Antimicrobial profile was determined by disk diffusion method. The prevalence of EAEC, ETEC, EPEC (eae), EIEC (ipaH) was 21.2%, 10.5%, 4.5%, and 0.6%, respectively, while that of mixed infection was 0.6%for ETEC/EAEC and 0.3%for EAEC/EPEC/ETEC. No EHEC strain was isolated. Pathogenetic analysis for EAEC showed that5.9% carried aspU,8.2% possessed both aspU and aggR and 7.1% had a combination of aspU, aggR andpcvd432 while that of ETEC was 2.3% for elt, 6.5% for both elt and est and 1.7% for est. The combination of aspU with aggR, elt and est, and pcvd432 with aggR, aspU and est was 0.3% for each case of ETEC/EAEC mixed infection. The aspU gene co-existed with aggR, pcvd432, eae and elt in the EAEC/EPEC/ETEC mixed infection. The prevalence of S. boydii , S. dysenteriae , S. flexneriand, S. sonnei was 0.8%, 0.6%, 1.7%, and 0.8%, respectively. No E. coli pathotype and shigella co-infection was detected. In addition, both E. coli pathotypes and Shigella species were resistant to ampicillin, trimethoprim/sulfamethoxazole, streptomycin, chloramphenicol and

  2. Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

    Science.gov (United States)

    Youmans, Bonnie P.; Ajami, Nadim J.; Jiang, Zhi-Dong; Petrosino, Joseph F.; DuPont, Herbert L.; Highlander, Sarah K.

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples. PMID:24189361

  3. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

    Science.gov (United States)

    Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

  4. Comparison of MI, Chromocult®coliform, and Compass CC chromogenic culture-based methods to detect Escherichia coli and total coliforms in water using 16S rRNA sequencing for colony identification.

    Science.gov (United States)

    Maheux, Andrée F; Bouchard, Sébastien; Bérubé, Ève; Bergeron, Michel G

    2017-06-01

    The MI, Chromocult ® coliform, and Compass CC chromogenic culture-based methods used to assess water quality by the detection of Escherichia coli and total coliforms were compared in terms of their specificity and sensitivity, using 16S rRNA sequencing for colony identification. A sewage water sample was divided in 2-μL subsamples for testing by all three culture-based methods. All growing colonies were harvested and subjected to 16S rRNA sequencing. Test results showed that all E. coli colonies were correctly identified by all three methods, for a specificity and a sensitivity of 100%. However, for the total coliform detection, the MI agar, Chromocult ® coliform agar, and Compass CC agar were specific for only 69.2% (9/13), 47.2% (25/53), and 40.5% (17/42), whereas sensitive for 97.8% (45/46), 97.5% (39/40), and 85.7% (24/28), respectively. Thus, given the low level of specificity of these methods for the detection of total coliforms, confirming the identity of total coliform colonies could help to take public health decisions, in particular for cities connected to a public drinking water distribution system since the growth of few putative total coliform colonies on chromogenic agar is problematic and can lead to unnecessary and costly boiling notices from public health authorities.

  5. A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

    Science.gov (United States)

    Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

    2014-07-01

    A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

  6. Comparison of four enrichment broths for the detection of non-O157 Shiga-toxin-producing Escherichia coli O91, O103, O111, O119, O121, O145 and O165 from pure culture and food samples.

    Science.gov (United States)

    Kanki, M; Seto, K; Harada, T; Yonogi, S; Kumeda, Y

    2011-08-01

    We compared the efficiency of universal pre-enrichment broth (UPB), modified Escherichia coli broth containing novobiocin (mEC + n), modified Tryptic Soy Broth (mTSB) and mTSB with novobiocin (mTSB + n) for the enrichment of non-O157 Shiga-toxin-producing E. coli (STEC). Freeze-injured and control non-O157 STEC (O91, O103, O111, O119, O121, O145 and O165) strains were used to artificially contaminate beef and radish sprout samples, which were then cultivated in each of the four enrichment media. After incubation, STEC strains were detected by loop-mediated isothermal amplification (LAMP) and plating assays. Enrichment in mEC + n was least effective for facilitating the detection of uninjured STEC strains in radish sprouts, while mTSB + n was least effective for enriching freeze-injured non-O157 STEC strains from beef samples for detection by LAMP assay. UPB and mTSB were superior to mEC + n and mTSB + n for the enrichment of non-O157 STEC from food samples. The enrichment of non-O157 STEC was negatively affected by the addition of novobiocin to enrichment broths. Novobiocin should not be added to media used for the enrichment of non-O157 STEC in food when cell injury is anticipated. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  7. Thioredoxin from Escherichia coli

    International Nuclear Information System (INIS)

    Holmgren, A.; Ohlsson, I.; Grankvist, M.L.

    1978-01-01

    A competition radioimmunoassay for Escherichia coli thioredoxin using 125 I-labeled thioredoxin-S 2 and a double antibody technique was developed. The method permits determination of picomole amounts of thioredoxin in crude cell extracts and was used to study the localization of thioredoxin cell fractions. E. coli B was calculated to have approximately 10,000 copies of thioredoxin per cell mainly located in the soluble fraction after separation of the membrane and soluble fractions by gentle lysis and centrifugation. E. coli B tsnC mutants which are defective in the replication of phage T7 DNA in vivo and in vitro were examined for their content of thioredoxin. E. coli B tsnC 7004 contained no detectable level of thioredoxin in cell-free extracts examined under a variety of conditions. The results strongly suggest that tsnC 7004 is a nonsense or deletion mutant. Two other E. coli tsnC mutants, 7007 and 7008, contained detectable levels of thioredoxin in crude extracts as measured by thioredoxin reductase and gave similar immunoprecipitation reactions as the parent strain B/1. By radioimmunoassay incompletely cross-reacting material was present in both strains. These results show that tsnC 7007 and 7008 belong to a type of thioredoxin mutants with missence mutations in the thioredoxin gene affecting the function of thioredoxin as subunit in phage T7 DNA polymerase

  8. A Dual Filtration-Based Multiplex PCR Method for Simultaneous Detection of Viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on Fresh-Cut Cantaloupe.

    Directory of Open Access Journals (Sweden)

    Ke Feng

    Full Text Available Fresh-cut cantaloupe is particularly susceptible to contamination with pathogenic bacteria, such as Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus. Therefore, development of rapid, yet accurate detection techniques is necessary to ensure food safety. In this study, a multiplex PCR system and propidium monoazide (PMA concentration were optimized to detect all viable pathogens in a single tube. A dual filtration system utilized a filtration membrane with different pore sizes to enrich pathogens found on fresh-cut cantaloupe. The results revealed that an optimized multiplex PCR system has the ability to effectively detect three pathogens in the same tube. The viable pathogens were simultaneously detected for PMA concentrations above 10 μg/ml. The combination of a nylon membrane (15 μm and a micro pore filtration membrane (0.22 μm formed the dual filtration system used to enrich pathogens. The achieved sensitivity of PMA-mPCR based on this dual filtration system was 2.6 × 103 cfu/g for L. monocytogenes, 4.3 × 10 cfu/g for E. coli O157:H7, and 3.1 × 102 cfu/g for S. aureus. Fresh-cut cantaloupe was inoculated with the three target pathogens using concentrations of 103, 102, 10, and 1 cfu/g. After 6-h of enrichment culture, assay sensitivity increased to 1 cfu/g for each of these pathogens. Thus, this technique represents an efficient and rapid detection tool for implementation on fresh-cut cantaloupe.

  9. Epithelial cells detect functional type III secretion system of enteropathogenic Escherichia coli through a novel NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Yael Litvak

    2017-07-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC, a common cause of infant diarrhea, is associated with high risk of mortality in developing countries. The primary niche of infecting EPEC is the apical surface of intestinal epithelial cells. EPEC employs a type three secretion system (TTSS to inject the host cells with dozens of effector proteins, which facilitate attachment to these cells and successful colonization. Here we show that EPEC elicit strong NF-κB activation in infected host cells. Furthermore, the data indicate that active, pore-forming TTSS per se is necessary and sufficient for this NF-κB activation, regardless of any specific effector or protein translocation. Importantly, upon infection with wild type EPEC this NF-κB activation is antagonized by anti-NF-κB effectors, including NleB, NleC and NleE. Accordingly, this NF-κB activation is evident only in cells infected with EPEC mutants deleted of nleB, nleC, and nleE. The TTSS-dependent NF-κB activation involves a unique pathway, which is independent of TLRs and Nod1/2 and converges with other pathways at the level of TAK1 activation. Taken together, our results imply that epithelial cells have the capacity to sense the EPEC TTSS and activate NF-κB in response. Notably, EPEC antagonizes this capacity by delivering anti-NF-κB effectors into the infected cells.

  10. Comparison of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms in water.

    Science.gov (United States)

    Maheux, Andrée F; Dion-Dupont, Vanessa; Bouchard, Sébastien; Bisson, Marc-Antoine; Bergeron, Michel G; Rodriguez, Manuel J

    2015-06-01

    The MI agar, Colilert(®), Chromocult coliform(®) agar, and DC with BCIG agar chromogenic culture-based methods used to assess microbiological quality of drinking water were compared in terms of their ubiquity, sensitivity, ease of use, growth of atypical colonies and affordability. For ubiquity, 129 total coliform (representing 76 species) and 19 Escherichia coli strains were tested. Then, 635 1-L well water samples were divided into 100 mL subsamples for testing by all four methods. Test results showed that 70.5, 52.7, 36.4, and 23.3% of the non-E. coli total coliform strains and 94.7, 94.7, 89.5, and 89.5% of the 19 E. coli strains yielded a positive signal with the four methods, respectively. They also yielded a total coliform positive signal for 66.5, 51.7, 64.9, and 55.0% and an E. coli positive signal for 16.1, 14.8, 17.3, and 13.4% of the 635 well water samples tested, respectively. Results showed that Colilert(®) is the most expensive method tested in terms of reactants, yet it is the easiest to use. Large numbers of atypical colonies were also often observed on Chromocult coliform(®) and DC with BCIG, thereby challenging the target microorganism count. Thus, the MI agar method seems to be the best option for the assessment of drinking water quality.

  11. Detection of coliform bacteria, determination of phylogenetic typing and antibiotic resistance profile of Escherichia coli in qanats and springs of East-Azerbaijan province

    Directory of Open Access Journals (Sweden)

    N. Shabani Lokarani

    2017-05-01

    Full Text Available Escherichia coli as a fecal contamination and is considered as an index in water. The aim of this study was to determine the phenotypic and genotypic characteristics of E. coli and antibiotic resistance of the isolates collected from qanats and springs in East-Azerbaijan province. For this purpose, 118 samples were selected from above mentioned area and examined by MPN method. The positive coliform samples were identified by phenotypic and genotypic methods. Afterwards, to determine the genetic diversity of E. coli isolates, phylogenetic typing we conducted by means of multiplex PCR. To determine the antibiotic resistance profile, antibiotic discs of Nalidixic Acid, Co-trimoxazol, Amoxicillin, Gentamaicin Ciprofloxacin, Chloramphenicol, Imipenem, Cefotaxime and Ceftazidime antibiogram were used. Based on results, 48% of the samples were evaluated as positive for coliform including 40% for E. coli and 19% for Klebsiella. Amongst 23 isolates confirmed as E. coli by PCR. Phylogenetic typing revealed  that 44% of E. coli strains belonged to type D and B2 and 56% belonged to A and B1 phylotypes. Antimicrobial susceptibility pattern showed that 92% of E. coli isolates were resistant to Amoxicillin. All E. coli isolates were sensitive to Imipenem. It was concluded that presence of pathogenic E. coli with high rate of antibacterial resistance in waters source could be considered as a human health hazard.

  12. Detection and characterization of the fimbrial sfp cluster in enterohemorrhagic Escherichia coli O165:H25/NM isolates from humans and cattle.

    Science.gov (United States)

    Bielaszewska, Martina; Prager, Rita; Vandivinit, Liz; Müsken, Anne; Mellmann, Alexander; Holt, Nicholas J; Tarr, Phillip I; Karch, Helge; Zhang, Wenlan

    2009-01-01

    The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an approximately 80-kb (six strains) or approximately 120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.

  13. An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

    Science.gov (United States)

    Hara-Kudo, Yukiko; Konishi, Noriko; Ohtsuka, Kayoko; Iwabuchi, Kaori; Kikuchi, Rie; Isobe, Junko; Yamazaki, Takumiko; Suzuki, Fumie; Nagai, Yuhki; Yamada, Hiroko; Tanouchi, Atsuko; Mori, Tetsuya; Nakagawa, Hiroshi; Ueda, Yasufumi; Terajima, Jun

    2016-08-02

    To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening

  14. A quantitative polymerase chain reaction assay for rapid detection of 9 pathogens directly from stools of travelers with diarrhea.

    Science.gov (United States)

    Antikainen, Jenni; Kantele, Anu; Pakkanen, Sari H; Lääveri, Tinja; Riutta, Jukka; Vaara, Martti; Kirveskari, Juha

    2013-10-01

    Every year, 80 million tourists traveling to tropical and subtropical areas contract traveler's diarrhea (TD). Forty percent to 80% of cases are caused by bacteria, yet clinical diagnostic tests are available to identify only a few of the strains that cause TD. We aimed to develop a quantitative polymerase chain reaction (qPCR) assay to identify all major pathogens in stool samples. We developed a low-cost, high-throughput, multiplex qPCR assay for simultaneous detection of 9 bacterial pathogens in stool samples: Salmonella, Yersinia, Campylobacter, and Vibrio cholerae, as well as Shigella or enteroinvasive Escherichia coli, enterohemorrhagic E coli, enterotoxigenic E coli (ETEC), enteroaggregative E coli (EAEC), and enteropathogenic E coli (EPEC). The assay was validated using positive (n = 245) and negative (n = 243) control strains, as well as preselected positive and negative stool samples. In addition, stool samples were collected from 96 returning travelers with TD. The findings were compared with those from routine diagnostic tests. The assay detected the bacterial strains with 100% sensitivity and specificity, compared with results from the reference tests. Of all stool samples collected from travelers with TD, EPEC was found in 47%, EAEC in 46%, ETEC in 22%, enterohemorrhagic E coli in 7%, Campylobacter in 6%, Shigella or enteroinvasive E coli in 2%, and Salmonella in 2%. Multiple pathogens were found in 37% of all samples. We developed a low-cost, high-throughput qPCR assay for use in routine diagnostic analysis and research. It detects the pathogenic bacteria most commonly associated with TD in stool samples with 100% sensitivity and specificity, compared with reference methods. The assay requires 4 hours, whereas current detection methods require 1 to 7 days. At least 1 TD pathogen was identified in stool samples from 76% of returning travelers, whereas conventional methods found a pathogen in only 17%. The most commonly detected bacteria were EPEC

  15. Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution

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    Moulin-Schouleur Maryvonne

    2009-07-01

    Full Text Available Abstract Background Extraintestinal pathogenic Escherichia coli (ExPEC strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. Results A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST, Pulsed Field Gel Electrophoresis (PFGE and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC virulence. APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001, whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000. Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006 were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. Conclusion Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides

  16. Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution

    Science.gov (United States)

    2009-01-01

    Background Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. Results A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence. APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. Conclusion Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some

  17. Rapid and simple method by combining FTA™ card DNA extraction with two set multiplex PCR for simultaneous detection of non-O157 Shiga toxin-producing Escherichia coli strains and virulence genes in food samples.

    Science.gov (United States)

    Kim, S A; Park, S H; Lee, S I; Ricke, S C

    2017-12-01

    The aim of this research was to optimize two multiplex polymerase chain reaction (PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli (STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Two multiplex polymerase chain reaction (PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli (STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in

  18. Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-lactamases from bovine mastitis isolates in the United Kingdom.

    Science.gov (United States)

    Timofte, Dorina; Maciuca, Iuliana E; Evans, Nicholas J; Williams, Helen; Wattret, Andrew; Fick, Jenny C; Williams, Nicola J

    2014-01-01

    Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.

  19. Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157:H7 by Propidium Monoazide Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2014-01-01

    Full Text Available Conventional methods to determine the efficacy of bacteriophage (phage for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA, a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01 phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7.

