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Sample records for aureus transcripts detection

  1. RT-PCR检测金黄色葡萄球菌%Reverse transcription-PCR assay for detection of viable Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    罗予; 李杰; 刘娜

    2011-01-01

    目的 探讨检测金黄色葡萄球菌及其活菌的RT-PCR方法 .方法 用RT-PCR方法 对金黄色葡萄球菌的spa基因进行检测,并做灵敏度和特异性测定,用RT-PCR检测细菌灭活前后的spa基因.结果 用spa基因检测金黄色葡萄球菌灵敏度为1.5×104 CFU/mL;Spa引物能特异性扩增出金黄色葡萄球菌的标准株和14株临床株的目的 片段,对大肠埃希菌、铜绿假单胞菌、表皮葡萄球菌和化脓性链球菌则无特异性扩增条带,而对白色念珠菌有较弱条带扩出;细菌灭活前可以检测出目的 基因,灭活后4℃放置24、48和72 h均无目的 基因片段扩出.结论可以用spa基因对金黄色葡萄球菌进行活菌检测.%Objective To observe the effect of reverse transcription-polymerase chain reaction (RT-PCR) in detecting viable Staphylococcus aureus using. Method The spa gene of Staphylococcus aweus was detected by mRNA-based RT-PCR both before and after inactivation. The sensitivity and specificity of the RT-PCR method were determined. Result The Special fragment of Staphylococcus aureus was extended by the pair of primers. There was no crossreaction with E. Coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Pyogenic streptococcus. The sensitivity of detection was 1.5 x 10* CFU/mL. mRNA from uninactivated cells was detected, while in inactivated cells,. mRNA became undetectable when dead cells were held at 4 ℃ temperature for over 24 h. Conclusion This gene can be used to detect viable Staphylococcus aureus.

  2. Tea Tree Oil-Induced Transcriptional Alterations in Staphylococcus aureus

    OpenAIRE

    Cuaron, Jesus A.; Dulal, Santosh; Song, Yang; Singh, Atul K; Montelongo, Cesar E.; Yu, Wanqin; Nagarajan, Vijayaraj; Jayaswal, Radheshyam K.; Wilkinson, Brian J; Gustafson, John E.

    2012-01-01

    Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and alt...

  3. Tea tree oil-induced transcriptional alterations in Staphylococcus aureus.

    Science.gov (United States)

    Cuaron, Jesus A; Dulal, Santosh; Song, Yang; Singh, Atul K; Montelongo, Cesar E; Yu, Wanqin; Nagarajan, Vijayaraj; Jayaswal, Radheshyam K; Wilkinson, Brian J; Gustafson, John E

    2013-03-01

    Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g. clpC, clpL, ctsR, dnaK, groES, groEL, grpE and hrcA) and cell wall metabolism (e.g. cwrA, isaA, sle1, vraSR and vraX). Inactivation of the heat shock gene dnaK or vraSR which encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in the S. aureus TTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO.

  4. Genome-Wide Transcriptional Profiling of the Response of Staphylococcus aureus to Cryptotanshinone

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    Haihua Feng

    2009-01-01

    Full Text Available Staphylococcus aureus (S. aureus strains with multiple antibiotic resistances are increasingly widespread, and new agents are required for the treatment of S. aureus. Cryptotanshinone (CT, a major tanshinone of medicinal plant Salvia miltiorrhiza Bunge, demonstrated effective in vitro antibacterial activity against all 21 S. aureus strains tested in this experiment. Affymetrix GeneChips were utilized to determine the global transcriptional response of S. aureus ATCC 25923 to treatment with subinhibitory concentrations of CT. Transcriptome profiling indicated that the antibacterial action of CT may be associated with its action as active oxygen radical generator; S. aureus undergoes an oxygen-limiting state upon exposure to CT.

  5. Rapid, Culture-Free Detection of Staphylococcus aureus Bacteremia

    Science.gov (United States)

    Burghardt, Elliot L.; Flenker, Katie S.; Clark, Karen C.; Miguel, Jeff; Ince, Dilek; Winokur, Patricia; Ford, Bradley; McNamara, James O.

    2016-01-01

    S. aureus bacteremia (SAB) is a common condition with high rates of morbidity and mortality. Current methods used to diagnose SAB take at least a day, and often longer. Patients with suspected bacteremia must therefore be empirically treated, often unnecessarily, while assay results are pending. In this proof-of-concept study, we describe an inexpensive assay that detects SAB via the detection of micrococcal nuclease (an enzyme secreted by S. aureus) in patient plasma samples in less than three hours. In total, 17 patient plasma samples from culture-confirmed S. aureus bacteremic individuals were tested. 16 of these yielded greater nuclease assay signals than samples from uninfected controls or individuals with non-S. aureus bacteremia. These results suggest that a nuclease-detecting assay may enable the rapid and inexpensive diagnosis of SAB, which is expected to substantially reduce the mortality and morbidity that result from this condition. PMID:27305148

  6. Highly sensitive detection of Staphylococcus aureus directly from patient blood.

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    Padmapriya P Banada

    Full Text Available BACKGROUND: Rapid detection of bloodstream infections (BSIs can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing-polymerase chain reaction (PCR platform as a model diagnostic system. METHODOLOGY/PRINCIPAL FINDINGS: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD of 5 genomic copies per reaction or 10 colony forming units (CFU per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs, plasma or whole blood, using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90% positive as opposed to cell-associated bacteria (in WBCs (71% samples positive or free bacterial DNA in plasma (62.5% samples positive. Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95% CI 0.75-0.96 and 0/59 negative controls with the sodA assay (specificity 100%, 95% CI 0.92-1. CONCLUSIONS: We have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.

  7. Effects of sub-lethal concentrations of mupirocin on global transcription in Staphylococcus aureus 8325-4 and a model for the escape from inhibition.

    Science.gov (United States)

    AlHoufie, Sari Talal S; Foster, Howard A

    2016-08-01

    Staphylococcus aureus is a major pathogen in both hospital and community settings, causing infections ranging from mild skin and wound infections to life-threatening systemic illness. Gene expression changes due to the stringent response have been studied in S. aureus using lethal concentrations of mupirocin, but no studies have investigated the effects of sub-lethal concentrations. S. aureus 8325-4 was exposed to sub-inhibitory concentrations of mupirocin. The production of ppGpp was assessed via HPLC and the effects on global transcription were studied by RNAseq (RNA sequencing) analysis. Growth inhibition had occurred after 1 h of treatment and metabolic analysis revealed that the stringent response alarmone ppGpp was present and GTP concentrations decreased. Transcriptome profiles showed that global transcriptional alterations were similar to those for S. aureus after treatment with lethal concentrations of mupirocin, including the repression of genes involved in transcription, translation and replication machineries. Furthermore, up-regulation of genes involved in stress responses, and amino acid biosynthesis and transport, as well as some virulence factor genes, was observed. However, ppGpp was not detectable after 12 or 24 h and cell growth had resumed, although some transcriptional changes remained. Sub-lethal concentrations of mupirocin induce the stringent response, but cells adapt and resume growth once ppGpp levels decrease.

  8. Genetic effects of an air discharge plasma on Staphylococcus aureus at the gene transcription level

    Science.gov (United States)

    Xu, Zimu; Wei, Jun; Shen, Jie; Liu, Yuan; Ma, Ronghua; Zhang, Zelong; Qian, Shulou; Ma, Jie; Lan, Yan; Zhang, Hao; Zhao, Ying; Xia, Weidong; Sun, Qiang; Cheng, Cheng; Chu, Paul K.

    2015-05-01

    The dynamics of gene expression regulation (at transcription level) in Staphylococcus aureus after different doses of atmospheric-pressure room-temperature air plasma treatments are investigated by monitoring the quantitative real-time polymerase chain reaction. The plasma treatment influences the transcription of genes which are associated with several important bio-molecular processes related to the environmental stress resistance of the bacteria, including oxidative stress response, biofilm formation, antibiotics resistance, and DNA damage protection/repair. The reactive species generated by the plasma discharge in the gas phase and/or induced in the liquid phase may account for these gene expression changes.

  9. Global transcriptional response of methicillin-resistant Staphylococcus aureus to thioridazine

    DEFF Research Database (Denmark)

    Bonde, Mette; Jacobsen, Kirstine; Kolmos, Hans Jørn

    is a phenothiazine derivate, which is known to restore in vitro susceptibility of MRSA strains to β-lactam antibiotics (e.g. oxacillin) [4]. Previously, we have demonstrated that thioridazine prevents the oxacillin-induced transcription of the resistance determinant mecA in a hospital-associated MRSA isolate leading......Few drugs are available against methicillin-resistant S. aureus (MRSA), and decreased susceptibility among staphylococci to newly introduced agents such as linezolid, daptomycin, and tigecycline has been observed [1-3]. Consequently, new treatment strategies are urgently needed. Thioridazine...... to examine the underlying mechanism we are conducting a global transcriptional analysis of the response of strain USA300 to thioridazine treatment with and without oxacillin. We show that thioridazine by itself elicits extensive transcriptional changes, including pathways involved in amino acid synthesis...

  10. Detection of Staphylococcus aureus in Milk Using Real-time Fluorescence Loop-mediated Isothermal Amplification

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    Ying Yu

    2015-07-01

    Full Text Available Staphylococcus aureus is a kind of worldwide food-borne pathogen. Recently, S. aureus has gained considerable attention because of the increasing alimentary toxicosis incidence. In this study, a Real-Time fluorescence Loop-Mediated isothermal Amplification (RT-LAMP was developed to detect S. aureus rapidly. The heat-stable nuclease (nuc gene of S. aureus, the target sequence, was selected to design four special primers. A rapid detection method for S. aureus was initially established under optimum reaction conditions. The assay, performed for 40 min at 61°C, did not show cross reactivity with other bacterial species. The specificity and sensitivity of RT-LAMP for detecting S. aureus were 100% and 8.0 CFU/mL, respectively. Results indicated that RT-LAMP was a potential field-usable molecular tool for detecting S. aureus This method can be an alternative to conventional LAMP in clinical applications and operational programs.

  11. Detection of enterotoxigenic Staphylococcus aureus isolates in domestic dairy products

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    HR Tavakoli

    2010-12-01

    Full Text Available Background and objectives: Staphylococcus aureusis a one of THE most frequent causes of food poisoning (FP in dairy products. The main etiologic agents of FP are staphylococcal enterotoxins (SE. There are different types of SE; types A (SEA and B (SEB are the most clinically important enterotoxins. Traditional dairy products are still produced in small batches and sold by some vendors without a permit from the Ministry of Health. This study focuses on the molecular and serological detection of enterotoxigenic Staphylococcus aureus SEA and SEB genes and its products, respectively from samples of such traditional products."nMaterials and Methods: 100 samples from dairy products were produced under sterile conditions via traditional methods and were transported to the laboratory. The samples were cultured and identified by routine bacteriological methods. The isolated bacteria were evaluated by PCR tests for detection of the genes encoding SEA and SEB. Subsequently, the ability of these strains to produce enterotoxin was examined by Sac's culture method and was confirmed by Sigel Radial Immounodiffussion (SRID."nResults: The results indicated that 32% of the dairy products were contaminated by S. aureus (cream 18% , cheese 10%, milk 4%. The PCR results showed that 15.6% of the S. aureus isolates possessed the SEA gene, 9.3% had the SEB gene, and 6.2% possessed both genes. The evaluation of enterotoxin production indicated that 80% of SEA and 33% of SEB genes were expressed."nConclusion: Enterotoxins SEA and SEB are heat stable and consequently; heating has no effect on dairy products contaminated by entertoxins. Subsequently, gastritis may occur within several hours after consumption. Our findings suggest that PCR is a rapid, sensitive, specific, and inexpensive method for detecting SE and can replace the traditional assays.

  12. Mechanism of hetero-erythromycin resistant Staphylococcus aureus and a comparison of detection methods

    Institute of Scientific and Technical Information of China (English)

    陈东科

    2014-01-01

    Objective To explore the phenotypes and genotypes of Staphylococcus aureus(S.aureus)hetero-resistant to erythromycin and clindamycin and compare their detection methods so as to report results accurately to guide clinical rational use of antibiotics.Methods D test was used to detect the phenotypes of S.aureus hetero-resistant to erythromycin.And then the results of two methods(automated instrument and disk diffusion)were analyzed.All strains were continuously passaged for 50 generations to

  13. The Novel Transcriptional Regulator SA1804 Is Involved in Mediating the Invasion and Cytotoxicity of Staphylococcus aureus

    OpenAIRE

    2015-01-01

    The two-component regulatory system, SaeRS, controls expression of important virulence factors, including toxins and invasins, which contribute to the pathogenicity of Staphylococcus aureus. Previously, we conducted a transcriptomics study for identification of SaeRS regulon and found that inactivation of SaeRS dramatically enhances the transcription of a novel transcriptional regulator (SA1804). This led us to question whether SA1804 is involved in bacterial pathogenicity by regulating the e...

  14. The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level.

    Science.gov (United States)

    Giraudo, A T; Cheung, A L; Nagel, R

    1997-07-01

    Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of alpha- and beta-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for alpha- and beta-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.

  15. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    Science.gov (United States)

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  16. Improved detection of methicillin-resistant Staphylococcus aureus using phenyl mannitol broth containing aztreonam and ceftizoxime.

    NARCIS (Netherlands)

    H.F.L. Wertheim (Heiman); H.A. Verbrugh (Henri); C. van Pelt (Cindy); P. de Man (Peter); A.F. van Belkum (Alex); M.C. Vos (Margreet)

    2001-01-01

    textabstractWe tested a phenyl mannitol broth containing ceftizoxime and aztreonam (PHMB(+)) for detection of methicillin-resistant Staphylococcus aureus (MRSA) with reference MRSA strains and, subsequently, with clinical samples (n = 1,098). All reference MRSA strains

  17. Rapid detection of viral antibodies based on multifunctional Staphylococcus aureus nanobioprobes.

    Science.gov (United States)

    Qiao, Jinjuan; Li, Yunpeng; Wei, Cuihua; Yang, Hang; Yu, Junping; Wei, Hongping

    2016-12-01

    Biosynthesis of nanoparticles inside S. aureus cells has enhanced the sensitivity of immunoassays based on the S. aureus nanoparticles. However, the current methods are limited to antigen detection by conjugating IgG antibodies on S. aureus nanoparticles. In this study, a simple way to conjugate antigens to the S. aureus nanobioparticles was developed by utilizing a cell wall binding domain (CBD) from a bacteriophage lysin PlyV12. Based on this novel design, simple agglutination tests of the IgG antibodies of Ebola virus (EBOV) nucleoprotein (NP) and Middle East Respiratory Virus (MERS) NP in rabbit sera were successfully developed by conjugating the S. aureus nanobioparticles with two fusion proteins EBOV NP- CBD and MERS NP-CBD, respectively. The conjugation was done easily by just mixing the fusion proteins with the S. aureus nanoparticles. The detection time was within 20 min without any special equipment or expertise. As far as we know, this is the first time to realize the detection of viral antibodies based on S. aureus nanoparticles.

  18. Detection of Staphylococcus Aureus Enterotoxin Genes A-E

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    Dadgar, T. (PhD

    2014-06-01

    Full Text Available Background and Objective: The main cause of spreading staphylococcal infections among patients is the healthy carriers working in hospitals. With the secretion of different sorts of toxins such as entrotoxin, this bacteria can provide the conditions for attacking on the host. The main objective of this study is identification of the characteristics and differences in the Staphylococcus aureus isolated from healthy carriers and from the patients on the basis of enterotoxin genes (sea-see. Material and Methods: One hundred and twenty of the patients and 80 of healthy carriers worked in health centers of Gorgan, north of Iran, were investigated for S. aureus isolate. The isolates were evaluated by PCR for Enterotoxin Genes A-E (SEA to SEE. Results: Enterotoxin genes (SEA to SEE was found in 87.5% of the total isolates and the most frequent one was enterotoxin gene sea (N= 124. The prevalence of these isolates in healthy carriers was significantly higher than those of the patients. Conclusion: Based on the results, the high percentage of S. aureus isolated from clinical samples contains enterotoxin genes. Therefore, Human as the source and carrier of S. aureus is paramount importance, which is due to significant relationship between being toxigenic strains and the source of isolation. Key words: Staphylococcus Aureus; Enterotoxin; Patient; Carrier

  19. Detection of vancomycin resistant Staphylococcus aureus: A comparative study of three different phenotypic screening methods

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    Bhateja P

    2005-01-01

    Full Text Available The objective of this study was to investigate screening methodologies, to detect Staphylococcus aureus strains with decreased susceptibility to vancomycin. Three methods were used to screen 160 Staphylococcus aureus clinical isolates along with ATCC quality control strains. Subsequently, MIC of all these 160 strains were determined by NCCLS methodology. The MIC of all the 160 clinical isolates was < 4µg/mL and were classified as vancomycin susceptible by NCCLS criteria but 23 strains were positive by Hiramatshu method, two grew on MHA (5µg/mL vancomycin while CDC method correctly identified no vancomycin intermediate S.aureus (VISA or vancomycin resistant S.aureus (VRSA strains with reference to there MIC. CDC method was found to be the most appropriate screening methodology for detection of VISA or VRSA for diagnostic laboratories.

  20. Detection of sea, sec and seq genes in Staphylococcus aureus nasal sampling acquiring from healthy carrier

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    Mojtaba Saadati

    2009-09-01

    Full Text Available Background: Various assays have been used to identify of enterotoxins produced by Staphylococcus aureus and because of antigenic similarities among enterotoxins, serological assay may not always be practical. The aim of this study was to detect of S. aureus enterotoxins (SEA, SEC and SEQ genes by multiplex PCR assay. Methods: Of 150 strains obtained from nasal carriers, 95 S. aureus were confirmed by biochemical test. Multiplex PCR assay for the detection of genes encoding staphylococcal enterotoxins A, C and Q genes (sea, c and q S. aureus was used. The nuc gene, which encodes thermonuclease was used as a target DNA to identify S. aureus.Results: DNA amplification fragments for the staphylococcal nuclease gene (nuc was 397 bp, 552 bp for staphylococcal enterotoxin A gene (sea, 271 bp for staphylococcal enterotoxin C gene (sec and 122 bp for staphylococcal enterotoxin Q gene (seq. S. epidermidis used as negative control and did not yield a PCR product. Among the 95 healthy human isolates from nasal carriage, forty one isolates (43/1% were diagnosed as sea, sec or seq-positive. Twenty four (25/3% isolates were sea gene, nine (9/5% isolates were the sec gene and eight (8/4% isolates were the seq gene and 54 (56/8% of them were other se genes. Conclusion: Because Staphylococcus aureus was isolated in nasal healthy carrier, so the PCR assay could be useful in the routine direct detection of staphylococcal enterotoxin A, C and Q genes.

  1. Pervasive transcription: detecting functional RNAs in bacteria.

    Science.gov (United States)

    Lybecker, Meghan; Bilusic, Ivana; Raghavan, Rahul

    2014-01-01

    Pervasive, or genome-wide, transcription has been reported in all domains of life. In bacteria, most pervasive transcription occurs antisense to protein-coding transcripts, although recently a new class of pervasive RNAs was identified that originates from within annotated genes. Initially considered to be non-functional transcriptional noise, pervasive transcription is increasingly being recognized as important in regulating gene expression. The function of pervasive transcription is an extensively debated question in the field of transcriptomics and regulatory RNA biology. Here, we highlight the most recent contributions addressing the purpose of pervasive transcription in bacteria and discuss their implications.

  2. D-Test Method for Detection of Inducible Clindamycin Resistance in Staphylococcus Aureus

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    Neda Pak

    2009-09-01

    Full Text Available Objective:Methicillin resistant staphylococcus aureus (MRSA is a frequent cause of infections in children. The purpose of this study was to determine the frequency of nasal colonization of S. aureus in children and detection of inducible clindamycin resistance (ICR by disk approximation test (D-test. Methods:This was a cross-sectional study conducted in Hamedan from 2007 to 2008. 520 nasal swabs were obtained from children under 12 years of age at the time of admission and 287 swabs at the time of discharge. Antibiogram was performed by method of disk diffusion for oxacillin, erythromycin, clindamycin, cefazolin and vancomycin as well as D-test. Chi-square test was applied for statistical analysis. Findings:Out of 520 patients, 118 (22.3% were colonized with S. aureus as community-acquired (CA-S. aureus. Of 287 patients, 64 (22.3% were colonized with isolates of S. aureus at discharge time. Of these 64 patients, 32 cases were colonized with hospital acquired (HA-S. aureus isolates after admission. Only one CA-MRSA isolate was resistant to clindamycin, 5% of 118 CA-S. aureus isolates and 6.3% of HA-S. aureus isolates had inducible clindamycin resistance (D-test. Also 37.5% of CA-MRSA isolates at the time of admission and 22.2% of HA-MRSA isolates at discharge had positive D-test. Conclusion:We emphasize that D-test should be used routinely and clindamycin should not be used in patients with infections caused by inducible resistant S. aureus.

  3. Methods of detection and typing of methicillin resistant Staphylococcus aureus isolated from animals

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    Radosavljević V.

    2014-01-01

    Full Text Available In this work there was evaluated the method of detection of methicillin resistant Staphylococcus aureus (MRSA by using two molecular and three phenotypic tests in investigation procedure of 70 strains of S.aureus isolated from animals. Recent findings of the new mecA homologue, mecALGA251, minimise the significance of mecA gene presence detection as a confirmation method of methicillin resistant Staphylococcus aureus identification. For this reason, along with multiplex PCR set of primers(165rDNK, nuc, mecA for detection mecA gene, there was also used multiplex PCR set of primers (spa, mecA, pvl, mecALGA251 for differentiation mecALGA251 from mecA, with simultaneous detection of luk-PV and spa gene fragments. In all 70 investigated isolates there was detected the presence of specific 16 SrDNK fragment and nuc gene which encodes a thermostable S. aureus nuclease, while in 5 out of 70 S. aureus isolates, there was proven mecA gene presence using two multiplex PCR tests. In the investigated strains there was determined neither mecC (mecALGA251gene presence, nor Panton Valentine Leukocidin encoding gene. By application cefoxitin disk-diffusion, latex-agglutination and two multiplex PCR tests, the identical results in identification 5 methicillin resistant out of 70 investigated S. aureus strains were obtained. In our investigation there was determined a complete correlation between the results of phenotypic and genotypic identification of methicillin resistant S. aureus. [Projekat Ministarstva nauke Republike Srbije, br. TR 31079

  4. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene.

    Science.gov (United States)

    Brakstad, O G; Aasbakk, K; Maeland, J A

    1992-07-01

    Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical

  5. Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

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    Dacheng Wang

    Full Text Available BACKGROUND: The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA. MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. CONCLUSIONS/SIGNIFICANCE: MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

  6. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  7. Rapid and low-cost biosensor for the detection of Staphylococcus aureus.

    Science.gov (United States)

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

    2017-04-15

    Staphylococcus aureus (S. aureus) is one of the most common etiological agents in hospital-acquired infections and food-borne illness. S. aureus toxins and virulence proteases often circulate in host blood vessels leading to life-threatening diseases. Standard identification approaches include bacterial culturing method, which takes several days. Other nucleic acid-based methods were expensive and required trained personnel. To surmount these limitations, a paper-based biosensor was developed. The sensing mechanism was based on the proteolytic activity of S. aureus proteases on a specific peptide substrate, sandwiched between magnetic nanobeads and gold surface on top of a paper support. An external magnet was fixed on the back of the sensor to accelerate the cleavage of the magnetic nanobeads-peptide moieties away from the sensor surface upon test sample dropping. The colour change resulting from the dissociation of the magnetic nanobeads moieties was detected by the naked eye and analysed using ImageJ analysis software for the purpose of quantitative measurement. Experimental results showed detection limits as low as 7, 40 and 100 CFU/mL for S. aureus in pure broth culture, and inoculated in food produces and environmental samples, respectively upon visual observation. The specificity of the sensor was examined by blind testing a panel of food-contaminating pathogens (Listeria monocytogenesis 19115 and E. coli O157:H7), clinical isolates (methicillin-resistant S. aureus (MRSA) and Candida albicans) and standard (Pseudomonas aeruginosa 15692) pathogens. Negative read-out was observed by the naked eye for all tested isolates except for MRSA. Moreover, this sensing tool requires minute's time to obtain the results. In conclusion, this sensing platform is a powerful tool for the detection of S. aureus as a potential point-of-care diagnostic platform in hospitals and for use by regulatory agencies for better control of health-risks associated with contaminated food

  8. Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR.

    Science.gov (United States)

    Schumacher, Maria A; Brennan, Richard G

    2003-03-01

    Multidrug transporters and their transcriptional regulators are key components of bacterial multidrug resistance (MDR). How these multidrug binding proteins can recognize such chemically disparate compounds represents a fascinating question from a structural standpoint and an important question in future drug development efforts. The Staphylococcus aureus multidrug binding regulator, QacR, is soluble and recognizes an especially wide range of structurally dissimilar compounds, properties making it an ideal model system for deciphering the molecular basis of multidrug recognition. Recent structures of QacR have afforded the first view of any MDR protein bound to multiple drugs, revealing key structural features of multidrug recognition, including a multisite binding pocket.

  9. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Wang, Xin-Ru; Wu, Li-Fen; Wang, Yan; Ma, Ying-Ying; Chen, Feng-Hua; Ou, Hong-Ling

    2015-01-01

    Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.

  10. The Novel Transcriptional Regulator SA1804 Is Involved in Mediating the Invasion and Cytotoxicity of Staphylococcus aureus

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    JUNSHU eYANG

    2015-03-01

    Full Text Available The two-component regulatory system, SaeRS, controls expression of important virulence factors, including toxins and invasins, which contribute to the pathogenicity of Staphylococcus aureus. Previously, we conducted a transcriptomics study for identification of SaeRS regulon and found that inactivation of SaeRS dramatically enhances the transcription of a novel transcriptional regulator (SA1804. This led us to question whether SA1804 is involved in bacterial pathogenicity by regulating the expression of virulence factors. To address this question, we created sa1804, saeRS, and sa1804/saeRS double deletion mutants in a USA300 community-acquired MRSA strain, 923, and determined their impact on the pathogenicity. The deletion of sa1804 dramatically increased the cytotoxicity and enhanced the capacity of bacteria to invade into the epithelial cells (A549, whereas the deletion of saeRS eliminated the cytotoxicity and abolished the bacterial ability to invade into the epithelial cells. Moreover, the double deletions of sa1804 and saeRS appeared a similar phenotype with the saeRS null mutation. Furthermore, we determined the regulatory mechanism of SA1804 using qPCR and gel-shift approaches. Our data indicate that the novel virulence repressor SA1804 is dependent on the regulation of SaeRS. This study sheds light on the regulatory mechanism of virulence factors and allows for us further elucidate the molecular pathogenesis of S. aureus.

  11. Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    YANG Yang; SU Xu-dong; YUAN Yao-wu; KANG Chun-yu; LI Ying-jun; ZHANG wei; ZHONG Xiao-ying

    2007-01-01

    A polymerase chain reaction (PCR) assay was employed for direct detection of Staphylococcus aureus without enrichment in dairy products. A solvent extraction procedure was successfully modified for the extraction of Staphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. A primer targeting the thermostable nuclease gene (nuc) was used in the PCR. A DNA fragment of 279 bp was amplified. The PCR product was confirmed by DNA sequencing. In this study, the PCR, GB- 4789.10-94, Perifilm RSA.Count Plate, and Baird-Parker + RPF Agar were compared.The sensitivity of the PCR was 10 CFU mL-1 of whole milk, skim milk, and 55 CFU g-1 of cheese. The developed methodology allowed for detection of Staphylococcus aureus in dairy products in less than 6 h. The time taken for the development of this PCR assay was 12-24 h, less than the time taken by the general PCR assay using the enrichment method, and the coincidence rate of this developed PCR was 94.3%, the sensitivity was 100%. It was a rapid, sensitive, and effective method for PCR to detect Staphylococcus aureus in milk and milk products.

  12. Detection of methicillin-resistant Staphylococcus aureus (MRSA) using the NanoLantern Biosensor

    Science.gov (United States)

    Strohsahl, Christopher M.; Miller, Benjamin L.; Krauss, Todd D.

    2009-02-01

    Staphylococcus aureus is a leading cause of human illness, and has developed the remarkable ability to resist the bactericidal capabilities of many of the world's leading antibiotics (i.e. MRSA). In an effort to enable rapid detection and treatment of MRSA infections, we have developed a DNA detection technology termed the NanoLantern(TM). The NanoLantern(TM) biosensor technology is based on the simple immobilization of a fluorophore-terminated DNA hairpin onto a gold chip. This produces a label-free sensor that allows for a positive response to be obtained without extensive processing of the sample, saving cost and increasing accuracy. We will also discuss a newly developed method of partial gene analysis, used to develop a DNA hairpin probe that is capable of detecting the presence of the mecR gene, a gene necessary for methicillin resistance to be present in S. aureus, with 100% sequence specificity. The successful incorporation of this probe into the NanoLantern(TM) platform, along with the concomitant development of the paired PCR assay has allowed for the successful detection of methicillin-resistance directly from a culture of S. aureus. These results represent an important step forward in terms of developing the ability to rapidly and effectively detect the presence of antibiotic resistance in bacterial infections.

  13. Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis

    Directory of Open Access Journals (Sweden)

    Zhang Tie

    2012-01-01

    Full Text Available To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP, four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1×102 CFU/mL and that of PCR was 1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.

  14. Electrochemical impedance immunosensor for rapid detection of stressed pathogenic Staphylococcus aureus bacteria.

    Science.gov (United States)

    Bekir, Karima; Barhoumi, Houcine; Braiek, Mohamed; Chrouda, Amani; Zine, Nadia; Abid, Nabil; Maaref, Abdelrazek; Bakhrouf, Amina; Ouada, Hafedh Ben; Jaffrezic-Renault, Nicole; Mansour, Hedi Ben

    2015-10-01

    In this work, we report the adaptation of bacteria to stress conditions that induce instability of their cultural, morphological, and enzymatic characters, on which the identification of pathogenic bacteria is based. These can raise serious issues during the characterization of bacteria. The timely detection of pathogens is also a subject of great importance. For this reason, our objective is oriented towards developing an immunosensing system for rapid detection and quantification of Staphylococcus aureus. Polyclonal anti-S. aureus are immobilized onto modified gold electrode by self-assembled molecular monolayer (SAM) method. The electrochemical performances of the developed immunosensor were evaluated by impedance spectroscopy through the monitoring of the charge transfer resistance at the modified solid/liquid interface using ferri-/ferrocyanide as redox probe. The developed immunosensor was applied to detect stressed and resuscitate bacteria. As a result, a stable and reproducible immunosensor with sensitivity of 15 kΩ/decade and a detection limit of 10 CFU/mL was obtained for the S. aureus concentrations ranging from 10(1) to 10(7) CFU/mL. A low deviation in the immunosensor response (±10 %) was signed when it is exposed to stressed and not stressed bacteria.

  15. A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

    Science.gov (United States)

    Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

    2015-07-07

    Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

  16. Detection of Antibiotic Resistant Staphylococcus aureus from Milk: A Public Health Implication.

    Science.gov (United States)

    Akindolire, Muyiwa Ajoke; Babalola, Olubukola Oluranti; Ateba, Collins Njie

    2015-08-25

    The aim of this study was to investigate the occurrence, antibiotic susceptibility profiles, and virulence genes determinants of S. aureus isolated from milk obtained from retail outlets of the North-West Province, South Africa. To achieve this, 200 samples of raw, bulk and pasteurised milk were obtained randomly from supermarkets, shops and some farms in the North-West Province between May 2012 and April 2013. S. aureus was isolated and positively identified using morphological (Gram staining), biochemical (DNase, catalase, haemolysis and rapid slide agglutination) tests, protein profile analysis (MALDI-TOF mass spectrometry) and molecular (nuc specific PCR) methods. The antimicrobial resistance profiles of the isolates were determined using the phenotypic agar diffusion method. Genes encoding enterotoxins, exfoliative toxins and collagen adhesins were also screened using PCR. Among all the samples examined, 30 of 40 raw milk samples (75%), 25 of 85 bulk milk samples (29%) and 10 of 75 pasteurised milk samples (13%) were positive for S. aureus. One hundred and fifty-six PCR-confirmed S. aureus isolates were obtained from 75 contaminated milk samples. A large proportion (60%-100%) of the isolates was resistant to penicillin G, ampicillin, oxacillin, vancomycin, teicoplanin and erythromycin. On the contrary, low level resistance (8.3%-40%) was observed for gentamicin, kanamycin and sulphamethoxazole. Methicillin resistance was detected in 59% of the multidrug resistant isolates and this was a cause for concern. However, only a small proportion (20.6%) of these isolates possessed PBP2a which codes for Methicillin resistance in S. aureus. In addition, 32.7% of isolates possessed the sec gene whereas the sea, seb sed, see, cna, eta, etb genes were not detected. The findings of this study showed that raw, bulk and pasteurised milk in the North-West Province is contaminated with toxigenic and multi-drug resistant S. aureus strains. There is a need to implement

  17. Detection of Intracellular Adhesion (ica Gene and Biofilm Formation Staphylococcus aureus Isolates from Clinical Blood Cultures

    Directory of Open Access Journals (Sweden)

    Mohsen Mirzaee

    2015-10-01

    Full Text Available Background: In fact the biofilms are composed of bacterial cells living inmulticellular structures such as tissues and organs embedded within a self-produced matrix of extracellular polymeric substance (EPS. Ability to attach and biofilm formation are the most important virulence factors Staphylococcus aureus isolates. The aims of this study were to detect intracellular adhesion (ica locus and its relation to the biofilm formation phenotype in clinical isolates of S. aureus isolated from bloodcultures.Methods: A total of 31 clinical S. aureus isolates were collected from Loghman Hospital of Tehran, Iran. In vitro biofilm formation ability was determined by microliter tissue culture plates. All clinical isolates were examined for determination the ica locus by using PCR method.Results: Twelve (38.7% of the isolates were strong biofilm producers. The results showed that 18(80.6% of the isolates carried icaD gene, whereas the prevalence of icaA, icaB and icaC were 51.6%, 45.1% and 77.4% respectively.Conclusions: S. aureus clinical isolates have different ability to form biofilm. This may be caused by the differences in the expression of biofilm related genes, genetic make-up and physiological conditions.