  20. Detection and Characterization of the Fimbrial sfp Cluster in Enterohemorrhagic Escherichia coli O165:H25/NM Isolates from Humans and Cattle ▿

    Science.gov (United States)

    Bielaszewska, Martina; Prager, Rita; Vandivinit, Liz; Müsken, Anne; Mellmann, Alexander; Holt, Nicholas J.; Tarr, Phillip I.; Karch, Helge; Zhang, Wenlan

    2009-01-01

    The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an ∼80-kb (six strains) or ∼120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown. PMID:18978078

  1. Influence of milk product type and its initial contamination on the efficiency of different methods for detection of Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli O157:H7

    Directory of Open Access Journals (Sweden)

    Boris Antunović

    2018-01-01

    Full Text Available This paper investigates differences in efficacy of isolating pathogenic bacteria Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli O157:H7 between conventional cultivation (ISO method and immunomagnetic separation (IMS method related to the types of dairy products and initial numbers of bacteria. Different milk products (dairy pudding- vanilla or chocolate; a mixture of yoghurt and pudding; solid, liquid and fruit yoghurt; AB culture - with or without fruit; cheese spread were intentionally contaminated with different numbers (≈10 and ≈30 of live cells of the observed bacteria per mL. The obtained results showed that the classical ISO procedure still represents an equally adequate method for the detection of S. Enteritidis and L. monocytogenes in dairy products as well as the IMS method. However, the ISO method was found to be inefficient for determination of E. coli O157:H7 when the initial contamination was low (≈10 live cells per mL. In such cases, even the IMS method appeared to be inefficient when used for fermented dairy product analysis. Fermented dairy products in contrast to the non-fermented ones, still represent a challenge for the development of routine detection methods, especially for S. Enteritidis, whilst the detection of L. monocytogenes and E. coli O157:H7 has improved by introducing the IMS method. The largest difference in the ability to detect bacteria in dairy product samples with reference to the initial number of bacteria by both methods was in the detection of E. coli O157:H7. The choice of broth (non-selective fluid broth vs. selective fluid broth did not matter in the in the detection of S. Enteritidis and L. monocytogenes by applying the IMS procedure. However, for the detection of E. coli O157:H7 the application of modified tripton-soya broth with novobiocin (mTSB+Nb has proved to be superior when compared to using the buffered peptone water (BPW. The presented results may be of importance as

  2. Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant Escherichia coli and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant E. coli Isolates

    Science.gov (United States)

    Wu, Beibei; Xia, Chun; Du, Xiangdang; Cao, Xingyuan; Shen, Jianzhong

    2006-01-01

    To detect florfenicol-resistant Escherichia coli isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione S-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromatography, respectively. Western blotting using the anti-FloR1 antibody showed specific binding of the antibody to the florfenicol-resistant FloR protein. Preincubation of florfenicol-resistant strains with the antibody significantly increased the intracellular accumulation of florfenicol and enhanced the bacterial susceptibility to florfenicol, suggesting that antibody binding to the FloR protein inhibited the activity of the efflux protein conferred by the floR gene. Analyses of florfenicol-resistant and -sensitive isolates by ELISA using the anti-FloR1 antibody showed good correlation between FloR protein expression and the floR genotype. The anti-FloR1 antibody-based ELISA is a useful tool for the detection of florfenicol-resistant bacteria harboring the floR gene. PMID:16455887

  3. Towards a Molecular Definition of Enterohemorrhagic Escherichia coli (EHEC): Detection of Genes Located on O Island 57 as Markers To Distinguish EHEC from Closely Related Enteropathogenic E. coli Strains

    Science.gov (United States)

    Delannoy, Sabine; Beutin, Lothar

    2013-01-01

    Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx1, stx2, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains. PMID:23325824

  4. Towards a molecular definition of enterohemorrhagic Escherichia coli (EHEC): detection of genes located on O island 57 as markers to distinguish EHEC from closely related enteropathogenic E. coli strains.

    Science.gov (United States)

    Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

    2013-04-01

    Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx1, stx2, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains.

  5. Taxonomy Icon Data: Escherichia coli [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Escherichia coli Escherichia coli Escherichia_coli_L.png Escherichia_coli_NL.png Escherichia..._coli_S.png Escherichia_coli_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+co...li&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NL http://biosciencedbc.jp/taxono...my_icon/icon.cgi?i=Escherichia+coli&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NS ...

  6. Covalently linked immunomagnetic separation/adenosine triphosphate technique (Cov-IMS/ATP) enables rapid, in-field detection and quantification of Escherichia coli and Enterococcus spp. in freshwater and marine environments.

    Science.gov (United States)

    Lee, C M; Griffith, J F; Kaiser, W; Jay, J A

    2010-07-01

    Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody-bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems. Covalently linked complexes were 58-89% more robust than antibody-bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov-IMS/ATP) and culture-based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli- 38 cells per 100 ml; Enterococcus spp. - 25 cells per 100 ml). Cov-IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov-IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence. Cov-IMS/ATP is a robust, in-field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels. To our knowledge, this is the first work to present a viable rapid, in-field assay for measuring FIB concentrations in marine water environments. Cov-IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  7. A double-taper optical fiber-based radiation wave other than evanescent wave in all-fiber immunofluorescence biosensor for quantitative detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Zhang, Zhonghuan; Hua, Fei; Liu, Ting; Zhao, Yong; Li, Jun; Yang, Ruifu; Yang, Changxi; Zhou, Lei

    2014-01-01

    Cylindrical or taper-and-cylinder combination optical fiber probe based on evanescent wave has been widely used for immunofluorescence biosensor to detect various analytes. In this study, in contrast to the contradiction between penetration depth and analyte diameter of optical fiber probe-based evanescent wave, we demonstrate that double-taper optical fiber used in a radiation wave-based all-fiber immunofluorescence biosensor (RWAIB) can detect micron-scale analytes using Escherichia coli O157:H7 as representative target. Finite-difference time-domain method was used to compare the properties of evanescent wave and radiation wave (RW). Ray-tracing model was formulated to optimize the taper geometry of the probe. Based on a commercial multi-mode fiber, a double-taper probe was fabricated and connected with biosensor through a "ferrule connector" optical fiber connector. The RWAIB configuration was accomplished using commercial multi-mode fibers and fiber-based devices according to the "all-fiber" method. The standard sample tests revealed that the sensitivity of the proposed technique for E. coli O157:H7 detection was 10(3) cfu · mL(-1). Quantitation could be achieved within the concentration range of 10(3) cfu · mL(-1) to 107 cfu · mL(-1). No non-specific recognition to ten kinds of food-borne pathogens was observed. The results demonstrated that based on the double-taper optical fiber RWAIB can be used for the quantitative detection of micron-scale targets, and RW sensing is an alternative for traditional evanescent wave sensing during the fabrication of fiber-optic biosensors.

  8. A double-taper optical fiber-based radiation wave other than evanescent wave in all-fiber immunofluorescence biosensor for quantitative detection of Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Zhonghuan Zhang

    Full Text Available Cylindrical or taper-and-cylinder combination optical fiber probe based on evanescent wave has been widely used for immunofluorescence biosensor to detect various analytes. In this study, in contrast to the contradiction between penetration depth and analyte diameter of optical fiber probe-based evanescent wave, we demonstrate that double-taper optical fiber used in a radiation wave-based all-fiber immunofluorescence biosensor (RWAIB can detect micron-scale analytes using Escherichia coli O157:H7 as representative target. Finite-difference time-domain method was used to compare the properties of evanescent wave and radiation wave (RW. Ray-tracing model was formulated to optimize the taper geometry of the probe. Based on a commercial multi-mode fiber, a double-taper probe was fabricated and connected with biosensor through a "ferrule connector" optical fiber connector. The RWAIB configuration was accomplished using commercial multi-mode fibers and fiber-based devices according to the "all-fiber" method. The standard sample tests revealed that the sensitivity of the proposed technique for E. coli O157:H7 detection was 10(3 cfu · mL(-1. Quantitation could be achieved within the concentration range of 10(3 cfu · mL(-1 to 107 cfu · mL(-1. No non-specific recognition to ten kinds of food-borne pathogens was observed. The results demonstrated that based on the double-taper optical fiber RWAIB can be used for the quantitative detection of micron-scale targets, and RW sensing is an alternative for traditional evanescent wave sensing during the fabrication of fiber-optic biosensors.

  9. Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli.

    Science.gov (United States)

    Abe, Cecilia M; Salvador, Fábia A; Falsetti, Ivan N; Vieira, Mônica A M; Blanco, Jorge; Blanco, Jesús E; Blanco, Miguel; Machado, Antônia M O; Elias, Waldir P; Hernandes, Rodrigo T; Gomes, Tânia A T

    2008-04-01

    To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.

  10. Diarrhoeagenic Escherichia coli are not a significant cause of diarrhoea in hospitalised children in Kuwait

    Directory of Open Access Journals (Sweden)

    Pacsa Alexander S

    2009-03-01

    Full Text Available Abstract Background The importance of diarrhoeagenic Escherichia coli (DEC infections in the Arabian Gulf including Kuwait is not known. The prevalence of DEC (enterotoxigenic [ETEC], enteropathogenic [EPEC], enteroinvasive [EIEC], enterohemorrhagic [EHEC] and enteroaggregative [EAEC] was studied in 537 children ≤ 5 years old hospitalised with acute diarrhoea and 113 matched controls from two hospitals during 2005–07 by PCR assays using E. coli colony pools. Results The prevalence of DEC varied from 0.75% for EHEC to 8.4% for EPEC (mostly atypical variety in diarrhoeal children with no significant differences compared to that in control children (P values 0.15 to 1.00. Twenty-seven EPEC isolates studied mostly belonged to non-traditional serotypes and possessed β and θ intimin subtypes. A total of 54 DEC isolates from diarrhoeal children and 4 from controls studied for antimicrobial susceptibility showed resistance for older antimicrobials, ampicillin (0 to 100%, tetracycline (33 to 100% and trimethoprim (22.2 to 100%; 43.1% of the isolates were multidrug-resistant (resistant to 3 or more agents. Six (10.4% DEC isolates produced extended spectrum β-lactamases and possessed genetic elements (blaCTX-M, blaTEM and ISEcp1 associated with them. Conclusion We speculate that the lack of significant association of DEC with diarrhoea in children in Kuwait compared to countries surrounding the Arabian Gulf Region may be attributable to high environmental and food hygiene due to high disposable income in Kuwait.

  11. Escherichia coli with virulence factors and multidrug resistance in the Plankenburg River

    Directory of Open Access Journals (Sweden)

    Corne Lamprecht

    2014-09-01

    Full Text Available Escherichia coli is a natural inhabitant of the gut and E. coli levels in water are considered internationally to be an indication of faecal contamination. Although not usually pathogenic, E. coli has been linked to numerous foodborne disease outbreaks, especially those associated with fresh produce. One of the most common ways through which E. coli can be transferred onto fresh produce is if contaminated water is used for irrigation. In this study, a total of 81 confirmed E. coli strains were isolated from the Plankenburg River as part of three separate studies over 3 years. During sampling, E. coli levels in the river were above the accepted levels set by the World Health Organization and the South African Department of Water Affairs and Forestry for safe irrigation of fresh produce, which indicates that transfer of E. coli during irrigation is highly probable. Multiplex polymerase chain reaction screening for pathogenic gene sequences revealed one enteroaggregative positive strain and four enteropathogenic positive strains. The four enteropathogenic strains were also found to be resistant to three or more critically and highly important antibiotics and were therefore classified as multidrug resistant strains. These results show that E. coli with enteropathogenic potential and multiple antimicrobial resistance properties has persisted over time in the Plankenburg River.

  12. [Principal characteristics and diagnosis of the pathogenic groups of Escherichia coli].

    Science.gov (United States)

    Rodríguez-Angeles, Guadalupe

    2002-01-01

    Escherichia coli colonizes the human intestinal tract within hours of birth and is considered a non-pathogenic member of the normal intestinal flora. However, there are six pathogenic groups that may produce diarrhea: enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC) and diffusely adherent (DAEC) groups. E. coli can be isolated and classified using traditional methods, by identifying its biochemical or serum characteristics. The pathogenic mechanisms may be studied in cell cultures and animal model assays, as well as more up to date molecular biology methods for study and diagnosis. The latter have proven that genes are involved in pathogenesis. The objective of the present work is to draw attention to the importance of E. coli as a pathogenic organism. This microorganism is an etiologic agent of sporadic cases of diarrhea, hemorrhagic colitis, dysentery, and hemolytic uremic syndromes and outbreaks. Diarrheic E. coli manifestations occur mainly among infants, and deep knowledge and understanding of this microorganism are crucial to better epidemiologic surveillance.

  13. Escherichia coli pathotypes in Pakistan from consecutive floods in 2010 and 2011.

    Science.gov (United States)

    Bokhari, Habib; Shah, Muhammad Ali; Asad, Saba; Akhtar, Sania; Akram, Muhammad; Wren, Brendan W

    2013-03-01

    This study compares Escherichia coli pathotypes circulating among children in Pakistan during the floods of 2010 and 2011 and from sporadic cases outside flood affected areas. Using multiplex polymerase chain reaction 115 of 205 stool samples (56.29%) were positive for diarrheagenic E. coli from specimens taken during the floods compared with 50 of 400 (12.5%) stool samples being positive for sporadic cases. The E. coli pathotypes were categorized as Enteropathogenic E. coli 33 (28.69%) and 13 (26%), Enterotoxigenic E. coli 29 (25.21%) and 15 (30%), Enteroaggregative E. coli 21 (18.2%) and 18 (36%), Enterohemorrhagic E. coli 5 (4.34%) and 1 (2%) from flood and sporadic cases, respectively. Furthermore, patients co-infected with more than one pathotype were 26 (22.60%) and 3 (6%) from flood and sporadic cases, respectively. The study shows an unexpectedly high rate of isolation of E. coli pathotypes suggesting Pakistan as an endemic region that requires active surveillance particularly during flood periods.

  14. Diarrheagenic Escherichia coli: Prevalence and Pathotype Distribution in Children from Peruvian Rural Communities.

    Science.gov (United States)

    Acosta, Gonzalo J; Vigo, Natalia I; Durand, David; Riveros, Maribel; Arango, Sara; Zambruni, Mara; Ochoa, Theresa J

    2016-09-07

    Diarrheagenic Escherichia coli (DEC) are common pathogens of childhood gastrointestinal infections worldwide. To date, research tracking DEC has mainly been completed in urban areas. This study aims to determine the prevalence and pathotype distribution of DEC strains in children from rural Peruvian communities and to establish their association with malnutrition. In this prospective cohort, 93 children aged 6-13 months from rural communities of Urubamba (Andes) and Moyobamba (jungle) were followed for 6 months. Diarrheal and control stool samples were analyzed using multiplex real-time polymerase chain reaction to identify the presence of virulence genes of DEC strains. The overall isolation rate of DEC was 43.0% (352/820). Enteroaggregative E. coli (EAEC, 20.4%), enteropathogenic E. coli (EPEC, 14.2%), and diffusely aggregative E. coli (DAEC, 11.0%) were the most prevalent pathotypes. EAEC was more frequently found in Moyobamba samples (P jungle of Peru. In addition, children with a greater decline in their growth rate had higher EAEC isolation rates, highlighting the importance of this pathogen in child malnutrition. © The American Society of Tropical Medicine and Hygiene.

  15. Evaluation of a multiplex real-time PCR method for detecting shiga toxin-producing Escherichia coli in beef and comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology laboratory guidebook method.

    Science.gov (United States)

    Fratamico, Pina M; Wasilenko, Jamie L; Garman, Bradley; Demarco, Daniel R; Varkey, Stephen; Jensen, Mark; Rhoden, Kyle; Tice, George

    2014-02-01

    The "top-six" non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 10(3) CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted

  16. Coliforms and Escherichia coli in waters

    International Nuclear Information System (INIS)

    Cavallari, E.; Stefanelli, Gian Piero; Lorenzini, T.

    2005-01-01

    The study shows the evaluation of a defined substrate method, Colilert 18/Quanty Tray, for the simultaneous detection of Coliforms bacteria and Escherichia coli in water. The results obtained indicate that this method represents a valid alternative to the traditional methods considering sensitivity, specificity, repeatability but also rapidity and simplicity of use [it

  17. [Detection and characterization of Shiga toxin-producing Escherichia coli in children treated at an inter-zonal pediatric hospital in the city of La Plata].

    Science.gov (United States)

    Oderiz, Sebastián; Leotta, Gerardo A; Galli, Lucía

    2018-01-11

    Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause watery diarrhea, bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). The objective of this study was to determine the phenotypic and genotypic profiles of STEC strains isolated from children with BD and HUS treated at a pediatric hospital in the city of La Plata in the period 2006-2012, and to establish the clonal relationship of O157:H7 isolates by pulsed field electrophoresis. The percentage of positive samples was 4.9% and 39.2% in patients with BD and HUS, respectively. Seventy-seven STEC strains from 10 different serotypes were isolated, with 100% colony recovery, O157:H7 being the most frequent (71.4%) serotype, followed by O145:NM (15.6%). An average of 98.2% of O157:H7 isolates belonged to biotype C and were sensitive to all the antibiotics tested. All of them (100%) carried genotype stx 2 , eae, fliC H7 , ehxA, iha, efa, toxB, lpfA1-3 and lpfA2-2. When the clonal relationship of the O157:H7 strains was studied, a total of 42 patterns with at least 88% similarity were identified, and 6 clusters with identical profiles were established. The eae-negative isolates belonged to serotypes O59:H19, O102:H6, O174:NM and O174:H21. The strains O59:H19 and O174:H21 were positive for the aggR gene. This study shows that STEC of different serotypes and genotypes circulate in the city of La Plata and surroundings. Despite the genetic diversity observed between the O157:H7 isolates, some were indistinguishable by the subtyping techniques used. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  18. lactamases genes among0 Escherichia coli from patients with ...

    African Journals Online (AJOL)

    -lactamases (ESBLs) that mediate resistance to b-lactam drugs among Escherichia coli and other uropathogens have been reported worldwide. However, there is little information on the detection of ESBLs genes in E. coli from patients with ...

  19. Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Novicki, T J; Daly, J A; Mottice, S L; Carroll, K C

    2000-02-01

    Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.

  20. Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

    Science.gov (United States)

    Apfel, C M; Evers, S; Hubschwerlen, C; Pirson, W; Page, M G; Keck, W

    2001-04-01

    An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

  1. Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

    Directory of Open Access Journals (Sweden)

    G.A. Leotta

    2005-03-01

    Full Text Available La infección por Escherichia coli productor de toxina Shiga (STEC es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.Shiga toxin-producing Escherichia coli (STEC cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The

  2. [Evaluation of usefulness of the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to lipopolysaccharides of Enterohemorrhagic Escherichia coli (EHEC) strains in patients with gastrointestinal disorders and patients with hemolytic uremic syndrome].

    Science.gov (United States)

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains are an important zoonotic food-borne and waterborne pathogens causing diarrhea and the severe hemolytic uremic syndrome (HUS) in humans. The aim of the study was to evaluate the usefulness of enzyme immunoassay ELISA for detection of antibodies to the lipopolysaccharides (LPS) of EHEC in patients with gastrointestinal disorders and patients with hemolytic-uremic syndrome. Sera obtained from 526 patients with gastrointestinal disorders, 26 patients with HUS and 74 patients with different bacterial gastroenteritis infections were screened by an LPS-based ELISA. The LPS antigens of EHEC belonging to serogroups O26, O103, O104, O111, O121, O145, and O157 were obtained by modified Boivin's method. Additionally, to determine the cut-off level, the 122 sera from healthy people were tested. Cellular extract from E. coli O14 were used to remove by absorption antibodies to the Enterobacteriaceae Common Antigen (ECA). Generally, seroprevalence of antibodies to the LPS of different EHEC serogroups in patients with gastrointestinal disorders was low. Additionally, interpretation of the some positive results was difficult to the fact of many serological mutual interactions. Particularly a lot of cross-reactions were seen in the group of sera obtained from patients with different bacterial gastroenteritis infections. The study showed also that in most cases the absorption of antibodies to the ECA had no significant effect on the cross-reactions observed in ELISA. On the other hand, the very high level of antibodies to the LPS antigen of E. coli O26 was found in 5 patients, to E. coli O157 in 4 patients, to E. coli O104 and O145 in 3 patients and E. coli O111 in 2 patients with HUS. Analysis of antibody levels in paired sera taken 2-3 weeks apart obtained from six HUS patients showed a rapid decline of antibody levels to the LPS antigens. The results showed the usefulness of the ELISA with lipopolysaccharides antigens to

  3. Laboratory and pilot-scale dead-end ultrafiltration concentration of sanitizer-free and chlorinated lettuce wash water for improved detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Magaña, Sonia; Schlemmer, Sarah M; Davidson, Gordon R; Ryser, Elliot T; Lim, Daniel V

    2014-08-01

    An automated dead-end (single pass, no recirculation) ultrafiltration device, the Portable Multi-use Automated Concentration System (PMACS), was evaluated as a means to concentrate Escherichia coli O157:H7 from 40 liters of simulated commercial lettuce wash water. The assessment included generating, sieving, and concentrating sanitizer-free lettuce wash water, either uninoculated or inoculated with green fluorescent protein-transformed E. coli O157:H7 at a high (1.00 log CFU/ml) or low (-1.00 log CFU/ml) concentration. Cells collected within the filters were recovered in approximately 400 ml of buffer to create lettuce wash retentates. The extent of concentration was determined by viable plate counts using a medium selective for the transformed E. coli O157:H7. The samples were qualitatively analyzed for E. coli O157:H7 according to the U. S. Food and Drug Administration Bacteriological Analytical Manual enrichment method and with an electrochemiluminescence immunoassay. This concentration method was then evaluated in a pilot-scale production line at Michigan State University using chlorinated (100, 30, and 10 ppm of available chlorine) lettuce wash water. The total PMACS processing times were 82 ± 6 and 65 ± 5 min for sanitizer-free and chlorinated washes, respectively. Overall, E. coli O157:H7 populations were approximately 2 log higher in retentates than in unconcentrated lettuce wash samples. The higher E. coli O157:H7 levels in the retentates enabled cultural and electrochemiluminescence immunoassay detection in some samples when the corresponding lettuce wash samples were negative. When combined with standard and rapid detection methods, the PMACS concentration method may provide a means to enhance pathogen monitoring of produce wash water.

  4. Detection of extended-spectrum β-lactamases (blaCTX-M-1 and blaTEM in Escherichia coli, Salmonella spp., and Klebsiella pneumoniae isolated from poultry in North Eastern India

    Directory of Open Access Journals (Sweden)

    H. Lalzampuia

    2014-11-01

    Full Text Available Aim: The present study was conducted to record the association of extended spectrum β-lactamases (ESBLs producing enteric bacteria with diarrhea of poultry birds in Mizoram, India. Materials and Methods: Fecal samples were collected from poultry birds with the history of diarrhea from different parts of Mizoram. Samples were processed for isolation and identification of Escherichia coli, Salmonella, and Klebsiella pneumoniae. All the isolates were subjected to antibiotic sensitivity assays. Phenotypically, ESBLs production ability was determined by double discs synergy test (DDST method. ESBLs producing isolates were subjected to polymerase chain reaction (PCR for detection of ESBLs genes. Plasmids were cured by acridine orange. Transfer of resistance from donor to recipient strains was done by in vitro horizontal method. Results: A total of 134 enteric bacteria was isolated, of which 102 (76.12%, 21 (15.67% and 11 (8.21% were E. coli, Salmonella spp. and K. pneumoniae, respectively. By DDST 7 (5.22% isolates (6 E. coli and 1 K. pneumoniae were ESBLs producer. PCR analysis confirmed 5 (3.73% (4 E. coli and 1 K. pneumoniae isolates harboured blaCTX-M-1 gene and/or blaTEM gene. All the isolates were carrying plasmids ranging between 0.9 kb and ~30 kb. Of the 4 isolates positive for blaCTX-M-1 and/or blaTEM, 2 (1.84% were confirmed for blaCTX-M-1 gene in their plasmid. No blaTEM gene was detected from plasmid. The resistance plasmid could not be transferred to the recipient by in vitro horizontal gene transfer method. Conclusion: ESBLs producing enteric bacteria are circulating in poultry in North Eastern Region of India. As poultry is one of the most common food animals in this region, these organisms may enter in human population through them.

  5. Rapid detection of amoxicillin-susceptible Escherichia coli in fresh uncultured urine: a new tool to limit the use of broad-spectrum empirical therapy of community-acquired pyelonephritis.

    Science.gov (United States)

    Chapelet, Guillaume; Corvec, Stéphane; Montassier, Emmanuel; Herbreteau, Guillaume; Berrut, Gilles; Batard, Eric; de Decker, Laure

    2016-06-01

    Because of the high prevalence of amoxicillin resistance among uropathogens, amoxicillin is not recommended as an empirical treatment of urinary tract infections (UTIs). Quick detection of an amoxicillin-susceptible Escherichia coli (ASEC) would allow prescribing amoxicillin without preliminary broad-spectrum empirical treatment in uncomplicated pyelonephritis. To quickly diagnose UTIs due to ASEC, we developed a real-time PCR that detects in fresh uncultured urine the E. coli-specific gene yccT as well as the blaTEM and blaCTX-M genes. The ASEC rapid test was considered positive if the PCR was positive for the yccT gene but negative for blaTEM and blaCTX-M. The test was compared with culture and susceptibility testing. Among 200 patients with a suspected community-acquired UTI, 61 (30.5%) had a monobacterial UTI due to ASEC. The ASEC rapid test result was obtained in 3 h 13 [95% confidence interval (CI) 3 h 12-3 h 15] and was positive for 43 patients (21.5%). Specificity and sensitivity were 97.8% (95% CI 95.8-99.8%) and 65.6% (95% CI 59.0-72.1%), respectively. Positive and negative predictive values were 93.0% (95% CI 89.5-96.5%) and 86.6% (95% CI 81.9-91.3%), respectively. Owing to its high specificity and positive predictive value, the ASEC rapid test allows the diagnosis of UTI due to ASEC only 3 h after urine sampling. A positive ASEC rapid test may be used to treat uncomplicated pyelonephritis with amoxicillin from the start, without preliminary broad-spectrum empirical treatment. The ASEC rapid test is a promising tool to spare fluoroquinolones and third-generation cephalosporins in UTIs. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  6. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs

    DEFF Research Database (Denmark)

    Weber, N. R.; Nielsen, J. P.; Hjulsager, Charlotte Kristiane

    2017-01-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were......: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order...... to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes...

  7. Detection of Class 1 and 2 Integrons, β-Lactamase Genes and Molecular Characterization of Sulfonamide Resistance in Escherichia coli Isolates Recovered from Poultry in China

    Directory of Open Access Journals (Sweden)

    Jam Kashif§, Rehana Buriro§, Javed Memon, Muhammad Yaqoob, Jamila Soomro§, Diao Dongxue, Huang Jinhu and Wang Liping*

    2013-07-01

    Full Text Available This study aimed to detect integrons, β-lactamase genes and to characterize sulfonamide resistant E. coli isolates recovered from poultry. All the isolates (n=38 were investigated for the presence of integrons, Sul1, Sul2, Sul3 genes by PCR. Class 1 and class 2 integron were present in 79 and 16%, respectively. Additional resistance gene cassette embedded in class 1 and 2 integrons was aadA1, aadA5, dfrA17 and aadA22, dfrA, respectively. Sul1 and Sul2 genes were detected in 42.1 and 60.5% isolates, respectively. Both the Sul1 and Sul2 were present in 23% isolates. However, Sul3 gene was not present. Co-existence of Sul1 and Sul2 with class 1 integrons was found in 28.9 and 60.5% of class 1 integron positive isolates, respectively. Whereas, a less percentage of isolates showed a low level of resistance to β-lactams and no blaCTX-M, blaSHV and blaTEM was found. The MIC results showed resistance to sulfadiazine and sulfamethoxazole-trimethoprim in 88 and 84% isolates, resistance to penicillin, ampicillin, amoxicillin was 52, 52 and 44%, respectively. Chloramphenicol, florfenicol, tetracycline and gentamycin resistance was found in 51, 5, 42 and 67% isolates, respectively. This study revealed high frequency of class 1 integrons, Sul genes among poultry E. coli isolates, therefore further spread of Sul genes and integrons is predictable.

  8. Antibody-based Detection of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium Grown in Low-shear Modeled Microgravity

    Science.gov (United States)

    Nyquist-Battie, Cynthia; Freeman, Laura; Leckband, Kristen; Martinez, Stephanie; Ansley, Ariel; Lund, Deanna; Lim, Daniel V.

    2008-06-01

    With the advent of prolonged spaceflights, it is important to determine if antibody-based assays can be used to monitor food and water for bacterial contaminants. In the present work, a ground-based high aspect ratio vessel (HARV) was used to determine if low shear modeled microgravity (LSMMG) alters antibody-binding to E. coli O157:H7 and Salmonella enterica serovar Typhimurium. Antibody-bacteria binding was similar under LSMMG and normal gravity because there was no difference in amount of captured bacteria measured by colony forming units (CFU) between assays conducted in the HARV and a conventional roller flask. The ability of E. coli O157:H7 and Salmonella Typhimurium grown in LSMMG to bind specific antibodies was also studied. After incubations of 4, 18 or 36 h in the HARV or a shaking incubator, bacteria were harvested for enzyme-linked immunosorbent assays (ELISA). In the E. coli O157:H7 ELISA using a goat polyclonal primary antibody, LSMMG did not alter the linear range of detection (105-107 cells/ml) nor the signal to noise ratio at any bacterial concentration. Although insignificant changes in signal to noise ratios were evident, LSMMG did not alter the range of detection (105-107 cells/ml) for Salmonella Typhimurium in ELISAs using either a polyclonal or a monoclonal antibody. These results suggest that immunoassays may be used in spacecrafts because LSMMG does not have significant deleterious effects on antibody-binding to bacteria nor does it significantly alter surface antigens necessary for antibody-based methods.

  9. Evaluation of a QIAamp DNA stool purification kit for Shiga-toxigenic Escherichia coli detection in bovine fecal swabs by PCR Evaluación del kit QIAamp DNA stool purification para la detección de Escherichia coli productor de toxina Shiga en hisopados de materia fecal bovina por PCR[

    Directory of Open Access Journals (Sweden)

    A. Gioffré

    2004-03-01

    Full Text Available A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC by polymersase chain reaction (PCR directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol, and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 % were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64% than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor, y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %, fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%. En conclusión, el uso del kit QIAamp DNA stool

  10. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    Science.gov (United States)

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Prevalence and pathogenic potential of Escherichia coli isolates from raw milk and raw milk cheese in Egypt.