  18. Metabolic profiling for detection of Staphylococcus aureus infection and antibiotic resistance.

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    Henrik Antti

    Full Text Available Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA and methicillin-sensitive S. aureus (MSSA were used in vitro and for infecting mice, while natural MSSA infection was studied in humans. Samples of bacterial growth media, the blood of infected mice and of humans were analyzed with combined Gas Chromatography/Mass Spectrometry. Multivariate data analysis was used to reveal the metabolic profiles of infection and the responses to different antibiotic treatments. In vitro experiments resulted in the detection of 256 putative metabolites and mice infection experiments resulted in the detection of 474 putative metabolites. Importantly, ineffective and effective antibiotic treatments were differentiated already two hours after treatment start in both experimental systems. That is, the ineffective treatment of MRSA using cloxacillin and untreated controls produced one metabolic profile while all effective treatment combinations using cloxacillin or vancomycin for MSSA or MRSA produced another profile. For further evaluation of the concept, blood samples of humans admitted to intensive care with severe sepsis were analyzed. One hundred thirty-three putative metabolites differentiated severe MSSA sepsis (n = 6 from severe Escherichia coli sepsis (n = 10 and identified treatment responses over time. Combined analysis of human, in vitro, and mice samples identified 25 metabolites indicative of effective treatment of S. aureus sepsis. Taken together, this

  19. Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

    Directory of Open Access Journals (Sweden)

    Suat Moi Puah

    2016-02-01

    Full Text Available Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52 of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5% and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.

  20. Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis.

    Science.gov (United States)

    Goerke, C; Fluckiger, U; Steinhuber, A; Zimmerli, W; Wolz, C

    2001-06-01

    The cytotoxic alpha-toxin (encoded by hla) of Staphylococcus aureus is regulated by three loci, agr, sarA and sae, in vitro. Here, we assess the regulation of hla in a guinea pig model of device-related infection by quantifying RNAIII (the effector molecule of agr) and hla directly in exudates accumulating in infected devices without subculturing of the bacteria. LightCycler reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the transcripts. Strains RN6390 and Newman expressed considerably smaller amounts of RNAIII in the guinea pig than during in vitro growth. The residual RNAIII expression decreased during the course of infection and was negatively correlated with bacterial densities. As with RNAIII, the highest hla expression was detected in both strains early in infection. Even in strain Newman, a weak hla producer in vitro, a pronounced expression of hla was observed during infection. Likewise, four S. aureus isolates from cystic fibrosis (CF) patients expressed Q1hla despite an inactive agr during device-related infection as in the CF lung. Mutation of agr and sarA in strain Newman and RN6390 had no consequence for hla expression in vivo. In contrast, the mutation in sae resulted in severe downregulation of hla in vitro as well as in vivo. In conclusion, S. aureus seems to be provided with regulatory circuits different from those characterized in vitro to ensure alpha-toxin synthesis during infections.

  1. Performance of CHROMagar Staph aureus and CHROMagar MRSA for detection of Staphylococcus aureus in seawater and beach sand--comparison of culture, agglutination, and molecular analyses.

    Science.gov (United States)

    Goodwin, K D; Pobuda, M

    2009-11-01

    Beach seawater and sand were analyzed for Staphylococcus aureus and methicillin resistant S. aureus (MRSA) for samples collected from Avalon, and Doheny Beach, CA. Membrane filtration followed by incubation on CHROMagar Staph aureus (SCA) and CHROMagar MRSA (C-MRSA) was used to enumerate S. aureus and MRSA, respectively. Media performance was evaluated by comparing identification via colony morphology and latex agglutination tests to PCR (clfA, 16S, and mecA genes). Due to background color and crowding, picking colonies from membrane filters and streaking for isolation were sometimes necessary. The specificity of SCA and C-MRSA was improved if colony isolates were identified by the presence of a matte halo in addition to mauve color; however routine agglutination testing of isolates did not appear warranted. Using the appearance of a colony on the membrane filter in conjunction with isolate appearance, the positive % agreement, the negative % agreement, and the % positive predictive accuracy for SCA was 84%, 95%, and 99% respectively, and for C-MRSA it was 85%, 98%, and 92%, respectively. Sensitivity and specificity of SCA and C-MRSA with membrane-filtered beach samples were optimized through identification experience, control of filter volume and incubation time, and isolation of colonies needing further identification. With optimization, SCA and C-MRSA could be used for enumeration of S. aureus and MRSA from samples of beach water and sand. For the sites studied here, the frequency of detection of S. aureus ranged from 60 to 76% and 53 to 79% for samples of beach seawater and sand, respectively. The frequency of detection of MRSA ranged from 2 to 9% and 0 to 12% for samples of seawater and sand, respectively.

  2. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus.

    Science.gov (United States)

    Lian, Yan; He, Fengjiao; Wang, Huan; Tong, Feifei

    2015-03-15

    A novel aptamer/graphene interdigitated gold electrode piezoelectric sensor was developed for the rapid and specific detection of Staphylococcus aureus (S. aureus) by employing S. aureus aptamer as a biological recognition element. 4-Mercaptobenzene-diazonium tetrafluoroborate (MBDT) salt was used as a molecular cross-linking agent to chemically bind graphene to interdigital gold electrodes (IDE) that are connected to a series electrode piezoelectric quartz crystal (SPQC). S. aureus aptamers were assembly immobilized onto graphene via the π-π stacking of DNA bases. Due to the specific binding between S. aureus and aptamer, when S. aureus is present, the DNA bases interacted with the aptamer, thereby dropping the aptamer from the surface of the graphene. The electric parameters of the electrode surface was changed and resulted in the change of oscillator frequency of the SPQC. This detection was completed within 60min. The constructed sensor demonstrated a linear relationship between resonance frequency shifts with bacterial concentrations ranging from 4.1×10(1)-4.1×10(5)cfu/mL with a detection limit of 41cfu/mL. The developed strategy can detect S. aureus rapidly and specifically for clinical diagnosis and food testing.

  3. Screening and detection of heterogenous vancomycin intermediate Staphylococcus aureus in Hospital Kuala Lumpur Malaysia, using the glycopeptide resistance detection Etest and population analysis profiling.

    Science.gov (United States)

    Ramli, Siti Roszilawati; Neoh, Hui-Min; Aziz, Muhammad Nazri; Hussin, Salasawati

    2012-01-02

    In a 3-month study done in Hospital Kuala Lumpur (HKL), 7 out of 320 methicillin resistant Staphylococcus aureus isolates were confirmed as heterogeneous vancomycin intermediate S. aureus (hVISA) using the glycopeptide resistance detection e-test and population analysis, giving a prevalence rate of 2.19%. This is the first report of hVISA in Malaysia.

  4. Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

    Science.gov (United States)

    den Reijer, P Martijn; Sandker, Marjan; Snijders, Susan V; Tavakol, Mehri; Hendrickx, Antoni P A; van Wamel, Willem J B

    2017-02-01

    Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

  5. Lab on a chip sensor for rapid detection and antibiotic resistance determination of Staphylococcus aureus.

    Science.gov (United States)

    Abeyrathne, Chathurika D; Huynh, Duc H; Mcintire, Thomas W; Nguyen, Thanh C; Nasr, Babak; Zantomio, Daniela; Chana, Gursharan; Abbott, Iain; Choong, Peter; Catton, Mike; Skafidas, Efstratios

    2016-03-21

    The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.

  6. Staphylococcus aureus detection in the mouth of housekeepers Detección de Staphylococcus aureus en la boca de trabajadores de la limpieza hospitalaria Detecção de Staphylococcus aureus na boca de trabalhadores da limpeza hospitalar

    Directory of Open Access Journals (Sweden)

    Elaine Drehmer de Almeida Cruz

    2011-02-01

    Full Text Available This study assessed the prevalence of colonization by Staphylococcus aureus in hospital housekeepers, and their knowledge and beliefs regarding this problem. Three saliva samples were collected and a questionnaire regarding knowledge and beliefs was applied. Of the 92 workers, 63 (68.5% participated in the study; 20 were not and 43 were colonized; 13 by methicillin resistant Staphylococcus aureus and 30 by methicillin sensitive Staphylococcus aureus. Persistent carrier status of methicillin resistant Staphylococcus aureus was detected in 15.4% of cases. Low knowledge and perception of occupational risk were observed. The mouth was identified as an important reservoir of methicillin resistant Staphylococcus aureus. Analyzing knowledge and beliefs, as well as the state of carrier, is an important strategy to be added to educational actions for the prevention of workers' colonization.Este estudio evaluó la prevalencia de la colonización por Staphylococcus aureus en trabajadores de limpieza hospitalaria, y su conocimiento y creencias acerca de la problemática. Fueron recolectadas tres muestras de saliva y aplicado un cuestionario referente al conocimiento y creencias. De 92 trabajadores, 63 (68,5% participaron del estudio; 20 se presentaron no colonizados y 43 colonizados; 13 para Staphylococcus aureus resistente a la meticilina y 30 para Staphylococcus aureus sensibles a la meticilina. El estado de portador persistente por Staphylococcus aureus resistente a la meticilina fue detectado en 15,4% de los casos. Bajo conocimiento y percepción del riesgo ocupacional fueron observados. La boca fue identificada como importante reservatorio de Staphylococcus aureus resistente a la meticilina. Analizar el conocimiento y creencias juntamente con la investigación del estado de portador es una importante estrategia a ser agregada a las acciones educativas para la prevención de la colonización de trabajadores.Este estudo avaliou a prevalência da coloniza

  7. Detection by multiplex PCR of Staphylococcus aureus , S. intermedius and S. hyicus in artificially contaminated milk

    Directory of Open Access Journals (Sweden)

    Eliezer Avila Gandra

    2016-01-01

    Full Text Available ABSTRACT: This research aimed to detect coagulase-positive Staphylococcus (CPS directly in samples of artificially contaminated milk, using multiplex PCR (mPCR. Standard and isolated bacterial strains of S. aureus, S. epidermidis, S. hyicus, S. intermedius, Listeria monocytogenes and Escherichia coli species were used, evaluating the specificity and detection limit of mPCR, for artificially contaminated UHT milk. Primers specific for the nuc gene (NUC1-NUC2 were used for S. aureus, NUC3-NUC4 for S. hyicus and NUC5-NUC6 for S. intermedius. It was possible to detect the three target species by mPCR, directly from bovine whole milk, with adequate specificity and acceptable detention limit for identification of coagulase-positive Staphylococcus (CPS in foods. The specificity was determined by the amplification of species-specific fragments, and the detection limit was assessed by the detection thresholds obtained for the three species (103 CFU mL-1. From these results, the mPCR described, with the proposed set of primers, has the potential for use in precise identification and differentiation between CPSs in milk samples.

  8. Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

    Science.gov (United States)

    Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

    2016-01-01

    Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

  9. Staphylococcus aureus methicillin resistance detected by HPLC-MS/MS targeted metabolic profiling.

    Science.gov (United States)

    Schelli, Katie; Rutowski, Joshua; Roubidoux, Julia; Zhu, Jiangjiang

    2017-03-15

    Recently, novel bioanalytical methods, such as NMR and mass spectrometry based metabolomics approaches, have started to show promise in providing rapid, sensitive and reproducible detection of Staphylococcus aureus antibiotic resistance. Here we performed a proof-of-concept study focused on the application of HPLC-MS/MS based targeted metabolic profiling for detecting and monitoring the bacterial metabolic profile changes in response to sub-lethal levels of methicillin exposure. One hundred seventy-seven targeted metabolites from over 20 metabolic pathways were specifically screened and one hundred and thirty metabolites from in vitro bacterial tests were confidently detected from both methicillin susceptible and methicillin resistant Staphylococcus aureus (MSSA and MRSA, respectively). The metabolic profiles can be used to distinguish the isogenic pairs of MSSA strains from MRSA strains, without or with sub-lethal levels of methicillin exposure. In addition, better separation between MSSA and MRSA strains can be achieved in the latter case using principal component analysis (PCA). Metabolite data from isogenic pairs of MSSA and MRSA strains were further compared without and with sub-lethal levels of methicillin exposure, with metabolic pathway analyses additionally performed. Both analyses suggested that the metabolic activities of MSSA strains were more susceptible to the perturbation of the sub-lethal levels of methicillin exposure compared to the MRSA strains.

  10. Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

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    Šramková Zuzana

    2016-06-01

    Full Text Available Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s and staphylococcal enterotoxin-like proteins (SEl-s. Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED, new SE-s (SEH, SEI, SEl-s (SEK, SEL and tsst-1 gene (toxic shock syndrome toxin. Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

  11. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

    Science.gov (United States)

    Kaito, Chikara; Saito, Yuki; Nagano, Gentaro; Ikuo, Mariko; Omae, Yosuke; Hanada, Yuichi; Han, Xiao; Kuwahara-Arai, Kyoko; Hishinuma, Tomomi; Baba, Tadashi; Ito, Teruyo; Hiramatsu, Keiichi; Sekimizu, Kazuhisa

    2011-02-03

    The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

  12. Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

    Directory of Open Access Journals (Sweden)

    Chikara Kaito

    Full Text Available The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA contains two bidirectionally overlapping open reading frames (ORFs, the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA strain, or into the MW2 (USA400 and FRP3757 (USA300 strains, which are community-acquired MRSA (CA-MRSA strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

  13. PCR primers for the detection of staphylococcal enterotoxins K, L, and M and survey of staphylococcal enterotoxin types in Staphylococcus aureus isolates from food poisoning cases in Taiwan.

    Science.gov (United States)

    Chiang, Yu-Cheng; Chang, Li-Tung; Lin, Chia-Wei; Yang, Chi-Yea; Tsen, Hau-Yang

    2006-05-01

    Staphylococcal enterotoxins (SEs) are important causative agents in gastroenteritidis and food poisoning cases. They are serologically grouped into five major classical types, i.e., SEA, SEB, SEC, SED, and SEE. In addition, new SEs, such as SEG through SEM, have recently been identified and characterized. In an attempt to survey the distribution of classical and new SEs in organisms responsible for staphylococcal infections in Taiwan, we developed PCR primers for the genes that define the SEK, SEL, and SEM types. Bacterial strains other than sek, sel, and sem Staphylococcus aureus, including strains of other Staphylococcus species, did not generate any false-positive results when examined with these primers. The expression potential for the sek, sel, and sem types were also determined by reverse transcription-PCR. Together with the PCR primers specific for the classical SEs and other new SEs, including SEG, SEH, SEI, and SEJ, we surveyed the SE genes in S. aureus strains isolated from food poisoning cases. For 147 S. aureus isolates originating from food poisoning cases, 109 (74.1%) were positive for one or more SE genes. Of them, the major classical enterotoxin type was sea (28.6%), followed by seb (20.4%), sec (8.2%), and sed (2.0%). For the new SE types, sei (30.6%) was detected the most often, followed by sek (18.4%), sem (12.9%), and sel (8.2%). Also, 64 (43.5%) of the total bacterial strains had more than one enterotoxin gene.

  14. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

    Science.gov (United States)

    Tawil, Nancy; Sacher, Edward; Mandeville, Rosemonde; Meunier, Michel

    2012-01-01

    Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.

  15. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

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    V Javid Khojasteh

    2011-12-01

    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  16. Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem); C. van Pelt (Cindy); A. Luijendijk (Ad); H.A. Verbrugh (Henri); W.H.F. Goessens (Wil)

    1999-01-01

    textabstractThe slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a

  17. An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection.

    Science.gov (United States)

    Hao, Liling; Gu, Huajie; Duan, Nuo; Wu, Shijia; Ma, Xiaoyuan; Xia, Yu; Tao, Zui; Wang, Zhouping

    2017-03-22

    A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of Staphylococcus aureus (S. aureus) with Co(2+) enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functional flowerlike gold nanoparticles (Co(2+)/ABEI-AuNFs) as donor and WS2 nanosheet as acceptor. In the presence of S. aureus, rolling circle amplification (RCA) can be started. Partially complementary sequence of RCA product functional ABEI-AuNFs (cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product to form duplex complex. This complex is less adsorbed onto the WS2 nanosheet, thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS2 nanosheet. In the absence of target S. aureus (and hence the absence of RCA and duplex formation), the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS2 nanosheet and chemiluminescence quenching ensues. Under optimal conditions, the logarithmic correlation between the concentration of S. aureus and the CL signal was found to be linear within the range of 50 cfu/mL to 1.5 × 10(5) cfu/mL (R(2) = 0.9913). The limits of detection of the developed method were found to be 15 cfu/mL for S. aureus. The selectivity and the capability of the biosensor in meat samples were also studied. Therefore, this simple and easy operation method can be used to detect S. aureus with high sensitivity and specificity.

  18. Transcription of the phage-encoded Panton-Valentine leukocidin of Staphylococcus aureus is dependent on the phage life-cycle and on the host background.

    Science.gov (United States)

    Wirtz, Christiane; Witte, Wolfgang; Wolz, Christiane; Goerke, Christiane

    2009-11-01

    Panton-Valentine leukocidin (PVL) is a pore-forming, bi-component toxin secreted by Staphylococcus aureus strains epidemiologically associated with diseases such as necrotizing pneumonia and skin and soft-tissue infections. Here we demonstrate that transcription of the phage-encoded PVL (encoded in the luk-PV operon) is dependent on two major determinants: the phage life-cycle and the host chromosomal background. Mitomycin C induction of PVL-encoding prophages from different community-acquired MRSA strains led to an increase in the amount of luk-PV mRNA as a result of read-through transcription from latent phage promoters and an increase in phage copy numbers. Failing prophage excision was reflected in a constant expression of luk-PV as in the case of strain USA300, suggesting that phi Sa2USA300 is a replication-defective prophage. Additionally, we could show that luk-PV transcription is influenced by the S. aureus global virulence regulators agr and sae. We found a strong impact of the host background on prophage induction and replication when analysing PVL phages in different S. aureus strains. For example phage phi Sa2mw was greatly induced by mitomycin C in its native host MW2 and in strain Newman but to a considerably lesser extent in strains 8325-4, RN6390 and ISP479c. This discrepancy was not linked to the SOS response of the bacteria since recA transcription did not vary between the strains. These results suggest a fine tuning between certain phages and their host, with major impact on the expression of phage-encoded virulence genes.

  19. Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator of tricarboxylic acid cycle activity in Staphylococcus aureus.

    Science.gov (United States)

    Hartmann, Torsten; Zhang, Bo; Baronian, Grégory; Schulthess, Bettina; Homerova, Dagmar; Grubmüller, Stephanie; Kutzner, Erika; Gaupp, Rosmarie; Bertram, Ralph; Powers, Robert; Eisenreich, Wolfgang; Kormanec, Jan; Herrmann, Mathias; Molle, Virginie; Somerville, Greg A; Bischoff, Markus

    2013-12-13

    The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

  20. Molecular Detection of Methicillin-Resistant Staphylococcus aureus by Non-Protein Coding RNA-Mediated Monoplex Polymerase Chain Reaction

    Science.gov (United States)

    Soo Yean, Cheryl Yeap; Selva Raju, Kishanraj; Xavier, Rathinam; Subramaniam, Sreeramanan; Gopinath, Subash C. B.; Chinni, Suresh V.

    2016-01-01

    Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50–200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays. PMID:27367909

  1. Simultaneous Identification and Susceptibility Determination to Multiple Antibiotics of Staphylococcus aureus by Bacteriophage Amplification Detection Combined with Mass Spectrometry.

    Science.gov (United States)

    Rees, Jon C; Pierce, Carrie L; Schieltz, David M; Barr, John R

    2015-07-01

    The continued advance of antibiotic resistance in clinically relevant bacterial strains necessitates the development and refinement of assays that can rapidly and cost-effectively identify bacteria and determine their susceptibility to a panel of antibiotics. A methodology is described herein that exploits the specificity and physiology of the Staphylococci bacteriophage K to identify Staphylococcus aureus (S. aureus) and determine its susceptibility to clindamycin and cefoxitin. The method uses liquid chromatography-mass spectrometry to monitor the replication of bacteriophage after it is used to infect samples thought to contain S. aureus. Amplification of bacteriophage K indicates the sample contains S. aureus, for it is only in the presence of a suitable host that bacteriophage K can amplify. If bacteriophage amplification is detected in samples containing the antibiotics clindamycin or cefoxitin, the sample is deemed to be resistant to these antibiotics, respectively, for bacteriophage can only amplify in a viable host. Thus, with a single work flow, S. aureus can be detected in an unknown sample and susceptibility to clindamycin and cefoxitin can be ascertained. This Article discusses implications for the use of bacteriophage amplification in the clinical laboratory.

  2. Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products.

    Science.gov (United States)

    Morandi, S; Brasca, M; Lodi, R; Cremonesi, P; Castiglioni, B

    2007-09-20

    Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

  3. Sensitive detection of viral transcripts in human tumor transcriptomes.

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    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  4. Application of a Novel "Pan-Genome"-Based Strategy for Assigning RNAseq Transcript Reads to Staphylococcus aureus Strains.

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    Diego Chaves-Moreno

    Full Text Available Understanding the behaviour of opportunistic pathogens such as Staphylococcus aureus in their natural human niche holds great medical interest. With the development of sensitive molecular methods and deep-sequencing technology, it is now possible to robustly assess the global transcriptome of bacterial species in their human habitat. However, as the genomes of the colonizing strains are often not available compiling the pan-genome for the species of interest may provide an effective method to reliably and rapidly compile the transcriptome of a bacterial species. The pan-genome of S. aureus and its associated core and accessory components were compiled based on 25 genomes and comprises a total of 65,557 proteins clustering into 4,198 Orthologous Groups (OGs. The generated gene catalogue was used to assign RNAseq-derived sequence reads to S. aureus in a variety of in vitro and in vivo samples. In all cases, the number of reads that could be assigned to S. aureus was greater using the OG database than using a reference genome. Growth of two S. aureus strains in synthetic nasal medium confirmed that both strains experienced strong iron starvation. Traits such as purine metabolism appeared to be more affected in a typical nasal colonizer than in a strain representative of the S. aureus USA300 lineage. Mapping sequencing reads from a metatranscriptome generated from the human anterior nares allowed the identification of genes highly expressed by S. aureus in vivo. The OG database generated in this study represents a useful tool to obtain a snapshot of the functional attributes of S. aureus under different in vitro and in vivo conditions. The approach proved to be advantageous to assign sequencing reads to bacterial strains when RNAseq data is derived from samples where strain information and/or the corresponding genome/s are unavailable.

  5. Use of the cobas 4800 system for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Moure, Raquel; Cañizares, Ángeles; Muíño, María; Lobato, Margarita; Fernández, Ana; Rodríguez, María; Gude, Maria José; Tomás, Maria; Bou, Germán

    2016-01-01

    The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively.

  6. Real-Time Detection of Staphylococcus Aureus Using Whispering Gallery Mode Optical Microdisks

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    Hala Ghali

    2016-05-01

    Full Text Available Whispering Gallery Mode (WGM microresonators have recently been studied as a means to achieve real-time label-free detection of biological targets such as virus particles, specific DNA sequences, or proteins. Due to their high quality (Q factors, WGM resonators can be highly sensitive. A biosensor also needs to be selective, requiring proper functionalization of its surface with the appropriate ligand that will attach the biomolecule of interest. In this paper, WGM microdisks are used as biosensors for detection of Staphylococcus aureus. The microdisks are functionalized with LysK, a phage protein specific for staphylococci at the genus level. A binding event on the surface shifts the resonance peak of the microdisk resonator towards longer wavelengths. This reactive shift can be used to estimate the surface density of bacteria that bind to the surface of the resonator. The limit of detection of a microdisk with a Q-factor around 104 is on the order of 5 pg/mL, corresponding to 20 cells. No binding of Escherichia coli to the resonators is seen, supporting the specificity of the functionalization scheme.

  7. In silico detection of sequence variations modifying transcriptional regulation.

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    Malin C Andersen

    2008-01-01

    Full Text Available Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers. The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

  8. RAPID MOLECULAR DETECTION OF METHICILLIN-RESISTANT S. AUREUS IN THE INTENSIVE CARE UNIT

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective Molecular detection of methicillin-resistant S. aureus(MRSA)in the Intensive Care Unit (ICU)will be helpful for the control of transmission among patients. Methods Both mecA and femB genes of 233 patients in ICU were examined ICU by polymerase chain reaction(PCR) for the presence of MRSA. Swabs were taken from various sites, such as axilla,nose , skin lesions and throat, and incubated over night in salt broth cultures. Results One hundred and five of patients(45.06%) were positive for mecA gene, twenty six(11. 16%)were femB positive and fifteen (6%) patients were positive for MRSA, and such screening data were available within 6~ 7 h fol- lowing admission. Conclusion Specific PCR approaches is helpful for routine conventional diagnosis of MRSA, mecA/femB PCR detection offers a rapid and specific alternative for screening MRSA from patients in high-risk ar- eas.

  9. Novel platform for the detection of Staphylococcus aureus enterotoxin B in foods.

    Science.gov (United States)

    Tallent, Sandra M; Degrasse, Jeffrey A; Wang, Ningyan; Mattis, Daiva M; Kranz, David M

    2013-03-01

    Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vβ domain of the T-cell receptor (Vβ-TCR) or polyclonal antibodies. The binding affinity of the Vβ-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vβ-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vβ-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.

  10. Transcriptional profiling of the two-component regulatory system VraSR in Staphylococcus aureus with low-level vancomycin resistance.

    Science.gov (United States)

    Chen, Hongbin; Xiong, Zhujia; Liu, Kuoyue; Li, Shuguang; Wang, Ruobing; Wang, Xiaojuan; Zhang, Yawei; Wang, Hui

    2016-05-01

    The objective of this study was to comprehensively identify the target genes regulated by the two-component regulatory system VraSR in Staphylococcus aureus and to clarify the role of VraSR in low-level vancomycin resistance. Expression of vraS was determined by real-time quantitative reverse transcriptase PCR (qRT-PCR). A clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain B6D and a vancomycin-intermediate S. aureus (VISA) strain D7 that was induced from a meticillin-resistant S. aureus strain were selected to construct vraSR null mutants by allelic replacement. The vraSR-complemented strain B6D_c was also constructed by allelic replacement. Genes differentially expressed in the wild-type, vraSR null mutant and complemented strains were detected using RNA-Seq and were validated by qRT-PCR. Compared with vancomycin-susceptible S. aureus strains, expression of vraS was upregulated in all four isogenic hVISA strains. Vancomycin minimum inhibitory concentrations (MICs) in the vraSR null mutants B6D-ΔvraSR and D7-ΔvraSR were significantly lower than in the wild-type strains B6D and D7 and the complemented strain B6D_c. RNA-Seq and qRT-PCR data showed that expression of genes encoding FmtA protein, foldase protein PrsA, capsular polysaccharide biosynthesis glycosyltransferase, TcaA, a putative membrane protein, and six hypothetical proteins was down regulated in both vraSR-null mutants B6D-ΔvraSR and D7-ΔvraSR. Most of these differentially expressed proteins are involved in cell wall biosynthesis, which is associated with vancomycin resistance in S. aureus. In conclusion, VraSR plays an important role in S. aureus strains with low-level vancomycin resistance. PrsA, FmtA, glycosyltransferase and TcaA are regulated directly or indirectly by VraSR.

  11. Detection of Methicillin Resistant Staphylococcus aureus and Determination of Minimum Inhibitory Concentration of Vancomycin for Staphylococcus aureus Isolated from Pus/Wound Swab Samples of the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal

    Directory of Open Access Journals (Sweden)

    Raghabendra Adhikari

    2017-01-01

    Full Text Available The present study was conducted to evaluate the performance of cefoxitin disc diffusion method and oxacillin broth microdilution method for detection of methicillin resistant S. aureus (MRSA, taking presence of mecA gene as reference. In addition, inducible clindamycin resistance and beta-lactamase production were studied and minimum inhibitory concentration (MIC of vancomycin for S. aureus isolates was determined. A total of 711 nonrepeated pus/wound swab samples from different anatomic locations were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Gram’s stain, and biochemical tests. A total of 110 (15.47% S. aureus isolates were recovered, of which 39 (35.50% isolates were identified as MRSA by cefoxitin disc diffusion method. By oxacillin broth microdilution method, 31.82% of the Staphylococcus aureus isolates were found to be MRSA. However, mecA gene was present in only 29.1% of the isolates. Further, beta-lactamase production was observed in 71.82% of the isolates, while inducible clindamycin resistance was found in 10% of S. aureus isolates. The MIC value of vancomycin for S. aureus ranged from 0.016 μg/mL to 1 μg/mL. On the basis of the absolute sensitivity (100%, both phenotypic methods could be employed for routine diagnosis of MRSA in clinical microbiology laboratory; however cefoxitin disc diffusion could be preferred over MIC method considering time and labour factor.

  12. Detection of Methicillin Resistant Staphylococcus aureus and Determination of Minimum Inhibitory Concentration of Vancomycin for Staphylococcus aureus Isolated from Pus/Wound Swab Samples of the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal

    Science.gov (United States)

    Adhikari, Raghabendra; Neupane, Sanjeev; Neupane, Mukesh; Bhattarai, Roshan; Bhatta, Sabita; Chaudhary, Raina; Lekhak, Binod

    2017-01-01

    The present study was conducted to evaluate the performance of cefoxitin disc diffusion method and oxacillin broth microdilution method for detection of methicillin resistant S. aureus (MRSA), taking presence of mecA gene as reference. In addition, inducible clindamycin resistance and beta-lactamase production were studied and minimum inhibitory concentration (MIC) of vancomycin for S. aureus isolates was determined. A total of 711 nonrepeated pus/wound swab samples from different anatomic locations were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Gram's stain, and biochemical tests. A total of 110 (15.47%) S. aureus isolates were recovered, of which 39 (35.50%) isolates were identified as MRSA by cefoxitin disc diffusion method. By oxacillin broth microdilution method, 31.82% of the Staphylococcus aureus isolates were found to be MRSA. However, mecA gene was present in only 29.1% of the isolates. Further, beta-lactamase production was observed in 71.82% of the isolates, while inducible clindamycin resistance was found in 10% of S. aureus isolates. The MIC value of vancomycin for S. aureus ranged from 0.016 μg/mL to 1 μg/mL. On the basis of the absolute sensitivity (100%), both phenotypic methods could be employed for routine diagnosis of MRSA in clinical microbiology laboratory; however cefoxitin disc diffusion could be preferred over MIC method considering time and labour factor.

  13. Detection and enumeration of Staphylococcus aureus and Streptococcus agalactiae in milk by Real-Time PCR

    OpenAIRE

    Aline Gerato Dibbern

    2015-01-01

    O presente trabalho foi organizado em dois estudos. O objetivo do primeiro estudo foi determinar o efeito da infecção intramamária (IIM) causada por S. aureus e S. agalactiae na contagem de células somáticas (CCS) e na composição do leite (gordura, proteína, lactose, sólidos totais e extrato seco desengordurado), o efeito da contagem de S. aureus e S. agalactiae sobre a composição do leite e a estimativa da frequência de vacas e de quartos infectados com S. aureus e S. agalactiae por meio da ...

  14. Optimization of triplex real time PCR for detecting Staphylococcus aureus mecA, pvl and nuc genes.

    Science.gov (United States)

    Vremeră, Teodora; Iancu, Luminiţa Smaranda; Logigan, Cătălina; Năstase, Eduard; Miftode, Egidia; Luncă, Cătălina; Dorneanu, Olivia

    2011-01-01

    Multiplex polymerase chain reaction (PCR) allows simultaneous detection of two or more genes, using the same reaction conditions, and so it is possible the rapid detection of methicillin resistant Staphylococcus aureus strains (MRSA) in clinical specimens. This study aimed to implement, for the first time in our laboratory, a triplex real time PCR (RT-PCR) technique for detection of genes encoding resistance to oxacillin and synthesis of Panton Valentine leukocidin (pvl), a pathogenicity factor characteristic for community acquired strains (CA-MRSA). The application of this method will permit the epidemiological surveillance of circulating strains and early application of prevention measures.

  15. Co-detection of Panton-Valentine Leukocidin and cotrimoxazole resistance in Staphylococcus aureus: Implications for HIV-patients' care

    Directory of Open Access Journals (Sweden)

    Christian eKraef

    2015-02-01

    Full Text Available Patients infected with the human immunodeficiency virus (HIV are frequently exposed to antimicrobial agents. This might have an impact on the resistance profile, genetic background and virulence factors of colonizing Staphylococcus aureus. Sub-Saharan Africa is considered to be endemic for Panton-Valentine leukocidin (PVL positive S. aureus which can be associated with skin and soft tissue infections. We compared S. aureus from nasal and pharyngeal swabs from HIV patients (n=141 and healthy controls (n=206 in Gabon in 2013, and analyzed determinants of colonization with PVL positive isolates in a cross-sectional study. S. aureus isolates were screened for the presence of selected virulence factors (incl. PVL and were subjected to antimicrobial susceptibility testing and genotyping. In HIV patients, S. aureus was more frequently detected (36.9 vs. 31.6% and the isolates were more frequently PVL positive than in healthy controls (42.1 vs. 23.2%. The presence of PVL was associated with cotrimoxazole resistance (OR=25.1, p<0.001 and the use of cotrimoxazole was a risk factor for colonization with PVL positive isolates (OR=2.5, p=0.06. PVL positive isolates were associated with the multilocus sequence types ST15 (OR=5.6, p<0.001 and ST152 (OR=62.1, p<0.001.Participants colonized with PVL positive isolates reported more frequently skin and soft tissue infection (SSTI in the past compared to carriers of PVL negative isolates (OR=2.7, p=0.01. In conclusion, the novelty of our study is that cotrimoxazole might increase the risk of SSTI in regions where cotrimoxazole resistance is high and associated with PVL. This finding needs to be confirmed in prospective studies.

  16. Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398.

    Science.gov (United States)

    Gómez, Paula; Lozano, Carmen; Benito, Daniel; Estepa, Vanesa; Tenorio, Carmen; Zarazaga, Myriam; Torres, Carmen

    2016-05-01

    The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1-5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that

  17. Methicillin-resistant Staphylococcus aureus: laboratory detection methods in use in the Republic of Ireland and Northern Ireland.

    LENUS (Irish Health Repository)

    Humphreys, H

    2002-01-01

    There is no universally agreed laboratory protocol for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and hence a variety of approaches are used. As part of an all-island survey of MRSA in the Republic of Ireland (the South) and Northern Ireland (the North), a questionnaire was circulated to 14 participating laboratories in the North and 49 in the South, to determine the methods used to isolate MRSA from clinical specimens, identify S. aureus and test for susceptibility to methicillin. Almost two-thirds (64%) of laboratories in the North but only 16% of laboratories in the South use enrichment culture. There is heavy reliance on commercial kits to confirm the identification of S. aureus in the South but all laboratories in the North use the staphylocoagulase test. More than 90% of all laboratories use a disc method for susceptibility testing and 71% of laboratories in the North supplement this with the E-test; however, a range of methicillin disk concentrations are in use. There is a need to review current laboratory methods used to detect MRSA, with follow-up audit on their implementation. Additional resources may be needed in some laboratories to comply with revised guidelines, and reference facilities are required to assess new commercially available techniques and to confirm the identification of unusual or difficult strains.

  18. Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

    KAUST Repository

    Wang, Zhijuan

    2011-05-01

    Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

  19. Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Julie Gagnaire

    Full Text Available The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF mass spectrometry (MS, correlate delta-toxin expression with accessory gene regulator (agr status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively. In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

  20. Detection of mecA- and mecC-Positive Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates by the New Xpert MRSA Gen 3 PCR Assay

    OpenAIRE

    Becker, Karsten; Denis, Olivier; Roisin, Sandrine; Mellmann, Alexander; Evgeny A Idelevich; Knaack, Dennis; van Alen, Sarah; Kriegeskorte, André; Köck, Robin; Schaumburg, Frieder; Peters, Georg; Ballhausen, Britta

    2015-01-01

    An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n = 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n = 160, including 10 mecC-positive MRSA) were accurately categ...