    Science.gov (United States)

    Ombarak, Rabee A; Hinenoya, Atsushi; Awasthi, Sharda Prasad; Iguchi, Atsushi; Shima, Ayaka; Elbagory, Abdel-Rahman M; Yamasaki, Shinji

    2016-03-16

    The objectives of this study were to investigate prevalence and pathogenic potential of Escherichia coli contaminating raw milk and its products in Egypt. Out of 187 dairy products including 72 raw milk samples, 55 Karish cheese and 60 Ras cheese, 222 E. coli isolates including 111, 89 and 22 were obtained from 55 raw milk samples (76.4%), 41 Karish cheese (74.5%), and 13 Ras cheese (21.7%), respectively. Isolated E. coli strains were examined for 24 representative virulence genes present in diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Among DEC and ExPEC virulence factors, genes for enteropathogenic E. coli (eaeA, bfpA, EAF), enterohemorrhagic E. coli (stx1, stx2, eaeA), enterotoxigenic E. coli (elt, est), enteroinvasive E. coli (invE), enteroaggregative E. coli (Eagg, astA), diffusely adherent E. coli (daaD), ExPEC (cdt-I to cdt-V, cnf1, cnf2, hlyA) and putative adhesins (efa1, iha, ehaA, saa, and lpfAO113) were screened by colony hybridization assay. Out of 222 E. coli strains, 104 (46.8%) isolated from 69 (36.9%) samples carried one or more virulence genes. The most prevalent gene detected was lpfAO113 (40.5%), followed by ehaA (32.4%,), astA (3.15%,), iha (1.80%), hlyA (1.35%), stx1 (0.90%), stx2 (0.90%), eaeA (0.45%), cdt-III (0.45%) and cnf2 (0.45%). Two strains isolated from Karish cheese harbored 5 virulence genes (stx1, stx2, iha, ehaA, lpfAO113). Stx subtype was determined to be stx1 (not stx1c or stx1d) and stx2d. Indeed, expression of hemolysin A, CDT-III, CNF-II, Stx1 and Stx2d was confirmed by blood agar plate, cytotoxicity assay and Western blotting, respectively. Among the 222 E. coli strains, 54 (48.6%), 38 (42.6%) and 12 (54.7%) isolated from raw milk, Karish cheese and Ras cheese were potentially virulent, respectively. O-genotyping indicated that most of the potentially virulent E. coli isolates did not belong to clinically important O serogroups except O75, O91 and O166, which have been associated with human

  12. Study on isolation, molecular detection of virulence gene and antibiotic sensitivity pattern of Escherichia coli isolated from milk and milk products

    Directory of Open Access Journals (Sweden)

    M. N. Brahmbhatt

    2013-06-01

    Full Text Available Aim: The study was undertaken to isolate pathogenic E. coli from milk and various milk products, detection of virulence gene using Polymerase chain reaction (PCR and investigate their antibiotic sensitivity pattern. Materials and Methods: Altogether 250 milk and various milk products samples consisting of raw milk (50, cheese (50, ice-cream (50, mawa (50 and dahi (50 were collected from milk vendors, retail shops located in Anand city, under aseptic precautions. For the enrichment of the organism from the collected samples, MacConkey broth was used and inoculation was carried out on MacConkey agar and EMB agar. Later on, to confirm the isolates, various biochemical tests such as IMViC test, Urease test were performed. Evaluation of antibiotic sensitivity pattern of E. coli was assessed by disk diffusion method. Finally the E. coli isolates were screened for the presence of virulence associated genes by PCR . Results: The prevalence of E. coli was observed 32 % in the samples comprising of milk (52.00%, cheese (28.00%, icecream (20.00%, mawa (44.00%, and dahi (16.00%. Antibiotic sensitivity was recorded high for Co-trimoxazole (100% followed by Gentamicin (96.73%, Trimithoprime (93.47% and Doxycycline hydochloride (92.39%. Least sensitivity was recorded for Ampicillin (8.69%. In this study, out of 80 E. coli isolates, 25 isolates (31.25% were positive for stx genes, of which 7 (8.75% isolates were positive for stx1 gene only, while 12 (15.00% isolates were positive for stx2 gene only and 5 (6.25% isolates were positive for both stx1 and stx2, 7 isolates (8.75% were positive for eaeA gene and all the isolate were negetive for rfb O157 gene. Conclusions: Current study supports the finding that raw milk and various milk products can be regarded as critical source of pathogenic E. coli This explains the need of strict monitoring and surveillance for effective measures of hygiene and sanitary practice during production of milk and various milk

  13. Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

    Science.gov (United States)

    Lindsey, Rebecca L; Garcia-Toledo, L; Fasulo, D; Gladney, L M; Strockbine, N

    2017-09-01

    Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies. Published by Elsevier B.V.

  14. 76 FR 20542 - Escherichia coli

    Science.gov (United States)

    2011-04-13

    ... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host bacteria... specific to Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic... exemption from the requirement of a tolerance for residues of Escherichia coli O157:H7 Specific...

  15. Evaluation of the Clinical and Laboratory Standards Institute phenotypic confirmatory test to detect the presence of extended-spectrum β-lactamases from 4005 Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates.

    Science.gov (United States)

    Morrissey, Ian; Bouchillon, Samuel K; Hackel, Meredith; Biedenbach, Douglas J; Hawser, Stephen; Hoban, Daryl; Badal, Robert E

    2014-04-01

    A subset of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates collected for the Study for Monitoring Antimicrobial Resistance Trends that were positive for the Clinical and Laboratory Standards Institute (CLSI) extended-spectrum β-lactamase (ESBL) phenotypic confirmatory test (n = 3245) or had an ertapenem MIC of ≥0.5 µg ml(-1) (n = 293), or both (n = 467), were analysed for ESBL genes. Most ESBL phenotype E. coli or K. pneumoniae possessed an ESBL gene (95.8 and 88.4 %, respectively), and this was 93.1 % if carbapenem-non-susceptible K. pneumoniae were removed. This rate was lower for P. mirabilis (73.4 %) and K. oxytoca (62.5 %). Virtually all ESBL-positive isolates (99.5 %) were cefotaxime non-susceptible [CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints)]. Fewer isolates (82 %) were ceftazidime non-susceptible (CLSI breakpoints). In addition, 21.1 % of E. coli, 25 % of K. oxytoca and 78.7 % of P. mirabilis isolates were ceftazidime susceptible but ESBL positive. This suggests that CLSI breakpoints for ceftazidime are too high to detect ESBLs. The lower EUCAST breakpoints detected ESBLs in E. coli and K. oxytoca better, but 59.6 % of ESBL-positive isolates of P. mirabilis were ceftazidime susceptible. For isolates with ertapenem MICs ≥0.5 µg ml(-1), more accurate ESBL phenotype analysis was observed for E. coli and K. pneumoniae (sensitivity >95 % for both, specificity 94.4 and 54.1 %, respectively). If carbapenemase-positive K. pneumoniae were excluded, the specificity increased to 78 %. The positive predictive values for the ESBL phenotypic test with E. coli and K. pneumoniae were 97.6 and 81.8 %, respectively, and negative predictive values were 75.9 and 95.2 %, respectively. We therefore suggest that it would be prudent to confirm phenotypic ESBL-positive P. mirabilis, K. pneumoniae and K. oxytoca with molecular analysis.

  16. Escherichia coli pathotypes

    Science.gov (United States)

    Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

  17. No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area

    Directory of Open Access Journals (Sweden)

    Wieler Lothar H

    2011-11-01

    Full Text Available Abstract Background Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC, but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC. Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4, STEC (stx1, stx2, escV, EAEC (pAA, aggR, astA, and ESBL-production (blaCTX-M, blaTEM, blaSHV. Results The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28% shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli. Conclusions While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.

  18. Higher atypical enteropathogenic Escherichia coli (a-EPEC) bacterial loads in children with diarrhea are associated with PCR detection of the EHEC factor for adherence 1/lymphocyte inhibitory factor A (efa1/lifa) gene.

    Science.gov (United States)

    Slinger, Robert; Lau, Kimberley; Slinger, Michael; Moldovan, Ioana; Chan, Francis

    2017-03-23

    Typical enteropathogenic Escherichia coli (t-EPEC) are known to cause diarrhea in children but it is uncertain whether atypical EPEC (a-EPEC) do, since a-EPEC lack the bundle-forming pilus (bfp) gene that encodes a key adherence factor in t-EPEC. In culture-based studies of a-EPEC, the presence of another adherence factor, called EHEC factor for adherence/lymphocyte activation inhibitor (efa1/lifA), was strongly associated with diarrhea. Since a-EPEC culture is not feasible in clinical laboratories, we designed an efa1/lifA quantitative PCR assay and examined whether the presence of efa1/lifA was associated with higher a-EPEC bacterial loads in pediatric diarrheal stool samples. Fecal samples from children with diarrhea were tested by qPCR for EPEC (presence of eae gene) and for shiga toxin genes to exclude enterohemorrhagic E. coli, which also contain the eae gene. EPEC containing samples were then tested for the bundle-forming pilus gene found in t-EPEC and efa1/lifA. The eae gene quantity in efa1/lifA-positive and negative samples was compared. Thirty-nine of 320 (12%) fecal samples tested positive for EPEC and 38/39 (97%) contained a-EPEC. The efa1/lifA gene was detected in 16/38 (42%) a-EPEC samples. The median eae concentration for efa1/lifA positive samples was significantly higher than for efa1/lifA negative samples (median 16,745 vs. 1183 copies/µL, respectively, p = 0.006). Atypical enteropathogenic E. coli-positive diarrheal stool samples containing the efa1/lifA gene had significantly higher bacterial loads than samples lacking this gene. This supports the idea that efa1/lifA contributes to diarrheal pathogenesis and suggests that, in EPEC-positive samples, efa/lifA may be a useful additional molecular biomarker.

  19. The effect of regions of interest and spectral pre-processing on the detection of non-O157 shiga-toxin producing escherichia coli serogroups on agar media by hyperspectral imaging

    Science.gov (United States)

    Food borne infection caused by Shiga toxin-producing Escherichia coli (STEC) is a major worldwide health concern. The best known STEC serotype is E. coli O157:H7, which can be easily identified when cultured on sorbitol-MacConkey (SMAC) agar. Recently, six non-O157 STEC serotypes have been found t...

  20. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Science.gov (United States)

    In, Julie; Lukyanenko, Valeriy; Foulke-Abel, Jennifer; Hubbard, Ann L; Delannoy, Michael; Hansen, Anne-Marie; Kaper, James B; Boisen, Nadia; Nataro, James P; Zhu, Chengru; Boedeker, Edgar C; Girón, Jorge A; Kovbasnjuk, Olga

    2013-01-01

    Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  1. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Julie In

    Full Text Available Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  2. Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains.

    Science.gov (United States)

    Nyholm, Outi; Halkilahti, Jani; Wiklund, Gudrun; Okeke, Uche; Paulin, Lars; Auvinen, Petri; Haukka, Kaisa; Siitonen, Anja

    2015-01-01

    Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor. The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied. The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only. This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics

  3. Complete Genome Sequence of Escherichia coli Strain WG5

    DEFF Research Database (Denmark)

    Imamovic, Lejla; Misiakou, Maria-Anna; van der Helm, Eric

    2018-01-01

    Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain.......Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain....

  4. Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing beta-lactamases de espectro ampliado em Klebsiella spp e em Escherichia coli obtidas em um hospital escola brasileiro: detecção, prevalência e tipagem molecular

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Peixoto de Freitas

    2003-12-01

    Full Text Available His study was performed to compare the methods of detection and to estimate the prevalence of extended-spectrum beta-lactamases (ESBL among Klebsiella spp and E.coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K.pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clínicas de Porto Alegre, using Kirby-Bauer zone diameter (KB, double-disk diffusion (DD, breakpoint for ceftazidime (MIC CAZ, increased zone diameter with clavulanate (CAZ/CAC and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC. Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E.coli, contrasting with all the other methods (p Este estudo foi desenvolvido para comparar métodos de detecção e para estimar a prevalência de Klebsiella spp e E.coli produtoras de beta-lactamases de espetro ampliado (ESBL em um Hospital Universitário no sul do Brasil. A correlação genética, determinada através de método molecular de tipagem, entre as amostras de K. pneumoniae também foi determinada. A produção de ESBL foi investigada em 95 amostras de Klebsiella spp e E.coli obtidas de pacientes no Hospital de Clínicas de Porto Alegre usando-se: medida do diâmetro a zona de inibição (KB, dupla-difusão de disco (DD, valores de concentração inibitória mínima da ceftazidima (MIC CAZ, aumento do diâmetro da zona de inibição com adição de clavulanato (CAZ/CAC e a relação entre o MIC da ceftazidima/MIC ceftazidima com clavulanato (MIC CAZ/CAC. A tipagem molecular foi realizada utilizando-se o método de macrorestrição de DNA e eletroforese em campo pulsado (PFGE. O método KB apresentou as maiores taxas de produção de ESBL (> 70% para Klebsiella e

  5. Comparison of Three Methods of Pour Plate (PP) ، Most Probable Number (MPN) and Membrane Filter (MF) for Detection of Escherichia coli in Well Water Samples in Tehran's Parks in 2010

    OpenAIRE

    Soltan Dallal, MM; Hosseini, M; Abedi Mohtaseb, TP; Tabatabaei Bafroei, A

    2010-01-01

    Background and objectives: Water-born diseases are typically causedby pathogens transmitted by orofecal way. Because it is no practical andno economical and also it is time-consuming to find water-bornpathogens in water reservoirs, the laboratory studies are performed on thebasis of indicator microorganism. Escherichia coli is considered as themost important indicator bacterium for water monitoring. The aim of thisstudy was to evaluate the three methods of Pour Plate (PP), MostProbable Number...

  6. A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E. coli (EHEC) to related enteropathotypes.

    Science.gov (United States)

    Monteiro, Ricardo; Ageorges, Valentin; Rojas-Lopez, Maricarmen; Schmidt, Herbert; Weiss, Agnes; Bertin, Yolande; Forano, Evelyne; Jubelin, Grégory; Henderson, Ian R; Livrelli, Valérie; Gobert, Alain P; Rosini, Roberto; Soriani, Marco; Desvaux, Mickaël

    2016-08-01

    Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Escherichia coli photoreactivating enzyme: purification and properties

    International Nuclear Information System (INIS)

    Snapka, R.M.; Sutherland, B.M.

    1980-01-01

    Researchers have purified large quantities of Escherichia coli photoreactivating enzyme to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36,800 and a S/sub 20,w/ 0 of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA containing uracil, adenine, guanine, and cytosine with no unusual bases detected

  8. EXPRESSION OF BACTERIOOPSIN GENES IN ESCHERICHIA COLI

    OpenAIRE

    TSUJIUCHI, Yutaka; IWASA, Tatsuo; TOKUNAGA, Fumio

    1994-01-01

    An inducible expression vector pUBO was constructed with native codons in order to express the gene of Bacteriorhodopsin (BOP) in Escherichia coli (E. coli). Vector pUBO contains lac-promoter followed by the partial structural gene of lacZ and the structural gene of BOP. The expression of this fusion protein was detected by ELISA with anti-BOP antiserum. The fusion protein obtained from E. coli trnsformed with pUBO formed approximately 0.1% of the total protein of the E. coli membrane fraction.

  9. Relationship between Phenotypic and Genotypic Florfenicol Resistance in Escherichia coli

    OpenAIRE

    Singer, Randall S.; Patterson, Sheila K.; Meier, Anne E.; Gibson, Jessica K.; Lee, Hannah L.; Maddox, Carol W.

    2004-01-01

    This study evaluated the relationship between florfenicol resistance and flo genotypes in 1,987 Escherichia coli isolates from cattle. The flo gene was detected in 164 isolates, all of which expressed resistance to florfenicol at MICs of ≥256 μg/ml. The florfenicol MICs for all isolates that lacked flo were ≤16 μg/ml.

  10. Relationship between Phenotypic and Genotypic Florfenicol Resistance in Escherichia coli

    Science.gov (United States)

    Singer, Randall S.; Patterson, Sheila K.; Meier, Anne E.; Gibson, Jessica K.; Lee, Hannah L.; Maddox, Carol W.

    2004-01-01

    This study evaluated the relationship between florfenicol resistance and flo genotypes in 1,987 Escherichia coli isolates from cattle. The flo gene was detected in 164 isolates, all of which expressed resistance to florfenicol at MICs of ≥256 μg/ml. The florfenicol MICs for all isolates that lacked flo were ≤16 μg/ml. PMID:15388477

  11. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presence...

  12. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome...

  13. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...