  1. Simplified screening in an emergency department detected methicillin-resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Mogensen, Christian Backer; Kjældgaard, Poul; Jensen, Charlotte;

    2016-01-01

    INTRODUCTION: All patients admitted to Danish hospitals are screened for methicillin-resistant Staphylococcus aureus (MRSA) by a questionnaire consisting of 19 questions issued by the Danish Health and Medicines Authority (DHMA). This study aimed to evaluate which of the questions were most useful...

  2. Comparison of Chromogenic Media to BD GeneOhm Methicillin-Resistant Staphylococcus aureus (MRSA) PCR for Detection of MRSA in Nasal Swabs▿

    OpenAIRE

    Bischof, Larry J.; Lapsley, Linda; Fontecchio, Karen; Jacosalem, Dollie; Young, Carol; Hankerd, Rosemary; Newton, Duane W.

    2009-01-01

    To select a method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs, we compared BD GeneOhm MRSA PCR and various culture media (mannitol salt agar with cefoxitin, MRSASelect, CHROMagar MRSA, and Spectra MRSA). While PCR detection of MRSA was more rapid, MRSASelect and Spectra MRSA demonstrated performance equivalent to that of PCR with maximal detection at 24 h.

  3. Detecting novel low-abundant transcripts in Drosophila

    DEFF Research Database (Denmark)

    Lee, Sanggyu; Bao, Jingyue; Zhou, Guolin;

    2005-01-01

    Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244...

  4. Detection of grlA and gyrA mutations in 344 Staphylococcus aureus strains.

    Science.gov (United States)

    Wang, T; Tanaka, M; Sato, K

    1998-02-01

    Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC-->TTC or TAC (Ser-80-->Phe or Tyr) and the gyrA mutation of TCA-->TTA (Ser-84-->Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT-->CAC (His-77 [silent]), TCA-->CCA (Ser-81-->Pro), and ATA-->ATT (Ile-100 [silent]) were novel, as was the GAC-->GGC (Asp-73-->Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC > or = 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC50] = 50 microg/ml). Those with the Ser80-->Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC50 < or = 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC < or = 6.25 microg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was < or = 6.25 microg/ml.

  5. BD Phoenix and Vitek 2 detection of mecA-mediated resistance in Staphylococcus aureus with cefoxitin.

    Science.gov (United States)

    Junkins, Alan D; Lockhart, Shawn R; Heilmann, Kristopher P; Dohrn, Cassie L; Von Stein, Diana L; Winokur, Patricia L; Doern, Gary V; Richter, Sandra S

    2009-09-01

    The BD Phoenix (BD Diagnostics, Sparks, MD) and Vitek 2 (bioMérieux, Durham, NC) automated susceptibility testing systems have implemented the use of cefoxitin to enhance the detection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). To assess the impact of this change, 620 clinically significant S. aureus isolates were tested in parallel on Phoenix PMIC/ID-102 panels and Vitek 2 AST-GP66 cards. The results for oxacillin and cefoxitin generated by the automated systems were compared to those generated by two reference methods: mecA gene detection and MICs of oxacillin previously determined by broth microdilution according to CLSI guidelines. Testing of isolates with discordant results was repeated to attain a majority or consensus final result. There was 100% final agreement between the results of the two reference methods. For the 448 MRSA and 172 methicillin-susceptible S. aureus isolates tested, the rates of categorical agreement of the results obtained with the automated systems with those obtained by the reference methods were 99.8% for the Phoenix panels and 99.7% for the Vitek 2 cards. A single very major error occurred on each instrument (0.2%) with different MRSA isolates. The only major error was attributed to the Vitek 2 system overcalling oxacillin resistance. In 16 instances (9 on the Phoenix system, 7 on the Vitek 2 system), an oxacillin MIC in the susceptible range was correctly changed to resistant by the expert system on the basis of the cefoxitin result. The inclusion of cefoxitin in the Phoenix and Vitek 2 panels has optimized the detection of MRSA by both systems.

  6. Comparison between a chimeric lysin ClyH and other enzymes for extracting DNA to detect methicillin resistant Staphylococcus aureus by quantitative PCR.

    Science.gov (United States)

    Hu, Yuanyuan; Yang, Hang; Wang, Jing; Zhang, Yun; Yu, Junping; Wei, Hongping

    2016-01-01

    Extracting DNA from Staphylococcus aureus cells is important for detecting MRSA by PCR. However, S. aureus cells are known to be difficult to disrupt due to their compact cell walls. Here, we systematically studied the efficiency of a highly active lysin ClyH for extracting DNA of S. aureus in comparison with commonly used enzymes, such as lysostaphin and achromopeptidase (ACP), and its compatibility in quantitative PCR (qPCR) detection of MRSA. qPCR analysis of S. aureus specific gene femB showed that ClyH was much faster than lysostaphin, ACP and lysozyme for releasing DNA. Five minutes disruption with ClyH at room temperature was enough to release all the DNA from S. aureus. Analysis of the spiked nasal swabs by a dual qPCR assay of the β-lactam resistance mecA gene and the staphylococcal cassette chromosome (SCCmec)-open reading frame X (orfX) junction (SCCmec-orfX) after ClyH lysis showed 100% sensitivity and specificity to the commercial BD GeneOhm™ MRSA test with ACP lysis, but the lysis time was reduced from 20 min by ACP to 5 min by ClyH. Our research shows that ClyH could be a better option than the currently used enzymes for DNA extraction from S. aureus, which can provide simpler and faster PCR detection of MRSA.

  7. Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis Detecção de resistência a meticilina e produção do fator slime por Staphylococcus aureus em mastite bovina

    Directory of Open Access Journals (Sweden)

    Alper Ciftci

    2009-06-01

    Full Text Available This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7% were positive for mecA gene. Although 22 of 59 (37.2% S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas

  8. Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples.

    Science.gov (United States)

    Sheet, O H; Grabowski, N T; Klein, G; Abdulmawjood, A

    2016-10-01

    Staphylococcus (S.) aureus is one of the most important animal pathogens causing bovine mastitis. Also, it is a major human pathogen that may produce a variety of toxins which cause staphylococcal food poisoning. In the present study a LAMP assay based on gene nuc to identify S. aureus was developed and validated. The specificity of the LAMP assay was confirmed by using 70 S. aureus isolates and 21 non-S. aureus strains. The optimal temperature-time combination to amplify gene nuc successfully was 65 °C and 30 min. The analytical sensitivity of the developed LAMP assay was 0.26 pg of S. aureus DNA per reaction. The limit of detection evaluated with milk spiked with S. aureus was 9 × 10(2) CFU mL(-1). The final results of this assay were available within less than 2 h. The present study showed that the LAMP assay based on gene nuc appeared to be rapid and simple, and could also be used to identify S. aureus isolates from mastitis milk of dairy cows.

  9. Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements.

    Science.gov (United States)

    Guntupalli, Rajesh; Sorokulova, Iryna; Olsen, Eric; Globa, Ludmila; Pustovyy, Oleg; Moore, Timothy; Chin, Bryan; Barbaree, James; Vodyanoy, Vitaly

    2012-09-01

    Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.

  10. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis

    Science.gov (United States)

    de Castro Melo, Poliana; Ferreira, Luciano Menezes; Filho, Antônio Nader; Zafalon, Luiz Francisco; Vicente, Hinig Isa Godoy; de Souza, Viviane

    2013-01-01

    Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well “U” bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production. PMID:24159293

  11. Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria

    Directory of Open Access Journals (Sweden)

    Muhammad Mustapha

    2016-05-01

    Full Text Available Aim: To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan. Materials and Methods: Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR techniques. Results: Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin. Conclusion: High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05 of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates.

  12. Detection of Oxacillin Resistance in Staphylococcus aureus Isolated from the Neonatal and Pediatric Units of a Brazilian Teaching Hospital

    Directory of Open Access Journals (Sweden)

    Valéria Cataneli Pereira

    2009-01-01

    Full Text Available Objective: To determine, by phenotypic and genotypic methods, oxacillin susceptibility in Staphylococcus aureus strains isolated from pediatric and neonatal intensive care unit patients seen at the University Hospital of the Botucatu School of Medicine.Methods: A total of 100 S. aureus strains isolated from the following materials were studied: 25 blood cultures, 21 secretions, 12 catheters, 3 cannulae and one chest drain from 62 patients in the neonatal unit, and 36 blood cultures, one pleural fluid sample and one peritoneal fluid sample from 38 patients in the pediatric unit. Resistance of the S. aureus isolates to oxacillin was evaluated by the disk diffusion method with oxacillin (1 μg and cefoxitin (30 μg, agar screening test using Mueller-Hinton agar supplemented with 6 μg/ml oxacillin and 4% NaCl, and detection of the mecA gene by PCR. In addition, the isolates were tested for β-lactamase production using disks impregnated with Nitrocefin and hyperproduction of β-lactamase using amoxicillin (20 μg and clavulanic acid (10 μg disks.Results: Among the 100 S. aureus strains included in the study, 18.0% were resistant to oxacillin, with 16.1% MRSA being detected in the neonatal unit and 21.0% in the pediatric unit. The oxacillin (1 μg and cefoxitin (30 μg disk diffusion methods presented 94.4% and 100% sensitivity, respectively, and 98.8% specificity. The screening test showed 100% sensitivity and 98.8% specificity. All isolates produced β-lactamase and one of these strains was considered to be a hyperproducer.Conclusions: The 30 μg cefoxitin disk diffusion method presented the best result when compared to the 1 μg oxacillin disk. The sensitivity of the agar screening test was similar to that of the cefoxitin disk diffusion method and higher than that of the oxacillin disk diffusion method. We observed variations in the percentage of oxacillin-resistant isolates during the study period, with a decline over the last years which

  13. Rapid detection of methicillin-resistant Staphylococcus aureus directly from clinical samples: methods, effectiveness and cost considerations

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    Stürenburg, Enno

    2009-07-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA isolates is a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin resistant. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (<1–3%. Central to these ‘search and destroy’ control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients (‘MRSA surveillance’. It has also been reported that the speed with which MRSA carriage is detected has an important role to play, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2–3 day delay before the final results are available, rapid detection techniques (commonly referred to as ‘MRSA rapid tests’ using PCR methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48–72 to 2–5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity however is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out any false-positive PCR results, a culture screen must always be carried out simultaneously.The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost-effectiveness of rapid MRSA tests is still sparse and thus the issue remains

  14. Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Rees, Jon C; Barr, John R

    2017-02-01

    Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories.

  15. Laboratory evaluation of phenotypic detection methods of methicillin-resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Arunava Kali

    2014-12-01

    Full Text Available Although conventional antibiotic susceptibility tests are most commonly performed for methicillin-resistant Staphylococcus aureus (MRSA, the results of these phenotypic tests are dependent on the standardization of the culture conditions. The aim of the study was to evaluate the conventional phenotypic screening tests in comparison to the mecA gene polymerase chain reaction (PCR. One hundred and two clinical isolates of MRSA identified by the oxacillin disk diffusion were subjected to PCR for the mecA gene and by the cefoxitin disk diffusion test and culture on oxacillin screen agar, mannitol salt agar, and methicillin-resistant Staphylococcus aureus Agar (MeReSA selective medium, for MRSA. Although all 102 isolates were resistant in oxacillin and cefoxitin disk diffusion, 92 (90.1% isolates were positive for the mecA gene. The sensitivities of the mannitol salt agar, MeReSA agar, and oxacillin screen agar were 89.13, 97.82, and 98.91%, respectively. The oxacillin screen agar may be recommended for confirming methicillin resistance in the disk diffusion test in resource-poor settings, where molecular methods are not available.

  16. Indirect diagnostic tests for the detection of subclinical mastitis in dairy goats experimentally infected with Staphylococcus aureus

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    Rodolfo de Moraes Peixoto

    2016-07-01

    Full Text Available ABSTRACT: The aim of the present study was to assess two diagnostic techniques (California mastitis test (CMT and the somatic cell count (SCC that can diagnose mastitis in dairy goats. Experimental infection was conducted using 20 mammary glands, a strain of Staphylococcus aureus, an infectious dose of 1.2x108CFU mL-1 and a volume of 1mL per mammary gland. The CMT and the SCC were used to detect subclinical mastitis. Bacterial culture (BC was performed immediately after milk collection and was used as the gold standard. Four experimental time points were established (0, 24, 48 and 72 hours post-inoculation. Analysis of the ROC curve confirmed that the best combination of sensitivity and specificity were obtained with a cutoff point of 405.5, 6030.0 and 729.5x103 cells mL-1, respectively at M1, M2 and M3. Furthermore, considering the drop in sensitivity throughout the experimental time points, the use of serial bacterial cultures are recommended, particularly in herds with a high prevalence of S. aureus.

  17. Potential use of {sup 68}Ga-apo-transferrin as a PET imaging agent for detecting Staphylococcus aureus infection

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Vijay, E-mail: vijay.kumar@swahs.health.nsw.gov.a [Department of Nuclear Medicine and PET, Westmead Hospital, Westmead, Sydney (Australia); Department of Nuclear Medicine, The Children' s Hospital at Westmead, Sydney (Australia); Discipline of Imaging, Sydney Medical School, University of Sydney (Australia); Boddeti, Dilip K. [Department of Nuclear Medicine and PET, Westmead Hospital, Westmead, Sydney (Australia); Department of Nuclear Medicine, The Children' s Hospital at Westmead, Sydney (Australia); Evans, Scott G. [Department of Nuclear Medicine and PET, Westmead Hospital, Westmead, Sydney (Australia); Roesch, Frank [Institute of Nuclear Chemistry, Johannes Gutenberg University Mainz (Germany); Howman-Giles, Robert [Department of Nuclear Medicine, The Children' s Hospital at Westmead, Sydney (Australia); Discipline of Imaging, Sydney Medical School, University of Sydney (Australia)

    2011-04-15

    Introduction: {sup 67}Ga citrate has been extensively used to detect infection and inflammation since 1971. However, its clinical utility is compromised due to several limitations. The present project explored whether {sup 68}Ga-apo-transferrin ({sup 68}Ga-TF), when prepared in vitro, is a useful agent for positron emission tomography (PET) imaging of bacterial infection. Methods: An infection was induced in male Wistar rats by injecting 5x10{sup 5} CFU units of Staphyococcus aureus in the right thigh muscle. {sup 68}Ga-TF was synthesized by mixing {sup 68}GaCl{sub 3} with apo-transferrin (TF, 2 mg) in sodium carbonate (0.1 M, pH 7.0) and incubating at 40{sup o}C for 1 h. Animals were injected with 10-15 MBq of {sup 68}Ga-TF containing approximately 0.2 mg TF and imaged at different time intervals using Siemens Biograph PET-CT. Results: When {sup 68}Ga-TF were injected in the infected rats, the infection lesion was detectable within 20 min post injection. The biodistribution showed the uptake at the lesion increased with time as shown by significantly increased standard uptake values for up to 4 h post injection. There was a considerable decrease in the background activity during the same period of study, giving higher target-to-muscle ratios. Blood pool activity at 3 h post injection was insignificant. {sup 68}GaCl{sub 3} (when not conjugated to TF) did not localize at the infection lesion up to 120 min post injection. Conclusion: The preliminary results suggest that {sup 68}Ga-TF is capable of detecting S. aureus infection in the rat model, within an hour after intravenous injection.

  18. Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks.

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    Alexander Mellmann

    2006-03-01

    Full Text Available BACKGROUND: The detection of methicillin-resistant Staphylococcus aureus (MRSA usually requires the implementation of often rigorous infection-control measures. Prompt identification of an MRSA epidemic is crucial for the control of an outbreak. In this study we evaluated various early warning algorithms for the detection of an MRSA cluster. METHODS AND FINDINGS: Between 1998 and 2003, 557 non-replicate MRSA strains were collected from staff and patients admitted to a German tertiary-care university hospital. The repeat region of the S. aureus protein A (spa gene in each of these strains was sequenced. Using epidemiological and typing information for the period 1998-2002 as reference data, clusters in 2003 were determined by temporal-scan test statistics. Various early warning algorithms (frequency, clonal, and infection control professionals [ICP] alerts were tested in a prospective analysis for the year 2003. In addition, a newly implemented automated clonal alert system of the Ridom StaphType software was evaluated. A total of 549 of 557 MRSA were typeable using spa sequencing. When analyzed using scan test statistics, 42 out of 175 MRSA in 2003 formed 13 significant clusters (p < 0.05. These clusters were used as the "gold standard" to evaluate the various algorithms. Clonal alerts (spa typing and epidemiological data were 100% sensitive and 95.2% specific. Frequency (epidemiological data only and ICP alerts were 100% and 62.1% sensitive and 47.2% and 97.3% specific, respectively. The difference in specificity between clonal and ICP alerts was not significant. Both methods exhibited a positive predictive value above 80%. CONCLUSIONS: Rapid MRSA outbreak detection, based on epidemiological and spa typing data, is a suitable alternative for classical approaches and can assist in the identification of potential sources of infection.

  19. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    Science.gov (United States)

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  20. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    OpenAIRE

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A.

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  1. 金黄色葡萄球菌检测方法的进展%Progress in detection of Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    李影; 周敏娟; 陈良安

    2016-01-01

    金黄色葡萄球菌尤其是耐甲氧西林金黄色葡萄球菌的感染日益增多,对人类健康造成巨大威胁.传统检测方法存在局限性,本文从分子生物学和免疫学方法两方面对目前金黄色葡萄球菌检测方法的进展进行综述,分析比较了各类方法的优缺点.%Staphylococcus aureus,especially methicillin-resistant Staphylococcus aureus infection is increasing,which becomes a great threat to human health.There are some limitations of the traditional detection methods.We used the methods of molecular biology and immunology for detection of Staphylococcus aureus,and analyzed the advantages and disadvantages of these two new methods.

  2. Localized surface plasmon resonance-based hybrid Au-Ag nanoparticles for detection of Staphylococcus aureus enterotoxin B

    Science.gov (United States)

    Zhu, Shaoli; Du, ChunLei; Fu, Yongqi

    2009-09-01

    A triangular hybrid Au-Ag nanoparticles array was proposed for the purpose of biosensing in this paper. Constructing the hybrid nanoparticles, an Au thin film is capped on the Ag nanoparticles which are attached on glass substrate. The hybrid nanoparticles array was designed by means of finite-difference and time-domain (FDTD) algorithm-based computational numerical calculation and optimization. Sensitivity of refractive index of the hybrid nanoparticles array was obtained by the computational calculation and experimental detection. Moreover, the hybrid nanoparticles array can prevent oxidation of the pure Ag nanoparticles from atmosphere environment because the Au protective layer was deposited on top of the Ag nanoparticles so as to isolate the Ag particles from the atmosphere. We presented a novel surface covalent link method between the localized surface plasmon resonance (LSPR) effect-based biosensors with hybrid nanoparticles array and the detected target molecules. The generated surface plasmon wave from the array carries the biological interaction message into the corresponding spectra. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin was directly detected at nanogramme per milliliter level using the triangular hybrid Au-Ag nanoparticles. Hence one more option for the SEB detection is provided by this way.

  3. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model

    Science.gov (United States)

    Lemmens-den Toom, N. A.; Willemse, J.; Koning, R. A.; Demmers, J. A. A.; Dekkers, D. H. W.; Rijkers, E.; El Ghalbzouri, A.; Nibbering, P. H.; van Wamel, W.

    2016-01-01

    Background & Aim The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. Results All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Conclusion Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections. PMID:26741798

  4. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

    Directory of Open Access Journals (Sweden)

    P M den Reijer

    Full Text Available The ability of Staphylococcus aureus to successfully colonize (abiotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant S. aureus strains on Leiden human epidermal models (LEMs and polystyrene surfaces (PS using a competitive Luminex-based assay.All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E, two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1, and two other proteins (lipase and LytM were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology.Functionally diverse virulence factors of (methicillin-resistant S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

  5. A Probe-Based Method for Confirmation of Methicillin-Resistant Staphylococcus Aureus and Detection of Panton-Valentine Leukocidin and TST Virulence Genes

    Science.gov (United States)

    Srinivasan, Ashok; Bankowski, Matthew J.; Seifried, Steven E.; Jinno, Sadao; Perkins, Rosalie; Singh, Seema; Ying, Claire.; Tice, Alan D.; Kim, Wesley; Hayden, Randall T.

    2016-01-01

    Probe-based detection of mecA, lukS/F-PV (Panton-Valentine leukocidin) and tst virulence genes in 435 isolates of Staphylococcus aureus had comparable sensitivity and specificity to end point polymerase chain reaction as a reference standard. PMID:21658873

  6. Microfluidics-Based PCR for Fusion Transcript Detection.

    Science.gov (United States)

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  7. First detection of methicillin-resistant Staphylococcus aureus ST398 and Staphylococcus pseudintermedius ST68 from hospitalized equines in Spain.

    Science.gov (United States)

    Gómez-Sanz, E; Simón, C; Ortega, C; Gómez, P; Lozano, C; Zarazaga, M; Torres, C

    2014-05-01

    Eight coagulase-positive staphylococci from equines with different pathologies obtained between 2005 and 2011 were investigated. Isolates were characterized by different molecular techniques (spa-, agr-, MLST), and clonal relatedness of strains was investigated by ApaI and SmaI PFGE. Anti-microbial resistance and virulence profiles were determined. Six isolates were identified as Staphylococcus aureus, and two as Staphylococcus pseudintermedius. Of these, four isolates were methicillin-resistant S. aureus (MRSA) ST398 and one S. pseudintermedius was mecA positive and typed as ST68. One MRSA ST398 strain was isolated in 2005 and might be one of the earliest MRSA ST398 descriptions in Spain. All 5 mecA-positive strains were multidrug resistant and were isolated from hospitalized equines. Three MRSA ST398 strains carried the recently described transposon Tn559 within the chromosomal radC gene. The mecA-positive S. pseudintermedius ST68 strain was also multidrug resistant and harboured the erm(B)-Tn5405-like element. This ST68 strain presented a clear susceptible phenotype to oxacillin and cefoxitin regardless of the presence of an integral and conserved mecA gene and mecA promoter, which enhances the need for testing the presence of this gene in routine analysis to avoid treatment failures. These data reflect the extended anti-microbial resistance gene acquisition capacities of both bacterial species and evidence their pathogenic properties. The first detection of MRSA ST398 and S. pseudintermedius ST68 in horses in Spain is reported.

  8. The Prevalence of Resistance to Methicillin in Staphylococcus aureus Strains Isolated from Patients by PCR Method for Detec-tion of mecA and nuc Genes.

    OpenAIRE

    Roxana Sahebnasagh; Horieh Saderi; Parviz Owlia

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the main cause of hospital infection emerged over the last decades. Rapid detection of MRSA is important for patient care and proper usage of infection control. Detection of mecA genes (encoding resistance to methicillin and other similar antibiotics) and nuc genes (encoding staphylococcal thermostable nuclease) by PCR method is now considered for rapid identification of MRSA strain. The aim of this study was to determine the prevalence of...

  9. The Prevalence of Resistance to Methicillin in Staphylococcus aureus Strains Isolated from Patients by PCR Method for Detec-tion of mecA and nuc Genes

    OpenAIRE

    SAHEBNASAGH, Roxana; Saderi, Horieh; Owlia, Parviz

    2014-01-01

    Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is the main cause of hospital infection emerged over the last decades. Rapid detection of MRSA is important for patient care and proper usage of infection control. Detection of mecA genes (encoding resistance to methicillin and other similar antibiotics) and nuc genes (encoding staphylococcal thermostable nuclease) by PCR method is now considered for rapid identification of MRSA strain. The aim of this study was to determi...

  10. Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal

    Directory of Open Access Journals (Sweden)

    R.H. Pokhrel

    2016-05-01

    Full Text Available Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone, while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA possessed a variant of elastin binding protein gene (ebpS with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.

  11. Evaluation of a real-time PCR assay for simultaneous detection of Kingella kingae and Staphylococcus aureus from synovial fluid in suspected septic arthritis.

    Science.gov (United States)

    Haldar, Malay; Butler, Meghan; Quinn, Criziel D; Stratton, Charles W; Tang, Yi-Wei; Burnham, Carey-Ann D

    2014-07-01

    Direct plating of synovial fluid (SF) on agar-based media often fails to identify pathogens in septic arthritis (SA). We developed a PCR assay for the simultaneous detection of Kingella kingae and Staphylococcus aureus from SF to evaluate molecular detection in SF and to estimate the incidence of K. kingae in SA in North America. The assay was based on detection of the cpn60 gene of K. kingae and the spa gene of S. aureus in multiplex real-time PCR. K. kingae was identified in 50% of patients between 0 and 5 yr of age (n=6) but not in any patients >18 yr old (n=105). Direct plating of SF on agar-based media failed to detect K. kingae in all samples. The PCR assay was inferior to the culture-based method for S. aureus, detecting only 50% of culture-positive cases. Our findings suggest that K. kingae is a common pathogen in pediatric SA in North America, in agreement with previous reports from Europe. PCR-based assays for the detection of K. kingae may be considered in children with SA, especially in those with a high degree of clinical suspicion.

  12. Rapid detection of Staphylococcus aureus in dairy and meat foods by combination of capture with silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification.

    Science.gov (United States)

    Chen, Xingxing; Wu, Xiaoli; Gan, Min; Xu, Feng; He, Lihua; Yang, Dong; Xu, Hengyi; Shah, Nagendra P; Wei, Hua

    2015-03-01

    Staphylococcus aureus is one of the main pathogens in dairy and meat products; therefore, developing a highly sensitive and rapid method for its detection is necessary. In this study, a quantitative detection method for Staph. aureus was developed using silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification. First, genomic DNA was extracted from lysed bacteria using silica-coated magnetic nanoparticles and amplified using thermophilic helicase-dependent isothermal amplification. After adding the nucleic-acid dye SYBR Green I to the amplicons, the fluorescence intensity was observed using a UV lamp or recorded using a fluorescence spectrophotometer. This detection system had a detection limit of 5×10(0) cfu/mL in pure culture and milk-powder samples and 5×10(1) cfu/mL in pork samples using a UV light in less than 2h. In addition, a good linear relationship was obtained between fluorescence intensity and bacterial concentrations ranging from 10(2) to 10(4) cfu/mL under optimal conditions. Furthermore, the results from contaminated milk powder and pork samples suggested that the detection system could be used for the quantitative analysis of Staph. aureus and applied potentially to the food industry for the detection of this pathogen.

  13. Detection of EWS-FLI1 fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors by nested reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Qixing Gong; Qinhe Fan; Zhihong Zhang; Weiming Zhang

    2005-01-01

    Objective: To assess the feasibility and significance of detecting EWS-FLIlfusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors (PNETs) by nested reverse transcription polymerase chain reaction (RT-PCR).Methods: Twelve formalin-fixed and paraffin-embedded (FFPE) samples of PNET were retrieved from archive and consultation materials,together with eight cases of controlled tumor. EWS-FLI1 fusion transcripts were detected by nested RT-PCR. Home-keeping gene β-actin was used to detect the quality of mRNA. Results: β-actin mRNA was detected in 9 of the 12 tumor cases. EWS-FLI1 fusion transcripts were detected in 6 cases, among which 4 had a "type 1" fusion transcript and 2 had a "type 2" fusion transcript. None of the controlled tumor was detected the fusion gene. Conclusion: RT-PCR is a feasible method for the detection of EWS-FLI1 fusion transcripts in FFPE tissues in PNET and the result is meaningful in differential diagnosis and prognostic evaluation.

  14. Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk.

    Science.gov (United States)

    Sung, Yun Ju; Suk, Ho-Jun; Sung, Hwa Young; Li, Taihua; Poo, Haryoung; Kim, Min-Gon

    2013-05-15

    We demonstrated the new antibody/gold nanoparticle/magnetic nanoparticle nanocomposites (antibody/AuNP/MNPs) and their application in the detection of the foodborne pathogen, Staphylococcus aureus (S. aureus), in milk. The nanocomposites were synthesized by coating the MNPs with bovine serum albumin (BSA) then adsorbing the AuNPs and anti-S. aureus antibodies on their surface. Using the completed immunomagnetic nanostructures, S. aureus inoculated in the milk sample was captured and isolated from the medium using the permanent magnet. The nanoparticle-bound cells as well as the unbound cells in the supernatant were enumerated via surface plating to evaluate the target binding capacity of the nanocomposites. The capture efficiencies of the antibody/AuNP/MNPs were 96% and 78% for S. aureus in PBS and the milk sample respectively, which were significantly higher than those of the antibody-coupled MNPs without any AuNP. The captured cells were also applied to the selective filtration system to produce color signals that were used for the detection of the target pathogen. During the filtration, the cells bound to the antibody/AuNP/MNPs remained on the surface of the membrane filter while unbound nanoparticles passed through the uniform pores of the membrane. After the gold enhancement, the cells-particles complex resting on the membrane surface rendered a visible color, and the signal intensity became higher as the target cell concentration increased. The detection limits of this colorimetric sensor were 1.5×10(3) and 1.5×10(5)CFU for S. aureus in PBS and the milk sample respectively. This sensing mechanism also had the high specificity for S. aureus over the other pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The assay required only 40min to obtain the results. With the use of the appropriate antibodies, our immunomagnetic nanocomposites-based detection strategy can provide an easy, convenient, and rapid sensing method for a

  15. New method for early detection of two random amplified polymorphic DNA (RAPD groups of Staphylococcus aureus causing bovine mastitis infection in Paraná State, Brazil

    Directory of Open Access Journals (Sweden)

    Dicezar Gonçalves

    2010-04-01

    Full Text Available The aim of this work was to develop a fast and accurate molecular approach to allow early detection of two RAPD groups of S. aureus causing bovine mastitis. Seventy five S. aureus isolates from infected animals were characterized by RAPD. Genomic fragments isolated from the unique bands present in either group were cloned and sequenced. Based on the DNA sequences, specific primers were designed to allow for the simultaneous detection of either group by multiplex PCR of S. aureus DNA isolated from clinical and subclinical bovine mastitis. Results showed that these proposed primers set could be used to detect various clinical and subclinical S. aureus isolates as well as the detection of the microorganism in bulk milk. Their use as a specific method for effective and early diagnostic tool for S. aureus infection in dairy herds is suggested.Esta pesquisa objetivou o desenvolvimento de técnica rápida e eficiente para diagnosticar precocemente diferentes linhagens de S. aureus causadoras de mastite bovina. Como resultados da metodologia empregada, foram isoladas duas linhagens destas bactérias que causam diferentes tipos de mastite bovina. Os fragmentos de DNA genômico caracterizando ambas as linhagens, por meio de RAPD foram inseridos em vetor plasmidial pGEM e clonados por meio de clones T10 F1 de Escherichia coli. As seqüências obtidas permitiram desenhar iniciadores específicos para o reconhecimento de ambas as linhagens, os quais foram testados com amostras de S. aureus e com outras linhagens próximas. O diagnóstico por meios moleculares, pode ser realizado diretamente de amostras coletadas de rebanhos leiteiros assim como dos equipamentos de ordenha. A significância deste estudo consiste em um rápido e acurado método para localizar animais infectados, representando importante ferramenta no manejo do rebanho, na redução de custos com tratamentos e, rápida recuperação de rebanhos infectados.

  16. Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

    KAUST Repository

    Hill-Cawthorne, Grant A.

    2014-06-27

    Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

  17. Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Grant A Hill-Cawthorne

    Full Text Available Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec. We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA, a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4% of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

  18. Detection of classical genes and enterotoxins of Staphylococcus aureus isolated from raw milk in the south-east region of Poland

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    Korpysa-Dzirba Weronika

    2014-12-01

    Full Text Available The aim of the study was to investigate if the enterotoxigenic strains of S. aureus isolated from raw milk are able to produce staphylococcal enterotoxins (SEs A - E. A total of 168 of S. aureus isolates from raw milk collected in the south - east region of Poland (Lubelskie Province were tested for SE production by the ELFA, while multiplex PCR was applied for detection of enterotoxin genes (sea, seb, sec, sed, see. It was found that 20 (11.9% out of 168 strains were positive for one or more classical SE markers and 19 of them produced a detectable level of enterotoxins. The results obtained by mPCR and ELFA were in agreement, when the presence of A, B, and D toxin types was tested; whereas SEC was not found by the ELFA although the S. aureus was positive for the respective gene. The results of the two methods showed that mPCR identified one more strain potentially producing enterotoxin than the ELFA, which may suggest that the enterotoxigenic S. aureus are not always able to express the toxin protein.

  19. Detection of All Species of the Genus Alphavirus by Reverse Transcription-PCR with Diagnostic Sensitivity▿

    OpenAIRE

    Grywna, K.; Kupfer, B.; Panning, M.; Drexler, J. F.; Emmerich, P.; Drosten, C.; Kummerer, B. M.

    2010-01-01

    Clinical arbovirus screening requires exclusion of a broad range of viruses with as few assays as possible. We present a reverse transcription-PCR (RT-PCR) for the detection of all species of the genus Alphavirus qualified for exclusion screening (limit of detection [LOD], 5 to 100 RNA copies per reaction across all Alphavirus species; detection of viremia down to ca. 10,000 copies per ml).

  20. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

  1. Development of a near-real-time procedure to detect Staphylococcus aureus enterotoxin A in military rations

    Science.gov (United States)

    Richardson, Michelle J.; Rand, Arthur G.; Senecal, Andre G.

    2002-02-01

    Using a chemiluminescent fiber optic biosensor and magnetic particles, a simple, sensitive and rapid method to determine Staphylococcus aureus enterotoxin A (SEA) in military ration components was developed. Anti-staphylococcal enterotoxin A (Anti-SEA) was immobilized on magnetic particles and incubated with SEA. The beads were then collected and rinsed on a membrane filter (0.45um). The captured toxin was then selectively labeled with a monoclonal-horseradish peroxidase (POD) conjugate. SEA concentration was detected with a luminometer and a chemiluminescent enhancing reagent. Total assay time was 1.25 hours. Chemiluminescent signal due to nonspecific binding was tested with various blocking agents. Phosphate buffered saline with casein had the lowest background signal. Primary antibody concentration, secondary labeled antibody concentration and chemiluminescent substrate type were also evaluated to optimize signal intensity. The chemiluminescent fiber optic biosensor assay was compared to the Analyte 2000, a commercial fluorescent fiber optic biosensor. This assay consisted of immobilizing Anti-SEA on polystyrene waveguides, and incubating the waveguides with the toxin. The waveguide was incubated with a selectively labeled monoclonal-CY5 Dye conjugate. The sensitivity of chemiluminescent and fluorescent immunoassays were 1 ng, significantly lower than the levels needed to cause illness.