  14. Enterohemorrhagic Escherichia coli (EHEC

    Directory of Open Access Journals (Sweden)

    Abdullah Kilic

    2011-08-01

    Full Text Available Escherichia coli is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless for human. E. coli O157:H7 is the most common member of a group of pathogenic E. coli strains known variously as enterohaemorrhagic, verocytotoxin-producing, or Shiga-toxin-producing organisms. EHEC bacterium is the major cause of haemorrhagic colitis and haemolytic uraemic syndrome. The reservoir of this pathogen appears to be mainly cattle and other ruminants such as camels. It is transmitted to humans primarily through consumption of contaminated foods. [TAF Prev Med Bull 2011; 10(4.000: 387-388

  15. Perfil de sensibilidade antimicrobiana e detecção do gene ISS pela reação em cadeia da polimerase na tipificação de Escherichia coli patogênica em codornas de corte sob inspeção sanitária Profile of antimicrobial resistance and detection of iss gene by the polymerase chain reaction in the typification of pathogenic Escherichia coli in meat type quails under sanitary inspection

    Directory of Open Access Journals (Sweden)

    Dayse Lima da Costa Abreu

    2010-05-01

    Full Text Available A patogenicidade das cepas de Escherichia coli está relacionada à expressão de fatores de virulência encontrados em elementos genéticos denominados plasmídios. O patotipo APEC, responsável por diferentes tipos de doenças em aves, pode apresentar o gene iss que aumenta a resistência das cepas de E. coli aos efeitos líticos do soro, além da resistência a diversos antimicrobianos. Este estudo foi conduzido para detectar E. coli em traquéias de codornas destinadas ao abate e avaliar, pela presença do gene iss e o perfil de susceptibilidade antimicrobiana, o potencial patogênico para aves e humanos dos isolados obtidos. Foram coletadas 180 traquéias de codornas para detecção de E. coli, determinação do perfil de resistência a agentes antimicrobianos e posterior detecção, por reação em cadeia da polimerase (PCR, do gene iss. Das traquéias analisadas, 8,9 % (16/180 foram positivas para E. coli, sendo obtidos 20 isolados deste agente. A maioria dos isolados foi resistente à Tetraciclina (16/20, seguida pela Ceftazidima (13/20 e Ácido Nalidíxico (12/20, sendo apenas um resistente à Amoxicilina. A detecção do gene iss ocorreu em 55% (11/20 dos isolados. A presença do gene iss e a resistência a múltiplos antimicrobianos dos isolados obtidos neste estudo pode indicar um possível potencial patogênico das cepas de E. coli tanto para codornas quanto para outros tipos de aves e animais e mesmo para o ser humano que fique em contato com as mesmas.The pathogenicity of Escherichia coli strains is partially related to the expression of virulence factors genes, present in genetic elements called plasmids. APEC strains responsible for diseases in birds may present the iss gene which increases the resistance of E. coli strains to the lityc effect of the host's serum, besides resistance to several antimicrobials. This study was conduced in order to detect E. coli in tracheae of meat-type quails and to evaluate, by the presence of

  16. Detección, aislamiento y caracterización de Escherichia coli productor de toxina Shiga a partir de carne molida fresca proveniente de carnicerías de Concepción, provincia de Tucumán Detection, isolation and characterization of Shiga toxin-producing Escherichia coli (STEC in fresh ground beef from butcher shops in Concepción, Tucumán Province

    Directory of Open Access Journals (Sweden)

    M. A. Jure

    2010-12-01

    Full Text Available Escherichia coli productor de toxina Shiga (STEC es un patógeno emergente transmitido por alimentos. Existen numerosos serotipos de STEC asociados a enfermedad en humanos, entre los cuales prevalece el serotipo O157:H7. La carne molida es el principal vehículo de transmisión. En la ciudad de Concepción, provincia de Tucumán, entre setiembre y diciembre de 2004 se diagnosticaron dos casos de síndrome urémico hemolítico (SUH. El objetivo de este trabajo fue detectar, aislar y caracterizar STEC O157 y no-O157 a partir de muestras de carne molida fresca obtenidas en las bocas de expendio. Entre los meses de setiembre y diciembre de 2004 se recolectaron 53 muestras de carne molida fresca en carnicerías de la ciudad de Concepción. Para la detección, el aislamiento y la caracterización de STEC O157:H7 se utilizó la metodología USDA-FSIS 2002. Para la detección de E. coli no-O157 se utilizaron dos técnicas de PCR; para el aislamiento y la caracterización se utilizó una metodología previamente validada en una etapa intralaboratorio. Siete muestras fueron positivas para el gen stx2, de las cuales 4 también fueron positivas para el gen rfbO157. Sin embargo, solo se aisló una cepa de E. coli O157:H7 biotipo C, portadora de los genes eae, stx2 y ehxA. El presente trabajo refleja la importancia de implementar técnicas que permitan detectar este grupo de patógenos emergentes a partir de productos cárnicos.Shiga toxin-producing Escherichia coli is an emerging foodborne pathogen. There are many STEC serotypes associated with human diseases, being the O157:H7 serotype the most prevalent. Ground beef is the main transmission vehicle. In Concepción city, Tucumán Province, between September and December 2004, two hemolytic uremic syndrome (HUS cases were diagnosed. The main objective of this work was to detect, isolate and characterize STEC O157 and non-O157 strains in fresh ground beef. Between September and December 2004, 53 fresh ground

  17. Escherichia coli Uropathogenesis In Vitro

    DEFF Research Database (Denmark)

    Andersen, Thomas E; Khandige, Surabhi; Madelung, Michelle

    2012-01-01

    Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation...

  18. Virulence - associated genes in Avian Pathogenic Escherichia coli of turkey

    Directory of Open Access Journals (Sweden)

    Antonio Camarda

    2010-01-01

    Full Text Available 50 Escherichia coli (APEC-Avian Pathogenic Escherichia coli strains and 15 E. coli (AFEC-Avian Faecal Escherichia coli from turkeys affected by colibacillosis and from healthy turkeys were tested for the presence of eight different virulence-associated genes. Besides, APEC were serotyped. O78 has been the most detected serotyped. The presence of the tested virulence genes was prevalently related to the APEC isolates. With reference to serogroup, all the tested O78 resulted iss and irp2 positive. Besides, tsh e cva/cvi were respectively present in 88.9 and 83.3% of O78. Nevertheless, the finding of a not typeable strains equipped with all the eight tested virulence genes among the APEC isolates suggest the importance of a careful and complete characterisation of the isolate to evaluate the real potential pathogenic attitude of the bacterium.

  19. A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp.

    Science.gov (United States)

    Park, Jungsun; Kim, Jin Seok; Kim, Soojin; Shin, Eunkyung; Oh, Kyung-Hwan; Kim, Yonghoon; Kim, Cheon Hyeon; Hwang, Min Ah; Jin, Chan Mun; Na, Kyoungin; Lee, Jin; Cho, Enhi; Kang, Byung-Hak; Kwak, Hyo-Sun; Seong, Won Keun; Kim, Junyoung

    2018-01-01

    In June 2015, a local public health laboratory was notified that students had developed gastroenteritis symptoms after attending a camp. An outbreak investigation was conducted to determine the extent and cause of the outbreak. A retrospective cohort study was conducted to determine the correlations between the illness and specific exposures at the school camp. All attendees were interviewed with a standard questionnaire that addressed clinical symptoms, food consumption, and environmental exposures. Clinical specimens were cultured using standard microbiological methods for bacterial and viral pathogens. The genetic relationships of all isolates were determined using pulsed-field gel electrophoresis (PFGE). A total 188 patients with symptoms of diarrhoea, abdominal pain, and nausea were identified. The completed questionnaires suggested that the consumption of drinking water was likely to be linked to this outbreak. Using microbiological methods, enterohaemorrhagic Escherichia coli, enteropathogenic E. coli, and enteroaggregative E. coli were isolated, and the isolates from both patient stool and environmental water samples displayed indistinguishable XbaI-PFGE patterns. The water system in the camp used groundwater drawn from a private underground reservoir for cooking and drinking. The environmental investigation revealed some problems with the water supply system, such as the use of inappropriate filters in the water purifier and a defect in the pipeline between the reservoir and the chlorination device. This outbreak points to the importance of drinking water quality management in group facilities where underground water is used and emphasizes the need for periodic sanitation and inspection to prevent possible waterborne outbreaks. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  20. Characterization of Escherichia coli isolates from laying hens with colibacillosis on 2 commercial egg-producing farms in Korea.

    Science.gov (United States)

    Oh, J Y; Kang, M S; Kim, J M; An, B K; Song, E A; Kim, J Y; Shin, E G; Kim, M J; Kwon, J H; Kwon, Y K

    2011-09-01

    The present study reports on layer chickens with colibacillosis in 2 commercial egg-producing farms (referred to as farm A and farm B, which were managed by the same owner and were about 1 km apart) in the middle region of the Korean peninsula. The 2 flocks were infected at the initiation of egg laying. They were characterized by no previous clinical signs but sudden mortality (2.7-4.0%), with severe lesions of septicemia and fibrinous polyserositis. Escherichia coli was isolated from the lesions of the infected birds. Serotyping tests identified isolates that belonged to somatic groups O1 (12/17), O46 (2/17), O78 (1/17), and O84 (1/17) or that were unidentified (1/17). Thirteen of 17 E. coli isolates (76.4%) obtained from 11 birds in the 2 flocks showed similar pulsed-field gel electrophoresis patterns that were arbitrarily designated as pattern A. The isolates had high frequencies of putative virulence genes including 100% [fimC (type 1 fimbriae), iucD (aerobactin synthesis), and iss (increased serum survival)], 94.1% [cva/cvi (structural genes of colicin V operon) and vat (vacuolating autotransporter toxin)], 88.2% [irp2, iron-repressible protein (yersinia bactin) synthesis, and fyuA, ferric yersinia uptake], and 82.3% [tsh (temperature-sensitive hemagglutinin)]; astA (encoding a heat-stable cytotoxin associated with enteroaggregative E. coli) was not associated with the enteric disorder. These data suggest that all chickens with colibacillosis on farms A and B were likely infected by E. coli strains that are highly pathogenic in avian species.

  1. Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion

    Science.gov (United States)

    Tozzoli, Rosangela; Grande, Laura; Michelacci, Valeria; Ranieri, Paola; Maugliani, Antonella; Caprioli, Alfredo; Morabito, Stefano

    2014-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) are pathogenic E. coli causing diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC are characterized by a constellation of virulence factors additional to Stx and have long been regarded as capable to cause HC and HUS when possessing the ability of inducing the attaching and effacing (A/E) lesion to the enterocyte, although strains isolated from such severe infections sometimes lack this virulence feature. Interestingly, the capability to cause the A/E lesion is shared with another E. coli pathogroup, the Enteropathogenic E. coli (EPEC). In the very recent times, a different type of STEC broke the scene causing a shift in the paradigm for HUS-associated STEC. In 2011, a STEC O104:H4 caused a large outbreak with more than 800 HUS and 50 deaths. Such a strain presented the adhesion determinants of Enteroaggregative E. coli (EAggEC). We investigated the possibility that, besides STEC and EAggEC, other pathogenic E. coli could be susceptible to infection with stx-phages. A panel of stx2-phages obtained from STEC isolated from human disease was used to infect experimentally E. coli strains representing all the known pathogenic types, including both diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC). We observed that all the E. coli pathogroups used in the infection experiments were susceptible to the infection. Our results suggest that the stx2-phages used may not have specificity for E. coli adapted to the intestinal environment, at least in the conditions used. Additionally, we could only observe transient lysogens suggesting that the event of stable stx2-phage acquisition occurs rarely. PMID:24999453

  2. Longitudinal Comparison of Antibiotic Resistance in Diarrheagenic and Non-pathogenic Escherichia coli from Young Tanzanian Children.

    Science.gov (United States)

    Seidman, Jessica C; Johnson, Lashaunda B; Levens, Joshua; Mkocha, Harran; Muñoz, Beatriz; Silbergeld, Ellen K; West, Sheila K; Coles, Christian L

    2016-01-01

    Enteroaggregative, enteropathogenic, and enterotoxigenic Escherichia coli contribute significantly to the burden of diarrheal infections particularly in developing countries. Antibiotic resistance is increasingly common among bacterial pathogens including pathogenic E. coli. We assessed the relationship between pathogenic E. coli carriage and resistance to six antibiotics in E. coli isolated from young children in rural Tanzania. We surveyed temporal stability in antibiotic resistance in 2492 E. coli isolated from fecal samples obtained from young children in rural Tanzania collected over a 6 months period. Approximately half of the 377 children sampled were exposed to an azithromycin mass treatment program for trachoma control and half resided in control villages. Children were sampled at baseline, 1-, 3-, and 6 months following azithromycin treatment. We compared resistance to six antibiotics in pathogenic and non-pathogenic strains at the population level, within fecal specimens, and within individuals over time using chi-square tests, paired odds ratios, and logistic regression, respectively. Resistance to ampicillin and trimethoprim/sulfamethoxazole was highly prevalent (>65%). Resistance to 5 of 6 antibiotics tested and multi-drug resistance occurred more frequently in pathogenic isolates (p ≤ 0.001) within fecal specimens and overall. Azithromycin mass treatment exposure was significantly associated with increased odds of carriage of isolates resistant to erythromycin (OR 3.64, p resistant to erythromycin, ampicillin, or trimethoprim/sulfamethoxazole compared to non-pathogenic isolates from the same fecal specimen. The potential linkage between resistance and virulence in E. coli suggests hygiene and sanitation interventions aimed at reducing disease burden could play a role in controlling transmission of antibiotic resistance.

  3. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    Science.gov (United States)

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  4. Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli.

    Science.gov (United States)

    Ooka, Tadasuke; Ogura, Yoshitoshi; Katsura, Keisuke; Seto, Kazuko; Kobayashi, Hideki; Kawano, Kimiko; Tokuoka, Eisuke; Furukawa, Masato; Harada, Seiya; Yoshino, Shuji; Seto, Junji; Ikeda, Tetsuya; Yamaguchi, Keiji; Murase, Kazunori; Gotoh, Yasuhiro; Imuta, Naoko; Nishi, Junichiro; Gomes, Tânia A; Beutin, Lothar; Hayashi, Tetsuya

    2015-11-03

    Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Serological evidence of asymptomatic infections during Escherichia coli O104:H4 outbreak in Germany in 2011.

    Directory of Open Access Journals (Sweden)

    Yanina Balabanova

    Full Text Available INTRODUCTION: The largest known outbreak caused by a rare hybrid strain of Shiga toxin-producing E.coli (STEC and enteroaggregative E. coli (EAEC (E.coli O104:H4 of serotype O104:H4 occurred in Germany in 2011. Fenugreek sprouts acted as a transmission vehicle and were widely consumed in the outbreak area at the time of the epidemic. In total 3,842 people developed a clinical illness caused by this strain; however the rates of asymptomatic infections remain unclear. We aimed to develop a serological assay for detection of E.coli O104 LPS specific antibodies and to establish the post-outbreak levels of seropositivity among people with documented exposure to contaminated sprouts. RESULTS AND DISCUSSION: Developed serological assays (ELISA with 84% sensitivity, 63% specificity and Western Blot with 100% sensitivity, 82.5% specificity identified 33% (16/49 level of asymptomatic infection. Relatively small sample size and a significant time- lapse between the onset of symptoms and serum samples collection (appr. 8 weeks might explain the assay variability. No association was found between clinical or demographic characteristics and assay positivity. Larger studies are needed to understand the complexity of human immune response and factors influencing development of clinical symptoms. Development of intra-outbreak research plans will substantially aid the conduct of more thorough scientific investigation during an outbreak period.

  6. Urinary tract infection: detection of Escherichia coli antigens in human urine with an ELIEDA immunoenzymatic assay Las infecciones urinarias: detección de antígenos de Escherichia coli en orina humana con un ensayo inmunoenzimático por electrodifusión (ELIEDA

    Directory of Open Access Journals (Sweden)

    Myriam T. N. Campanhã

    1999-08-01

    Full Text Available Escherichia coli is the most common causative agent of urinary tract infection (UTI, and diagnosing this infection usually relies on bacteriologic methods. Nevertheless, screening methods can be useful for a rapid presumptive diagnosis even though some of these screening methods have low sensitivity or are expensive. To investigate a possible new alternative approach, an antigen-based immunoassay-enzyme-linked immunoelectrodiffusion assay (ELIEDA-was standardized for screening for this bacterial infection. Combining counter immunoelectrophoresis with an immunoenzymatic assay, the ELIEDA requires concentrated urine specimens, a cellulose acetate membrane, polyclonal antibodies to E. coli raised in rabbits, and peroxidase-labeled sheep antibodies to rabbit immunoglobulin G (IgG. This ELIEDA technique was evaluated using 244 urine specimens, 76 of them with E. coli, 47 with heterologous bacteria, and 121 without bacteria. In comparison to bacteriologic methods, the sensitivity, specificity, and positive and negative predictive values for the ELIEDA were 93.4%, 98.2%, 95.9%, and 97.1%, respectively. The data obtained suggest that this assay is useful for routine diagnostic screening for UTI caused by E. coli. In addition, since the ELIEDA stained membranes can be stored, this assay makes retrospective studies possible.Escherichia coli es el agente causal más frecuente de las infecciones urinarias (IU, cuyo diagnóstico suele basarse en métodos bacteriológicos. No obstante, los métodos de tamizaje pueden ser útiles para hacer un diagnóstico preliminar con rapidez, pese a que algunos de ellos tienen poca sensibilidad y son caros. Con el fin de investigar la posibilidad de usar otra técnica de diagnóstico, se estandarizó un inmunoensayo de tipo antigénico-ensayo inmunoenzimático por electrodifusión (ELIEDA, por enzyme-linked immunoelectrodiffusion assay-para hacer el tamizaje de este tipo de infección. El ELIEDA, que consiste en el uso

  7. Detección y caracterización de Escherichia coli productor de toxina Shiga a partir de casos clínicos y de alimentos en Uruguay Detection and characterization of Shiga toxin - producing Escherichia coli from clinical cases and food in Uruguay

    Directory of Open Access Journals (Sweden)

    G. Varela

    2008-06-01

    Full Text Available Establecimos la frecuencia de aislamiento de Escherichia coli productor de toxina Shiga (STEC a partir de muestras clínicas y de alimentos, así como las características fenotípicas y genotípicas de las cepas recuperadas. Se analizaron 198 muestras fecales de niños con diarrea sanguinolenta (DS, 14 muestras fecales de niños con síndrome urémico hemolítico (SUH y 220 muestras de carne picada. También se estudiaron 4 cepas STEC aisladas de alimentos embutidos. Se recuperó STEC de 3 (1,5% de los niños con DS, de 1 (7% niño con SUH y de 4 (1,8% de las muestras de carne picada. Todas las cepas fueron eae y ehxA positivas. Los serotipos detectados fueron: O157:H7 (9 cepas, O26:H11 (2 cepas, O111:NM (1 cepa y O145:HNT (1 cepa. Todas las cepas O157:H7 portaron el subtipo eae-g1; las cepas O26:H11 y O145:HNT portaron el subtipo eae-b1 y la cepa O111:NM portó el subtipo eae-g2/q. Las cepas STEC del mismo serogrupo mostraron alta diversidad genética. En Uruguay STEC no sería agente frecuente de diarrea con sangre en niños. Sin embargo, las cepas recuperadas presentaron los genes asociados con enfermedad severa y 2 de los 3 niños infectados con STEC evolucionaron a SUH. La carne picada y otros alimentos serían vehículos importantes de O157:H7.We have assessed the frequency of Shiga toxin-producing Escherichia coli (STEC in clinical and food samples as well as studied the genotypic and phenotypic characteristics of the recovered strains. One hundred ninety eight fecal samples from children with bloody diarrhea (BD, 14 from children with hemolytic uremic syndrome (HUS, 220 ground beef samples and 4 STEC isolates from other beef-derived products were analyzed. The STEC strains were isolated from 3 (1.5% children with bloody diarrhea, 1 (7% from a child with HUS and 4 (1.8% from ground beef samples. All strains were eae and ehxA positive. The serotypes found were: O157:H7 (9 strains, O26:H11 (2, O111: NM (1 and O145:HNT (1. All O157:H7 STEC

  8. Inactivation of Escherichia coli O157:H7 in hamburgers by gamma irradiation

    OpenAIRE

    Chirinos, Rodolfo R.O.; Vizeu, Dirceu M.; Destro, Maria Teresa; Franco, Bernadette D.G.M.; Landgraf, Mariza

    2002-01-01

    Escherichia coli O157:H7 causes bloody diarrhoea, haemorrhagic colitis and life-threatening complications like haemolytic uremic syndrome and thrombotic thrombocitopenic purpura. Among foods associated with outbreaks caused by this pathogen, hamburger is the most common one. The aim of this research was to determine the radiation dose to reduce the population of E. coli O157:H7 in hamburgers to non-detectable levels in order to render a safer product. Hamburgers, inoculated with Escherichia c...

  9. Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC Strains.

    Directory of Open Access Journals (Sweden)

    Outi Nyholm

    Full Text Available Shigatoxigenic Escherichia coli (STEC and enterotoxigenic E. coli (ETEC cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the

  10. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Science.gov (United States)

    2009-01-01

    Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead to unwanted secondary

  11. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Penn Charles W

    2009-12-01

    Full Text Available Abstract Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead

  12. Principales características y diagnóstico de los grupos patógenos de Escherichia coli Diagnosis and main characteristics of Escherichia coli pathogenic groups

    Directory of Open Access Journals (Sweden)

    Guadalupe Rodríguez-Angeles

    2002-09-01

    Full Text Available Escherichia coli coloniza el intestino del hombre pocas horas después del nacimiento y se considera de flora normal, pero hay descritos seis grupos de E. coli productora de diarrea: enterotoxigénica (ETEC, enterohemorrágica (EHEC, enteroinvasiva (EIEC, enteropatógena (EPEC, enteroagregativa (EAEC y de adherencia difusa (DAEC. La bacteria se puede aislar e identificar tradicionalmente con base en sus características bioquímicas o serológicas, pero también se pueden estudiar sus mecanismos de patogenicidad mediante ensayos en cultivos celulares o modelos animales y, más recientemente, empleando técnicas de biología molecular que evidencian la presencia de genes involucrados en dichos mecanismos. La intención del presente trabajo es resaltar la importancia del estudio y diagnóstico de E. coli como patógeno capaz de causar casos aislados o brotes de diarrea, síndrome urémico hemolítico, colitis hemorrágica y cuadros de disentería, principalmente en niños; por esto es necesario conocer mejor a la bacteria y mantener la vigilancia epidemiológica.Escherichia coli colonizes the human intestinal tract within hours of birth and is considered a non-pathogenic member of the normal intestinal flora. However, there are six pathogenic groups that may produce diarrhea: enterotoxigenic (ETEC, enterohemorrhagic (EHEC, enteroinvasive (EIEC, enteropathogenic (EPEC, enteroaggregative (EAEC and diffusely adherent (DAEC groups. E. coli can be isolated and classified using traditional methods, by identifying its biochemical or serum characteristics. The pathogenic mechanisms may be studied in cell cultures and animal model assays, as well as more up to date molecular biology methods for study and diagnosis. The latter have proven that genes are involved in pathogenesis. The objective of the present work is to draw attention to the importance of E. coli as a pathogenic organism. This microorganism is an etiologic agent of sporadic cases of diarrhea

  13. A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits.

    Science.gov (United States)

    Gustavsson, Tobias; Trane, Maria; Moparthi, Vamsi K; Miklovyte, Egle; Moparthi, Lavanya; Górecki, Kamil; Leiding, Thom; Arsköld, Sindra Peterson; Hägerhäll, Cecilia

    2010-08-01

    Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

  14. Expression of maize prolamins in Escherichia Coli

    International Nuclear Information System (INIS)

    Wang, Szu-zhen; Esen, Asim

    1985-01-01

    We have constructed a cDNA expression library of developing corn (Zea manys L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250-900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with 32 P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed. (author)

  15. Synergistic effects in mixed Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Reisner, A.; Holler, B.M.; Molin, Søren

    2006-01-01

    the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from...... the cocultivation of 403 undomesticated E. coli strains with a characterized E. coli K-12 strain was detected at a significant frequency. The survey suggests that different mechanisms underlie the observed stimulation, yet synergistic development of biofilm within the subset of E. coli isolates (n = 56) exhibiting...... the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes...

  16. Whole Genome Epidemiological Typing of Escherichia coli

    DEFF Research Database (Denmark)

    Kaas, Rolf Sommer

    Escherichia coli (E. coli) is of huge importance in global health both as a commensal organism living within its host or as a pathogen causing millions of infections each year. Infections occur both sporadic and as outbreaks with sometimes up to thousands of infected people. To limit the number...... of infections it is important to monitor pathogenic E. coli in order to detect outbreaks as quickly as possible and find the source of the outbreak. The effectiveness of monitoring and tracking of pathogens is very dependent on the typing methods that are employed. Classical typing methods employed for E. coli......D thesis attempts to take the first steps toward such a method. In Kaas I all publicly available E. coli genomes sequenced (186) are analyzed. 1,702 core genes were found in all genomes. 3,051 genes were found in 95% of the genomes. The pan genome was found to consist of 16,373 genes. The overall phylogeny...

  17. Escherichia coli as a probiotic?

    NARCIS (Netherlands)

    Jansen, GJ; Wildeboer-Veloo, ACM; van der Waaij, D; Degener, JE

    1998-01-01

    The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II(R)) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in

  18. ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS

    African Journals Online (AJOL)

    DR. AMINU

    ABSTRACT. The bio-effects of the ethanol extracts from the leaf and stem of Momordica charantia were studied with the view to ascertain the medical usefulness ascribed to the plant by the locals. The plant parts, stem and leaf, revealed remarkable activity against Escherichia coli and Staphlococcus aureus. The leaves ...

  19. Conjugal Pairing in Escherichia Coli

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 8. Conjugal Pairing in Escherichia Coli. Joshua Lederberg. Classics Volume 13 Issue 8 August 2008 pp 793-794. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/013/08/0793-0794 ...

  20. Conjugal Pairing in Escherichia Coli

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 8. Conjugal Pairing in Escherichia Coli. Joshua Lederberg. Classics Volume 13 Issue 8 August 2008 pp 793-794. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/013/08/0793-0794 ...

  1. Comparison of U.S. Environmental Protection Agency and U.S. Composting Council microbial detection methods in finished compost and regrowth potential of Salmonella spp. and Escherichia coli O157:H7 in finished compost.

    Science.gov (United States)

    Reynnells, Russell; Ingram, David T; Roberts, Cheryl; Stonebraker, Richard; Handy, Eric T; Felton, Gary; Vinyard, Bryan T; Millner, Patricia D; Sharma, Manan

    2014-07-01

    Bacterial pathogens may survive and regrow in finished compost due to incomplete thermal inactivation during or recontamination after composting. Twenty-nine finished composts were obtained from 19 U.S. states and were separated into three broad feedstock categories: biosolids (n=10), manure (n=4), and yard waste (n=15). Three replicates of each compost were inoculated with ≈ 1-2 log CFU/g of nonpathogenic Escherichia coli, Salmonella spp., and E. coli O157:H7. The U.S. Environmental Protection Agency's (EPA) protocols and U.S. Composting Council's (USCC) Test Methods for the Examination of Composting and Compost (TMECC) were compared to determine which method recovered higher percentages of inoculated E. coli (representing fecal coliforms) and Salmonella spp. from 400-g samples of finished composts. Populations of Salmonella spp. and E. coli O157:H7 were determined over 3 days while stored at 25°C and compared to physicochemical parameters to predict their respective regrowth potentials. EPA Method 1680 recovered significantly (p=0.0003) more inoculated E. coli (68.7%) than TMECC 07.01 (48.1%) due to the EPA method using more compost in the initial homogenate, larger transfer dilutions, and a larger most probable number scheme compared to TMECC 07.01. The recoveries of inoculated Salmonella spp. by Environmental Protection Agency Method 1682 (89.1%) and TMECC 07.02 (72.4%) were not statistically significant (p=0.44). The statistically similar recovery percentages may be explained by the use of a nonselective pre-enrichment step used in both methods. No physicochemical parameter (C:N, moisture content, total organic carbon) was able to serve as a sole predictor of regrowth of Salmonella spp. or E. coli O157:H7 in finished compost. However, statistical analysis revealed that the C:N ratio, total organic carbon, and moisture content all contributed to pathogen regrowth potential in finished composts. It is recommended that the USCC modify TMECC protocols to test

  2. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Rui [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China)

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H{sub 2}O{sub 2}) and H{sub 2}O{sub 2}-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H{sub 2}O{sub 2} and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H{sub 2}O{sub 2}, as well as the incubation time between H{sub 2}O{sub 2} and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10{sup 2} CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10{sup 3} CFU/mL to 1.18 × 10{sup 6} CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. - Highlights: • A novel fluorescence immunoassay was developed for the ultrasensitive detection of E. coli O157:H7. • This detection was achieved through the combination of the high bioactivity of CAT and H{sub 2}O{sub 2}-sensitive QDs. • The activity of CAT to H{sub 2}O{sub 2} is 1000 folds higher than that of the HRP

  3. Natural DNA uptake by Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Sunita Sinha

    Full Text Available Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.

  4. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

    Science.gov (United States)

    Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Escherichia coli in broiler chickens with airsacculitis

    Directory of Open Access Journals (Sweden)

    Leandro S. Machado

    2014-09-01

    Full Text Available ABSTRACT. Machado L.S., do Nascimento E.R., Pereira V.L.A., Abreu D.L.C., Gouvea R. & Santos L.M.M. 2014. [Escherichia coli in broiler chickens with airsacculitis.] Escherichia coli em frangos de corte com aerossaculite. Revista Brasileira de Medicina Veterinária, 36(3:261-265, 2014. Departamento de Medicina Veterinária Preventiva e Saúde Pública, Faculdade de Veterinária, Universidade Federal Fluminense, Rua Dr. Vital Brazil Filho 64, Vital Brazil, Niterói, RJ 24230-340, Brazil. E-mail: leandromachadovet@yahoo.com.br The Brazilian poultry industry grows each year and becomes increasingly representative in the production and export of products. The health care with poultry have accompanied and favored this evolution, however, respiratory agents that affect the weight and carcass quality, continue to cause great damage to the poultry industry. Airsacculitis is considered the main cause of total and partial condemnation of carcasses of broilers, and has been attributed to Mycoplasmosis mostly caused by Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS and Escherichia coli. The aim of this study was to relate the positivity of MG / MS and E. coli detected by PCR as a risk factor for airsacculitis in condemnation of broilers in Health Inspection Service. We studied 30 broiler poultry slaughtered in a slaughterhouse under Federal Sanitary Inspection, located in the State of Rio de Janeiro. 30 chickens were randomly collected from different lots and tracheas obtained in each PCR. DNA was extracted by phenol-chloroform method and amplified using pairs of “primer”specific for MG, MS and E. coli. Of the 30 chickens analyzed by PCR, 30% (9/30 had lesions in air sacs. None of the birds showed infection with MG and/or MS PCR, however 33.3% (3/9 birds were positive for airsacculitis iss gene from E.coli. E.coli found in broiler chickens that were negative for mycoplasma airsacculitis, implying the presence of such bacteria may be sufficient

  6. Molecular characterization of enterohemorrhagic Escherichia coli hemolysin gene (EHEC-hlyA)-harboring isolates from cattle reveals a diverse origin and hybrid diarrheagenic strains.

    Science.gov (United States)

    Askari Badouei, Mahdi; Morabito, Stefano; Najafifar, Arash; Mazandarani, Emad

    2016-04-01

    In the present study we investigated the occurrence of Escherichia coli strains harboring the gene encoding enterohemorrhagic E. coli hemolysin (EHEC-HlyA) in cattle and the association of this gene with various diarrheagenic E. coli (DEC) pathotypes. First, the bovine E. coli isolates were screened for EHEC-hlyA gene by PCR, and then they were characterized for the phylogenetic groups and the presence of the major virulence genes of different DEC pathotypes. In total, 25 virulence gene profiles were observed in 54 EHEC-hlyA+ isolates that reflect a considerable heterogeneity. The EHEC-hlyA+ strains were mostly associated with EHEC (72%), while only 7.4% were enteropathogenic E. coli (EPEC). We also showed the presence of estA gene of enterotoxigenic E. coli (ETEC) in 6 isolates (11.1%). Interestingly, two of the estA+ strains showed hybrid pathotypes with one carrying eae/estA (EPEC/ETEC), and the other one stx2/astA/estA (EHEC/ETEC). None of the isolates were related to enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and necrotoxigenic E. coli (NTEC). The EHEC-plasmid encoded genes occurred in seven different combinations with EHEC-hlyA/saa/subA/espP being the most prevalent (46.3%). All stx-/eae+ strains carried O island 57 (OI-57) molecular marker(s) that may indicate these to be the progenitors of EHEC or strains losing stx. The most prevalent phylogroup was B1 (61.1%), but the most heterogeneous strains including the hybrid strains belonged to A phylogroup. Overall, our results indicate that cattle EHEC-hlyA encoding E. coli isolates consist of diverse diarrheagenic strains with the possible existence of hybrid pathotypes. Future studies are required to clarify the evolutionary aspects and clinical significance of these strains in humans and domestic animals. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Comparative genomic analysis of two novel sporadic Shiga toxin-producing Escherichia coli O104:H4 strains isolated 2011 in Germany.

    Science.gov (United States)

    Tietze, Erhard; Dabrowski, Piotr Wojciech; Prager, Rita; Radonic, Aleksandar; Fruth, Angelika; Auraß, Philipp; Nitsche, Andreas; Mielke, Martin; Flieger, Antje

    2015-01-01

    A large outbreak of gastrointestinal disease occurred in 2011 in Germany which resulted in almost 4000 patients with acute gastroenteritis or hemorrhagic colitis, 855 cases of a hemolytic uremic syndrome and 53 deaths. The pathogen was an uncommon, multiresistant Escherichia coli strain of serotype O104:H4 which expressed a Shiga toxin characteristic of enterohemorrhagic E. coli and in addition virulence factors common to enteroaggregative E. coli. During post-epidemic surveillance of Shiga toxin-producing E. coli (STEC) all but two of O104:H4 isolates were indistinguishable from the epidemic strain. Here we describe two novel STEC O104:H4 strains isolated in close spatiotemporal proximity to the outbreak which show a virulence gene panel, a Shiga toxin-mediated cytotoxicity towards Vero cells and aggregative adherence to Hep-2 cells comparable to the outbreak strain. They differ however both from the epidemic strain and from each other, by their antibiotic resistance phenotypes and some other features as determined by routine epidemiological subtyping methods. Whole genome sequencing of these two strains, of ten outbreak strain isolates originating from different time points of the outbreak and of one historical sporadic EHEC O104:H4 isolate was performed. Sequence analysis revealed a clear phylogenetic distance between the two variant strains and the outbreak strain finally identifying them as epidemiologically unrelated isolates from sporadic cases. These findings add to the knowledge about this emerging pathogen, illustrating a certain diversity within the bacterial core genome as well as loss and gain of accessory elements. Our results do also support the view that distinct new variants of STEC O104:H4 repeatedly might originate from yet unknown reservoirs, rather than that there would be a continuous diversification of a single epidemic strain established and circulating in Germany after the large outbreak in 2011.