  2. In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

    Directory of Open Access Journals (Sweden)

    Ka L. Hong

    2015-01-01

    Full Text Available Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

  3. Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis

    Science.gov (United States)

    Andrews, Laura M.; Jones, Mark R.; Digman, Michelle A.; Gratton, Enrico

    2013-03-01

    Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

  4. Noninvasive Detection of TMPRSS2:ERG Fusion Transcripts in the Urine of Men with Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Bharathi Laxman

    2006-10-01

    Full Text Available We recently reported the identification of recurrent gene fusions in the majority of prostate cancers involving the 5V untranslated region of the androgenregulated gene TMPRSS2, the ETS family members ERG, ETV1, ETV4. Here we report the noninvasive detection of these gene fusions in the urine of patients with clinically localized prostate cancer. By quantitative polymerase chain reaction, we assessed the expression of ERG, TMPRSS2:ERG transcripts in urine samples obtained after prostatic massage from 19 patients (11 prebiopsy, 8 pre-radical prostatectomy with prostate cancer. We observed a strong concordance between ERG overexpression, TMPRSS2:ERG expression, with 8 of 19 (42% patients having detectable TMPRSS2:ERG transcripts in their urine. Importantly, by fluorescence in situ hybridization, we confirmed the presence or the absence of TMPRSS2:ERG gene fusions in matched prostate cancer tissue samples from three of three patients with fusion transcripts in their urine, from two of two patients without fusion transcripts in their urine. These results demonstrate that TMPRSS2:ERG gene fusions can be detected in the urine of patients with prostate cancer, support larger studies on prospective cohorts for noninvasive detection of prostate cancer.

  5. Ikaros family transcription factors expression in rat thymus: detection of impaired development.

    Science.gov (United States)

    Paradzik, M; Novak, S; Mokrovic, G; Bordukalo Niksic, T; Heckel, D; Stipic, J; Pavicic Baldani, D; Cicin-Sain, L; Antica, M

    2012-01-01

    The expression of Ikaros family transcription factors and consequently their signalling pathway is limiting for hematopoietic and lymphocyte development in mice and human. Due to their importance, these transcription factors are highly homologous between species. As an initial approach to examining the possible involvement of Ikaros transcription factors in pathogenesis of rat lymphoid development, we analyzed the expression of all known Ikaros family members, Ikaros, Aiolos, Helios, Eos and Pegasus in the rat thymus. We established a semi-quantitative RT-PCR to detect mRNA of each transcription factor. For the first time we give evidence of the expression of Ikaros family transcription factors in the rat thymus. Further, we evaluated whether their mRNA expression was succumbed to changes when the rats were exposed to ethanol, as a known debilitating agent during development. Therefore we analyzed the thymus of adult rats whose mothers were forced to drink ethanol during gestation, to detect possible changes in thymus mRNA expression levels of Ikaros, Aiolos, Helios, Eos and Pegasus. We found that rats prenatally exposed to ethanol show a slightly higher expression of Ikaros family transcription factors in the adult thymus when compared to control rats, but these differences were not statistically significant. We further studied the distribution of the major lymphocyte subpopulations in the rat thymus according to CD3, CD4 and CD8 expression by four color flow cytometry. We found a higher incidence of CD3 positive cells in the double positive, CD4+CD8+ thymic subpopulation of rats prenatally exposed to ethanol when compared to non-exposed animals. Our findings indicate that ethanol exposure of pregnant rats might influence the development of CD3 positive cells in the thymus of the offspring but this result should be further tackled at the level of transcription factor expression.

  6. Detection of mecC-positive Staphylococcus aureus (CC130-MRSA-XI in diseased European hedgehogs (Erinaceus europaeus in Sweden.

    Directory of Open Access Journals (Sweden)

    Stefan Monecke

    Full Text Available Recently, a novel mec gene conferring beta-lactam resistance in Staphylococcus aureus has been discovered. This gene, mecC, is situated on a SCCmec XI element that has to date been identified in clonal complexes 49, 130, 425, 599 and 1943. Some of the currently known isolates have been identified from animals. This, and observations of mecA alleles that do not confer beta-lactam resistance, indicate that mec genes might have a reservoir in Staphylococcus species from animals. Thus it is important also to screen wildlife isolates for mec genes. Here, we describe mecC-positive Staphylococcus aureus (ST130-MRSA-XI and the lesions related to the infection in two diseased free-ranging European hedgehogs (Erinaceus europaeus. One was found dead in 2003 in central Sweden, and suffered from S. aureus septicaemia. The other one, found on the island of Gotland in the Baltic Sea in 2011, showed a severe dermatitis and was euthanised. ST130-MRSA-XI isolates were isolated from lesions from both hedgehogs and were essentially identical to previously described isolates from humans. Both isolates carried the complete SCCmec XI element. They lacked the lukF-PV/lukS-PV and lukM/lukF-P83 genes, but harboured a gene for an exfoliative toxin homologue previously described from Staphylococcus hyicus, Staphylococcus pseudintermedius and other S. aureus of the CC130 lineage. To the best of our knowledge, these are the first reported cases of CC130-MRSA-XI in hedgehogs. Given that one of the samples was taken as early as 2003, this was the earliest detection of this strain and of mecC in Sweden. This and several other recent observations suggest that CC130 might be a zoonotic lineage of S. aureus and that SCCmec XI/mecC may have originated from animal pathogens.

  7. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

    Science.gov (United States)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi-Pol, Magdalena M; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-09-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

  8. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

    NARCIS (Netherlands)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi - Pol, Magda; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-01-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but r

  9. Detection of the intercellular adhesion gene cluster (ica in clinical Staphylococcus aureus isolates

    Directory of Open Access Journals (Sweden)

    Namvar, Amirmorteza Ebrahimzadeh

    2013-04-01

    Full Text Available [english] is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in men. The ability of microorganisms to produce biofilm facilitates them to withstand the host immune response and is recognized as one factor contributing to chronic or persistent infections. It was demonstrated that the -encoded genes lead to the biosynthesis of polysaccharide intercellular adhesion (PIA molecules, and may be involved in the accumulation phase of biofilm formation. Different studies have shown the decisive role of the gene as virulence factors in staphylococcal infections. This study was carried out to demonstrate the relationship between gene and production of slime layer in strains. Sixty strains were isolated from patients. The isolates were identified morphologically and biochemically following standard laboratory methods. After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB, to which 15% glycerol was added, and stored at –20°C. Slime formation and biofilm assay was monitored. A PCR assay was developed to identify the presence of (intercellular adhesion gene gene in all isolates. Thirty-nine slime producing colonies with CRA plates (65% formed black colors, the remaining 21 isolates were pink (35%. In the quantitative biofilm assay 35 (58% produced biofilm while 25 (42% isolates did not exhibit this property. All isolates were positive for detection of gene by PCR method. The interaction of and in the investigated isolates may be important in slime layer formation and biofilm phenomena.We propose PCR detection of the gene locus as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, with implications for therapeutic and preventive measures pertainin to the management of colonized indwelling catheters.

  10. Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus

    Science.gov (United States)

    Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

    2017-01-01

    The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers. PMID:28239371

  11. Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health.

    Science.gov (United States)

    Colombo, Andrea V; Barbosa, Graziela M; Higashi, Daniela; di Micheli, Giorgio; Rodrigues, Paulo H; Simionato, Maria Regina L

    2013-10-01

    Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33% of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15% of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6% of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57% and 50% of patients, respectively, with probing depth and

  12. Evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test for detection of MRSA nasal colonization.

    Science.gov (United States)

    Yam, W C; Siu, Gilman K H; Ho, P L; Ng, T K; Que, T L; Yip, K T; Fok, Cathie P K; Chen, Jonathan H K; Cheng, Vincent C C; Yuen, K Y

    2013-09-01

    Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization is crucial for the prevention and control of MRSA infections in health care settings. The LightCycler MRSA Advanced Test (Roche Diagnostics) is a commercially available real-time PCR assay for direct detection of MRSA nasal colonization by targeting of the staphylococcal cassette chromosome mec (SCCmec)-orfX junction. The diagnostic performance of the assay was compared with that of ChromID MRSA agar (bioMérieux) culture and an in-house duplex real-time PCR assay. Among 1,246 nasal swab specimens collected from 2 general hospitals in Hong Kong, 174 (14%) were considered true positive for MRSA. Chromogenic culture and the in-house real-time PCR assay identified 147 (84.5%) and 133 (76.4%) true-positive cases with specificities of 100% and 98.6%, respectively. Based on the target melting temperature (Tm) values (57.0 to 62.0 °C) defined by the manufacturer, the LightCycler MRSA Advanced Test identified only 85 (48.9%) true-positive specimens. Interestingly, an additional 60 (34.5%) true-positive specimens were detected despite atypical Tm values of 55 °C, providing overall sensitivity and specificity values of 83.3% and 99%, respectively. Among isolates with Tm values of 55 °C, most were typed as clonal complex 45 (CC45). By sequence analysis of the SCCmec-orfX junction, characteristic single-nucleotide polymorphisms (SNPs) were identified only in isolates with Tm values of 55°C and not in those with typical Tm values. It is conceivable that those SNPs were located inside the target region of the proprietary hybridization probes, which resulted in a Tm shift in the melting curve analysis. Our study highlights the importance of a global evaluation of commercial kits so that the interpretation algorithm covers different lineages of MRSA clones prevalent in various geographical regions.

  13. Methicillin-Resistant Staphylococcus aureus (MRSA Detection from the Hands of Jatinangor Community Health Center’s Health Care Providers

    Directory of Open Access Journals (Sweden)

    Jeevanisha Patmanathan

    2015-06-01

    Full Text Available Background: Methicillin-resistant Staphylococcus aureus is a bacterium that is resistance towards β-lactam antibiotics, and it seems to be one of the leading causes of nosocomial infections. Hands of the health care workers are said to be the main source for the nosocomial transmission. Thus, the study aims to determine methicillin-resistant S. aureus from the hands of Jatinangor Community Health Center’s health care workers. Methods: Samples were taken from the hands of 30 Jatinangor Community Health Center’s staffs, including medical and paramedical; from October 2012 to November 2012. Then, these samples underwent further laboratory examinations, starting from culture, identification and susceptibility test towards cefoxitin, in identifying methicillin-resistant S. aureus. Results: Out of the 30 samples taken, 6 samples (20% were positive for S. aureus isolates. In which, 4 (13.33% of the samples were positive for methicillin-resistant S. aureus. Conclusions: Since, health care workers are the main people in contact with patients and maintaining proper hand hygiene makes a huge difference; hand hygiene should be given adequate attention for the benefit of all.

  14. The Prevalence of Resistance to Methicillin in Staphylococcus aureus Strains Isolated from Patients by PCR Method for Detec-tion of mecA and nuc Genes.

    Directory of Open Access Journals (Sweden)

    Roxana Sahebnasagh

    2014-01-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA is the main cause of hospital infection emerged over the last decades. Rapid detection of MRSA is important for patient care and proper usage of infection control. Detection of mecA genes (encoding resistance to methicillin and other similar antibiotics and nuc genes (encoding staphylococcal thermostable nuclease by PCR method is now considered for rapid identification of MRSA strain. The aim of this study was to determine the prevalence of MRSA isolated from patients in Tehran, Iran by PCR method for detection of mecA and nuc genes.Phenotypic method such as microscopic and colony morphology and catalase and coagulase tests were used for identification of S. aureus isolates. DNA was extracted from all isolates and the presence of nuc and mecA gene was detected by PCR method. For determination of MRSA by phenotypic methods, oxacillin disk diffusion test were used. Data were analyzed by SPSS software.Out of 126 clinical sample identified by phenotypic method, 101 isolates had nuc gene. In disk diffusion tests by oxacillin disk, 78.2% of isolates were considered to be MRSA, but in PCR method for mecA gene, 69% isolates were positive.The results showed a high prevalence of methicillin-resistance among S. aureus isolates. Identifying MRSA strains, isolating MRSA-positive patients and carrier's treatment in a hospital to prevent MRSA infection is important in limiting the spread of MRSA. The PCR method for detection of nuc and mecA genes has potential for rapid and accurate diagnosis of MRSA strains.

  15. IN SITU BY BRIDIZATION DETECTING L- FORM DNA OF STAPHYLOCOCCUS AUREUS OF LARYNGOCARCINOMA%原位杂交检测喉癌中金葡菌L型DNA

    Institute of Scientific and Technical Information of China (English)

    徐淑秀; 汪万英; 姚敏

    2000-01-01

    目的 探讨喉癌细胞中是否有金葡菌L型DNA原位杂交阳性表达。方法 应用原位核酸杂交技术检测20例喉癌细胞核内金葡菌L型DNA的表达。结果 60%癌细胞核、浆内显示金葡茵L型DNA阳性信号。免疫组化染色(S-P法)金葡菌L型抗原阳性率为75%。革兰氏染色金葡菌L型阳性率为70%。结论 金葡菌L型DNA已进入喉癌细胞内,极有可能与宿主细胞DNA整合,从基因水平上影响细胞增生和癌变,提示金葡菌及其L型感染与喉癌发生和发展可能有密切的关系[6]。%Aim To study whetger there is positive express in situ hybridization of L-form DNA or Staphylococcus aureus in laryngocarcinoma cell. Methods 20 cases of expressnon of L-form DNA of Staphylococcus aureus in laryngocarcinoma karyon were detected with nucleic acid in situ hybridization. Results 60% cancer karyon, cytoplasm display positive signal of Staphylococcus aureus L-form DNA. Positive rate of Staphylococcus aureus L-form antigen is 75% with immunohischemical (s-p) stainipg. The rate is 70% with gram staining. Conclusion Staphylococcus aureus L-form DNA had get into laryngocarcinoma ceells, is great possibly to integrate into host ce11, and influence hyperplasis and canceration of cell from gene level. Staphylococcus aureus and its L-form infection have possibly close relation to happen and development of laryngocarcinoma.

  16. Detection of Human Picornaviruses by Multiplex Reverse Transcription-PCR and Liquid Hybridization

    OpenAIRE

    Jokela, Pia; Joki-Korpela, Päivi; Maaronen, Marita; Glumoff, Virpi; Hyypiä, Timo

    2005-01-01

    A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5′ untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with g...

  17. A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus (Colorado strain).

    Science.gov (United States)

    Ali, A; Reynolds, D L

    1999-01-01

    A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

  18. An international, prospective, multicenter evaluation of the combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for detection of methicillin-resistant Staphylococcus aureus isolates from positive blood cultures.

    Science.gov (United States)

    Salimnia, H; Fairfax, M R; Lephart, P; Morgan, M; Gilbreath, J J; Butler-Wu, S M; Templeton, K E; Hamilton, F J; Wu, F; Buckner, R; Fuller, D; Davis, T E; Abdelhamed, A M; Jacobs, M R; Miller, A; Pfrommer, B; Carroll, K C

    2014-11-01

    Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.

  19. Genotypic and phenotypic detection of capsular polysaccharides in Staphylococcus aureus isolated from bovine intramammary infections in Argentina

    Directory of Open Access Journals (Sweden)

    C. Camussone

    2012-09-01

    Full Text Available Staphylococcus aureus (n=157 isolated from intramammary infections in Argentine dairy areas were evaluated for presence of cap5 and cap8 loci. Isolates carrying cap5 and cap8 were serotyped using specific antisera. Sixty four percent of the isolates were genotyped as cap5 or cap8 and 50% of them expressed CP5 or 8.

  20. Detection of alpha-toxin and other virulence factors in biofilms of staphylococcus aureus on polystyrene and a human epidermalmodel

    NARCIS (Netherlands)

    P.M. Den Reijer (P. Martijn); J.A. Haisma (Janneke); N. Lemmens-den Toom; J. Willemse (José); R.A. Koning; J.A.A. Demmers (Jeroen); D.H. Dekkers (Dick); E.J. Rijkers; A. El Ghalbzouri (Abdoelwaheb); P.H. Nibbering (Peter); W.J.B. van Wamel (Willem)

    2016-01-01

    textabstractBackground & Aim: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as al

  1. Label-free detection of Staphylococcus aureus in skin using real-time potentiometric biosensors based on carbon nanotubes and aptamers.

    Science.gov (United States)

    Zelada-Guillén, Gustavo A; Sebastián-Avila, José Luis; Blondeau, Pascal; Riu, Jordi; Rius, F Xavier

    2012-01-15

    In this paper we report the first biosensor that is able to detect Staphylococcus aureus in real-time. A network of single-walled carbon nanotubes (SWCNTs) acts as an ion-to-electron potentiometric transducer and anti-S. aureus aptamers are the recognition element. Carbon nanotubes were functionalized with aptamers using two different approaches: (1) non-covalent adsorption of drop-casted pyrenil-modified aptamers onto the external walls of the SWCNTs; and (2) covalent bond formation between amine-modified aptamers and carboxylic groups previously introduced by oxidation at the ends of the SWCNTs. Both of these approaches yielded functional biosensors but there were large differences in the minimum detectable bacteria concentration and sensitivity values. With covalent functionalization, the minimum concentration detected was 8×10(2)colony-forming units (CFU)/mL and the sensitivity was 0.36 mV/Decade. With the non-covalent approach, the sensitivity was higher (1.52 mV/Decade) but the minimum concentration detected was greatly affected (10(7) CFU/mL). In both cases, potential as a function of Decade of bacteria concentration was linear. Functional biosensors were used to test real samples from freshly excised pig skin, contaminated with the target microorganism, as a surrogate for human skin.

  2. Detection of E6/E7 HPV oncogene transcripts as biomarker of cervical intaepithelial displasia

    Directory of Open Access Journals (Sweden)

    Mauro Carcheri

    2009-09-01

    Full Text Available It is widely accepted that only persistent infection with high risk types of Human Papillomavirus (HPV HR is a significant risk factor for the development of an invasive squamous cervical cancer. The overexpression of viral oncogenes E6/E7 of HPV is considered a necessary process for incurring in a malignant phenotype.A HPV infection can be identified by detection of HPV DNA in biological samples, but the DNAbased tests cannot delineate between transient or persistent and potentially transforming infection. Instead there is many evidence to suggest that detection of HPV gene expression may constitute a more specific approach to highlight a clinically significant infection. Especially seems that the detection of E6/E7 transcripts can be usefully used for identify the women with a persistent HPV infection that will can induce a future cervical cancer. The aim of our study is to investigate if the detection of oncogenic viral gene activity by detecting transcripts of the E6 and E7 genes can be most usefull of HPV-DNA test in the triage of ASCUS or low grade cervical lesions. Our results confirm that HPV E6/E7 mRNA test can be considered a promising method to stratify HPV positive women for risk of future high-grade cervical lesions or cervical intaepithelial neoplasia.

  3. Clonal dynamics of nasal Staphylococcus aureus and Staphylococcus pseudintermedius in dog-owning household members. Detection of MSSA ST(398.

    Directory of Open Access Journals (Sweden)

    Elena Gómez-Sanz

    Full Text Available The objective of this study was to investigate the dynamics of nasal carriage by Staphylococcus aureus (SA and Staphylococcus pseudintermedius (SP among healthy dog-owning household members involved in 7 previous index cases of suspected anthropozoonotic (n = 4 and zoonotic (n = 3 interspecies transmission [4 direct cases, identical SA (n = 3 or SP (n = 1 in owner and dog; three indirect, SP in owner (n = 2 or SA in dog (n = 1]. Co-carriage with methicillin-resistant coagulase-negative staphylococci (MRCoNS was also evaluated. Sixteen owners and 10 dogs were sampled once every three months for one year. In total, 50 SA and 31 SP were analysed by MLST, and SA also by spa typing. All isolates were subjected to ApaI/SmaI-PFGE and antimicrobial resistance and virulence profiles were determined. All index owners were persistent SA carriers in all direct-anthropozoonotic transmission cases, while only one dog was persistent SA carrier. Owner and dog exhibited a persistent SP carriage status in the direct-zoonotic transmission case. SP was maintained in the index human over time in one indirect-zoonotic transmission case. Only one SP was methicillin-resistant. SA belonged to genetic backgrounds of MRSA pandemic clones: CC45, CC121, CC30, CC5 and CC398. Three individuals carried a MSSA t1451-ST398 clone with the erm(T-cadD/cadX resistance genes. SA or SP were persistently detected in the nasal cavity of 7 (43.8% and 2 (12.5% owners, and in one and 2 dogs, respectively. SA was recovered as the single species in 10 owners and in one dog; SP in 3 owners and 4 dogs; and both bacterial species in one owner and 4 dogs. Co-carriage of SA or SP with MRCoNS isolates was common (30.7%. This is the first study on the dynamics of nasal carriage of SA and SP in healthy pet-owning household members. Dog-contact may play a role in the staphylococcal species distribution of in-contact individuals.

  4. Clonal dynamics of nasal Staphylococcus aureus and Staphylococcus pseudintermedius in dog-owning household members. Detection of MSSA ST(398).

    Science.gov (United States)

    Gómez-Sanz, Elena; Torres, Carmen; Ceballos, Sara; Lozano, Carmen; Zarazaga, Myriam

    2013-01-01

    The objective of this study was to investigate the dynamics of nasal carriage by Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP) among healthy dog-owning household members involved in 7 previous index cases of suspected anthropozoonotic (n = 4) and zoonotic (n = 3) interspecies transmission [4 direct cases, identical SA (n = 3) or SP (n = 1) in owner and dog; three indirect, SP in owner (n = 2) or SA in dog (n = 1)]. Co-carriage with methicillin-resistant coagulase-negative staphylococci (MRCoNS) was also evaluated. Sixteen owners and 10 dogs were sampled once every three months for one year. In total, 50 SA and 31 SP were analysed by MLST, and SA also by spa typing. All isolates were subjected to ApaI/SmaI-PFGE and antimicrobial resistance and virulence profiles were determined. All index owners were persistent SA carriers in all direct-anthropozoonotic transmission cases, while only one dog was persistent SA carrier. Owner and dog exhibited a persistent SP carriage status in the direct-zoonotic transmission case. SP was maintained in the index human over time in one indirect-zoonotic transmission case. Only one SP was methicillin-resistant. SA belonged to genetic backgrounds of MRSA pandemic clones: CC45, CC121, CC30, CC5 and CC398. Three individuals carried a MSSA t1451-ST398 clone with the erm(T)-cadD/cadX resistance genes. SA or SP were persistently detected in the nasal cavity of 7 (43.8%) and 2 (12.5%) owners, and in one and 2 dogs, respectively. SA was recovered as the single species in 10 owners and in one dog; SP in 3 owners and 4 dogs; and both bacterial species in one owner and 4 dogs. Co-carriage of SA or SP with MRCoNS isolates was common (30.7%). This is the first study on the dynamics of nasal carriage of SA and SP in healthy pet-owning household members. Dog-contact may play a role in the staphylococcal species distribution of in-contact individuals.

  5. Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

    Science.gov (United States)

    Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

    2015-07-01

    A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

  6. Comparison of air samples, nasal swabs, ear-skin swabs and environmental dust samples for detection of Methicillin Resistant Staphylococcus aureus (MRSA) in pig herds

    DEFF Research Database (Denmark)

    Agersø, Yvonne; Vigre, Håkan; Cavaco, Lina;

    2014-01-01

    To identify a cost-effective and practical method for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds, the relative sensitivity of four sample types: nasal swabs, ear-skin (skin behind the ears) swabs, environmental dust swabs and air was compared. Moreover, dependency...... detection by air sampling is easy to perform, reduces costs and analytical time compared to existing methods, and is recommended for initial testing of herds. Ear-skin swab sampling may be more sensitive for MRSA detection than air sampling or nasal swab sampling.......-herd prevalence ⩾25%]. The results indicate that taking swabs of skin behind the ears (ten pools of five) was even more sensitive than taking nasal swabs (ten pools of five) at the herd level and detected significantly more positive samples. spa types t011, t034 and t4208 were observed. In conclusion, MRSA...

  7. Complementary Post Transcriptional Regulatory Information is Detected by PUNCH-P and Ribosome Profiling

    Science.gov (United States)

    Zur, Hadas; Aviner, Ranen; Tuller, Tamir

    2016-01-01

    Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time. Ribo-Seq is based on sequencing all the ribosome protected mRNA fragments in a cell, while PUNCH-P is based on mass-spectrometric analysis of only newly synthesized proteins. Here we describe the first Ribo-Seq/PUNCH-P comparison via the analysis of mammalian cells during the cell-cycle for detecting relevant differentially expressed genes between G1 and M phase. Our analyses suggest that the two approaches significantly overlap with each other. However, we demonstrate that there are biologically meaningful proteins/genes that can be detected to be post-transcriptionally regulated during the mammalian cell cycle only by each of the approaches, or their consolidation. Such gene sets are enriched with proteins known to be related to intra-cellular signalling pathways such as central cell cycle processes, central gene expression regulation processes, processes related to chromosome segregation, DNA damage, and replication, that are post-transcriptionally regulated during the mammalian cell cycle. Moreover, we show that combining the approaches better predicts steady state changes in protein abundance. The results reported here support the conjecture that for gaining a full post-transcriptional regulation picture one should integrate the two approaches. PMID:26898226

  8. A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis

    Directory of Open Access Journals (Sweden)

    Ribeiro Franclim R

    2009-01-01

    Full Text Available Abstract Background The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings. Results We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as TCF3:PBX1, ETV6:RUNX1, and TMPRSS2:ERG from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies. Conclusion This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.

  9. A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection.

    Science.gov (United States)

    Li, Chunxiang; Qiu, Xiyang; Hou, Zhaohui; Deng, Keqin

    2015-02-15

    Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, quantitative, and inexpensive detection of TF. The strategy consists of a hairpin DNA probe containing a TF binding sequence for target TF, a dumbbell-shaped probe, a primer DNA probe designed partly complementary to hairpin DNA probe, and a dumbbell probe. In the presence of target TF, the binding of the TF with hairpin DNA probe will prohibit the hybridization of the primer DNA probe with the "stem" and "loop" region of the hairpin DNA probe, then the unhybridized region of the primer DNA will hybridize with dumbbell probe, subsequently promote the ligation reaction and the rolling circle amplification (RCA), finally, the RCA products are quantified via the fluorescent intensity of SYBR Green I (SG). Using TATA-binding protein (TBP) as a model transcription factor, the proposed assay system can specifically detect TBP with a detection limit as low as 40.7 fM, and with a linear range from 100 fM to 1 nM. Moreover, this assay related DNA probe does not involve any modification and the whole assay proceeds in one tube, which makes the assay simple and low cost. It is expected to become a powerful tool for bioanalysis and clinic diagnostic application.

  10. An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

    CERN Document Server

    Wollman, Adam J M; Foster, Simon; Leake, Mark C

    2016-01-01

    Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphological...

  11. Detection of Airborne Methicillin-Resistant Staphylococcus aureus Inside and Downwind of a Swine Building, and in Animal Feed: Potential Occupational, Animal Health, and Environmental Implications.

    Science.gov (United States)

    Ferguson, Dwight D; Smith, Tara C; Hanson, Blake M; Wardyn, Shylo E; Donham, Kelley J

    2016-01-01

    Aerosolized methicillin-resistant Staphylococcus aureus (MRSA) was sampled inside and downwind of a swine facility. Animal feed was sampled before and after entry into the swine facility. Aerosolized particles were detected using an optical particle counter for real-time measurement and with an Andersen sampler to detect viable MRSA. Molecular typing and antimicrobial susceptibility testing were performed on samples collected. Viable MRSA organisms isolated inside the swine facility were primarily associated with particles >5 µm, and those isolated downwind from the swine facility were associated with particles airborne MRSA organisms. Air samples collected after power washing with a biocide inside the swine facility resulted in no viable MRSA organisms detected. This pilot study showed that the ecology of MRSA is complex. Additional studies are warranted on the maximum distance that viable MRSA can be emitted outside the facility, and the possibility that animal feed may be a source of contamination.

  12. Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand.

    Science.gov (United States)

    Wongboot, Warawan; Chomvarin, Chariya; Engchanil, Chulapan; Chaimanee, Prajuab

    2013-07-04

    The aims of this study were to develop multiplex PCR for simultaneous detection of five superantigenic toxin genes (sea, seb, sec, sed and tst-1) in Staphylococcus aureus isolated from 149 clinical samples and nasal swabs from 201 healthy subjects in Thailand, and to compare prevalence and expression of those genes between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The sensitivity of multiplex PCR was 10(3) CFU/ml (60 CFU/PCR reaction) for DNA templates extracted by both boiling and extraction methods. S. aureus strains from patients (65%) harbored more superantigenic toxin genes than healthy subjects (54%). MRSA (80%) isolated from patients harbored more superantigenic toxin genes than MSSA (52%). Sea was the most frequently found gene in S. aureus strains from patients and carriers. MRSAisolates harbored sea and produced SEA more frequently than MSSA isolates (p <0.05) and MRSA isolates (59%) from blood samples consisted of a higher number of superantigenic toxin producers than MSSA (9%) (p < 0.05). More S. aureus strains isolated from patients with severe septicemia contained superantigenic toxin genes (94%) and produced toxins (82%) than those from non-severe patients (64% and 57%, respectively). The multiplex PCR method described here offers a reliable tool for simultaneous detection of various staphylococcal toxin genes.

  13. 金黄色葡萄球菌肠毒素的快速检测%Rapid Detection of Staphylococcus Aureus Enterotoxin

    Institute of Scientific and Technical Information of China (English)

    魏晓敏

    2016-01-01

    Staph ylococcus aureus toxins strain often causes poisoning and mainly occurs in the high temperatures of the season, foods easily contaminated are meat, jelly, leftovers, rice wine, eggs and egg products. Immunological methods are often used to detect staphylococcal enterotoxin, which is sensitive, specific, simple and fast. Wherein the reverse latex agglutination method, enzyme-linked immunosorbent assay (ELISA) method and colloidal gold method are more practical, which are commonly used in rapid detection of staphylococcus aureus enterotoxin.%金黄色葡萄球菌中的毒素型菌株常会引起人中毒,且多发于气温较高的季节,易被污染的食物有肉馅、凉粉、剩饭、米酒、蛋及蛋制品等。常采用免疫学方法检测葡萄球菌肠毒素,该方法不仅敏感、特异,而且简便快速。其中反向乳胶凝集、酶联免疫吸附(ELISA)和胶体金法3种方法较为实用,常用于金黄色葡萄球菌肠毒素的快速检测。

  14. Recent Advance on the Detection of Staphylococcus Aureus Enterotoxins%金黄色葡萄球菌肠毒素检测的研究进展

    Institute of Scientific and Technical Information of China (English)

    云盛; 索晓敏; 徐正挺; 杨俊平; 李宇星

    2012-01-01

    Staphylococcus aureus is a major human and zoonotie pathogen widely spreading in the nature. It can produce staphylococcal enterotoxins (SE) and induce food poisoning. Detection of SE is listed in food safety inspection regulations with the improving of people's awareness about food safety. It is necessary to establish a rapid and sensitive method for detection of SE to insure food safety.%金黄色葡萄球菌(Staphylococculs aureus)是一种重要的人畜共患致病菌,广泛存在于自然界中,其产生的肠毒素(staphylococcal enterotoxins,SE)可通过污染食物而导致食物中毒.目前,随着人们对食品安全重视度的加深,国际上已将肠毒素的检测列入食品检验法规,因此建立灵敏、快速的肠毒素检测方法,是食品安全检测的一项重要研究内容.

  15. Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.

  16. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.

  17. Rapid detection of Staphylococcus aureus by a multiplex PCR assay%金黄色葡萄球菌的多重PCR快速鉴定方法

    Institute of Scientific and Technical Information of China (English)

    吕国平; 王苋; 秦丽云

    2012-01-01

    Objective To establish a multiplex PCR method for identifying Staphylococcus aureus and to study the distribution of thermonuclease genes in foodbome Staphylococcus aureus. Methods Three primers were designed for detecting nuc, nuc A and 16S rDNA genes. The specificities of three primers were tested by single PCR method and sequencing. The specificity and sensitivity of the multiplex PCR method were tested. Results The specificities of three primers met the test requirement. The specificity met the test requirement, and the detection limits for DNA template were 100 cfu/μl. All the Staphylococcus aureus strains had nuc and nucA genes. Conclusion The multiplex PCR method is fast, specific, rigorous, sensitive and can be used for identifying a large number of Staphylococcus aureus strains simultaneously.%目的 建立多重PCR快速鉴定金黄色葡萄球菌检测方法,并了解nuc和nucA基因在食源性金黄色葡萄球菌中的分布状况.方法 设计nuc、nucA和16S rDNA基因的引物,利用单重PCR方法和测序验证引物的特异性;建立多重PCR快速检测方法,并应用此方法对23株食源性金黄色葡萄球菌、15株其他种属细菌以及一起食物中毒样品的增菌液进行检验,以评价该方法的特异性和灵敏性.结果 利用单对引物对实验室的4个菌株进行盲筛,出现的条带均为单一条带,且与目的片段长度一致,测序结果显示扩增片段为目的基因片段.建立的多重PCR方法对金黄色葡萄球菌有很好的特异性和灵敏性,灵敏度达100 cfu/μl,研究所涉及的23株食源性金黄色葡萄球菌nuc和nucA基因均为阳性.结论 该方法简便、快速、特异性好、灵敏度高,适用于金黄色葡萄球菌的快速鉴定.

  18. Rapid and Specific Detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by Transcription-Reverse Transcription Concerted Reaction with an Automated System

    OpenAIRE

    Nakaguchi, Yoshitsugu; Ishizuka, Tetsuya; Ohnaka, Satoru; Hayashi, Toshinori; Yasukawa, Kiyoshi; Ishiguro, Takahiko; Nishibuchi, Mitsuaki

    2004-01-01

    Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, ...

  19. Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes.

    Science.gov (United States)

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan

    2008-01-01

    Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

  20. Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR.

    Science.gov (United States)

    Chiefari, Amy K; Perry, Michael J; Kelly-Cirino, Cassandra; Egan, Christina T

    2015-12-01

    To minimize specimen volume, handling and testing time, we have developed two TaqMan(®) multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8-20 cfu per rtPCR for the clinical stool matrix.

  1. GFP tracking transcriptional activity endogenous p53: vector construction and application in genotoxicity detection

    Institute of Scientific and Technical Information of China (English)

    ZENG Wei-sen; LUO Chen; XIE Wei-bing; CHEN Han-yuan

    2001-01-01

    To establish a sensitive.and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H202 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H202, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP, hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.

  2. Direct detection of transcription factors in cotyledons during seedling development using sensitive silicon-substrate photonic crystal protein arrays.