  8. Escherichia fergusonii Associated with Pneumonia in a Beef Cow

    Directory of Open Access Journals (Sweden)

    Guillermo M. Rimoldi

    2013-01-01

    Full Text Available An adult Angus cow developed hyperthermia, prostration, and respiratory distress, dying 36 hours after the onset of clinical signs. The main finding during postmortem examination was a severe focally extensive pneumonia. Icterus and a chronic mastitis were also noticed. Histologic examination of the lungs detected fibrinonecrotic pneumonia, with large number of oat cells and intralesional Gram-negative bacterial colonies. Samples from lung lesions were collected, and a pure growth of Escherichia fergusonii was obtained. E. fergusonii is a member of Enterobacteriaceae, related to Escherichia coli and Salmonella sp. In veterinary medicine, E. fergusonii has been reported in calves and sheep with clinical cases suggestive of salmonellosis; in a horse and a goat with enteritis and septicemia; and in ostriches with fibrinonecrotic typhlitis. To our knowledge, this report represents the first description of E. fergusonii associated with an acute pneumonia in cattle.

  9. Identifying New Small Proteins in Escherichia coli.

    Science.gov (United States)

    VanOrsdel, Caitlin E; Kelly, John P; Burke, Brittany N; Lein, Christina D; Oufiero, Christopher E; Sanchez, Joseph F; Wimmers, Larry E; Hearn, David J; Abuikhdair, Fatimeh J; Barnhart, Kathryn R; Duley, Michelle L; Ernst, Sarah E G; Kenerson, Briana A; Serafin, Aubrey J; Hemm, Matthew R

    2018-04-12

    The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Towson University.

  10. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...... to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...

  11. Original Paper Prevalence of Arcobacter, Escherichia coli ...

    African Journals Online (AJOL)

    2011-04-12

    Apr 12, 2011 ... Prevalence of Arcobacter, Escherichia coli, Staphylococcus aureus and ... species by selective cultural procedures and for Escherichia coli, Salmonella species and Staphylococcus aureus enriched ... Point System monitoring of critical contamination points used in meat production to ensure food safety in.

  12. PATHOGENIC POTENTIALS OF ESCHERICHIA COLI ISOLATED ...

    African Journals Online (AJOL)

    Electrolyte and haematological parameters in rabbits infected with pathogenic isolates of Escherichia coli from rural water supplies in Rivers State, Nigeria, where monitored. Rabbits were orally infected with suspension containing 3x107 cfu /ml of Escherichia coli to induce diarrhoea, and the electrolyte (sodium, potassium ...

  13. Characterization of Escherichia coli Phylogenetic Groups ...

    African Journals Online (AJOL)

    Keywords: Drug resistance, Escherichia coli, Extraintestinal infections, Polymerase chain reaction,. Phylogenetic group, Virulence. Access this .... performed by two methods: A carbapenem–EDTA combined disk method and MBL E-test ..... Enterobacteriaceae: Escherichia, Klebsiella, Proteus and other genera. In: Collee JG ...

  14. Inactivation of Escherichia coli O157:H7 in hamburgers by gamma irradiation Inativação de Escherichia coli O157:H7 em hambúrgueres submetidos à irradiação gama

    OpenAIRE

    Rodolfo R.O. Chirinos; Dirceu M. Vizeu; Maria Teresa Destro; Bernadette D.G.M. Franco; Mariza Landgraf

    2002-01-01

    Escherichia coli O157:H7 causes bloody diarrhoea, haemorrhagic colitis and life-threatening complications like haemolytic uremic syndrome and thrombotic thrombocitopenic purpura. Among foods associated with outbreaks caused by this pathogen, hamburger is the most common one. The aim of this research was to determine the radiation dose to reduce the population of E. coli O157:H7 in hamburgers to non-detectable levels in order to render a safer product. Hamburgers, inoculated with Escherichia c...

  15. Frequency and Distribution of Diarrhoeagenic Escherichia Coli Strains Isolated from Pediatric Patients with Diarrhoea in Bosnia and Herzegovina

    Directory of Open Access Journals (Sweden)

    Amela Dedeić-Ljubović

    2009-05-01

    Full Text Available Diarrhoeal disease is a major cause of illness and death among infants and young children worldwide. Among the Escherichia coli (E. coli causing intestinal diseases, there are six well-described categories: enteroaggregative E. coli (EAEC, diffusely adherent E. coli (DAEC, enteroinvasive E. coli (EIEC, entero-pathogenic E. coli (EPEC, enterohaemorrhagic E. coli (EHEC and enterotoxigenic E. coli (ETEC.The aim of the present study was to investigate the relative contribution of different groups of diarrhoe-agenic E. coli (DEC in paediatric patients with diarrhoea. Clinical stool specimens from 380 children with diarrhoea, with ages ranging from birth to < 12 years, were selected for the study over a period of 17 months (August 2007 to December 2008. The study showed that 85/380 children (22% had diarrhoea due to diarrhoeagenic E. coli. The most prevalent was enteropathogenic E. coli (EPEC isolated from 46/85 paediatric patients (54%, followed by enterotoxigenic (ETEC isolated from 19/85 (22.3%, en-terohaemorrhagic (EHEC from 18/85 (21.1% and enteroinvasive (EIEC from 2/85 patients (2.3%. The most prevalent serotypes of EPEC were O86:K61 and O44:K74 isolated from 10/46 (21.7%, O128:K67 from 6/46 patients (13%, followed by O158:K- and O126:K71 isolated from 4/46 patients (8.6%. Among the ETEC the most prevalent serotypes were O78:K80 isolated from 10/19 (56.7% and O25:K11 from 9/19 patients (47.3%, especially during the first twelve months: 9/19 patients (47.3%. The most prevalent EHEC strain found in this study was O145:K- and O103:K-: 5/18 patients (27.8%. Two isolated strains of EIEC belong to serotype O164:K-. The average age of the patients was 2 years. Two patients with bloody diarrhoea had EHEC serotype O157:H7 which progressed to haemolytic-uremic syndrome (HUS.Our study shows that diarrhoeagenic E. coli is a significant causal agent of diarrhoeal diseases in paediatric patients in Bosnia and Herzegovina. This study is the first report about

  16. Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4: Implications for Virulence in Humans.

    Directory of Open Access Journals (Sweden)

    Wenlan Zhang

    Full Text Available Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC and enteroaggregative (EAEC E. coli. The strain displays "stacked-brick" aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.E. coli O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1% patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (P≤0.01. This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (P  = 0.001.The pAA plasmid can be lost by E. coli O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.

  17. Genotoxicity of Graphene in Escherichia coli

    Science.gov (United States)

    Sharma, Ananya

    Rapid advances in nanotechnology necessitate assessment of the safety of nanomaterials in the resulting products and applications. One key nanomaterial attracting much interest in many areas of science and technology is graphene. Graphene is a one atom thick carbon allotrope arranged in a two-dimensional honeycomb lattice. In addition to being extremely thin, graphene has several extraordinary physical properties such as its exceptional mechanical strength, thermal stability, and high electrical conductivity. Graphene itself is relatively chemically inert and therefore pristine graphene must undergo a process called functionalization, which is combination of chemical and physical treatments that change the properties of graphene, to make it chemically active. Functionalization of graphene is of crucial importance as the end application of graphene depends on proper functionalization. In the field of medicine, graphene is currently a nanomaterial of high interest for building biosensors, DNA transistors, and probes for cancer detection. Despite the promising applications of graphene in several areas of biomedicine, there have been only few studies in recent years that focus on evaluating cytotoxicity of graphene on cells, and almost no studies that investigate how graphene exposure affects cellular genetic material. Therefore, in this study we used a novel approach to evaluate the genotoxicity, i.e., the effects of graphene on DNA, using Escherichia coli as a prokaryotic model organism.

  18. Multi-faceted Approach to Vaccine Development Against Escherichia coli O157:H7

    Science.gov (United States)

    2006-03-15

    serotypes, O157:H7 does not ferment sorbital and thus forms colorless colonies on sorbital-containing MacConkey (SMAC) agar (March et al., 1986). Culturing...toxin 2 B subunit. J.Infect.Dis. 183: 435-443. March, S. B. and Ratnam, S. (1986) Sorbitol- MacConkey medium for detection of Escherichia coli O157

  19. Comparison of methods for the enumeration of enterohemorrhagic Escherichia coli from veal hides and carcasses

    Science.gov (United States)

    The increased association of enterohemorrhagic Escherichia coli (EHEC) with veal calves has led the United States Department of Agriculture Food Safety and Inspection Service to report results of veal meat contaminated with the Top 7 serogroups separately from beef cattle. However, detection methods...

  20. An OXA-48-producing Escherichia coli isolated from a Danish patient with no hospitalization abroad

    DEFF Research Database (Denmark)

    Gedebjerg, Anne; Hasman, Henrik; Sorensen, Christian Moller

    2015-01-01

    Carbapenemase-producing organisms are disseminating globally and are now emerging as a worrying threat in Scandinavia. Before August 2013, OXA-48-producing organisms had not been detected in Danish patients. Here we report the isolation of an ST746 OXA-48-producing Escherichia coli with the plasm...

  1. Fluorogenic membrane overlays to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and seawater

    Science.gov (United States)

    Three assays were developed to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and other foods and in seawater and other environmental samples. Assays involve membrane overlays of overnight colonies on non-selective agar plates to detect ß-glucuronidase and lysyl am...

  2. Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands

    NARCIS (Netherlands)

    van den Beld, Maaike J. C.; Friedrich, Alexander W.; van Zanten, Evert; Reubsaet, Frans A. G.; Kooistra-Smid, Mirjam A. M. D.; Rossen, John W. A.

    2016-01-01

    An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coil (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic

  3. Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

    Science.gov (United States)

    Povolotsky, Tatyana L; Hengge, Regine

    2016-01-01

    The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c

  4. Mild Illness during Outbreak of Shiga Toxin-Producing Escherichia coli O157 Infections Associated with Agricultural Show, Australia.

    Science.gov (United States)

    Vasant, Bhakti R; Stafford, Russell J; Jennison, Amy V; Bennett, Sonya M; Bell, Robert J; Doyle, Christine J; Young, Jeannette R; Vlack, Susan A; Titmus, Paul; El Saadi, Debra; Jarvinen, Kari A J; Coward, Patricia; Barrett, Janine; Staples, Megan; Graham, Rikki M A; Smith, Helen V; Lambert, Stephen B

    2017-10-01

    During a large outbreak of Shiga toxin-producing Escherichia coli illness associated with an agricultural show in Australia, we used whole-genome sequencing to detect an IS1203v insertion in the Shiga toxin 2c subunit A gene of Shiga toxin-producing E. coli. Our study showed that clinical illness was mild, and hemolytic uremic syndrome was not detected.

  5. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray

    Science.gov (United States)

    Antibody array was developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli O serogroups. Sensitivity of the array was 10**5 CFU, and the limit of detection of serogroups in ground beef was 1-10 CFU following 12 h of enrichment. The array utilized a minimal amount...

  6. A Novel Integron in the Genome of Escherichia Coli Isolated From Indonesian Monitor Lizard (Varanus Spp).

    OpenAIRE

    PUSPITASARI, ESTI; SUWANTO, ANTONIUS; MALIK, AMARILA; ERDELEN, WALTER

    2001-01-01

    The genotype of antibiotic resistance in natural isolates of Escherichia coli was determined through integron detection and characterization of the associated antibiotic resistance. E. coli SG2 isolated from Varanus salvator of Java demonstrated resistance to spectinomycin (50ng/ml) and streptomycin (SOng/ml). Integron detection indicated that eight isolates out of nine E. coli isolates possessed a conserved segment of the integron. Amplification of the inserted cassette of the int...

  7. Detection and enumeration of Coliforms and Escherichia coli in water

    African Journals Online (AJOL)

    Water samples from eighteen different sources in Nairobi were analyzed for coliform and E. coli content using three microbiological techniques, namely: Multiple tube or Most Probable Number (MPN) technique, Defined Substrate Technique (DST) or Colilert technique and Petrifilm(TM) Plate Count Techniques. All the three ...

  8. The propagation of Escherichia Coli and of conservative tracers. A comparison

    International Nuclear Information System (INIS)

    Alexander, I.; Seiler, K.P.

    1982-01-01

    The propagation of Escherichia Coli (ATCC 11229, Gelsenkirchen) is compared with that of conservative tracers in groundwater. The experiments were performed with injection quantities of 10 7 , 10 8 , 10 10 and 10 11 of Escherichia Coli. Both, bacteria and conservative tracers pass their maximum at the same instant in the observation gauges. With injection quantities of more than 10 8 , the propagation of the Escherichia Coli sets in at the same time as it begins with the dyes. When the quantities range below 10 8 , the propagation begins after that of conservative tracers, because Coli bacteria were measured with a lower degree of detecting sensitivity than the tracers. With Coli injection quantities ranging above 10 10 , an increased filtering of these bacteria can be observed. Coli bacteria propagate more laterally than conservative tracers, however it could not be proved that this lateral propagation depends on the bacteria concentration. (orig.) [de

  9. Detection of mastitis pathogens by analysis of volatile bacterial metabolites

    NARCIS (Netherlands)

    Hettinga, K.A.; Valenberg, van H.J.F.; Lam, T.J.G.M.; Hooijdonk, van A.C.M.

    2008-01-01

    The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In

  10. Detection and Enumeration of Microorganisms in Water and Waste Waters.

    Science.gov (United States)

    Andrews, S.

    1980-01-01

    Presents and explains analytical procedures for the detection and quantitative enumeration of bacteria which constitute or are indicators of fecal contamination of aquatic environments. Tests are given for Escherichia coli, fecal Streptococci, Clostridium perfringens, and Salmonella. (WB)

  11. Genetic Structure and Antimicrobial Resistance of Escherichia coli and Cryptic Clades in Birds with Diverse Human Associations.

    Science.gov (United States)

    Blyton, Michaela D J; Pi, Hongfei; Vangchhia, Belinda; Abraham, Sam; Trott, Darren J; Johnson, James R; Gordon, David M

    2015-08-01

    The manner and extent to which birds associate with humans may influence the genetic attributes and antimicrobial resistance of their commensal Escherichia communities through strain transmission and altered selection pressures. In this study, we determined whether the distribution of the different Escherichia coli phylogenetic groups and cryptic clades, the occurrence of 49 virulence associated genes, and/or the prevalence of resistance to 12 antimicrobials differed between four groups of birds from Australia with contrasting types of human association. We found that birds sampled in suburban and wilderness areas had similar Escherichia communities. The Escherichia communities of backyard domestic poultry were phylogenetically distinct from the Escherichia communities sourced from all other birds, with a large proportion (46%) of poultry strains belonging to phylogenetic group A and a significant minority (17%) belonging to the cryptic clades. Wild birds sampled from veterinary and wildlife rehabilitation centers (in-care birds) carried Escherichia isolates that possessed particular virulence-associated genes more often than Escherichia isolates from birds sampled in suburban and wilderness areas. The Escherichia isolates from both the backyard poultry and in-care birds were more likely to be multidrug resistant than the Escherichia isolates from wild birds. We also detected a multidrug-resistant E. coli strain circulating in a wildlife rehabilitation center, reinforcing the importance of adequate hygiene practices when handling and caring for wildlife. We suggest that the relatively high frequency of antimicrobial resistance in the in-care birds and backyard poultry is due primarily to the use of antimicrobials in these animals, and we recommend that the treatment protocols used for these birds be reviewed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  13. Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

    OpenAIRE

    Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

    2012-01-01

    We explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx ...

  14. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2012-02-21

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145). This new date..., that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121...

  15. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

  16. Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104:H4 outbreak strain and EHEC O157:H7.