    Science.gov (United States)

    Jones, Sarah I; Tan, Yafang; Shamimuzzaman, Md; George, Sherine; Cunningham, Brian T; Vodkin, Lila

    2015-03-01

    Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

  3. Reliability of a Tissue Microarray in Detecting Thyroid Transcription Factor-1 Protein in Lung Carcinomas

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Bai; Hong Shen

    2007-01-01

    OBJECTIVE To compare the expression of the thyroid transcription factor-1 (TTF-1) in human normal adult type Ⅱ alveolar epithelial cells,embryonic pneumocytes and cancer cells of lung carcinoma and metastatic lymph nodes using a tissue microarray (TMA) along with paired conventional full sections.and to jnvestigate the reliability of tissue microarrays in detecting protein expression in lung carcinoma.METHODS A lung carcinoma TMA including 765 cores was constructed.TTF-1 protein expression in both TMA and paired conventional full sections were detected by yhe immunohistochemical SP method using a monoclonal antibody to TTF-1.A PU (Positive Unit) of TTF-1 protein was assessed quantitatively by the Leica Q500MC image analysis system with results from the paired conventional full sections as controls.RESULTS There was no signifcance between TMA and paired conven tional full sections in TTF-1 expression in difierent nuclei of the lung tissue.CONCLUSION TTF-1 protein expression in lung carcinoma detected by TMA was highly concordanl with that of paired full sections.TMA is a reliable method in detecting protein expression.

  4. Profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of Methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods

    Directory of Open Access Journals (Sweden)

    Shahla Abbas Poor

    2014-10-01

    Full Text Available Background: Hospital-acquired infections are a major challenge to patient. A range of gram-negative organisms are responsible for hospital-acquired infections, the Enterobacteriaceae family being the most commonly identified group overall. Infections by ESBL producers are associated with severe adverse clinical outcomes that have led to increased mortality, prolonged hospitalization, and rising medical costs. The aim of this study was to survey profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods. Material and Methods: In this study participants were patients hospitalized in PICU part of Bahrami Hospital, Tehran, with attention to involved organ. For isolation of bacteria from patient’s samples, culture performed on different selective and differential media. After confirmation of bacteria by biochemical tests, susceptibility testing was performed by disc diffusion method. Phenotypic detection of MRSA strains was performed using cefoxcitin disc. ESBL producing strains were detected by ceftazidime (CAZ and ceftazidime/clavulanic acid (CAZ/CLA discs. Results: Among all isolated organisms from clinical samples, the most common isolated organisms were Escherichia coli (24 cases, Pseudomonas areoginosa (9 cases and Staphylococcus aureus (8 cases, respectively. Among eight MRSA isolated strains from different clinical samples, six strains (75% were MRSA. Among 52 isolated gram negative organisms, 5 strains (9/6% were ESBL. Conclusion: Standard interventions to prevent the transmission of antimicrobial resistance in health care facilities include hand hygiene, using barrier precautions in the care of colonized and infected patients, using dedicated instruments and equipment for these patients. The colonized or infected patients should be isolated in single rooms, multibed rooms or areas

  5. In silico PCR Verification and Simplex Real-Time PCR Detection of Methicillin-Resistant Staphylococcus aureus (MRSA from East Coast Malaysian Clinical Isolates

    Directory of Open Access Journals (Sweden)

    Zarizal Bin SUHAILI

    2013-06-01

    Full Text Available The aims of this study were to validate the primers developed for molecular-based detection and identification of Malaysian clinical isolates of methicilin-resistance Staphylococcus aureus (MRSA using in-silico Polymerase Chain Reaction (PCR and real-time PCR SYBR with Green I. Rapid molecular diagnostic and risk assessment of the MRSA are possible by real-time PCR SYBR Green I. However, validation of such primers for real-life samples is expensive and time consuming. Hence, development and verification of real-time PCR primers by in-silico PCR can be the first step in the selection of the most appropriate primers. Three species-specific markers were chosen targeting coa (staphylocoagulase, nuc (thermonuclease and mecA (methicillin-resistance and were specifically verified against 35 selected S. aureus strains by using in-silico PCR. For the actual laboratory verification, all of the 3 genes were detected with a single specific melting curve peak (Tm at 76.16 ± 0.8 °C, 78.50 ± 0.4 °C and 74.41 ± 0.6 °C for a coa, nuc and mecA respectively in 32 bacterial strains including ATCC reference strains. Thus, there is no disagreement between both in-silico PCR and real-time PCR verification and validation of the primers designed for the detection and identification of MRSA in this study. The potential of using a bioinformatics approach (in-silico PCR before selecting primer pairs for a given study may enable researchers to accept or reject the potential primer pairs for downstream experimental (in vitro PCR without wasting any chemicals as well as related cost.

  6. Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

    Science.gov (United States)

    Savage, Harry M.

    2017-01-01

    We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days. PMID:28075325

  7. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization.

    Science.gov (United States)

    Olschläger, Stephan; Günther, Stephan

    2012-07-01

    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

  8. Detection and characterization of enterotoxin genes in Staphyloccocus aureus%金黄色葡萄球菌肠毒素基因的检测与分型

    Institute of Scientific and Technical Information of China (English)

    傅丹青; 陈棋炯; 丁水军; 戴城钢

    2011-01-01

    Objective:To detect enterotoxin genes ( SEA、SEB、SEC、SED、SEE ) in Staphylococcus aureus isolates from different sources and characterize genotype that of isolates. Methods: 46 S. aureus isolates were isolated from hospital disinfection/ public/food monitoring sample and the vomit and excreta from food poisoning patient, then the enterotoxin genes were detected by VIDAS and characterized genotype by PCR. Results: The positive rate of the enterotoxin gene in isolates was 47.83%, and the most frequendy found genes were SEA(31.82% ) and SEB(27.27% ). Enterotoxin gene from different sources had different detection rates. The enterotoxin genes were positive in 10 of 24 strain from hospital disinfection monitoring samples, 2 of 4 strain from public monitoring sample, 4 of 12 strain from food monitoring sample and 6 of 6 strain from the vomit and excreta from food poisoning patient, then the positive rate were 41.76%, 50. 00%, 33.33%, and 100%, respectively. Conclusion: 22 of 46 strains can be detected kinds of S. aureus enterotoxin genes, then the positive rate was high. VIDAS and PCR can be applied to detect enterotoxin genes simply and rapidly, then the methods could be used in grass root CDC conveniently.%目的:了解不同来源的金黄色葡萄球菌(Staphylococcus aureus,SA)分离株产肠毒素的情况,以及主要肠毒素SEA、SEB、SEC、SED、SEE基因携带状况.方法:采用小型VIDAS全自动荧光酶标免疫法对46株分别从医院消毒监测样品、公共场所监测样品、食品监测样品以及食物中毒病人粪便和呕吐物中分离到的SA菌株进行肠毒素检测,再采用PCR技术对产肠毒素的菌株进行基因分型.结果:46株SA菌株中,携带肠毒素的为22株,检出率为47.83%.其中以SEA、SEB检出率较高,分别为31.82%和27.27%.不同来源SA分离株携带肠毒素基因的比例不同,24株来自医院消毒监测样品分离株中检出率为41.67%(10/24);4株来自公共场所监测

  9. In situ detection of transcripts of the myogenic factor MyoD in whole chicken embryos

    Directory of Open Access Journals (Sweden)

    Jane E. Gabriel

    2000-03-01

    Full Text Available In situ hybridization has provided insights into the molecular basis of skeletal myogenesis during embryonic development. In situ detection of different muscle-specific regulatory factors in whole embryos has been described. Spatial and temporal expression patterns of these factors differed among species. The expression pattern of MyoD in whole chicken embryos was studied via in situ hybridization using a probe obtained by the reverse transcription - polymerase chain reaction (RT-PCR technique. In newly formed somites (embryos of stage 12, MyoD mRNA transcripts were detected along the anterior to posterior axis of somites immediately adjacent to the neural tube, whereas in mature somites (embryos of stage 24, MyoD transcripts were detected throughout the entire somite. These results indicate that MyoD expression is important for initiating and maintaining the avian myogenic system.A detecção in situ de diferentes fatores regulatórios músculo-específicos vem sendo uma prática muito empregada para o entendimento das bases moleculares envolvidas no processo de formação do tecido muscular esquelético durante o desenvolvimento embrionário em diferentes organismos. O objetivo deste estudo foi investigar o padrão de expressão dos transcritos do fator miogênico MyoD em embriões inteiros de galinha por análises de hibridização in situ, usando uma sonda obtida a partir de ensaios de transcrição reversa e reação em cadeia da polimerase (RT-PCR. Nos somitos recém-formados (embriões no estádio 12, os transcritos de MyoD foram detectados na porção mediana dos somitos posicionados imediatamente adjacentes ao tubo neural, enquanto nos somitos maduros (embriões no estádio 24, a expressão do gene do MyoD foi observada em toda a região dos somitos. Tais resultados indicam que a expressão do MyoD é importante no processo de determinação e manutenção da miogênese nas aves.

  10. PHENOTYPIC AND GENOTYPIC DETECTION OF ENTEROTOXINS, TOXIC SHOCK SYNDROME TOXIN-1 AND OF METHICILLIN RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATED FROM RETAIL READY-TO-EAT FOODS IN NORTHEASTERN THAILAND.

    Science.gov (United States)

    Wongboot, Warawan; Chomvarin, Chariya; Namwat, Wises

    2015-01-01

    Toxigenic Staphylococcus aureus contamination of ready-to-eat (RTE) foods is a leading cause of foodborne illness in Thailand. From 151 RTE food samples randomly collected from food vendors and food shops in Khon Kaen municipality, Thailand and after culture-based identification of S. aureus isolates, pentaplex PCR was used for simultaneous detection of super-antigenic toxin (SE) genes (sea, seb, sec, sed and tst-1) and presence of their toxins by reversed passive latex agglutination assay. S. aureus was identified in 57 isolates, of which 60% and 25% was positive for presence of super-antigenic toxin genes and toxins, respectively; and among the former isolates sea was the most common (46%), as well as its product (SEA) (14%) among the latter group. Methicillin resistance S. aureus mecA was not found in any of the isolates using both PCR and disk diffusion methods. These results showed that pentaplex PCR is a useful tool for detection of SE-encoding genes in S. aureus isolates from RTE food.

  11. Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA from Screening Specimens ▿

    OpenAIRE

    Rossney, Angela S.; Herra, Celine M, Dr; Brennan, Gráinne I.; Morgan, Pamela M.; O'Connell, Brian

    2008-01-01

    The need for rapid methods to accurately detect methicillin-resistant Staphylococcus aureus (MRSA) is widely acknowledged, and a number of molecular assays are commercially available. This study evaluated the Xpert MRSA assay, which is run on the GeneXpert real-time PCR platform (Cepheid) for use in a clinical laboratory. The following parameters were investigated: (i) the limits of detection (LoDs) for four MRSA strains; (ii) the ability to detect isolates of MRSA from a collection represent...

  12. A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    张宁; 徐永健; 张珍祥; 熊维宁

    2003-01-01

    To determine the feasibility of a nonradioactive electrophoresis mobility shift assay fordetecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus tar-get sequence of NF-κB were labled with DIG by terminal transferase. After nuclear protein stimula-ted with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDT C)electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, theywere electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemilu-minescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded spe-cifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method andthe activity of NF-κB in PMA group was more than that in PMA+PDTC group. It is suggestedthat detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, whichcan be performed in ordinary laboratories.

  13. Molecular detection of EWS-Ets fusion transcripts and their clinicopathologic significance in Ewing's sarcoma/peripheral primitive neuroectodermal tumor

    Institute of Scientific and Technical Information of China (English)

    WANG Hua; ZHENG Jie; WANG Yu-ping; YANG Yu; YOU Jiang-feng

    2005-01-01

    Background Ewing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficult to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Fli1 and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET.Methods Thirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features (including rhabdomyosarcoma, neuroblastoma, lymphoma, small cell carcinoma, and desmoplastic small round cell tumor) were used as negative controls. Β-actin and β2-microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay was performed to detect the EWS-Fli1 and EWS-ERG fusion transcripts.Results β-actin and β2-microglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS-Fli1 fusion transcripts were detected in 11 of 13 (85%) ES/pPNETs. Three chimeric transcripts, all EWS-Fli1, were detected in ES/pPNET samples. Among 11 EWS-Fli1-positive cases, 7 cases had a typeⅠfusion transcript involving fusion of EWS exon 7 with Fli1 exon 6, 2 cases had a typeⅡfusion transcript involving EWS exon 7 with Fli1 exon 5, and 2 cases expressed fusion transcripts involving EWS exon 7 and Fli1 exon 8. Type Ⅰ EWS-Fli1 fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 cases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Fli1 fusion transcripts and CD99 expression. Conclusions Molecular detection of EWS-Fli1 fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is

  14. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

    Directory of Open Access Journals (Sweden)

    Ravi Prakash

    2014-12-01

    Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

  15. Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.

    Science.gov (United States)

    McCann, Chase D; Jordan, Jeanne A

    2014-04-01

    Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (PDNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.

  16. Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by transcription-reverse transcription concerted reaction with an automated system.

    Science.gov (United States)

    Nakaguchi, Yoshitsugu; Ishizuka, Tetsuya; Ohnaka, Satoru; Hayashi, Toshinori; Yasukawa, Kiyoshi; Ishiguro, Takahiko; Nishibuchi, Mitsuaki

    2004-09-01

    Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10(3) to 10(7) copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.

  17. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species.

    Science.gov (United States)

    Ramsey, Matthew M; Freire, Marcelo O; Gabrilska, Rebecca A; Rumbaugh, Kendra P; Lemon, Katherine P

    2016-01-01

    Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.

  18. Rapid bench identification of methicillin-sensitive and methicillin-resistant Staphylococcus aureus: A multicenter comparative evaluation of Alere PBP2a Culture Colony Test (Alere) Versus Slidex MRSA detection (bioMérieux).

    Science.gov (United States)

    Tasse, Jason; Dupieux, Céline; Caillon, Jocelyne; Lanotte, Philippe; Lamy, Brigitte; Aissa, Nejla; Bemer, Pascale; Mereghetti, Laurent; Michon, Anne-Laure; Lozniewski, Alain; Bes, Michèle; Trouillet-Assant, Sophie; Laurent, Frédéric

    2016-08-01

    Using 30 clinical isolates of Staphylococcus aureus representative of the most prevalent clones circulating in France, the performance of the Alere™ PBP2a Culture Colony Test (CCT) and the Slidex(®) MRSA detection kit (SMD) were compared in 5 different labs. CCT demonstrated better performance and was easier to conduct in routine.

  19. Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.

    Science.gov (United States)

    Kilic, Abdullah; Basustaoglu, A Celal

    2011-12-01

    We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.

  20. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang [Asan Medical Center, University of Ulsan, Seoul (Korea, Republic of)

    1994-07-15

    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  1. Correlation between the VITEK2 system and cefoxitin disk diffusion for the daily detection of oxacillin resistance in a large number of clinical Staphylococcus aureus isolates.

    Science.gov (United States)

    Bemer, P; Juvin, M E; Le Gargasson, G; Drugeon, H; Reynaud, A; Corvec, S

    2010-06-01

    The aim of the present study was to compare the performance of the new VITEK2 AST-P551 card with the cefoxitin disk diffusion method for the daily detection of methicillin resistance with a high number of Staphylococcus aureus clinical isolates. Detection of the PBP2a protein or mecA gene was performed for each discordant case. Seventy (3.3%) isolates out of 2,107 clinical strains showed discordant results, two very major errors, four major errors and 64 minor errors. Fifty-nine (84%) discordant results were resolved, with a final overall agreement of 99.5%. Eleven (0.5%) strains remained discordant (minor error [mE]). Four of 370 MRSA strains were misclassified as susceptible in daily practice by the cefoxitin disk diffusion method. All of these strains were resistant to aminoglycosides and/or fluoroquinolones. The VITEK2 system is highly reliable for methicillin resistance detection at the routine level. Oxacillin-susceptible classified clinical strains with associated resistance patterns required attention.

  2. A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Maria W Smith

    Full Text Available PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP. At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i any mutation of the restriction site reduced the signal to zero; (ii double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same

  3. Staphylococcus aureus shifts towards commensalism in response to Corynebacterium species

    Directory of Open Access Journals (Sweden)

    Matthew M Ramsey

    2016-08-01

    Full Text Available Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence towards a commensal state when exposed to commensal Corynebacterium species.

  4. A multi-center blinded study on the efficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcus aureus (GISA and heterogeneous GISA (h-GISA

    Directory of Open Access Journals (Sweden)

    Sloos Jacobus H

    2007-09-01

    Full Text Available Abstract Backgrounds To determine the true incidence of hGISA/GISA and its consequent clinical impact, methods must be defined that will reliably and reproducibly discriminate these resistant phenotypes from vancomycin susceptible S. aureus (VSSA. Methods This study assessed and compared the ability of eight Dutch laboratories under blinded conditions to discriminate VSSA from hGISA/GISA phenotypes and the intra- and inter-laboratory reproducibility of agar screening plates and the Etest method. A total of 25 blinded and unique strains (10 VSSA, 9 hGISA and 6 GISA were categorized by the PAP-AUC method and PFGE typed to eliminate clonal duplication. All strains were deliberately added in quadruplets to evaluate intra-laboratory variability and reproducibility of the methods. Strains were tested using three agar screening methods, Brain Heart Infusion agar (BHI + 6 μg/ml vancomycin, Mueller Hinton agar (MH + 5 μg/ml vancomycin and MH + 5 μg/ml teicoplanin and the Etest macromethod using a 2 McFarland inoculum. Results and Discussion The ability to detect the hGISA/GISA phenotypes varied significantly between methods and phenotypes. BHI vancomycin and MH vancomycin agar screens lacked the ability to detect hGISA. The MH teicoplanin agar screen was more sensitive but still inferior to Etest that had a sensitivity of 98.5% and 99.5%, for hGISA and GISA, respectively. Intra- and inter-laboratory reproducibility varied between methods with poorest performance seen with BHI vancomycin. Conclusion This is the first multi-center blinded study to be undertaken evaluating various methods to detect GISA and hGISA. These data showed that the ability of clinical laboratories to detect GISA and hGISA varied considerably, and that screening plates with vancomycin have a poor performance in detecting hGISA.

  5. Multicenter evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test as a rapid method for detection of MRSA in nasal surveillance swabs.

    Science.gov (United States)

    Peterson, Lance R; Liesenfeld, Oliver; Woods, Christopher W; Allen, Stephen D; Pombo, David; Patel, Parul A; Mehta, Maitry S; Nicholson, Bradly; Fuller, DeAnna; Onderdonk, Andrew

    2010-05-01

    The rate of methicillin-resistant Staphylococcus aureus (MRSA) infection continues to rise in many health care settings. Rapid detection of MRSA colonization followed by appropriate isolation can reduce transmission and infection. We compared the performance of the new Roche LightCycler MRSA advanced test to that of the BD GeneOhm MRSA test and culture. Double-headed swabs were used to collect anterior nasal specimens from each subject. For both tests, DNA was extracted and real-time PCR was performed according to manufacturer's instructions. For culture, one swab of the pair was plated directly to CHROMagar MRSA. The swab paired with the BD GeneOhm MRSA test was also placed into an enrichment broth and then plated to CHROMagar MRSA. Colonies resembling staphylococci were confirmed as S. aureus by standard methods. Discrepant specimens had further testing with additional attempts to grow MRSA as well as sample amplicon sequencing. Agreement between results for the two swabs was 99.3% for those with valid results. A total of 1,402 specimens were tested using direct culture detection of MRSA as the gold standard; 187 were culture positive for MRSA. The LightCycler MRSA advanced test had relative sensitivity and specificity of 95.2% (95% confidence interval [CI]: 91.1% to 97.8%) and 96.4% (95% CI: 95.2% to 97.4%), respectively. The BD GeneOhm assay had relative sensitivity and specificity of 95.7% (95% CI: 91.7% to 98.1%) and 91.7% (95% CI: 90.0% to 93.2%), respectively. Following discrepancy analysis, the relative sensitivities of the LightCycler MRSA advanced test and the BD GeneOhm MRSA assay were 92.2 and 93.2%, respectively; relative specificities were 98.9 and 94.2%, respectively. Specificity was significantly better (Padvanced test. The sensitivity of direct culture was 80.4%. The LightCycler MRSA advanced test is a useful tool for sensitive and rapid detection of MRSA nasal colonization.

  6. Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Alice Kar Lai, E-mail: s0907465@cuhk.mail.serv.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Lu, Haifei, E-mail: hflu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Wu, Shu Yuen, E-mail: sywu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Kwok, Ho Chin, E-mail: hckwock@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Ho, Ho Pui, E-mail: hpho@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Yu, Samuel, E-mail: samscyu@gmail.com [The MacDiarmid Institute for Advanced Materials and Nanotechnology, Christchurch (New Zealand); Izon Science, PO Box 39-168, Harewood, Christchurch 8545 (New Zealand); Cheung, Anthony Ka Lun, E-mail: kalun2004@hotmail.com [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Kong, Siu Kai, E-mail: skkong@cuhk.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong)

    2013-06-11

    Graphical abstract: -- Highlights: •A novel diagnostic assay is developed to detect the MRSA's Panton-Valentine Leukocidin toxin. •Detection is based on target DNA amplification at one single temperature at 65 °C by LAMP. •Amplicons are then hybridized with 2 Au-nanoparticles with specific DNA probes for sensing. •The supra-assemblies are subsequently sensed by resistive pulse sensing. •Detection limit: ∼200 copies of DNA; time for detection: completed within 2 h. -- Abstract: This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55 ± 2 nm) through biotin–avidin binding. A second AuNP2 (30 ± 1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.

  7. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a s...

  8. Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

    NARCIS (Netherlands)

    Hansenova Manaskova, S.; Bikker, F.J.; Veerman, E.C.I.; van Belkum, A.; van Wamel, W.J.B.

    2013-01-01

    The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In thi

  9. Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negative Staphylococcus aureus and coagulase negative staphylococci.

    Science.gov (United States)

    Panda, Sasmita; Kar, Sarita; Choudhury, Ranginee; Sharma, Savitri; Singh, Durg V

    2014-03-01

    We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S. aureus and CoNS isolated from infected eyes and five S. aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S. aureus and CoNS from a variety of ocular samples.

  10. Frequent detection of transcriptionally active Felis catus papillomavirus 2 in feline cutaneous squamous cell carcinomas.

    Science.gov (United States)

    Thomson, Neroli A; Munday, John S; Dittmer, Keren E

    2016-05-01

    Felis catus papillomavirus 2 (FcaPV-2) causes premalignant skin lesions in cats and has also been found in a proportion of cutaneous squamous cell carcinomas (SCCs) - a common and potentially fatal cancer of cats. Whilst this could suggest a role of the virus in cancer development, FcaPV-2 has also been detected in skin swabs of normal cats, making it difficult to discern whether the papillomavirus is causing the cancer or merely an 'innocent bystander'. To distinguish between these two possibilities, real-time PCR was used to determine the viral copy number and the transcriptional activity of FcaPV-2 infections present in 70 formalin-fixed paraffin-embedded skin lesions including 10 papillomavirus-induced premalignant lesions and 60 SCCs. FcaPV-2 gene expression was found in 21 of 60 (35 %) SCCs, all 10 premalignant lesions and none of 10 normal skin samples. The results showed two distinct subsets of SCCs. The majority of the SCCs had low copy numbers of FcaPV-2 DNA (mean of 17 copies per copy of reference gene DNA) and no FcaPV-2 gene expression, suggesting the virus was an incidental finding. In contrast, 20 SCCs had detectable FcaPV-2 E6/E7 gene expression and very high copy numbers of FcaPV-2 DNA, with a mean of 32 930 copies per copy of reference gene DNA. The relative quantity of E6/E7 gene expression and the viral copy number in this group were similar to those found in the papillomavirus-induced premalignant lesions, suggesting that FcaPV-2 may play a role in the development of a subset of feline cutaneous SCCs.

  11. 快速检测食品中金黄色葡萄球菌及其肠毒素型的研究进展%Research Advance in Rapid Detection of Staphylococcus aureus in Food and Its Enterotoxin

    Institute of Scientific and Technical Information of China (English)

    李荔枝; 胡萍

    2011-01-01

    Some methods for the rapid detection of Staphylococcus aureus in food were summarized, including Petrifilm RSA detection method, Baird - Parker + RPF Agar detection method, colloidal gold immune - chromatographic assay and polymerase chain reaction (PCR), and PCR was mostly used for assaying the enterotoxin of Staphylococcus aureus.%综述了食品中金黄色葡萄球菌各种快速检测方法,包括Petrifilm RSA检测法、Baird - Parker+ RPF Agar检测法、胶体金免疫层析方法及聚合酶链反应(PCR),其中聚合酶链反应大多是用来检测金黄色葡萄球茵的肠毒素型.

  12. Real-Time PCR Detection of Host-Mediated Cyanophage Gene Transcripts during Infection of a Natural Microcystis aeruginosa Population

    OpenAIRE

    Yoshida, Mitsuhiro; Yoshida, Takashi; Yoshida-Takashima, Yukari; Kashima, Aki; Hiroishi, Shingo

    2010-01-01

    The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method ...

  13. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    Science.gov (United States)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  14. An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

    Science.gov (United States)

    Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

    2016-10-01

    Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

  15. Staphylococcus aureus and Pregnancy

    Science.gov (United States)

    Staphylococcus aureus and Pregnancy In every pregnancy, a woman starts out with a 3-5% chance of having a ... This sheet talks about whether exposure to staphylococcus aureus may increase the risk for birth defects over ...

  16. Rapid detection of tdh and trh mRNAs of Vibrio parahaemolyticus by the transcription-reverse transcription concerted (TRC) method.

    Science.gov (United States)

    Masuda, Noriyoshi; Yasukawa, Kiyoshi; Isawa, Yuichi; Horie, Ryuichi; Saitoh, Juichi; Ishiguro, Takahiko; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki; Hayashi, Toshinori

    2004-01-01

    We developed a novel method named the transcription-reverse transcription concerted (TRC) method and an instrument that allowed rapid and completely homogeneous real-time monitoring of RNA isothermal sequence amplification without any post-amplification analysis in our previous study [Ishiguro et al., Anal. Biochem., 314, 77-86 (2003)]. In this study, we newly established rapid and sensitive TRC systems for the detection of the mRNAs transcribed from two major virulence genes of Vibrio parahaemolyticus: the tdh gene encoding the thermostable direct hemolysin (tdh) and the trh gene encoding the thermostable direct hemolysin-related hemolysin. Examination of the standard RNAs prepared in vitro showed that a positive result, increase in the fluorescence intensity to the cut-off value within 25 min, was obtained for as few as 100 copies of RNA. The TRC method specific to the trh mRNA detected the mRNAs transcribed from the trh1 and trh2 genes, two representative trh variants sharing 84% sequence identity. The detection time gave a linear relationship to the logarithm of starting RNA copies ranging from 10(3) to 10(7) copies, showing that quantitative analysis is possible. The detection time for 10(3) copies of the standard RNAs ranged from 11 to 15 min. Examination of the total RNAs extracted from the standard strains of V. parahaemolyticus demonstrated that the new TRC systems are sufficiently sensitive to detect as few as 100 CFUs of the strains carrying the target genes. Total RNA preparations extracted from 24 strains of V. parahaemolyticus, 52 strains belonging to 31 other species of the genus Vibrio and 11 strains belonging to 8 species of non-Vibrio genera were examined. The results of the detection of tdh- and trh-specific mRNAs by the two TRC systems and those of the respective genes by the DNA colony hybridization method agreed. We conclude that the new TRC systems are rapid, highly sensitive, easy to manipulate, and are suitable for routine examination of

  17. Triclosan promotes Staphylococcus aureus nasal colonization.

    Science.gov (United States)

    Syed, Adnan K; Ghosh, Sudeshna; Love, Nancy G; Boles, Blaise R

    2014-04-08

    The biocide triclosan is used in many personal care products, including toothpastes, soaps, clothing, and medical equipment. Consequently, it is present as a contaminant in the environment and has been detected in some human fluids, including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the noses and throats of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin, as well as inanimate surfaces such as plastic and glass. Lastly, triclosan-exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization. IMPORTANCE Triclosan has been used as a biocide for over 40 years, but the broader effects that it has on the human microbiome have not been investigated. We demonstrate that triclosan is present in nasal secretions of a large portion of a test population and its presence correlates with Staphylococcus aureus nasal colonization. Triclosan also promotes the binding of S. aureus to human proteins and increases the susceptibility of rats to nasal colonization by S. aureus. These findings are significant because S. aureus colonization is a known risk factor for the development of several types of infections. Our data demonstrate the unintended consequences of unregulated triclosan use and contribute to the growing body of research demonstrating inadvertent effects of triclosan on the environment and human health.

  18. Microbiological quality and safety of raw milk and soft cheese and detection of autochthonous lactic acid bacteria with antagonistic activity against Listeria monocytogenes, Salmonella Spp., and Staphylococcus aureus.

    Science.gov (United States)

    Ortolani, Maria Beatriz Tassinari; Yamazi, Anderson Keizo; Moraes, Paula Mendonça; Viçosa, Gabriela Nogueira; Nero, Luís Augusto

    2010-02-01

    This study aimed to characterize the microbiological quality and safety of raw milk and soft cheese, verifying possible associations between microbial populations, and the detection of lactic acid bacteria (LAB) with antagonistic activity against foodborne pathogens. Raw milk (n = 36) and soft cheese (n = 18) samples were collected and submitted for the analysis of mesophilic aerobes, total coliforms, Escherichia coli, LAB, coagulase-positive Staphylococcus (CPS), Listeria monocytogenes, and Salmonella spp. In all, 389 LAB isolates were randomly selected and submitted for antagonistic tests against L. monocytogenes, St. aureus, Salmonella Typhimurium, and Lactobacillus sakei. The samples presented high counts of mesophilic aerobes, total coliforms, and LAB, and also high and significant correlation indices between these populations. Low levels of CPS and E. coli were observed, as well as an absence of Salmonella spp. and L. monocytogenes. A substantial portion of the analyzed samples presented LAB cultures with antagonistic activity, but not against Salmonella Typhimurium. The obtained results indicate the antimicrobial potential of the autochthonous microbiota of raw milk and soft cheese. Despite the spoilage potential, the LAB present in the studied food products can be isolated and properly characterized as antagonistic cultures, to be used in bioconservation studies for pathogen control in foods.

  19. S100A7/psoriasin expression in the human lung: unchanged in patients with COPD, but upregulated upon positive S. aureus detection

    Directory of Open Access Journals (Sweden)

    Branscheid Detlev

    2011-02-01

    Full Text Available Abstract Background Progressive airway inflammation and susceptibility to the airway colonisation and infection are characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD. Antimicrobial peptides (AMPs are central to the function of the innate host immune response against microbial pathogens and are regulators of inflammation and immunity. S100A7/psoriasin, a recently described AMP, is an essential component of the human epithelia against invading pathogens and acts as an effector molecule of the host innate defence in the skin. We hypothesized that S100A7/psoriasin is involved in the airway mucosal immunity and differently regulated and expressed in the lung during progression of COPD. Methods S100A7/psoriasin gene expression was assessed in bronchial biopsies and bronchoalveolar lavage (BAL fluid cells of healthy controls and COPD patients. Using confocal microscopy and immunohistochemistry, the protein expression of S100A7/psoriasin was investigated. Results Here, we report that S100A7/psoriasin, the major antimicrobial peptide of the human skin, is constitutively expressed in perinuclear granules of human bronchial epithelial cells and alveolar macrophages. Whereas typical activators of the innate immune response like TLR ligands and cytokines induced the upregulation of CXCL-8 mRNA and release of CXCL-8 by epithelial cells, S100A7/psoriasin mRNA expression was not modulated. To investigate a potential association of S100A7/psoriasin with COPD, S100A7/psoriasin mRNA expression was assessed in bronchial biopsies and BAL fluid cells of patients at different stages of COPD and controls. Overall, 10 healthy individuals and 34 COPD patients were enrolled in this study. We found an association of S100A7/psoriasin mRNA expression with bacterial detection in the tracheobronchial system (p = 0.0304, which was the strongest in individuals positive for with S. aureus (p = 0.0005. However, S100A7/psoriasin mRNA expression

  20. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

    Science.gov (United States)

    Guan, Li; Zhao, Lin-Qing; Zhou, Hang-Yu; Nie, Kai; Li, Xin-Na; Zhang, Dan; Song, Juan; Qian, Yuan; Ma, Xue-Jun

    2016-07-01

    Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

  1. Electrochemical DNA biosensor with chitosan-Co(3)O(4) nanorod-graphene composite for the sensitive detection of Staphylococcus aureus nuc gene sequence.

    Science.gov (United States)

    Qi, Xiaowei; Gao, Hongwei; Zhang, Yuanyuan; Wang, Xiuzhen; Chen, Ying; Sun, Wei

    2012-12-01

    In this paper a novel nanocomposite material prepared by Co(3)O(4) nanorods (nano-Co(3)O(4)), graphene (GR) and chitosan (CTS) was fabricated and further modified on carbon ionic liquid electrode (CILE), which was used as the substrate electrode to construct a new electrochemical DNA biosensor. The single-stranded DNA (ssDNA) probe was immobilized on the CTS-Co(3)O(4)-GR/CILE surface by electrostatic attraction, which could hybridize with the target ssDNA sequence under the selected conditions. By using methylene blue (MB) as the electrochemical indicator, the hybridization reactions were monitored with the reduction peak current. By combining the biocompatibility of Co(3)O(4) nanorods, excellent electron transfer ability and big surface of GR, good film-forming ability of CTS and the high conductivity of CILE, the amount of ssDNA adsorbed on the electrode surface was increased and the electrochemical response of MB was accelerated. Under the optimal conditions differential pulse voltammetric responses of MB were in linear with the specific target ssDNA sequence in the concentration range from 1.0×10(-12) to 1.0×10(-6)M with the detection limit as 4.3×10(-13)M (3σ). Good discrimination ability to the one-base and three-base mismatched ssDNA sequences could be achieved and the polymerase chain reaction (PCR) amplification products of Staphylococcus aureus nuc gene sequence were detected with satisfactory results.

  2. Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile.

    Science.gov (United States)

    Senoh, Mitsutoshi; Kato, Haru; Murase, Tomoko; Hagiya, Hideharu; Tagashira, Yasuaki; Fukuda, Tadashi; Iwaki, Masaaki; Yamamoto, Akihiko; Shibayama, Keigo

    2014-11-01

    The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 × 10(2) colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.

  3. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    OpenAIRE

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplif...

  4. Multiplex PCR for rapid detection of enterotoxin genes in foodborne Staphylococcus aureus%食源性金黄色葡萄球菌肠毒素基因的多重PCR快速检测法

    Institute of Scientific and Technical Information of China (English)

    秦丽云; 魏绣萍; 吕国平

    2012-01-01

    Objective To establish a multiplex polymerase chain reaction (PCR) method for screening the foodborne Staphylococcus aureus enterotoxin(SE) genes. Methods Four primers were designed according to the published Staphylococcus aureus toxin genes conserved sequence. The specificity of four primers were tested by single PCR method and sequencing. A multiplex PCR method was established and used for detecting 165 strains of Staphylococcus aureus isolated from food risk monitoring and food poisoning events. Results A total of 165 strains of Staphylococcus aureus were detected with their typeA-typeD enterotoxin genes in this study,in which 95 (57.58%) strains carried at least one kind of enterotoxin genes; 42.42%(72/165) of strains carried one enterotoxin gene type, while 13.94%(23/165) of strains carried two or more types of enterotoxin gene. Conclusion This multiplex PCR method, which has been gradually applied to foodborne Staphylococcus aureus and food poisoning events, is rapid, sensitive, specific and is applicable for the detection of Staphylococcus aureus enterotoxin genes in food risk monitoring and food poisoning diagnosis.%目的 建立食源性金黄色葡萄球菌肠毒素基因的多重PCR快速检测方法.方法 根据公布的金黄色葡萄球菌肠毒素基因A~D型序列,分别设计引物并验证引物的特异性,建立多重PCR方法,检测食品风险监测和食物中毒事件中分离得到的165株金黄色葡萄球菌肠毒素基因A~D型.结果 165株金黄色葡萄球菌有95株携带肠毒素基因,毒素基因携带率为57.6%(95/165),携带一种基因型的占42.4%(72/165),同时携带两种以上毒素基因的占13.9%(23/165).结论 该方法具有简便快速、灵敏度高、特异性强等特点,适用于食源性金黄色葡萄球菌和食物中毒中金黄色葡萄球菌肠毒素的检测.

  5. Detecção de Staphylococcus aureus utilizando a técnica de REP-PCR no monitoramento da qualidade do leite de cabra Detection of Staphylococcus aureus using the REP-PCR technique to monitor goat milk quality

    Directory of Open Access Journals (Sweden)

    Cellyneude de Souza Olivindo

    2009-07-01

    Full Text Available Este estudo foi realizado com o objetivo de aplicar a técnica de REP-PCR no monitoramento da qualidade do leite de cabra por meio da detecção de Staphylococcus aureus em amostras de mãos de ordenhador, tetos das cabras, leite, ordenhadeira e água para estabelecimento e implantação do sistema de Análise de Perigos e Pontos Críticos de Controle (APPCC. Verificaram-se vários fingerprints de todos os isolados coletados das diferentes fontes estudadas (mãos de ordenhador, tetos das cabras, leite, ordenhadeira e água. Observaram-se comportamentos muito similares das bandas, o que indica que os isolados podem ser relatados como clones epidemiológicos. As mãos do ordenhador caracterizaram-se como ponto crítico de controle, pois se destacaram como iniciador de contaminação nas amostras Staphylococcus aureus. A técnica demonstrou ser eficiente para a análise da similaridade entre indivíduos da espécie Staphylococcus aureus e, portanto, constitui ferramenta útil para investigação de falhas no manejo e na busca de controle mais eficiente para evitar ou reduzir a disseminação de microrganismos patogênicos causadores de sérias enfermidades em humanos e animais, que muitas vezes podem ser transmitidas por produtos como o leite e seus derivados.The objective of the present study was to apply REP-PCR sequences in the monitoring goat milk quality, by detecting Staphylococcus aureus, in samples of from milker hands, goat teats, milk, milking machine and water, for the future establishment and implantation of the system of Hazard Analysis Critical Control Points (HACCP. Several fingerprints were verified of all the isolates collected from the different sources studied (milker hands, goat teats, milk, milking machine and water. Very similar band behaviors were observed that indicated that the isolates can be reported as epidemic clones. Milker´s hands were was characterized as a critical control point (CCP, because it stands out as an

  6. Staphylococcus aureus toxins.

    Science.gov (United States)

    Otto, Michael

    2014-02-01

    Staphylococcus aureus is a dangerous pathogen that causes a variety of severe diseases. The virulence of S. aureus is defined by a large repertoire of virulence factors, among which secreted toxins play a preeminent role. Many S. aureus toxins damage biological membranes, leading to cell death. In particular, S. aureus produces potent hemolysins and leukotoxins. Among the latter, some were recently identified to lyse neutrophils after ingestion, representing an especially powerful weapon against bacterial elimination by innate host defense. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. Several further toxins add to this multi-faceted program of S. aureus to evade elimination in the host. This review will give an overview over S. aureus toxins focusing on recent advances in our understanding of how leukotoxins work in receptor-mediated or receptor-independent fashions.

  7. Use of Existing Diagnostic Reverse-Transcription Polymerase Chain Reaction Assays for Detection of Ebola Virus RNA in Semen.

    Science.gov (United States)

    Pettitt, James; Higgs, Elizabeth S; Adams, Rick D; Jahrling, Peter B; Hensley, Lisa E

    2016-04-15

    Sexual transmission of Ebola virus in Liberia has now been documented and associated with new clusters in regions previously declared Ebola free. Assays that have Emergency Use Authorization (EUA) and are routinely used to detect Ebola virus RNA in whole blood and plasma specimens at the Liberian Institute for Biomedical Research were tested for their suitability in detecting the presence of Ebola virus RNA in semen. Qiagen AVL extraction protocols, as well as the Ebola Zaire Target 1 and major groove binder quantitative reverse-transcription polymerase chain reaction assays, were demonstrably suitable for this purpose and should facilitate epidemiologic investigations, including those involving long-term survivors of Ebola.

  8. Detecting PML-RARα transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    ZHU Hong-hu; LIU Yan-rong; QIN Ya-zhen; JIANG Bin; SHAN Fu-xiang; WU Shu-lan; YANG Ping-di; ZHAO Jie; LU Dao-pei

    2007-01-01

    Background Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005.Methods All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7500platform. The quantitation of PML-RARα transcripts was represented by the normalized quotient, that is, PML-RARα transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n=23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n=13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.Results The sensitivity of RQ-PCR was 1 per 105 cells and 5 copies of the PML-RARα transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P=0.034). The rate of molecular remission within 70days was higher in group 1 than in group 2 (75.00% vs 38.46%, P=0.036). Compared with pretreatment, median reduction of the PML-RARα transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2

  9. Real-time PCR detection of host-mediated cyanophage gene transcripts during infection of a natural Microcystis aeruginosa population.

    Science.gov (United States)

    Yoshida, Mitsuhiro; Yoshida, Takashi; Yoshida-Takashima, Yukari; Kashima, Aki; Hiroishi, Shingo

    2010-01-01

    The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL(-1). The highest relative abundance of phage g91 RNA (10(-2) per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10(5) cells mL(-1)); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.

  10. Induction Conditions and EMA-qPCR Detection of Staphyloccocus aureus in Viable but Non-Culturable State%金黄色葡萄球菌VBNC状态的诱导条件和EMA-qPCR检测

    Institute of Scientific and Technical Information of China (English)

    余以刚; 田聪; 肖性龙; 陶文扬; 黎金霞; 吴晖

    2013-01-01

    In order to establish a model for the determination of induction conditions of Staphyloccocus aureus in viable but non-culturable (VBNC) state and to reveal the effects of temperature, salt concentration and pH value on the culturable cell number of Staphyloccocus aureus, orthogonal experiments were carried out to optimize the induction conditions. Then, the culturable cells were monitored and a method based on the DNA-binding dye EMA ( Ethidium Bromide Monoazide) and the qPCR technology was proposed to detect Staphyloccocus aureus in VBNC state. The results show that pH value greatly influences the culturable cell number, and that the cultivation in a nutrient broth medium with 15% (mass fraction) NaCl and 0. 3% (volume fraction) acetic acid at 4℃ for 12 d is best for the induction of Staphyloccocus aureus in VBNC state. Moreover, it is indicated that EMA-qPCR is an effective method to detect Staphyloccocus aureus in VBNC state under different induction conditions, with a Ct value improvement (cycle threshold) varying from 1. 29 to 8. 56.%为建立金黄色葡萄球菌(Staph yloccocus aureus)活的非可培养(VBNC)状态的诱导条件模型,考察了温度、盐度和酸度3个因素对金黄色葡萄球菌细菌可培养数的影响,通过正交试验优化得到了VBNC状态的诱导条件,同时观察细菌可培养数的变化,建立了DNA结合染料叠氮溴乙锭(EMA)与qPCR技术相结合检测VBNC金黄色葡萄球菌的方法.实验结果表明:细菌可培养数受酸度的影响最大,VBNC状态的最佳诱导条件为菌液在含15% NaCl和0.3%乙酸的营养肉汤培养基中于4℃下培养12d;通过正交试验诱导后的不可培养茵可由EMA-qPCR方法有效检出,其与qPCR法的Ct值(荧光信号达到设定的阈值所经历的循环数)之差在1.29 ~ 8.56之间变化.

  11. Epidemiological features, resistance genes, and clones among community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates detected in northern Spain.

    Science.gov (United States)

    González-Domínguez, María; Seral, Cristina; Sáenz, Yolanda; Salvo, Soledad; Gude, María José; Porres-Osante, Nerea; Torres, Carmen; Castillo, Francisco Javier

    2012-12-01

    Twenty-nine community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates were prospectively selected according to epidemiological criteria among 374 MRSA isolates collected in our laboratory during 2009-2010 in order to determine which community-associated MRSA (CA-MRSA) and healthcare-associated MRSA (HA-MRSA) clones are circulating in the community in northern Spain. PVL genes were detected in 5 strains (17.2%) that belonged to SCCmec type IV or V and to the agr group I (ST8 and ST2050), agr group II (ST121), and agr group III (ST30 and ST852). These strains were isolated from patients with different clinical manifestations such as urinary tract infection, abscess, or pneumonia, and most of them belonged to emergency department patients with no history of visits to General Practitioners (GPs) in the year before the isolation. We considered that the prevalence of CA-MRSA in community-onset isolates was low (17.2%). A high proportion of the CO-MRSA strains (58.6%) were ST125-MRSA-IVc (CC5), responsible for most of the infections caused by HA-MRSA strains in Spain. This endemic clone is also circulating in the community of northern Spain as we could demonstrate in this study. Antimicrobial resistance was found in spa type t067 isolates linked to the presence of ant(4')-Ia and msr(A). Most of the CO-MRSA isolates in this study corresponded to spa types more associated to the hospital environment, suggesting the interchange of genetic lineages of MRSA among community and hospital niches.

  12. Evaluation of {sup 99m}Tc-UBI 29-41 scintigraphy for specific detection of experimental Staphylococcus aureus prosthetic joint infections

    Energy Technology Data Exchange (ETDEWEB)

    Sarda-Mantel, Laure; Meulemans, Alain; Vrigneaud, Jean-Marc; Raguin, Olivier; Lebtahi, Rachida; Guludec, Dominique Le [Universite Denis Diderot-Paris 7, UMR S773, Paris (France); Service de Medecine Nucleaire, AP-HP, Groupe Hospitalier Bichat-Beaujon, Paris (France); INSERM, U773, Paris (France); Saleh-Mghir, Azzam [Universite Versailles-St-Quentin, EA 3647, Garches (France); Welling, Mick M. [Leiden University Medical Center (LUCM), Department of Radiology, Section of Nuclear Medicine, Leiden (Netherlands); Hervatin, Florence [Universite Denis Diderot-Paris 7, UMR S773, Paris (France); CEA, DSV/DRM/SHFJ, Orsay (France); Martet, Genevieve [Universite Denis Diderot-Paris 7, UMR S773, Paris (France); INSERM, U773, Paris (France); Chau, Francoise [Universite Denis Diderot-Paris 7, UMR S773, Paris (France); Universite Denis Diderot Paris 7, EA 3964, Paris (France)

    2007-08-15

    {sup 99m}Tc-UBI 29-41 (UBI), an antimicrobial peptide, specifically targets bacteria. We tested the ability of UBI to discriminate between infected and uninfected prosthetic joints using a rabbit model previously validated. Left knee arthroplasty was performed on 20 New Zealand rabbits, then 10{sup 7} cfu of S. aureus (n = 12) or sterile saline (n = 8) was injected into the joint. On days 9 and 20 after surgery, planar UBI scintigraphy was performed in six infected and four uninfected rabbits, 1 h and 4 h p.i. (150 MBq), on a gamma camera. Operated-to-normal knee activity ratio (ONKR) was calculated on each scintigram. Then, after sacrifice, tissue samples of both knees were counted in a gamma counter. One rabbit injected with sterile saline had cutaneous infection at sacrifice and was excluded from analysis. ONKR was higher in infected than in uninfected animals 4 h p.i. 20 days after surgery: 1.75 {+-} 0.48 vs 1.13 {+-} 0.11, p = 0.04. From 1 h to 4 h p.i., ONKR increased in 9/12 infected and 0/7 uninfected animals. According to UBI uptake intensity and kinetics, scintigraphy was truly positive in all infected cases on day 9 and in four of six infected cases on day 20. It was truly negative in two of three sterile inflamed prosthetic knees on day 9, and in all cases on day 20. Biodistribution studies revealed increased UBI uptake in periprosthetic tissues in all animals 9 days after surgery, and only in infected animals on day 20. In this experimental study, {sup 99m}Tc-UBI 29-41 scintigraphy permitted the early detection of acute prosthetic joint infection, and exclusion of infection in chronic sterile prosthetic joint inflammation. (orig.)

  13. Detection of antibiotic genes and toxic genes of Staphylococcus aureus%院内外环境分离金黄色葡萄球菌的耐药性及毒素基因检测

    Institute of Scientific and Technical Information of China (English)

    袁梦; 袁月明; 陈辉; 黄锐敏; 鞠长燕; 俞慕华

    2012-01-01

    目的 检测医院及环境样中金黄色葡萄球菌(Staphylococcus aureus,SA)耐药基因及毒素基因,了解其分布情况.方法 采用多重聚合酶链反应(multiplex polymeras chain reaction,PCR)技术分别检测耐药基因mecA、femA与毒素基因TSST、PVL.结果 83株金黄色葡萄球菌,仅3株(3.61%)菌检出mecA基因,22株菌(26.50%)检出femA基因,15株菌(18.D7%)同时检出mecA与femA基因.医院样本检出12株耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA),3株甲氧西林耐药凝固酶阴性的金黄色葡萄球菌(methieillin-resistant and coagulase negative Staphylococcus aureus,MRCNS).83株分离自医院及环境样本SA,2株分离自咽拭子的样本检出TSST基因,检出率4.54%(2/44);1株分离自血液的样本检出PVL基因,检出率2.27%(1/44),其余菌株未检出毒素基因.结论 院内样本中,部分菌株携带mecA基因,环境样本未发现携带mecA菌株,两者存在差异.83株SA菌,毒素基因携带率较低.检测耐药、毒素基因携带率,为临床治疗及院内感染控制,食物中毒溯源提供依据.%Objective To detect the antibiotic and toxic genes of Slaphylococcus aureus in hospital and environment. Methods The antibiotic genes mecA, femA and toxic genes TSST, PVL of Staphylococcus aureus were detected separately by multiplex polymerase chain reaction (PCR). Results In all S3 Staphylococcus aureus strains,only 3 strains(3.61%) carried mecA genes,22 strains(26.50%) carried femA genes,15 strains(18.07%) carried mecA and femA genes. 12 MRSA strains and 3 MSCNS strains were detected from samples from hospital,44 strains Staphylococcus aureus were isolated from samples of hospital and environment, strains from samples of Throat swabs carried TSST gene (4.54%, 2/44) and 1 strains from sample of blood carried PVL gene (2.27%, 1/44),all of the rest strains did not carry the two toxic genes. Conclusion Some strains from hospital samples carry mec

  14. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

    Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

  15. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

  16. A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

    Science.gov (United States)

    Mayr, Reinhard; Haider, Michaela; Thünauer, Roland; Haselgrübler, Thomas; Schütz, Gerhard J; Sonnleitner, Alois; Hesse, Jan

    2016-04-15

    Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

  17. Evaluación de cuatro métodos para la detección de Staphylococcus aureus meticilino-resistente de muestras clínicas en un hospital regional Evaluation of four methods for detecting methicillin-resistant Staphylococcus aureus isolates from clinical specimens at a regional hospital in Mexico

    Directory of Open Access Journals (Sweden)

    Gabriel Acosta-Pérez

    2012-02-01

    Full Text Available OBJETIVO: Investigar la prevalencia de Staphylococcus aureus meticilino-resistente (MRSA en aislados clínicos y determinar la concordancia entre los métodos de detección de MRSA en un laboratorio con recursos y personal limitado. MATERIAL Y MÉTODOS: Se analizaron 140 cepas de Staphylococcus aureus aisladas de muestras clínicas de diferentes departamentos mediante pruebas convencionales: producción de β-lactamasa, sensibilidad a oxacilina con MIC-Vitek 2-XL, ChromID MRSA, difusión en agar para discos de 30 μg de cefoxitina, detección de PBP2a y PCR para el gen mecA. Se determinó el índice kappa de Cohen, para evaluar la concordancia entre los diferentes métodos utilizados. RESULTADOS: La prevalencia encontrada fue de 90.7%. La sensibilidad y especificidad para los diferentes métodos de detección fue: difusión en disco para cefoxitina 97 y 92% respectivamente, MIC Vitek 2-XL 97 y 69%, ChromoID MRSA 97 y 85% y detección de PBP2a 98 y 100%. CONCLUSIONES: Todos los métodos son muy buenos para la detección de MRSA; la elección en el uso de cada método dependerá de la infraestructura de cada laboratorio.OBJETIVE: To estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA in clinical isolates and to compare different methods for detection of MRSA in a lab with limited available personnel and resources. MATERIAL AND METHODS: 140 Staphylococcus aureus strains isolated from patients in several departments were assayed for β-lactamase production, MIC-Vitek 2 oxacillin, ChromID MRSA, disk diffusion in agar for cefoxitin 30 μg and PBP2a detection. The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. Cohen´s kappa index was also calculated in order to evaluate the intra assay agreement between the used methods. RESULTS: The found prevalence was 90.7%. Sensitivity and specificity were: disk diffusion for cefoxitin 97 and 92% respectively, MIC Vitek 2-XL 97 and 69%, Chromo

  18. Feasibility of rapid polymerase chain reaction for detection of methicillin-resistant Staphylococcus aureus colonization among emergency department patients with abscesses

    Directory of Open Access Journals (Sweden)

    Pulia MS

    2013-10-01

    Full Text Available Michael S Pulia,1 Mary Calderone,2 Brad Hansen,2 Christine E Stake,3 Mark Cichon,3 Zhanhai Li,4 Nasia Safdar51Division of Emergency Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2Loyola University Stritch School of Medicine, Maywood, IL, USA; 3Department of Emergency Medicine, Loyola University Medical Center, Maywood, IL, USA; 4University of Wisconsin Department of Biostatistics and Medical Informatics, Madison, WI, USA; 5William S Middleton Veterans Affairs Medical Center and the Department of Medicine, Division of Infectious Disease, University of Wisconsin School of Medicine and Public Health, Madison, WI, USAPurpose: In the era of community-associated methicillin-resistant Staphylococcus aureus (MRSA, clinicians face a difficult challenge when selecting antibiotics to treat abscesses. The lack of rapid diagnostics capable of identifying the causative organism often results in suboptimal antibiotic stewardship practices. Although not fully elucidated, the association between MRSA colonization and subsequent infection represents an opportunity to enhance antibiotic selectivity. Our primary objective was to examine the feasibility of utilizing a rapid polymerase chain reaction (PCR system (Cepheid’s GeneXpert® to detect MRSA colonization prior to patient discharge in the emergency department (ED.Methods: This feasibility study was conducted at a tertiary care, urban, academic ED. Patients presenting with a chief complaint related to a potential abscess during daytime hours over an 18-week period were screened for eligibility. Subjects were enrolled into either the PCR swab protocol group (two-thirds or traditional care group (one-third. PCR swabs were obtained from known MRSA carriage sites (nasal, pharyngeal and the superficial aspect of the wound.Results: The two groups were similar in terms of demographics, abscess location, and MRSA history. The PCR results were available prior to patient

  19. Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among Swiss veterinary health care providers: detection of livestock- and healthcare-associated clones.

    Science.gov (United States)

    Wettstein Rosenkranz, K; Rothenanger, E; Brodard, I; Collaud, A; Overesch, G; Bigler, B; Marschall, J; Perreten, V

    2014-07-01

    We screened a total of 340 veterinarians (including general practitioners, small animal practitioners, large animal practitioners, veterinarians working in different veterinary services or industry), and 29 veterinary assistants for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) at the 2012 Swiss veterinary annual meeting. MRSA isolates (n = 14) were detected in 3.8 % (95 % CI 2.1 - 6.3 %) of the participants whereas MRSP was not detected. Large animal practitioners were carriers of livestock-associated MRSA (LA-MRSA) ST398-t011-V (n = 2), ST398-t011-IV (n = 4), and ST398-t034-V (n = 1). On the other hand, participants working with small animals harbored human healthcare-associated MRSA (HCA-MRSA) which belonged to epidemic lineages ST225-t003-II (n = 2), ST225-t014-II (n = 1), ST5-t002-II (n = 2), ST5-t283-IV (n = 1), and ST88-t186-IV (n = 1). HCA-MRSA harbored virulence factors such as enterotoxins, β-hemolysin converting phage and leukocidins. None of the MRSA isolates carried Panton-Valentine leukocidin (PVL). In addition to the methicillin resistance gene mecA, LA-MRSA ST398 isolates generally contained additional antibiotic resistance genes conferring resistance to tetracycline [tet(M) and tet(K)], trimethoprim [dfrK, dfrG], and the aminoglycosides gentamicin and kanamycin [aac(6')-Ie - aph(2')-Ia]. On the other hand, HCA-MRSA ST5 and ST225 mainly contained genes conferring resistance to the macrolide, lincosamide and streptogramin B antibiotics [erm(A)], to spectinomycin [ant(9)-Ia], amikacin and tobramycin [ant(4')-Ia], and to fluoroquinolones [amino acid substitutions in GrlA (S84L) and GyrA (S80F and S81P)]. MRSA carriage may represent an occupational risk and veterinarians should be aware of possible MRSA colonization and potential for developing infection or for transmitting these strains. Professional exposure to animals should be reported upon hospitalization and before medical

  20. Rapid detection of sacbrood virus (SBV by one-step reverse transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Jin-Long Yang

    2012-02-01

    Full Text Available Abstract Background Sacbrood virus (SBV primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

  1. 应用实时荧光PCR技术检测金黄色葡萄球菌肠毒素基因%Application of real-time fluorescent PCR technique in detection of enterotoxin genes in Staphyloccocus aureus

    Institute of Scientific and Technical Information of China (English)

    葛小萍; 陈棋炯; 孙永祥; 傅丹青; 丁水军; 戴城钢

    2012-01-01

    目的:建立一种简便、特异的荧光PCR检测方法,用于金黄色葡萄球菌肠毒素的检测.方法:按金黄色葡萄球菌SEA ~ SEE型肠毒素基因序列设计引物,在普通PCR检测体系中,加入SYBR Green Ⅰ荧光染料,建立荧光PCR检测体系.结果:46株金黄色葡萄球菌中检出22株携带肠毒素基因,阳性率为47.83%.以SEA、SEB检出率较高,分别为31.82%和27.27%;不同来源的分离株携带肠毒素基因的比例不同,同时携带2种及以上毒素基因的菌株占27.27%.结论:荧光PCR检测金黄色葡萄球菌肠毒素的方法具有快速、敏感、特异性高的特点,适用于肠毒素基因的分型与分布的研究,适合基层疾控部门使用.%Objective: Using real - time fluorescent PCR method to detect enterotoxin genes in Staphylococcus aureus (S. aureus) isolates from different sources. Methods: Primers were designed based on enterotoxin SEA ~ SEE genes sequences from 5. aureus and applied with SYBR Green I fluorescent dye to establish real - time PCR system. Results : 22 strains carrying enterotoxin genes were detected from 46 5. aureus strains, and the positive rate of enterotoxin gene in isolates was 47. 83% . The most frequently found genes were SEA (31. 82% ) and SEB (27. 27% ) . The detection rates of enterotoxin gene from different sources were different, and the isolates with more than one toxin gene accounted for 27.27% . Conclusion: SYBR Green I incorporation assay of real - time PCR can be applied in gene typing of S. aureus enterotoxin, which is more rapid, sensitive and specific. The typing and screening method could be promoted to use in grass roots CDC conveniently.

  2. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    Science.gov (United States)

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  3. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV.

  4. Diagnostic accuracy of culture-based and PCR-based detection tests for methicillin-resistant Staphylococcus aureus : a meta-analysis

    NARCIS (Netherlands)

    Luteijn, J. M.; Hubben, G. A. A.; Pechlivanoglou, P.; Bonten, M. J.; Postma, M. J.

    2011-01-01

    P>A systematic review and meta-analysis were performed to determine and compare the sensitivity and specificity of PCR-based and culture-based diagnostic tests for methicillin-resistant Staphylococcus aureus (MRSA). Our analysis included 74 accuracy measurements from 29 publications. Nine tests were

  5. A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome

    Directory of Open Access Journals (Sweden)

    Lei Mei G

    2012-01-01

    Full Text Available Abstract Background Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. Findings We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP and chromosome (attB attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2 that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2 was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site. Conclusions In this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for

  6. Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

    Directory of Open Access Journals (Sweden)

    Ji Yeon Kwon

    2013-09-01

    Full Text Available A detection system based on a multiplex reverse transcription (RT polymerase chain reaction (PCR was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV, lily mottle virus (LMoV, lily symptomless virus (LSV. Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

  7. 金黄色葡萄球菌食物中毒菌株肠毒素检测及基因分析%Enterotoxin detection and gene analysis in food poisoning strains of Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    张金芳; 陈棋炯; 丁水军; 孙永祥; 傅丹青

    2012-01-01

    Objective: To detect Staphylococcus aureus isolated from a food poisoning, then analyse biological characteristics and characterize enterotoxin genotype of the isolates. Methods; Biochemical characteristics and drug sensitive test were conducted by VITEK - 32 automated microbial identification/susceptibility analysis system, then enterotoxin genes were detected by mini - VIDAS and characterized genotype by PCR. Results; 19 strains of S. aureus were isolated from samples. All S. aureus were resistant to penicillin G and can secrete β- lactamase. The enterotoxin gene types were SEA and SEE. Conclusion: The enterotoxin gene type of S. aureus isolates was mixed type of SEA and SEE. That is a better explanation about food poisoning.%目的:了解一起食物中毒中分离到的金黄色葡萄球菌(Staphylococcus aureus,SA)的生物学特性及其肠毒素基因检测.方法:采用VITEK-32全自动微生物鉴定/药敏分析系统对分离到的金黄色葡萄球菌菌株进行生化鉴定和药敏试验,并采用mini-VIDAS全自动荧光酶标免疫仪对菌株进行肠毒素检测,再采用PCR技术对产肠毒素的菌株进行基因分型.结果:总共分离到的19株金黄色葡萄球菌,所有分离株均对青霉素G耐药,均分泌β-内酰胺酶,肠毒素基因分型检测均为SEA与SEE.结论:19株分离株均携带肠毒素,经鉴定均为SEA与SEE肠毒素混合型,由此对食物中毒的原因有一个较圆满的解释.

  8. Rapid and Sensitive Salmonella Typhi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Fan, Fenxia; Yan, Meiying; Du, Pengcheng; Chen, Chen; Kan, Biao

    2015-09-01

    Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

  9. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

    Science.gov (United States)

    Kiedrowski, Megan R; Crosby, Heidi A; Hernandez, Frank J; Malone, Cheryl L; McNamara, James O; Horswill, Alexander R

    2014-01-01

    Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

  10. Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

    Directory of Open Access Journals (Sweden)

    Megan R Kiedrowski

    Full Text Available Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2 that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

  11. Establish and apply a rapid detection system of Staphylococcus aureus enterotoxin genes%金黄色葡萄球菌肠毒素快速分型体系的建立及应用

    Institute of Scientific and Technical Information of China (English)

    王丽; 王冰; 黎桂莲; 林一曼; 贺连华; 石晓路; 吴平芳; 陈妙玲

    2012-01-01

    Objective To establish a new rapid and highly sensitive detection system for Staphylococcus aureus(S. Aureus) ente-rotoxin genes using SYBR green real-time PCR and its application of rapid diagnosis for alimentary toxicosis caused by S. Aureus and related food risks. Methods Primers specific for S. Aureus enterotoxin A,B,C,D and E genes were designed according to Gen-Bank data. PCR systems based on SYBR-Green were constructed for each gene and performed for the detection of A - E genes in 185 wild strains of S. Aureus. Results For the real time PCR assay,the DNA sensitivity achieved 1. 34 - 2. 80 ng/μL and the bacterial solution sensitivity was 46 - 96 CFU/mL. 58. 38% of the 185 wild strains of S. Aureus were with single gene type of A - E genes,and 4. 86% were with tow type of toxin genes, when being detected by the constructed PCR systems. Conclusion A rapid PCR system with high sensitivity and specificity was successfully established,and could be used for the accurate typing of S. Aureus entertoxin genes,for the diagnosis of alimentary toxicosis and for the monitoring of food risks.%目的 建立SYBR-Green荧光PCR体系金黄色葡萄球菌肠毒素快速分型体系,应用于金黄色葡萄球菌食物中毒和食品风险监测快速诊断.方法 根据GenBank公布的金黄色葡萄球菌肠毒素A、B、C、D、E基因的保守序列设计特异性引物,分别建立SYBR-Green荧光PCR体系.应用该系列体系检测185株金黄色葡萄球菌野生株的肠毒素基因A~E型.结果 建立的SYBR-Green荧光PCR系列体系其DNA灵敏度为1.34~2.80 ng/μL,菌液灵敏度为46~96 CFU/mL;用以检测185株金黄色葡萄球菌的肠毒素基因A~E型,携带一种基因型的为58.38%,同时携带两种以上毒素基因占4.86%.结论 建立的荧光PCR反应体系快速、灵敏度高、特异性强,能应用于金黄色葡萄球菌肠毒素基因的准确分型,对食物中毒诊断和食品风险监测很有意义.

  12. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    Science.gov (United States)

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  13. Detection of sasX gene in Staphylococcus aureus and molecular epidemiological characteristics%金黄色葡萄球菌sasX基因的检测及其分子流行特征

    Institute of Scientific and Technical Information of China (English)

    钟一鸣; 丁健生; 刘文恩; 李虹玲; 晏群; 刘清霞

    2016-01-01

    目的:调查医院金黄色葡萄球菌 sasX 基因的携带情况并探究其分子流行特征,为揭示该基因在本地区的流行状况提供依据。方法收集中南大学湘雅医院2012年1-12月临床分离鉴定的金黄色葡萄球菌128株,采用 PCR 检测 sasX 基因,对该基因阳性的菌株采用多重 PCR 检测 SCCmec 分型、PCR 扩增 pvl 毒素基因,并进行多位点序列分型(MLST )和葡萄球菌 A 蛋白序列分析。结果128株金黄色葡萄球菌中共检测出2株 sasX 基因阳性菌株,检出率为1.6%,经头孢西丁鉴定这2株菌株均为耐甲氧西林金黄色葡萄球菌(MRSA),SCCmec Ⅲ分型均为 SCCmec Ⅲ型,pv l 基因均阴性,MLST 分型为 ST239,spa 分型为 t037型。结论首次在湖南地区临床分离的金黄色葡萄球菌中发现存在 sasX 基因,sasX 基因可能是导致医院持续感染的毒力因素之一,应密切关注其流行发展趋势。%OBJECTIVE To investigate the prevalence of sasX gene in Staphylococcus aureus (S .aureus) strains and explore the molecular epidemiological characteristics so as to reveal the prevalence of the gene in this area . METHODS A total of 128 clinical S .aureus isolates were collected from Xiangya Hospital ,Central South Universi-ty from Jan 2012 to Dec 2012 ,then the sasX gene was detected by using PCR ,the SCCmec Ⅲ genotypes were de-tected for the sasX-positive strains with the use of multiple PCR ,the pvl toxin gene was amplified by PCR ,the multilocus sequence typing (MLST) was conducted to determine the sequence types(STs) ,and the staphylococcal protein A sequence typing (spa) of the sasX-positive strains was performed .RESULTS Of the 128 strains of S .au-reus ,2 were detected positive for the sasX gene ,with the detection rate 1 .6% ;both of the 2 strains were identi-fied as methicillin-resistant S .aureus (MRSA) by cefoxitin ,all of the SCCmec Ⅲ genotypes were SCCmec Ⅲ ,and the pvl gene was detected

  14. 食源性金黄色葡萄球菌9种肠毒素基因的多重PCR检测%Multiplex PCR for the detection of the 9 enterotoxin genes in foodborne staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    卫沛楠; 吕国平; 徐保红; 周吉坤

    2013-01-01

    目的 建立多重PCR方法检测食源性金黄色葡萄球菌9种肠毒素基因,了解其在食源性金黄色葡萄球菌中的分布状况.方法 建立多重PCR方法检测144株食源性金黄色葡萄球菌的9种肠毒素基因,对其分布状况进行研究.结果 建立的多重PCR方法特异、高效,9种肠毒素基因在144株食源性金黄色葡萄球菌中的检出率由高到低依次为SEU (25.69%)、SEG (22.22%)、SEM (20.83%)、SEK (20.14%)、SEQ (18.06%)、SEH (15.97%)、SEN (10.42%)、SEJ (8.33%)、SEL(5.56%);52.78%的菌株含有该9种肠毒素基因中的至少1种,34.03%的菌株含有9种肠毒素基因中的两种或两种以上.结论 该多重PCR方法特异性高,快速简便,可用于金黄色葡萄球菌肠毒素基因的分布研究;144株食源性金黄色葡萄球菌中9种肠毒素基因均有检出,其中以SEU、SEG、SEM、SEK检出率较高.%OBJECTIVE To establish a multiplex PCR method for detecting 9 enterotoxin genes in foodborne staphylococcus aureus,and study the distribution of 9 enterotoxin genes in foodborne staphylococcus aureus.METHODS Two multiplex PCR methods were established to detect 9 enterotoxin genes in 144 foodborne staphylococcus aureus strains,and analyzed their distribution.RESULTS The multiplex PCR method was specific,efficient; The detection rates of 9 enterotoxin genes in 144 foodborne staphylococcus aureus strains were in descending order for SEU (25.69%),SEG (22.22%),SEM (20.83%)SEK (20.14%),SEQ (18.06%),SEH (15.97%),SEN (10.42%),SEJ (8.33%),SEL (5.56%); 52.78% of the strains contained at least one gene of these 9 enterotoxin genes,34.03% of the strains contained two or more genes of these 9 enterotoxin genes.CONCLUSION The multiplex PCR method is highly specific,simple and convenient; It can be used for the study of the enterotoxin genes distribution in staphylococcus aureus; All 9 enterotoxin genes have been detected in 144foodborne staphylococcus

  15. Improved detection of Lassa virus by reverse transcription-PCR targeting the 5' region of S RNA.

    Science.gov (United States)

    Olschläger, Stephan; Lelke, Michaela; Emmerich, Petra; Panning, Marcus; Drosten, Christian; Hass, Meike; Asogun, Danny; Ehichioya, Deborah; Omilabu, Sunday; Günther, Stephan

    2010-06-01

    The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.