    Science.gov (United States)

    Wöchtl, Bettina; Gunzer, Florian; Gerner, Wilhelm; Gasse, Hagen; Koch, Michaela; Bagó, Zoltán; Ganter, Martin; Weissenböck, Herbert; Dinhopl, Nora; Coldewey, Sina M; von Altrock, Alexandra; Waldmann, Karl-Heinz; Saalmüller, Armin; Zimmermann, Kurt; Steinmann, Jörg; Kehrmann, Jan; Klein-Hitpass, Ludger; Blom, Jochen; Ehricht, Ralf; Engelmann, Ines; Hennig-Pauka, Isabel

    2017-01-01

    Shiga toxin (Stx) producing Escherichia coli ( E. coli ) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome. We observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease. Unexpectedly, E

  17. Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli.

    Science.gov (United States)

    Holowko, Maciej B; Wang, Huijuan; Jayaraman, Premkumar; Poh, Chueh Loo

    2016-11-18

    Cholera is a potentially mortal, infectious disease caused by Vibrio cholerae bacterium. Current treatment methods of cholera still have limitations. Beneficial microbes that could sense and kill the V. cholerae could offer potential alternative to preventing and treating cholera. However, such V. cholerae targeting microbe is still not available. This microbe requires a sensing system to be able to detect the presence of V. cholera bacterium. To this end, we designed and created a synthetic genetic sensing system using nonpathogenic Escherichia coli as the host. To achieve the system, we have moved proteins used by V. cholerae for quorum sensing into E. coli. These sensor proteins have been further layered with a genetic inverter based on CRISPRi technology. Our design process was aided by computer models simulating in vivo behavior of the system. Our sensor shows high sensitivity to presence of V. cholerae supernatant with tight control of expression of output GFP protein.

  18. Polarity effects in the lactose operon of Escherichia coli.

    Science.gov (United States)

    Li, Yong; Altman, Sidney

    2004-05-21

    An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene.

  19. Polyamines modulate streptomycin-induced mistranslation in Escherichia coli.

    Science.gov (United States)

    Nastri, H G; Fastame, I G; Algranati, I D

    1993-12-14

    The effects of intracellular levels of polyamines on both the in vivo inhibition of protein synthesis and the decrease of translation accuracy induced by streptomycin have been studied in polyamine-auxotrophic strains of Escherichia coli infected with the MS2 bacteriophage. The amount of viral coat protein formed was strongly reduced upon addition of increasing concentrations of streptomycin to polyamine-supplemented bacteria. In contrast, the antibiotic almost did not inhibit coat protein synthesis in polyamine-starved cells. The increase of mistranslation frequency elicited by streptomycin was only observed in bacteria grown with putrescine. In these cells several coat protein-satellites were detected after two-dimensional gel electrophoresis. These proteins, more basic than the normal MS2 coat protein, contain multiple substitutions of lysine for asparagine.

  20. Occurrence of pathogenic and faecal Escherichia coli in layer hens

    Directory of Open Access Journals (Sweden)

    Silvia Tagliabue

    2010-01-01

    Full Text Available A total of 117 Escherichia coli from colibacillosis affected (APEC and clinically healthy birds (AFEC were serotyped and tested for the presence of virulence genes: iss, tsh, cva. A total of 54.5% E. Coli were typeable and 15 different serogroups were identified. The most common serogroups among APEC strains were O78, O2 and O128, whereas O139 was predominant in faecal strains from healthy birds. Iss, tsh e cva were more frequently detected among the septicaemic E. coli strains. The association of virulence genes was observed. Particularly, the pathotype iss-tsh-cva was present in 46.5% of APEC strains. Referring to serogroups, E. coli O78 and O2 originating from colibacillosis affected birds were always isstsh- cva positive but did not share virulence genes when they came from healthy birds.

  1. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...... in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome....

  2. Adaptation of Escherichia coli traversing from the faecal environment to the urinary tract

    DEFF Research Database (Denmark)

    Nielsen, Karen L.; Stegger, Marc; Godfrey, Paul A.

    2016-01-01

    The majority of extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections (UTI) are found in the patient’s own gut flora, but only limited knowledge is available on the potential adaptation that may occur in the bacteria in order to traverse the perineum and successfull......, the majority showed no detectable differences with respect to mutations or mobilome when compared to their faecal counterpart. The results show that UPECs are successful in colonizing both the bladder and gut without adaptation....

  3. Virulence factors, antimicrobial resistance, and plasmid content of Escherichia coli isolated in swine commercial farms

    OpenAIRE

    Costa,M.M.; Drescher,G.; Maboni,F; Weber,S.S.; Schrank,A.; Vainstein,M.H.; Schrank,I.S.; Vargas,A.C.

    2010-01-01

    Virulence factors and antimicrobial resistance patterns of Escherichia coli isolates were evaluated. A total of 80 E. coli isolates were evaluated, being 64 from clinical samples (intestinal content and fragments of organs from diarrheic piglets), seven from feces of clinically healthy piglets and sows, and nine environmental samples (five from facilities, two from feed, one from insect, and one from waste). Molecular characterization was performed by PCR detection of fimbriae and toxin genes...

  4. Emergence of colistin-resistant Escherichia coli clinical isolates harboring mcr-1 in Vietnam

    Directory of Open Access Journals (Sweden)

    Tatsuya Tada

    2017-10-01

    Full Text Available The mcr-1 was first detected on a plasmid in colistin-resistant Escherichia coli from livestock and patients in China. We described here the emergence of colistin-resistant E. coli clinical isolates harboring mcr-1 on the chromosomes in Vietnam. To our knowledge, this is the first report of hospital-acquired E. coli isolates harboring mcr-1 in a medical setting in Vietnam.

  5. Escherichia coli is not a suitable fecal indicator to assess water fecal contamination by otters

    OpenAIRE

    M. Oliveira; D. Freire; N. M. Pedroso

    2017-01-01

    Abstract The detection of pathogenic microorganisms in aquatic environments is extremely relevant in terms of public health. As these laboratorial methodologies are usually difficult, expensive and time-consuming, they are frequently replaced by the assessment of fecal indicator bacteria, such as Escherichia coli. This study aimed to assess the presence of E. coli in fecal samples from Neotropical otters, to evaluate its potential as fecal indicator to be applied to the determination of water...

  6. The prevalence of OqxAB multidrug efflux pump amongst olaquindox resistant Escherichia coli in pigs

    DEFF Research Database (Denmark)

    Hansen, L. H.; Sørensen, S. J.; Jørgensen, H. S.

    2005-01-01

    to olaquindox as well as resistance to other antimicrobials like chloramphenicol. In this study, 10 of the 556 (1.8%) previously isolated Escherichia coli strains were shown to have an MIC >or= 64 microg/ml olaquindox. In nine of the ten strains, the oqxA gene was detected. Sequencing of an internal fragment......, resistant strains. Furthermore, horizontal transfer of olaquindox resistance from three olaquindox-resistant isolates was achieved using an olaquindox-sensitive E. coli as recipient....

  7. Emergence of colistin-resistant Escherichia coli clinical isolates harboring mcr-1 in Vietnam.

    Science.gov (United States)

    Tada, Tatsuya; Nhung, Pham Hong; Shimada, Kayo; Tsuchiya, Mitsuhiro; Phuong, Doan Mai; Anh, Nguyen Quoc; Ohmagari, Norio; Kirikae, Teruo

    2017-10-01

    The mcr-1 was first detected on a plasmid in colistin-resistant Escherichia coli from livestock and patients in China. We described here the emergence of colistin-resistant E. coli clinical isolates harboring mcr-1 on the chromosomes in Vietnam. To our knowledge, this is the first report of hospital-acquired E. coli isolates harboring mcr-1 in a medical setting in Vietnam. Copyright © 2017. Published by Elsevier Ltd.

  8. Characterization of Escherichia coli causing community acquired urinary tract infections in Mexico City.

    Science.gov (United States)

    Belmont-Monroy, Laura; Ribas-Aparicio, Rosa María; Navarro-Ocaña, Armando; Manjarrez-Hernández, H Ángel; Gavilanes-Parra, Sandra; Aparicio-Ozores, Gerardo; Cauich-Sánchez, Patricia Isidra; Garza-Ramos, Ulises; Molina-López, José

    2017-02-01

    The O25-ST131 clone was identified within 169 uropathogenic Escherichia coli (UPEC) strains. The 44.8% of the 29 O25-ST131 clones detected were positive to least to one extended-spectrum β-lactamase gene. The phylogroup D was mainly found. The O25-ST131 clone appeared to be associated with community-acquired UTI in Mexico City. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Plasmid-Mediated Florfenicol Resistance Encoded by the floR Gene in Escherichia coli Isolated from Cattle

    Science.gov (United States)

    Cloeckaert, Axel; Baucheron, Sylvie; Flaujac, Geraldine; Schwarz, Stefan; Kehrenberg, Corinna; Martel, Jean-Louis; Chaslus-Dancla, Elisabeth

    2000-01-01

    A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104. PMID:10991873

  10. CONFIRMATIONAL IDENTIFICATION OF ESCHERICHIA COLI, A COMPARISON OF GENOTYPIC AND PHENOTYPIC ASSAYS FOR GLUTAMATE DECARBOXYLASE AND B-D-GLUCURONIDASE

    Science.gov (United States)

    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  11. Conditional function of autoaggregative protien cah and common cah mutations in Shiga toxin-producing Escherichia coli

    Science.gov (United States)

    Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 super-shedder strain SS17, a large d...

  12. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    Science.gov (United States)

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  13. Leaner and meaner genomes in Escherichia coli

    DEFF Research Database (Denmark)

    Ussery, David

    2006-01-01

    A 'better' Escherichia coli K-12 genome has recently been engineered in which about 15% of the genome has been removed by planned deletions. Comparison with related bacterial genomes that have undergone a natural reduction in size suggests that there is plenty of scope for yet more deletions....

  14. Inhibition of Escherichia Coli, Salmonella and Staphylococcus ...

    African Journals Online (AJOL)

    Escherichia coli O157:H7, Salmonella typhimurium and Staphylococcus. aureus are of great concern to the food industry, especially in foods stored under refrigerated conditions where, unlike most food-borne pathogens are able to multiply. This investigation was conducted to study the inhibitory effect of some spice ...

  15. Strategies for Protein Overproduction in Escherichia coli.

    Science.gov (United States)

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  16. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, Jonathan P; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome...

  17. The eclipse period of Escherichia coli

    DEFF Research Database (Denmark)

    von Freiesleben, Ulrik; Krekling, Martin A.; Hansen, Flemming G.

    2000-01-01

    The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse...

  18. Drug-resistant Escherichia coli, Rural Idaho

    OpenAIRE

    Hannah, Elizabeth L.; Angulo, Frederick J.; Johnson, James R.; Haddadin, Bassam; Williamson, Jacquelyn; Samore, Matthew H.

    2005-01-01

    Stool carriage of drug-resistant Escherichia coli in home-living residents of a rural community was examined. Carriage of nalidixic acid–resistant E. coli was associated with recent use of antimicrobial agents in the household. Household clustering of drug-resistant E. coli was observed. Most carriers of drug-resistant E. coli lacked conventional risk factors.

  19. Escherichia Coli Removal from Water Using Electrophotocatalytic ...

    African Journals Online (AJOL)

    The aim of this experimental applied study is to evaluate the removal of Escherichia Coli, as the microbial contamination indicator of water, from drinking water using electrophotocatalytic method. The contaminated water in an electrophotocatalytic reactor were prepared by adding 102-103 cell of E. coli bacteria to drinking ...

  20. Fimbrial adhesins from extraintestinal Escherichia coli

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

  1. (ESBL) producing Escherichia coli and Klebsiella pneumoniae

    African Journals Online (AJOL)

    Emerging antibiotic resistance due to extended spectrum β-lactamase (ESBL) production limited the use of β-lactam antibiotics against Escherichia coli and Klebsiella pneumoniae. This observational study was conducted at the Microbiology department of the Children's Hospital, Lahore Pakistan, from June, 2009 to ...

  2. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...

  3. Antimicrobial resistance among commensal Escherichia coli from ...

    African Journals Online (AJOL)

    Commensal bacteria contribute to the distribution and persistence of antimicrobial resistance in the environment. This study monitored antimicrobial resistance in commensal Escherichia coli from the faeces of on-farm and slaughter cattle and beef. A total of 342 (89.5%) E. coli isolates were obtained from 382 samples.

  4. Shiga toxin-producing Escherichia coli

    DEFF Research Database (Denmark)

    Pedersen, Rune Micha; Nielsen, Marc Trunjer Kusk; Möller, Sören

    2018-01-01

    OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal...

  5. Control of Ribosome Synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Molin, Søren; Meyenburg, K. von; Måløe, O.

    1977-01-01

    The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue {alpha...

  6. Escherichia coli survival in waters: Temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  7. Emergence of Quinolone Resistance amongst Escherichia coli ...

    African Journals Online (AJOL)

    Two hundred and seventy three isolates of Escherichia coli obtained from 7 hospitals in Lagos were screened for Fluoroquinolone resistance (FQR). Rate of resistance was 22.3% showing an increase in quinolone resistance when compared with resistant rates between 1994 and 1999 which ranged from 0 – 2% then.

  8. Progressive segregation of the Escherichia coli chromosome

    DEFF Research Database (Denmark)

    Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

    2006-01-01

    We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth...

  9. Characterization of Escherichia coli Phylogenetic Groups ...

    African Journals Online (AJOL)

    Background: Escherichia coli strains mainly fall into four phylogenetic groups (A, B1, B2, and D) and that virulent extra‑intestinal strains mainly belong to groups B2 and D. Aim: The aim was to determine the association between phylogenetic groups of E. coli causing extraintestinal infections (ExPEC) regarding the site of ...

  10. Mutagenic DNA repair in Escherichia coli

    International Nuclear Information System (INIS)

    Bridges, B.A.; Sharif, Firdaus

    1986-01-01

    The authors report a study of the misincorporation step in excision proficient umuC Escherichia coli as revealed by delayed photoreversal and show that it parallels the loss of photoreversibility of mutations induced in isogenic umu + bacteria; in both cases the end-point was mutation to streptomycin resistance. (author)

  11. Multiplex Genome Editing in Escherichia coli

    DEFF Research Database (Denmark)

    Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2018-01-01

    Lambda Red recombineering is an easy and efficient method for generating genetic modifications in Escherichia coli. For gene deletions, lambda Red recombineering is combined with the use of selectable markers, which are removed through the action of, e.g., flippase (Flp) recombinase. This PCR...

  12. Antibiotic resistance properties of uropathogenic Escherichia coli ...

    African Journals Online (AJOL)

    Purpose: To investigate the antibiotic resistance pattern of uropathogenic Escherichia coli (UPEC) strains isolated from pregnant women with history of recurrent urinary tract infections (RUTIs) and healthy pregnant women. Methods: A total of 485 high vaginal swab specimens were collected from pregnant women with ...

  13. Antibiotic resistance properties of uropathogenic Escherichia coli ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, ..... and Argentina [28]. CONCLUSION. As far as we know, the present study is the first prevalence report on antibiotic resistance pattern of UPEC strains in ... serogroups profiles of uropathogenic Escherichia coli isolated ...

  14. Altered membrane permeability in multidrug resistant Escherichia ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... involvement during the transport of β - lactams in multidrug resistant Escherichia coli isolated from extra-intestinal infections. Also, the ... lactam resistance in multidrug resistant E. coli in ESBL and non-ESBL isolates. .... and decreased susceptibility to carbapenems, particularly ertapenem (Perez et al.,.

  15. (ESBL) producing Escherichia coli and Klebsiella pneumoniae

    African Journals Online (AJOL)

    use

    2011-11-21

    Nov 21, 2011 ... carbapenems (Patricia, 2001; Bush, 2001). The β-lactamases produced by bacteria are ... among clinical isolates of the family Enterobacteriaceae. (Sanders and Sanders, 1992). ESBLs have ..... lactamases among multidrug resistant Escherichia coli and Klebsiella species causing urinary tract infections in ...

  16. Altered membrane permeability in multidrug resistant Escherichia ...

    African Journals Online (AJOL)

    The study was conducted with the objective of examining the outer membrane proteins and their involvement during the transport of β - lactams in multidrug resistant Escherichia coli isolated from extra-intestinal infections. Also, the response of gram negative bacterial biomembrane alteration was studied using extended ...

  17. Optimization of plasmid electrotransformation into Escherichia coli ...