  16. External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7% were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL. In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection

  17. Detecção da toxina-1 da síndrome do choque tóxico em amostras de Staphylococcus aureus isoladas de mastite bovina Detection of toxic shock syndrome toxin by Staphylococcus aureus strains isolated from bovine mastitis

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    H.F.T. Cardoso

    2000-02-01

    Full Text Available Este trabalho teve por objetivo caracterizar a produção da toxina da síndrome do choque tóxico (TSST-1 e de enterotoxinas estafilocócicas (SE A, B, C e D em 127 amostras de S. aureus, isoladas de amostras de leite proveniente de vacas com mastite no Estado de Minas Gerais, entre 1994 e 1997. A verificação da produção de toxinas foi feita pela técnica de sensibilidade ótima em placa. Das 127 amostras testadas, 60 (47% eram produtoras de TSST-1 e 54 (43% produtoras de SE, 38 amostras produziram SED (30%, 24 SEB (19%, 8 SEC (6% e 4 SEA (3%. Estes resultados trazem preocupações quanto à saúde pública pela alta prevalência de amostras de S. aureus produtoras de TSST-1 e de enterotoxinas em isolamentos a partir de leite de vacas com mastite.A total of 127 strains of Staphylococcus aureus were examined for the production of toxic shock syndrome toxin (TSST-1 and staphylococcal enterotoxins (SE A, B, C and D. The strains were isolated from milk samples from cows with mastitis in dairy herds of Minas Gerais State, Brazil, from 1994 to 1997. The toxins were detected using the optimum-sensitivity plate method. Of 127 isolates, 60 (47% produced TSST-1 and 54 (43% produced SE, 38 (30% produced SED, 24 (19% SEB, 8 (6% SEC and 4 (3% enterotoxin A..

  18. Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors.

    Science.gov (United States)

    Steinhuber, Andrea; Goerke, Christiane; Bayer, Manfred G; Döring, Gerd; Wolz, Christiane

    2003-11-01

    We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

  19. Rapid and sensitive electrochemiluminescence detection of rotavirus by magnetic primer based reverse transcription-polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Zhan Fangfang; Zhou Xiaoming [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Xing Da, E-mail: xingda@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2013-01-25

    Graphical abstract: In this work, we have developed and demonstrated a magnetic primer based RT-PCR assay for ECL detection of rotavirus. In the presence of two functional primers (magnetic primer and TBR-primer) and PCR reagents, cDNA from RT was amplified directly onto MPs during PCR cycles of denaturation, annealing and extension. The resulting MPs-TBR complexes were easily loaded on the electrode surface and produced a concentrated ECL signal. The figure shows the schematic illustration of magnetic primer RT-PCR based ECL assay for rotavirus detection. Highlights: Black-Right-Pointing-Pointer A novel method for detection of rotavirus has been developed. Black-Right-Pointing-Pointer In the presence of magnetic primer, TBR-primer and PCR reagents, cDNA form RT was amplified directly onto MPs. Black-Right-Pointing-Pointer To obtain the best sensing and efficient performance, important parameters associated with the efficiency were investigated carefully. Black-Right-Pointing-Pointer The proposed method will find numerous applications in food safety field and clinical diagnosis. - Abstract: A novel method for detection of rotavirus has been developed by integrating magnetic primer based reverse transcription-polymerase chain reaction (RT-PCR) with electrochemiluminescence (ECL) detection. This is realized by accomplishing RT of rotavirus RNA in traditional way and performing PCR of the resulting cDNA fragment on the surface of magnetic particles (MPs). In order to implement PCR on MPs and achieve rapid ECL detection, forward and reverse primers are bounded to MPs and tris-(2,2 Prime -bipyridyl) ruthenium (TBR), respectively. After RT-PCR amplification, the TBR labels are directly enriched onto the surface of MPs. Then the MPs-TBR complexes can be loaded on the electrode surface and analyzed by magnetic ECL platform without any post-modification or post-incubation process. So some laborious manual operations can be avoided to achieve rapid yet sensitive detection

  20. A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

    Science.gov (United States)

    Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole; Essangui, Estelle; Diesburg, Steven; Mouliom, Abas; Melingui, Bernard; Manga, Jeanne; Donkeu, Christiane; Epote, Annie; Texier, Gaëtan; LaBarre, Paul; Burton, Robert

    2016-01-01

    Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in

  1. DETECTION OF MICROMETASTASES OF LUNG CANCER BY USING LUNX mRNA SPECIFIC REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

    Institute of Scientific and Technical Information of China (English)

    朱广迎; 刘德林; 王绪; 彭猛青; 刘惠; 沈万华; 张海舟; 王伟; 陈杰

    2002-01-01

    Objective: To detect of lung cancer micrometastases in peripheral blood and regional lymphatic nodes by using lunx mRNA specific reverse transcription-polymerase chain reaction (RT-PCR). Methods: RT-PCR was used to detect lunx mRNA in peripheral blood of 26 patients with lung cancer. We also detected 44 regional lymphatic nodes obtained from 25 patients with lung cancer who underwent curative lobectomy. All the 44 regional lymphatic nodes were also examined by histopathology. Micrometastatic tumor cells in the peripheral blood and regional lymphatic nodes were semiquantitatively determined with the ratio of lunx band intensity to the glyceraldehydes-3-phosphate dehydrogenase band intensity. Results: The positive detection rate of lunx mRNA in peripheral blood for non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients were 60% (12/20) and 67% (4/6) respectively. 16 (36.4%) of regional lymphatic nodes from 44 lung cancer patients were positive by RT-PCR while 6 (13.6%) were positive by histopathology (x2=6.06, P=0.014). However, no blood samples and lymphatic nodes from patients with benign pulmonary diseases or normal volunteers were positive for lunx mRNA. The positive detection rate of lunx mRNA in bone marrow of NSCLC amd SCLC patients were 65% (13/20) and 67% (4/6) respectively. Conclusion: RT-PCR amplification of lunx mRNA is an sensitive and specific means to detect early haematogenous and regional lymphatic nodes dissemination of cancer cells for patients with lung cancer.

  2. Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

    Institute of Scientific and Technical Information of China (English)

    HU Xin-xi; LEI Yan; WANG Pei; TANG Lin-fei; HE Chang-zheng; SONG Yong; XIONG Xing-yao; NIE Xian-zhou

    2015-01-01

    A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse tran-scription-polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yel ow dwarf virus (OYDV), Leek yel ow stripe virus (LYSV), Shal ot latent virus (SLV) and al exiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected al exivirus amplicons revealed the al exivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97%sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting al exiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (al exiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. Al primer pairs produced virus-speciifc fragments in both simplex and multiplex formats, thus conifrming the efifcacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples col ected in two geographic locations in Hunan. Al exiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5%of leaf samples, respectively;and mixed infections with two or more viruses accounted for 54%of the garlic samples. The results obtained by mRT-PCR were conifrmed by simplex RT-PCR assays. In conclusion, this newly devel-oped mRT-PCR provides a rapid, sensitive and reliable method for the detection and identiifcation of major garlic viruses.

  3. Transcriptome MicroRNA Profiling of Bovine Mammary Glands Infected with Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Rui Li

    2015-03-01

    Full Text Available MicroRNAs are small non-coding RNA molecules that are important regulators of gene expression at the post-transcriptional level. miRNAs impact the processes of cell proliferation, differentiation and apoptosis. Thus, the regulation of miRNA expression profiles associated with mastitis will be conducive for its control. In this study, Staphylococcus aureus (S. aureus was administered to the mammary gland of Chinese Holstein cows to construct a bacteria-type mastitis model. Total RNA was isolated from bovine mammary gland tissue samples from the S. aureus-induced mastitis group and controls. miRNAs were analyzed using Solexa sequencing and bioinformatics processing for the experimental group and control group. Two miRNA libraries were constructed respectively. A total of 370 known bovine miRNAs and 341 novel mi RNAs were detected for the S. aureus and 358 known bovine miRNAs and 232 novel miRNAs for control groups. A total of 77 miRNAs in the S. aureus group showed significant differences compared to the control group. GO (Gene Ontology analysis showed these target genes were involved in the regulation of cells, binding, etc., while KEGG (Kyoto Encyclopedia of Genes and Genomes analysis showed that these genes were enriched in endocytosis, and olfactory transduction pathways involved in cancer. These results provide an experimental basis to reveal the cause and regulatory mechanism of mastitis and also suggest the potential of miRNAs to serve as biomarkers for the diagnosis of mastitis in dairy cows.

  4. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    Science.gov (United States)

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material.

  5. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Camp, Jeremy V; Nowotny, Norbert

    2016-10-01

    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detectionisothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses.

  6. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A

    2014-03-01

    Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.

  7. Staphylococcus aureus reservoirs during traditional Austrian raw milk cheese production.

    Science.gov (United States)

    Walcher, Georg; Gonano, Monika; Kümmel, Judith; Barker, Gary C; Lebl, Karin; Bereuter, Othmar; Ehling-Schulz, Monika; Wagner, Martin; Stessl, Beatrix

    2014-11-01

    Sampling approaches following the dairy chain, including microbiological hygiene status of critical processing steps and physicochemical parameters, contribute to our understanding of how Staphylococcus aureus contamination risks can be minimised. Such a sampling approach was adopted in this study, together with rapid culture-independent quantification of Staph. aureus to supplement standard microbiological methods. A regional cheese production chain, involving 18 farms, was sampled on two separate occasions. Overall, 51·4% of bulk milk samples were found to be Staph. aureus positive, most of them (34·3%) at the limit of culture-based detection. Staph. aureus positive samples >100 cfu/ml were recorded in 17·1% of bulk milk samples collected mainly during the sampling in November. A higher number of Staph. aureus positive bulk milk samples (94·3%) were detected after applying the culture-independent approach. A concentration effect of Staph. aureus was observed during curd processing. Staph. aureus were not consistently detectable with cultural methods during the late ripening phase, but >100 Staph. aureus cell equivalents (CE)/ml or g were quantifiable by the culture-independent approach until the end of ripening. Enterotoxin gene PCR and pulsed-field gel electrophoresis (PFGE) typing provided evidence that livestock adapted strains of Staph. aureus mostly dominate the post processing level and substantiates the belief that animal hygiene plays a pivotal role in minimising the risk of Staph. aureus associated contamination in cheese making. Therefore, the actual data strongly support the need for additional sampling activities and recording of physicochemical parameters during semi-hard cheese-making and cheese ripening, to estimate the risk of Staph. aureus contamination before consumption.

  8. Performance of reversed transcription loop-mediated isothermal amplification technique detecting EV71: a systematic review with meta-analysis.

    Science.gov (United States)

    Lei, Xiaoying; Wen, Hongling; Zhao, Li; Yu, Xuejie

    2014-04-01

    Human enterovirus 71 (EV71) is the major etiological agent of hand, foot and mouth disease (HFMD), which is a common infectious disease in young children. Studies in the past have shown that reversed transcription loop-mediated isothermal amplification (RT-LAMP) was a rapid approach for the detection of EV71 in HFMD. This meta-analysis study is to evaluate the diagnostic role of RT-LAMP in detecting EV71 infection. A comprehensive literature research of PubMed, Embase, Wan Fang Data, and Chinese National Knowledge Infrastructure databases was conducted on articles aiming at the diagnostic performance of RT-LAMP in EV71 detection published before February 10, 2014. Data from selected studies were pooled to yield the summary sensitivity, specificity, positive and negative likelihood ratio (PLR, NLR), diagnostic odds ratio (DOR), and receiver operating characteristic (SROC) curve by using STATA VERSION 12.0 software. Ten studies including a total of 907 clinical samples were of high quality in this meta-analysis. Overall, the pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve was 0.99 (0.97, 1.00), 0.97 (0.94, 1.00), 5.90 (95% CI: 3.90-8.94), 0.20 (95% CI: 0.14-0.29), and 1.00 (95% CI: 0.99-1.00), respectively. The univariate analysis of potential variables showed some changes in the diagnostic performance, but none of the differences reached statistical significance. Despite inter-study variability, the test performance of RT-LAMP was consistent with real-time RT-PCR in detecting EV71. This meta-analysis suggests that RT-LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting EV71.

  9. Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Oloniniyi, Olamide K; Kurosaki, Yohei; Miyamoto, Hiroko; Takada, Ayato; Yasuda, Jiro

    2017-03-26

    Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

  10. Staphylococcus aureus Transcriptome Architecture

    DEFF Research Database (Denmark)

    Mäder, Ulrike; Nicolas, Pierre; Depke, Maren;

    2016-01-01

    to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19...... antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination...

  11. Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates.

    Directory of Open Access Journals (Sweden)

    Jiazhang Qiu

    Full Text Available BACKGROUND: Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in Staphylococcus aureus. However, there are no data on the effect of thymol on the production of other exotoxins (e.g., alpha-hemolysin and enterotoxins by S. aureus. METHODOLOGY/PRINCIPAL FINDINGS: Secretion of alpha-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant S. aureus isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF release assays were performed to elucidate the biological relevance of changes in alpha-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of hla, sea, and seb (the genes encoding alpha-hemolysin, SEA and SEB, respectively was analyzed by quantitative RT-PCR. Thymol inhibited transcription of hla, sea and seb in S. aureus, resulting in a reduction of alpha-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities. CONCLUSIONS/SIGNIFICANCE: Subinhibitory concentrations of thymol decreased the production of alpha-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of S. aureus infections when used in combination with beta-lactams and glycopeptide antibiotics, which induce expression of alpha-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors.

  12. Detection of vanC 1 gene transcription in vancomycin-susceptible Enterococcus faecalis

    Directory of Open Access Journals (Sweden)

    Tiane Martin de Moura

    2013-06-01

    Full Text Available Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

  13. Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis.

    Science.gov (United States)

    Moura, Tiane Martin de; Cassenego, Ana Paula Vaz; Campos, Fabrício Souza; Ribeiro, Andrea Machado Leal; Franco, Ana Cláudia; d'Azevedo, Pedro Alves; Frazzon, Jeverson; Frazzon, Ana Paula Guedes

    2013-06-01

    Here we report the presence and expression levels of the vanC1 and vanC(2/3) genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC1 and vanC(2/3) genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

  14. Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis

    Science.gov (United States)

    de Moura, Tiane Martin; Cassenego, Ana Paula Vaz; Campos, Fabrício Souza; Ribeiro, Andrea Machado Leal; Franco, Ana Cláudia; d'Azevedo, Pedro Alves; Frazzon, Jeverson; Frazzon, Ana Paula Guedes

    2013-01-01

    Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis. PMID:23828012

  15. Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Directory of Open Access Journals (Sweden)

    Wang Xiang

    2012-07-01

    Full Text Available Abstract Background Human metapneumovirus (hMPV is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP assay for the detection of hMPV and applied to the clinical samples. Results In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB, and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3, two inner primers (FIP, BIP and two loop primers (LF and LB, were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV, human respiratory syncytial Virus (RSV, or influenza virus A/PR/8/34 (H1N1. The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1% were conformed as hMPV positive by RT-LAMP, but 18 (10.2% positive by RT-PCR. Conclusion Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.

  16. A one-step reverse transcription loop-mediated isothermal amplification for detection and discrimination of infectious bursal disease virus

    Directory of Open Access Journals (Sweden)

    Qi Xiaole

    2011-03-01

    Full Text Available Abstract Background Infectious bursal disease (IBD is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV. It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP method for the detection and discrimination of IBDV. Results In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR. Conclusion RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.

  17. Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.

    Science.gov (United States)

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-10-21

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay's specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  18. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2014-10-01

    Full Text Available Papaya ringspot virus (PRSV, Papaya leaf distortion mosaic virus (PLDMV, and Papaya mosaic virus (PapMV produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%, 93/341 (27.3%, and 3/341 (0.9%, for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3% of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  19. Aphids preserved in propylene glycol can be used for reverse transcription-polymerase chain reaction detection of Potato virus Y.

    Science.gov (United States)

    Nie, Xianzhou; Pelletier, Yvan; Mason, Nicola; Dilworth, Andrea; Giguère, Marie-Andrée

    2011-08-01

    The effectiveness of propylene glycol on the retention of RNA target of Potato virus Y (PVY), an aphid stylet-borne virus, in Myzus persicae was investigated in comparison to ethanol and liquid nitrogen/-80°C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the PVY targets from the propylene glycol/ethanol/liquid nitrogen preserved single aphids after a 5min acquisition period from infected potato plants. In the liquid nitrogen/-80°C and 70% ethanol treatments, 55.6% and 38.8% aphids tested PVY-positive, respectively. In the 0-75% propylene glycol treatments, 12.2-44.7% aphids tested PVY-positive. The lowest detection rate was in the 0% (positive rate, 15.2%) and the 10% propylene glycol (positive rate, 12.2%). As the propylene glycol concentration increased to 25%, 29.8% aphids tested positive. A high PVY-positive rate was also found in 35-75% propylene glycol treatments at 44.7% (35% propylene glycol), 36.7% (50% propylene glycol) and 34.8% (75% propylene glycol), which is comparable to the rate shown in 70% ethanol. No significant difference in the positive detection rate was observed in aphids preserved in 50% propylene glycol at room temperature for 2, 4 and 10 days. These results demonstrate that propylene glycol at 25-75% can retain PVY targets effectively in aphids for an extended time period, and thus can be used in aphid traps to preserve viruliferous aphids for later RT-PCR detection of PVY.

  20. Staphylococcus aureus resistente a la meticilina (SARM)

    Centers for Disease Control (CDC) Podcasts

    2007-10-22

    Datos importantes sobre las infecciones por SARM en Estados Unidos, en las escuelas y los entornos médicos. (Title: Methicillin-resistant Staphylococcus aureus (MRSA)Created: 10/2007).  Created: 10/22/2007 by National Center for Preparedness, Detection, and Control of Infectious Diseases.   Date Released: 11/9/2007.

  1. Castanea sativa (European Chestnut Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance.

    Directory of Open Access Journals (Sweden)

    Cassandra L Quave

    Full Text Available The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes, against all Staphylococcus aureus accessory gene regulator (agr alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 μg mL-1, as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.

  2. Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance

    Science.gov (United States)

    Quave, Cassandra L.; Lyles, James T.; Kavanaugh, Jeffery S.; Nelson, Kate; Parlet, Corey P.; Crosby, Heidi A.; Heilmann, Kristopher P.; Horswill, Alexander R.

    2015-01-01

    The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56–25 μg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract’s lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies. PMID:26295163

  3. 牛源耐甲氧西林金黄色葡萄球菌的检测%Detection of Methicillin-resistant Staphylococcus aureus Strains Isolated from Bovine Mastitis

    Institute of Scientific and Technical Information of China (English)

    苏洋; 蒲万霞; 邓海平; 李春慧; 梁红雁; 陈智华

    2013-01-01

    本研究旨在了解甘肃地区奶牛乳房炎金黄色葡萄球菌的耐药性和耐甲氧西林金黄色葡萄球菌的感染情况,为奶牛乳房炎的防制提供理论依据.采用KB纸片扩散法,检测17株金黄色葡萄球菌对8种不同抗菌药物的敏感性;再用琼脂稀释法检测了苯唑西林、万古霉素对金黄色葡萄球菌的最小抑菌浓度(MICs);头孢西丁纸片扩散法和PCR扩增特异性mecA耐药基因对所有受试菌株进行全面的耐甲氧西林金黄色葡萄球菌检测.结果表明,菌株对青霉素、磺胺异恶唑具有较强抗性,而对环丙沙星、头孢唑啉、万古霉素和苯唑西林全敏感;头孢西丁纸片扩散法未能检测出表型为MRSA的阳性菌株,而PCR方法却检测出8株mecA基因阳性菌株,且这些菌株的苯唑西林MIC均小于2μg/mL.菌株的耐药情况较严重,对甲氧西林敏感而携带mecA基因的菌株高频存在于被调查地区的奶牛场中.%The aim of present study was to investigate the antimicrobial resistance, and the prevalence of methicillin-resistant Staphylococcus aureus (S. aureus) isolated from bovine mastitis in Gansu province, to provide credible theory evidence for prevention and treatment on bovine mastitis. Eight commonly used antimicrobial agents were used for determining antimicrobial susceptibility of 17 total S. aureus strains by disk diffusion method. Agar screen method was used for determining the oxacil-lin and vancomycin minimum inhibitory concentration value as well. Disk diffusion method using the cephamycin antibiotics ce-foxitin and detection of mecA gene by PCR assay were performed to detect the presence of MRSA. Most of strains showed a high resistance for penicillin and sulfafurazole, yet keeping complete sensitivity for ciprofloxacin, cefazolin, vancomycin and ox-acillin. None MRS A isolate was identified by the phenotypic detection method, but eight MRSA isolates with the MIC of oxac-illin lower than 2 μg/mL were

  4. Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1 is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg mice that carry HRE/ODD-luciferase (HOL gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

  5. Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity.

    Science.gov (United States)

    Tang, Yue; Xiang, Wei; Hawkins, Steve A C; Kretzschmar, Hans A; Windl, Otto

    2009-09-01

    Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

  6. Design and evaluation of reverse transcription nested PCR primers for the detection of betanodavirus in finfish.

    Science.gov (United States)

    Rajan, J Joseph Sahaya; Praveena, P Ezhil; Bhuvaneswari, T; Jithendran, K P

    2016-06-01

    Viral encephalopathy and retinopathy otherwise known as viral nervous necrosis (VNN) is a neuropathological condition affecting more than 50 fish species worldwide, mostly marine. Different PCR protocols with specific primers were reported from many countries for confirmation of VNN in fishes. In the present study, two pairs of primers were designed and evaluated for the diagnosis of clinical and subclinical cases of infections from field. These primers designated as BARL-F1/BARL-R1 amplified a 902 bp product in the variable region (T4) of the coat protein gene by first step PCR. Nested PCR primers BARL-F2/BARL-R2 amplified a fragment of 313 bp. The results were comparable with other commonly used primer sets such as F2/R3 and RG668f/RG919r primers. These new primers could detect betanodavirus in standard reference samples containing low, moderate and high viral load. Known positive and negative control samples of fish also revealed a predictive value of 100 % by RT-PCR diagnosis.

  7. Toxin-Antitoxin Systems of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Christopher F. Schuster

    2016-05-01

    Full Text Available Toxin-antitoxin (TA systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  8. Toxin-Antitoxin Systems of Staphylococcus aureus.

    Science.gov (United States)

    Schuster, Christopher F; Bertram, Ralph

    2016-05-05

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  9. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    Science.gov (United States)

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  10. The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals.

    Science.gov (United States)

    Geiger, Tobias; Goerke, Christiane; Mainiero, Markus; Kraus, Dirk; Wolz, Christiane

    2008-05-01

    The two-component system SaeRS of Staphylococcus aureus is closely involved in the regulation of major virulence factors. However, little is known about the signals leading to saeRS activation. A total of four overlapping transcripts (T1 to T4) from three different transcription starting points are expressed in the sae operon. We used a beta-galactosidase reporter assay to characterize the putative promoter regions within the saeRS upstream region. The main transcript T2 is probably generated by endoribonucleolytic processing of the T1 transcript. Only two distinct promoter elements (P1 and P3) could be detected within the saeRS upstream region. The P3 promoter, upstream of saeRS, generates the T3 transcript, includes a cis-acting enhancer element and is repressed by saeRS. The most distal P1 promoter is strongly autoregulated, activated by agr, and repressed by sigma factor B. In strain Newman a mutation within the histidine kinase SaeS leads to a constitutively activated sae system. Evaluation of different external signals revealed that the P1 promoter in strain ISP479R and strain UAMS-1 is inhibited by low pH and high NaCl concentrations but activated by hydrogen peroxide. The most prominent induction of P1 was observed at subinhibitory concentrations of alpha-defensins in various S. aureus strains, with the exception of strain ISP479R and strain COL. P1 was not activated by the antimicrobial peptides LL37 and daptomycin. In summary, the results indicate that the sensor molecule SaeS is activated by alteration within the membrane allowing the pathogen to react to phagocytosis related effector molecules.

  11. Antimicrobial Resistance Analysis and Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)Among Staphylococcus aureus Strains Isolated from Bovine Mastitis%牛源金黄色葡萄球菌的耐药性及耐甲氧西林金黄色葡萄球菌的检测

    Institute of Scientific and Technical Information of China (English)

    苏洋; 蒲万霞; 陈智华; 邓海平

    2012-01-01

    [目的]了解内蒙古地区奶牛乳房炎金黄色葡萄球菌耐药性和耐甲氧西林金黄色葡萄球菌(MRSA)感染的情况,为奶牛乳房炎的防治提供理论依据.[方法]采用K-B纸片扩散法,检测分离自内蒙古地区38株金黄色葡萄球菌对17种药物的敏感性,同时用琼脂稀释法检测苯唑西林、万古霉素对金黄色葡萄球菌的最小抑菌浓度(MIC);再用头孢西丁、苯唑西林纸片扩散法、苯唑西林盐琼脂筛选法和PCR方法扩增mecA耐药基因对分离菌株进行全面MRSA检测.[结果]分离菌株对每种抗生素都有不同程度抗性,对氨苄西林、头孢拉丁、青霉素、复方新诺明、新生霉素和链霉素的耐药率都高于45%,而对氧氟沙星、丁胺卡那霉素、万古霉素、环丙沙星、庆大霉素和头孢唑林的敏感性高于90%,2株细菌的万古霉素MIC≥16 μg.mL-1;其中8株细菌的苯唑西林MIC≥8μg·mL-1,而其它菌株的苯唑西林MIC≤2μg·mL-1,分离菌株多重耐药情况严重,耐受3种及3种以上药物的菌株占84.21%,其中4株细菌能同时耐受9种不同抗菌药物;16 (42.11%)株细菌被检测携带mecA耐药基因,而仅有其中7株的苯唑西林MIC≥4 μg·mL-1;头孢西丁、苯唑西林纸片扩散法和苯唑西林盐琼脂筛选法分别检出7株、10株和7株表型为MRSA的菌株.[结论]分离菌株的耐药性和多重耐药现象较为严重,被调查地区奶牛场中已经存在MRSA和OS-MRSA感染情况,且感染率高.%[Objective] The aim of the study is to investigate the antimicrobial resistance of S. aureus (Science) and to detect the presence of methicillin-resistant S. aureus (MRSA) among S. aureus strains isolated from bovine mastitis in Inner Mongolia, and to provide credible theory evidence for prevention and treatment of bovine mastitis. [Method] K-B.disk diffusion method was used to test drug sensitivity of 38 total strains of S.aureus to 17 commonly used antibiotics

  12. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette;

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains...

  13. Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus

    Science.gov (United States)

    Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to the infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for a sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to th...

  14. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Science.gov (United States)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  15. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

    Science.gov (United States)

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

  16. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Science.gov (United States)

    This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...

  17. Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae captured in Manaus, Amazonas

    Directory of Open Access Journals (Sweden)

    Valéria CS Pinheiro

    2005-12-01

    Full Text Available The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1% were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

  18. Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

    NARCIS (Netherlands)

    Ahmadi, S.; Almasi, A.M.; Fatehi, F.; Struik, P.C.; Moradi, A.

    2013-01-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll vi

  19. 荧光原位杂交法(FISH)快速检测尿液中金黄色葡萄球菌%Fluorescence in Situ Hybridization Rapidly Detects Staphylococcus Aureus in Urinary Tract Infection Samples

    Institute of Scientific and Technical Information of China (English)

    吴青; 李艳; 汪明; 顾剑; 胡慧霞; 徐万洲; 韦传东; 吴泽刚

    2011-01-01

    目的 利用荧光原位杂交法快速检测尿液中的金黄色葡萄球菌,筛查金黄色葡萄球菌所致的尿路感染.方法 针对金黄色葡萄球菌16sRNA设计荧光标记的核苷酸探针,利用荧光原位杂交技术(FISH)对132例疑似尿路感染患者中段尿标本进行检测;同时进行中段尿培养.结果 荧光原位杂交法检测阳性的有9例,与中段尿培养比较,其敏感度为100.0%,特异度为99.2%,阳性预期值为90.0%,阴性预期值为100.0%.结论 荧光原位杂交能快速检测尿液中的金黄色葡萄球菌,对金黄色葡萄球菌所致的尿路感染进行快速诊断.%Objective The rapid detection of Staphylococcus aureus in urine by fluorescence in situ hybridization (FISH)method. Screening urinary tract infection (UTIs) caused by Staphylococcus aureus. Methods Probes that were specific for Staphylococcus aureus were designed based on the conserved 16 s RNA sequences. Detected a total of 132 urine samples using FISH method. And all of tbese samples tested via traditional culturing techniques. Results 9 of these samples tested positive for a UTI via FISH analysis. Compared to conventional methods of bacterial identification,the FISH method had a sensitivity of 100.0% ,specificity of 99. 2%,positive expected value of 90. 0% and negative expected value of 100. 0%. Conclusion FISH had the potential to become an extremely useful diagnostic tool for UTIs because it has a quick turnaround time and high accuracy.

  20. Loss of variation of state detected in soybean metabolic and human myelomonocytic leukaemia cell transcriptional networks under external stimuli.

    Science.gov (United States)

    Sakata, Katsumi; Saito, Toshiyuki; Ohyanagi, Hajime; Okumura, Jun; Ishige, Kentaro; Suzuki, Harukazu; Nakamura, Takuji; Komatsu, Setsuko

    2016-10-24

    Soybean (Glycine max) is sensitive to flooding stress, and flood damage at the seedling stage is a barrier to growth. We constructed two mathematical models of the soybean metabolic network, a control model and a flooded model, from metabolic profiles in soybean plants. We simulated the metabolic profiles with perturbations before and after the flooding stimulus using the two models. We measured the variation of state that the system could maintain from a state-space description of the simulated profiles. The results showed a loss of variation of state during the flooding response in the soybean plants. Loss of variation of state was also observed in a human myelomonocytic leukaemia cell transcriptional network in response to a phorbol-ester stimulus. Thus, we detected a loss of variation of state under external stimuli in two biological systems, regardless of the regulation and stimulus types. Our results suggest that a loss of robustness may occur concurrently with the loss of variation of state in biological systems. We describe the possible applications of the quantity of variation of state in plant genetic engineering and cell biology. Finally, we present a hypothetical "external stimulus-induced information loss" model of biological systems.

  1. Loss of variation of state detected in soybean metabolic and human myelomonocytic leukaemia cell transcriptional networks under external stimuli

    KAUST Repository

    Sakata, Katsumi

    2016-10-24

    Soybean (Glycine max) is sensitive to flooding stress, and flood damage at the seedling stage is a barrier to growth. We constructed two mathematical models of the soybean metabolic network, a control model and a flooded model, from metabolic profiles in soybean plants. We simulated the metabolic profiles with perturbations before and after the flooding stimulus using the two models. We measured the variation of state that the system could maintain from a state–space description of the simulated profiles. The results showed a loss of variation of state during the flooding response in the soybean plants. Loss of variation of state was also observed in a human myelomonocytic leukaemia cell transcriptional network in response to a phorbol-ester stimulus. Thus, we detected a loss of variation of state under external stimuli in two biological systems, regardless of the regulation and stimulus types. Our results suggest that a loss of robustness may occur concurrently with the loss of variation of state in biological systems. We describe the possible applications of the quantity of variation of state in plant genetic engineering and cell biology. Finally, we present a hypothetical “external stimulus-induced information loss” model of biological systems.

  2. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV.

    Science.gov (United States)

    Lee, Se Hee; Baek, Yun Hee; Kim, Yang-Hoon; Choi, Young-Ki; Song, Min-Suk; Ahn, Ji-Young

    2016-01-01

    Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.

  3. Duplex Identification of Staphylococcus aureus by Aptamer and Gold Nanoparticles.

    Science.gov (United States)

    Chang, Tianjun; Wang, Libo; Zhao, Kexu; Ge, Yu; He, Meng; Li, Gang

    2016-06-01

    Staphylococcus aureus is the top common pathogen causing infections and food poisoning. Identification of S. aureus is crucial for the disease diagnosis and regulation of food hygiene. Herein, we report an aptamer-AuNPs based method for duplex identification of S. aureus. Using AuNPs as an indicator, SA23, an aptamer against S. aureus, can well identify its target from Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, we find citrate-coated AuNPs can strongly bind to S. aureus, but not bind to Salmonella enterica and Proteus mirabilis, which leads to different color changes in salt solution. This colorimetric response is capable of distinguishing S. aureus from S. enteritidis and P. mirabilis. Thus, using the aptasensor and AuNPs together, S. aureus can be accurately identified from the common pathogens. This duplex identification system is a promising platform for simple visual identification of S. aureus. Additionally, in the aptasensing process, bacteria are incubated with aptamers and then be removed before the aptamers adding to AuNPs, which may avoid the interactions between bacteria and AuNPs. This strategy can be potentially applied in principle to detect other cells by AuNPs-based aptasensors.

  4. SAMMD: Staphylococcus aureus Microarray Meta-Database

    Directory of Open Access Journals (Sweden)

    Elasri Mohamed O

    2007-10-01

    Full Text Available Abstract Background Staphylococcus aureus is an important human pathogen, causing a wide variety of diseases ranging from superficial skin infections to severe life threatening infections. S. aureus is one of the leading causes of nosocomial infections. Its ability to resist multiple antibiotics poses a growing public health problem. In order to understand the mechanism of pathogenesis of S. aureus, several global expression profiles have been developed. These transcriptional profiles included regulatory mutants of S. aureus and growth of wild type under different growth conditions. The abundance of these profiles has generated a large amount of data without a uniform annotation system to comprehensively examine them. We report the development of the Staphylococcus aureus Microarray meta-database (SAMMD which includes data from all the published transcriptional profiles. SAMMD is a web-accessible database that helps users to perform a variety of analysis against and within the existing transcriptional profiles. Description SAMMD is a relational database that uses MySQL as the back end and PHP/JavaScript/DHTML as the front end. The database is normalized and consists of five tables, which holds information about gene annotations, regulated gene lists, experimental details, references, and other details. SAMMD data is collected from the peer-reviewed published articles. Data extraction and conversion was done using perl scripts while data entry was done through phpMyAdmin tool. The database is accessible via a web interface that contains several features such as a simple search by ORF ID, gene name, gene product name, advanced search using gene lists, comparing among datasets, browsing, downloading, statistics, and help. The database is licensed under General Public License (GPL. Conclusion SAMMD is hosted and available at http://www.bioinformatics.org/sammd/. Currently there are over 9500 entries for regulated genes, from 67 microarray

  5. Isorhamnetin Attenuates Staphylococcus aureus-Induced Lung Cell Injury by Inhibiting Alpha-Hemolysin Expression.

    Science.gov (United States)

    Jiang, Lanxiang; Li, Hongen; Wang, Laiying; Song, Zexin; Shi, Lei; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

    2016-03-01

    Staphylococcus aureus, like other gram-positive pathogens, has evolved a large repertoire of virulence factors as a powerful weapon to subvert the host immune system, among which alpha-hemolysin (Hla), a secreted pore-forming cytotoxin, plays a preeminent role. We observed a concentration-dependent reduction in Hla production by S. aureus in the presence of sub-inhibitory concentrations of isorhamnetin, a flavonoid from the fruits of Hippophae rhamnoides L., which has little antibacterial activity. We further evaluate the effect of isorhamnetin on the transcription of the Hla-encoding gene hla and RNAIII, an effector molecule in the agr system. Isorhamnetin significantly down-regulated RNAIII expression and subsequently inhibited hla transcription. In a co-culture of S. aureus and lung cells, topical isorhamnetin treatment protected against S. aureus-induced cell injury. Isorhamnetin may represent a leading compound for the development of anti-virulence drugs against S. aureus infections.

  6. Quantitative detection of Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae in patients with new influenza A (H1N1)/2009 and influenza A/2010 virus infection.

    Science.gov (United States)

    Safaeyan, Firouzeh; Nahaei, Mohammad Reza; Seifi, Sirus Jedary; Kafil, Hossein Samadi; Sadeghi, Javid

    2015-01-01

    Einleitung: Die Virusgrippe ist eine saisonale Infektionskrankheit, die mit ausgeprägterer Morbidität und Mortalität einhergeht. In den USA werden jährlich mehr als 35.000 Todesfälle und 200.000 Krankenhausbehandlungen erfasst. Die mit der viralen Primärinfektion assoziierte bakterielle Superinfektion oder Ko-Infektion verursacht schwere Krankheitsverläufe speziell bei Hochrisikogruppen wie alten Menschen und Kleinkindern. Zielsetzung: Die Zielsetzung der Studie bestand in der quantitativen Bestimmung von S. aureus, S. pneumoniae and H. influenzae bei Patienten mit saisonaler Influenza A bzw. pandemischer Influenza A (H1N1) und Symptomen respiratorischer Infektionen, im Vergleich zu je einer Kontrollgruppe. Methode: Insgesamt wurden von April 2009 bis April 2010 625 Patienten mit Verdacht auf eine respiratorische Infektion untersucht, davon 58 Patienten mit Nachweis von Influenza A (H1N1). Vom November 2010 bis zum Februar 2011 wurden 158 Patienten mit respiratorischen Symptomen auf das Vorkommen der saisonalen Influenza A untersucht, davon erwiesen sich 25 als positiv. Zur Ermittlung der bakteriellen Kolonisation wurden parallel 62 gesunde Personen untersucht (Kontrollgruppe). Bei der verwendeten Real-time PCR wurde als Cutoff zur Unterscheidung von Koloniation und Infektion im Respirationstrakt 10(4) CFU/ml eingeführt. Ergebnisse:S. aureus, S. pneumoniae und H. influenzae wurden bei 12%, 26% bzw. 33% der Patienten mit Nachweis von Influenzavirus A (H1N1) gefunden; die Häufigkeit in der Kontrollgruppe betrug 9%, 19% bzw. 31%. Bei der saisonalen Influenza A waren bei 12%, 24% bzw. 32% die Erreger nachweisbar, in der parallelen Kontrollgruppe bei 5%, 11% bzw. 10%. Schlussfolgerung: Die Ergebnisse zeigen, dass der Serotyp der pandemischen Influenza A (H1N1) die Inzidenz der bakteriellen Superinfektion für die drei untersuchten Bakterienspecies nicht erhöht hat. Die quantitative Detektion einer sekundären bakteriellen Infektion mittels Real-time PCR ist

  7. Detection of HSP mRNA Transcription in Transport Stressed Pigs by Fluorescence Quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Yu-bao; BAO En-dong; WANG Zhi-liang; ZHAO Ru-qian

    2007-01-01

    The RNA transcripted in vitro was used as the standard quantitative template to make the standard curve and establish the fluorescence quantitative RT-PCR (FQ-PCR) method. By means of FQ-PCR, the transcription changes of HSP70 and HSPg0 mRNA in the livers and hearts of transport stressed pigs were studied. The level of HSP70 mRNA transcription increased continuously from the beginning of transportation. The inductions of HSP70 mRNA transcription in the livers and hearts of 10 h transport stressed pigs were 2.5 and 4.1 times higher than that of the un-transport stressed pigs (P<0.01).However, the transcription levels of HSPg0 mRNA in the livers and hearts decreased with the transport stress.

  8. THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

    Science.gov (United States)

    A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...

  9. Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    April M Sapp

    Full Text Available Nitric oxide (NO is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and

  10. 金黄色葡萄球菌活的非可培养状态复苏及PMA-qPCR检测%Resuscitation and PMA-qPCR Detection of Staphylococcus aureus in the Viable but Non-culturable State

    Institute of Scientific and Technical Information of China (English)

    田聪; 余以刚; 肖性龙; 吴晖; 赖富饶; 杨锡洪

    2013-01-01

    In order to study the resuscitation of Staphylococcus aureus in the viable but non-culturable state (VBNC),three factors (temperature,salt concentrations and pH) were investigated to induce VBNC Staphylococcus aureus.Four different resuscitation methods were attempted and a new detection method using qPCR and PMA in combination with qPCR was established to detect Staphyloccocus aureus.The results showed that the VBNC samples could resuscitate in 48 h after disposed in 8% Tween80.In addition,PMA-qPCR was an effective method for detection ofVBNC Staphyloccocus aureus,which efficiently overcome the shortcomings of plate counts.%为研究金黄色葡萄球菌活的非可培养(VBNC)状态的复苏问题,采取温度、盐度和酸度3个因素复合诱导制备VBNC菌,尝试四种复苏方法,并以荧光定量PCR和PMA-qPCR技术结合的方法进行检测.结果证明:8%无菌Tween80可以使VBNC状态金黄色葡萄球菌48 h后复苏,同时PMA-qPCR能够有效检出VBNC状态金黄色葡萄球菌,克服传统平板计数法对于VBNC菌的漏检.

  11. Staphylococcus aureus bacteremia.

    Science.gov (United States)

    Jensen, Allan Garlik

    2003-11-01

    Staphylococcus aureus bacteremia (SAB) is still associated with a high mortality, and knowledge on risk factors and the clinical and the therapeutic aspects of SAB is still limited. This thesis focuses on the clinical aspects of SAB and its metastatic infections. In a study of all patients with bacteremia in Copenhagen County October 1992 through April 1993 (study I) we emphasized previous findings, that S. aureus is one of the most frequent pathogens in bacteremia, and in a case control study also in Copenhagen County 1994-95 (study II) we demonstrated, that not only an inserted central venous catheter and nasal S. aureus carriage but also hyponatremia and anemia are important risk factors for hospital-acquired SAB (study II). Studies on the treatment of SAB have pointed out, that the eradication of a primary is important, but there are only limited clinical studies dealing with antibiotic treatment. By logistic regression analysis, we were able to demonstrate that focus eradication is essential, but also that treatment with dicloxacillin 1 g x 4 or 2 g x 3 are superior to 1 g x 3 (studie III), indicating that the time for serum concentration above the Minimal Inhibitory Concentration (MIC) for the bacteria plays a role in the outcome of SAB treatment. S. aureus osteomyelitis secondary to SAB is frequently observed. No other countries, however, have a centralized registration, which make it possible to evaluate a large number of these patients. Since 1960, The Staphylococcal Laboratory, Statens Serum Institut in Copenhagen, has registrated selected clinical informations from nearly all patients with positive blood cultures of S. aureus. Based on this registration, we were able to show an increased number of S. aureus osteomyelitis among older patients and a decreased number of S. aureus osteomyelitis of femur and tibia among younger infants in the period 1980-90 (study IV). By reviewing the records of a large number of patients with vertebral S. aureus

  12. Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

    2014-09-01

    The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers.

  13. Methicillin-Resistant Staphylococcus aureus (MRSA) Detection: Comparison of Two Molecular Methods (IDI-MRSA PCR Assay and GenoType MRSA Direct PCR Assay) with Three Selective MRSA Agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for Use with Infection-Control Swabs▿

    OpenAIRE

    van Hal, S. J.; Stark, D.; Lockwood, B.; Marriott, D; Harkness, J.

    2007-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected an...

  14. Detecting of viable Staphylococcus aureus by loop-mediated isothermal amplification coupling with propidium monoazide in dairy products%PMA—LAMP方法检测灭菌乳中金黄色葡萄球菌的研究

    Institute of Scientific and Technical Information of China (English)

    胡惠秩; 满朝新; 董鑫悦; 刘珊珊; 薛玉清; 杨士芹; 谢鲲昊; 刘颖; 卢雁; 姜毓君

    2012-01-01

    In recent years, the increasing number of Staphylococcus aureus outbreaks linked to food product highlighted the need to develop technique with rapid, simple and accurate.The conventional technologies for rapid and sensitive detection of S. aureus viable cells in produce had several limitations.The signal from viable versus dead ceils could not be distinguished due to the persistence of DNA after the cell death,resulting in overestimate the number of cells.In the present study,some problems had been solved by developing a new concept with DNA -intercalating dye propidium membrane( PMA) combining with loop-mediated isothermal amplification(LAMP). The test was used to analyse S. aureus and routine dairy products, a series of primers targeted six distinct sequences of nuc gene were designed, which was characteristic of S. aureus. Also, the LAMP assay and the performance of PMA-LAMP for detecting viable S.aureus were optimized.Moreover,they improved the method for extraction DNA from dairy samples. In pure culture, the detection limit of PMA- LAMP was 3.2CFU/mL, up to 100-fold more sensitive than PCR.In dariy products, PMA-LAMP assay could detect as less as 5×101CFU/mL compared to that of PCR-PMA was 5×103CFU/mL. The complete LAMP-PMA assay for took about 6h, demonstrating the method was rapid and convenient. In conclusion, PMA-LAMP offers a novel DNA-based detection method for distinction between viable and dead cells with wide application in food products.%近年来,金黄色葡萄球菌引起的食品安全事件频发,这就需要建立一种快速准确地检测食品中金黄色葡萄球菌的方法。传统方法检测食品中金黄色葡萄球菌活菌存在很多缺点。由于细胞死亡后其DNA依然能够存活许久,所以传统方法不能有效区分DNA来自死菌还是活菌。通过荧光染料PMA与快速检测技术LAMP相结合的方法快速、灵敏的检测灭菌乳中金黄色葡萄球菌,并对死/活菌进行区分。根据金

  15. Identification of the ClpX Regulon in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Thomsen, Line Elnif; Ingmer, Hanne;

    Staphyloccous aureus is a major human pathogen capable of causing a wide spectrum of infections ranging from superficial wound infections to life-threatening endocarditis and toxic shock syndrome. Essential for S. aureus virulence is a large number of cell-surface-associated proteins and secreted...... proteins. Results from our group have shown that the ClpXP proteolytic complex and the ClpX chaperone play central roles in regulating expression of many of these factors (2;3). By using DNA microarrays to compare transcription of strain 8325-4 (wt) and the isogenic ¿clpX strain during the transition phase...

  16. Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato.

    Science.gov (United States)

    Suzuki, Ryoji; Fukuta, Shiro; Matsumoto, Yuho; Hasegawa, Toru; Kojima, Hiroko; Hotta, Makiko; Miyake, Noriyuki

    2016-10-01

    For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection.

  17. Subinhibitory concentrations of punicalagin reduces expression of virulence-related exoproteins by Staphylococcus aureus.

    Science.gov (United States)

    Mun, Su-Hyun; Kong, Ryong; Seo, Yun-Soo; Zhou, Tian; Kang, Ok-Hwa; Shin, Dong-Won; Kwon, Dong-Yeul

    2016-11-01

    Staphylococcus aureus produces a number of virulence factors. The major virulence factors exhibited by S aureus include various antigens, enzymes, cytotoxins and exotoxins (e.g. hemolysins, enterotoxins and toxic shock syndrome toxin). In this report, we show the influence of punicalagin on the secretion of exoprotein from S aureus by western blotting, tumor necrosis factor (TNF) release assay and quantitative RT-PCR. When added to S aureus cultures at an OD600 of 0.9, graded subinhibitory concentrations of punicalagin reduced the production of α-toxin, SEA and SEB in methicillin-resistant Staphylococcus aureus in a dose-dependent manner. Consistently, punicalagin reduced TNF-inducing activity by S aureus culture supernatants. Here, the transcriptional level of agr (accessory gene regulator) in S aureus was inhibited by punicalagin, suggesting that the reduced transcription may affect the secretion of exotoxins. These findings suggest that the expression of α-toxin and enterotoxins in S aureus is sensitive to the action of punicalagin, which may be an advantageous candidate in the treatment of toxigenic staphylococcal disease.

  18. Isolation and Identification of Staphylococcus aureus in Raw Milk and Rapid Detection of Toxigenic Strains%原料奶中金黄色葡萄球菌分离鉴定及产毒菌株的快速检测

    Institute of Scientific and Technical Information of China (English)

    覃艳华; 谢和; 胡萍; 李荔枝

    2011-01-01

    Strains of Staphylococcus aureus were isolated and identified from raw milk by traditional taxonomic identification and polymerase chain reaction (PCR), and their toxin producing ability was detected by mini-VIDAS to study the S. aureus contamination in raw milk. The results showed that S. aureus was the main contamination bacteria, 9 strains of them were positive which detected by miniVIDAS, and the detection rate of toxigenic strains was 12.86% in the 70 strains of S. aureus isolated. The PCR designed targeting the primer of thermonuclease(nuc) gene of S. aureus had coincidence rate of 100% with traditional taxonomic identification, and it had higher specificity and sensitivity.%为了蟹原料奶中产毒型金黄色葡萄球菌的污染情况,采用传统分类学鉴定方法与PCR鉴定方法结令,对原料奶中金黄色葡萄球菌进行分离鉴定,并运用全自动荧光免疫分析仪检测了分离.菌株的产毒情况.结果表明,分离鉴定的70株金黄色葡萄球菌中,mini-VIDAS仪分析为阳性的有9株,产毒型金黄色葡萄球菌的检出率为12.86%,金黄色葡萄球菌为原料奶的主要污染菌之一.针对金黄色葡萄球菌耐热核酸酶基因设计的引物进行的PCR鉴定方法,与传统分类学方法的符合率为100%,并且具有较高的特异性及敏感性.

  19. Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Delbecchi Louis

    2007-10-01

    Full Text Available Abstract Background In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT. This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. Results The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P P Conclusion This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray, the RT step is critical and should be examined with care.

  20. 乳品中金黄色葡萄球菌肠毒素B免疫传感检测方法的研究%Research of an Electrochemical Immunosensor for Detecting Staphylococcus Aureus Enterotoxin B in Dairy Products

    Institute of Scientific and Technical Information of China (English)

    高博; 孙秀兰; 张银志; 顾小红

    2012-01-01

    Staphylococcus aureus enterotoxin B(SEB) is a kind of extracellular toxins which is secreted by Staphylococcus aureus and mainly exists in high protein foods, such as milk. In this paper, we constructed an self-assembly immunosensor with self-made SEB antibody, double-layer L-cysteine and gold nanoparticle. The sensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. We also optimized the experimeutal conditions, and established the SEB standard curve. The linear ranges were 2~10 ng/mL and 10 ~ 100 ng/mL with a correlation coeficient of 0. 9939 and 0.9926, respectively,and the detection limit was 0. 667 ng/mL (S/N = 3). The sensor shows good specificity,reusability and stability,and can be used in dairy products for the rapid detection of SEB.%本文利用自制的金黄色葡萄球菌肠毒素B(SEB)抗体构建了一种L-半胱氨酸和纳米金的双层自组装免疫传感器,采用循环伏安法及交流阻抗法对传感器进行表征与测定,并对各项相关条件进行优化,最终建立了SEB检测的标准曲线,线性范围分别在2~10 ng/mL和10~100 ng/mL,相关系数分别为0.9939和0.9926,检出限(S/N=3)为0.667 ng/mL,乳品检测回收率在84.3%~93.2%之间.该传感器特异性良好,稳定性好,可再生使用,可应用于乳品中SEB的快速检测.

  1. Cellular immune responses of filaria (Litomosoides sigmodontis) infected BALB/c mice detected on the level of cytokine transcription.

    Science.gov (United States)

    Taubert, A; Zahner, H

    2001-08-01

    Cellular immune responses of BALB/c mice infected with 80 or 160 L3 of Litomosoides sigmodontis were studied over a period of 200 days postinfection (p.i.) by stimulating spleen cells with specific microfilariae and adult antigens and Concanavalin A (Con A). Effects were determined as the level of transcription of cytokine genes [interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-10, IL-13] employing a semiquantitative reverse transcriptase-polymerase chain reaction technique. Con A stimulation resulted in generally enhanced transcription levels in infected animals. Exposure to filarial antigens stimulated T cells of infected animals dependent on time p.i. There was a general strong response in the early prepatency (24 days p.i.), a temporary almost complete downregulation of cytokine gene transcription except IL-10 towards the end of prepatency (45 days p.i.), and subsequently strong reactions particularly concerning IFN-gamma and IL-13 during patency and postpatency. The dose of infection as well as the mode of antigenic stimulation had generally only small effects on the cytokine gene transcription: following the same type of kinetics, infection with 160 L3 as well as the use of microfilarial antigen generally induced lower levels of cytokine gene transcription compared with infection with 80 L3 and stimulation with female antigen, respectively.

  2. Extensive phage dynamics in Staphylococcus aureus contributes to adaptation to the human host during infection.

    Science.gov (United States)

    Goerke, Christiane; Wirtz, Christiane; Flückiger, Ursula; Wolz, Christiane

    2006-09-01

    Bacteriophages serve as a driving force in microbial evolution, adaptation to new environments and the pathogenesis of human bacterial infections. In Staphylococcus aureus phages encoding immune evasion molecules (SAK, SCIN, CHIPS), which integrate specifically into the beta-haemolysin (Hlb) gene, are widely distributed. When comparing S. aureus strain collections from infectious and colonizing situations we could detect a translocation of sak-encoding phages to atypical genomic integration sites in the bacterium only in the disease-related isolates. Additionally, significantly more Hlb producing strains were detected in the infectious strain collection. Extensive phage dynamics (intragenomic translocation, duplication, transfer between hosts, recombination events) during infection was shown by analysing cocolonizing and consecutive isolates of patients. This activity leads to the splitting of the strain population into various subfractions exhibiting different virulence potentials (Hlb-production and/or production of immune evasion molecules). Thus, phage-inducing conditions and strong selection for survival of the bacterial host after phage movement are typical for the infectious situation. Further in vitro characterization of phages revealed that: (i) SAK is encoded not only on serogroup F phages showing a conserved tropism for hlb but also on serogroup B phages which always integrate in a distinct intergenic region, (ii) the level of sak transcription correlates to phage inducibility but is independent of the phage localization in the chromosome, and (iii) phages can be stabilized extra-chromosomally during their life cycle.

  3. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  4. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Science.gov (United States)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  5. High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Jungwirth, Britta; Sala, Claudia; Kohl, Thomas A;

    2013-01-01

    The transcriptional regulator GlxR has been characterized as a global hub within the gene-regulatory network of Corynebacterium glutamicum. Chromatin immunoprecipitation with a specific anti-GlxR antibody and subsequent high-throughput sequencing (ChIP-seq) was applied to C. glutamicum to get new...

  6. Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method.

    Science.gov (United States)

    Kurosaki, Yohei; Grolla, Allen; Fukuma, Aiko; Feldmann, Heinz; Yasuda, Jiro

    2010-07-01

    Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.

  7. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    Science.gov (United States)

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

  8. Influence of Magnolol on the Secretion of α-Toxin by Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Xu-Ming Deng

    2010-03-01

    Full Text Available In this study we investigated the antimicrobial activity of magnolol on Staphylococcus aureus. The minimal inhibitory concentrations of magnolol against 31 S. aureus strains ranged from 4–32 μg/mL. In addition, hemolysin assays, Western blotting, and real-time RT-PCR were performed to investigate the effect of magnolol on α-toxin secretion by both methicillin-sensitive S. aureus (MSSA and methicillin-resistant S. aureus (MRSA. The results indicated that sub-inhibitory concentrations of magnolol dose-dependently inhibited the transcription of hla (the gene encoding α-toxin in S. aureus, resulting in a reduction of α-toxin secretion and, thus, hemolytic activities.

  9. Quantitative detection of Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae in patients with new influenza A (H1N1/2009 and influenza A/2010 virus infection

    Directory of Open Access Journals (Sweden)

    Safaeyan, Firouzeh

    2015-04-01

    Full Text Available Introduction: Viral influenza is a seasonal infection associated with significant morbidity and mortality. In the United States more than 35,000 deaths and 200,000 hospitalizations are recorded annually due to influenza. Secondary bacterial infections or co-infections associated with cases of influenza are a leading cause of severe morbidity and mortality, especially among high-risk groups such as the elderly and young children. Aim: The aim of the present study was the quantitative detection of and in a group of patients with seasonal influenza A, influenza A ( pandemic 2009, and patients with symptoms of respiratory infection, but the negative for serving as control group.Method: In total, 625 patients suspected respiratory infection from April 2009 to April 2010 were studied. There were 58 patients with influenza A and 567 patients negative for influenza A . From November 2010 to February 2011, 158 patients with respiratory symptoms were analyzed for seasonal influenza A. There were 25 patients with seasonal influenza A. To check the colonization status among the healthy individuals 62 healthy persons were further investigated. Individual were screened in parallel. The choices of special genes were amplified from clinical specimens using real-time PCR with a cutoff of 10 CFU/mL to differentiate colonization from infection in respiratory tract.Results: and were detected in 12%, 26% and 33% of patients with , while the corresponding figures were 9%, 19%, and 31% for negative patients. Among patients with seasonal influenza A 12% 24% , and 32% co-infections were detected, while influenza negative control group yielded 5% , 11% , and 10% , respectively. Conclusion: The results of this study indicated that the serotype of pandemic 2009 did not increase incidence of secondary infection with and . Quantitative detection of secondary bacterial infection by QR-PCR can help us for distinguishing colonization from infection and controlling misuse of

  10. Crustin protein Amk1 from black tiger shrimp (Penaeus monodon inhibits Vibrio harveyi and Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Moltira Tonganunt1*

    2008-05-01

    Full Text Available A crustin gene (Amk1 was identified from a haemocyte library of the black tiger shrimp, Penaeus monodon. The full-length cDNA consists of 411 bp encoding a deduced precursor of 136 amino acids with a signal peptide of 17 aminoacids. Amk1 contains a hydrophobic and a Gly-rich region at the N-terminus and a 12 conserved cysteine domain (6-DSC at the C-terminus. Transcripts of Amk1 are mainly detected in haemocytes and gills by RT-PCR analysis. A recombinant Amk1was overexpressed and purified from Escherichia coli. This has a molecular mass of 43.66 kDa with a predicted pI of 8.23. Antibacterial assays demonstrated that recombinant Amk1 exhibited antibacterial activity against Gram-positive and Gramnegativebacteria with strong inhibition against Staphylococcus aureus and Vibrio harveyi.

  11. Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

    Science.gov (United States)

    Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

    2009-10-01

    Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

  12. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.;

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal....... (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals...

  13. Improvement and optimization of a multiplex real-time reverse transcription polymerase chain reaction assay for the detection and typing of Vesicular stomatitis virus.

    Science.gov (United States)

    Hole, Kate; Velazquez-Salinas, Lauro; Velazques-Salinas, Lauro; Clavijo, Alfonso

    2010-05-01

    An improvement to a previously reported real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the detection of Vesicular stomatitis virus (VSV) is described. Results indicate that the new assay is capable of detecting a panel of genetically representative strains of VSV present in North, Central, and South America. The assay is specific for VSV and allows for simultaneous differentiation between Vesicular stomatitis Indiana virus and Vesicular stomatitis New Jersey virus. This real-time RT-PCR is able to detect current circulating strains of VSV and can be used for rapid diagnosis of VSV and differentiation of VSV from other vesicular diseases, such as foot-and-mouth disease.

  14. Identification of single nucleotide polymorphisms associated with hyperproduction of alpha-toxin in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Xudong Liang

    Full Text Available The virulence factor α-toxin (hla is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.

  15. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

    Science.gov (United States)

    Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-06-01

    In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

  16. Design and feasibility of a novel, rapid, and simple fluorescence 26-plex rt-PCR assay for simultaneous detection of 24 fusion transcripts in adult acute myeloid leukemia.

    Science.gov (United States)

    Laforêt, Marie-Pierre; Turlure, Pascal; Lippert, Eric; Cornillet-Lefebvre, Pascale; Pigneux, Arnaud; Pradeau, Rachel; Feuillard, Jean; Gachard, Nathalie

    2013-03-01

    Identification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%.

  17. Isolation of nuc mutant isolates of Staphylococcus aureus from bovine clinical mastitis.

    Science.gov (United States)

    Zastempowska, E; Orczykowska-Kotyna, M; Lassa, H

    2014-06-01

    Isolates of Staphylococcus aureus with a mutation in the nuclease (nuc) gene were recovered from cases of bovine mastitis in Poland. Three S. aureus isolates from cows in one herd had a 42 base pair duplication in the nuc gene. These isolates belonged to sequence type 97 (ST97) and clonal complex 97 (CC97). They had a different spa type and multiple-locus variable-number tandem-repeat fingerprinting (MLVF) subtype than a S. aureus isolate without the nuc mutation from the same herd. Isolation of nuc mutant S. aureus strains from cases of bovine mastitis may confound diagnostic PCRs based on detection of the nuc gene.

  18. [Examination of Staphylococcus aureus survival and growth during cheese-making process].

    Science.gov (United States)

    Aoyama, Kenji; Takahashi, Chitose; Yamauchi, Yoshihiko; Sakai, Fumihiko; Igarashi, Hideo; Yanahira, Syuichi; Konishi, Hiroaki

    2008-04-01

    Inoculation tests of Staphylococcus aureus were performed to evaluate the risk of toxic hazard in cheese manufacturing processes. S. aureus was inoculated into pasteurized milk or cheese curd, and the survival and growth were examined. S. aureus grew only slightly or decreased in cell number under the manufacturing condition of semi-hard type cheese or soft-type cheese. Under the conditions of the fresh cheese making process, S. aureus slightly increased in cell number, though no enterotoxin was detected. In processed cheese, S. aureus did not grow at all. Growth inhibition of S. aureus by lactic acid produced from starter culture was suggested to be the cause of growth inhibition in the natural cheese.

  19. Be alert to the alterations in the biological characteristics in heterogeneous vancomycin-intermediate Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    X Zhou

    2012-01-01

    Full Text Available The development of reduced vancomycin susceptibility in Staphylococcus aureus in many cases appears to be associated with characteristic changes. These changes may have pitfall of identifying S. aureus by automated testing methods like Vitek 32. In this study, we retested 24 heterogeneous vancomycin-intermediate Staphylococcus haemolyticus (h-VISH collected in 2008-2010 at the Department of Clinical Microbiology by conventional biochemical tests and polymerase chain reaction (PCR. The heterogeneous vancomycin-intermediate S. aureus (hVISA reversion test and electron microscopic examination were also used. Six isolates of 24 h-VISH possessed nuc, coa, and 16S rRNA genes, and could be reversed into S. aureus. It suggested that biochemical and morphological changes in hVISA and vancomycin-intermediate S. aureus (VISA should be considered, and the detection of S. aureus, especially reduced vancomycin susceptibility isolates, requires more attention and different techniques.

  20. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Hongmei Bao

    2014-01-01

    Full Text Available A novel influenza A (H7N9 virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans. No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9 virus from different resource samples.

  1. Inhibition of β-lactamase-mediated oxacillin resistance in Staphylococcus aureus by a deoxyribozyme

    Institute of Scientific and Technical Information of China (English)

    Zheng HOU; Jing-ru MENG; Jin-rong ZHAO; Ben-quan HU; Jie LIU; Xiao-jun YAN; Min JIA; Xiao-xing LUO

    2007-01-01

    Aim:To investigate the oxacillin susceptibility restoration of methicillin-resistant Staphylococcus aureus (MRSA) by targeting the signaling pathway of blaR1blaZ with a DNAzyme. Methods:A DNAzyme (named PS-DP,z602) targeting blaR1 mRNA was designed and synthesized. After DRz602 was introduced into a MRSA strain WHO-2,the colony-forming units of WHO-2 on the Mueller-Hinton agar containing 6 mg/L oxacillin and the minimum inhibitory concentrations of oxacillin were determined. The inhibitory effects of DRz602 on the expressions of antibiotic-resistant gene blaR1 and its downstream gene blaZ were detected by real time RT-PCR. Results:PS-DRz602 significantly decreased the transcription of blaR1 mRNA and led to the significant reduction of blaZ in a concentrationdependent manner. Consequently,the resistance of S aureus WHO-2 to the β-lactam antibiotic oxacillin was significantly inhibited. Conclusion:Our results indicated that blocking the blaRl-blaZ signaling pathway via DNAzyme might provide a viable strategy for inhibiting the resistance of MRSA to β-lactam antibiotics and that BIaR1 might be a potential target for pharmacological agents combating MRSA.

  2. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    Science.gov (United States)

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus.

  3. Presence of Staphylococcus aureus on university dance studio floors and barres: a preliminary investigation.

    Science.gov (United States)

    Unsworth, Desiree A; Russell, Jeffrey A; Martiny, Adam C

    2014-01-01

    Staphylococcus aureus (S. aureus) is a bacterium associated with various infectious diseases. Not only has the bacterium been detected in sports environments, the reported incidences of S. aureus infections have steadily increased in athletic teams. However, in spite of similarities between sports and dance facilities, to our knowledge no previous study has examined the presence of this bacterium in the dance environment. We hypothesized that S. aureus would be present in a university's dance studios, and that it would be extant in higher concentrations inside versus outside the studios. Using common microbiological culturing methods, samples were gathered from floors and barres in three studios of a single university, as well as from outside floors and railings near the studios and a conference room used by dancers. Confirming our hypothesis, we detected S. aureus in every dance studio sample (0.03 to 0.38 cfu/cm 2 ). Supporting our second hypothesis, we found that average S. aureus concentrations from the three studios were significantly higher compared to both outside and conference room samples (P ≤ 0.001). The latter two locations did not yield any S. aureus concentrations. Control samples developed as expected. The results of this study suggest that S. aureus bacteria are common on the flooring and barres of university dance studios, with the bacterial concentrations possibly dependent on the hours of usage of these surfaces. Whether the presence of S. aureus in dance studios presents a health risk to dancers should be studied further.

  4. Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach

    Science.gov (United States)

    Reports of Staphylococcus aureus detected in marine environments have occurred since the early 1990’s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible s...

  5. Characterization and comparative analysis of a second thermonuclease in Staphylococcus aureus

    Science.gov (United States)

    Staphylococcal nuclease (here termed as NUC1) is considered an important virulence factor and a unique marker widely used in detection of Staphylococcus aureus. A novel functional thermostable nuclease (here termed as NUC2) in S. aureus was characterized after recombinant expression in Escherichia...

  6. Linezolid resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Pavani Gandham

    2014-08-01

    Full Text Available Linezolid is the only antibiotic available as an oral formulation for resistant staphylococcal infections. It is effective in skin and soft tissue infections, nosocomial pneumonias including VAP, infective endocarditis and MRSA meningitis. It is also effective in the eradication of both nasal and throat colonization of MRSA. Its high bioavailability and post antibiotic effect, ease of switching to oral therapy during its use and the fact that it can be used in patients of all ages, also in patients with liver disease and poor kidney function and its increased effectiveness over glycopeptides makes this drug a precious drug in the treatment of resistant staphylococcal infections. Linezolid resistance in staphylococcus is defined as a linezolid MIC of and #8805;8 mg/L. Reported Linezolid resistance in India and elsewhere is 2-20%. There is clonal dissemination of Linezolid Resistant Staphylococcus aureus (LRSA within or across health care settings which demands continuous surveillance to determine the emergent risk of resistance strains and to establish guidelines for appropriate use. Clinical laboratories should confirm any LRSA preferably by a second method, prior to using linezolid for serious infections. Effective surveillance, more judicious use of this antibiotic, avoiding linezolid usage for empiric therapy in hospital acquired staphylococcus infections, optimization of the pharmacological parameters of the antibiotics in specific clinical situation, decreasing bacterial load by timely surgical debridement or drainage of collections, use of combination therapies would prevent the emergence of resistance to linezolid in staphylococcus aureus. [Int J Res Med Sci 2014; 2(4.000: 1253-1256

  7. Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus.

    Science.gov (United States)

    Beavers, William N; Skaar, Eric P

    2016-08-01

    Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets.

  8. Líquen aureus "algesiogênico" "Algesiogenic" lichen aureus

    Directory of Open Access Journals (Sweden)

    Roberto Rheingantz da Cunha Filho

    2006-03-01

    Full Text Available Descreve-se caso de líquen aureus em paciente do sexo feminino, com 23 anos de idade que apresentava há dois anos lesão dolorosa, purpúrica, acastanhada tendendo por semelhante a cor de ferrugem e de aspecto liquenóide no antebraço. O exame anatomopatológico revelou denso infiltrado linfo-histiocitário na derme superior papilar, com extravasamento de hemácias. O líquen aureus é relativamente raro, sendo ainda mais raro o sintoma de dor.A case is described of lichen aureus in a 23 year old female with a 2-year history of painful, purpuric, rust-coloured to tan, lichenous lesion on forearm. A biopsy specimen demonstrated a dense lymphohistiocytic infiltrate in the upper dermis, with extravasation of red cells. The "algesiogenic" lichen aureus is a very rare dermatosis.

  9. Staphylococcus aureus: methicillin-susceptible S. aureus to methicillin-resistant S. aureus and vancomycin-resistant S. aureus.

    Science.gov (United States)

    Rehm, Susan J; Tice, Alan

    2010-09-15

    The evolution of methicillin-resistant and vancomycin-resistant Staphylococcus aureus has demanded serious review of antimicrobial use and development of new agents and revised approaches to prevent and overcome drug resistance. Depending on local conditions and patient risk factors, empirical therapy of suspected S. aureus infection may require coverage of drug-resistant organisms with newer agents and novel antibiotic combinations. The question of treatment with inappropriate antibiotics raises grave concerns with regard to methicillin-resistant S. aureus selection, overgrowth, and increased virulence. Several strategies to reduce the nosocomial burden of resistance are suggested, including shortened hospital stays and outpatient parenteral antimicrobial therapy of the most serious infections.

  10. Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions.

    Directory of Open Access Journals (Sweden)

    Ulrike Mäder

    2016-04-01

    Full Text Available Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

  11. Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

    Directory of Open Access Journals (Sweden)

    C Coelho

    2003-06-01

    Full Text Available Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR was applied for the simultaneous detection of hepatitis A virus (HAV, poliovirus (PV and simian rotavirus (RV-SA11 and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp, PV (394 bp and RV (278 bp were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation.

  12. Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

    Science.gov (United States)

    Thanarajoo, Sathis Sri; Kong, Lih Ling; Kadir, Jugah; Lau, Wei Hongi; Vadamalai, Ganesan

    2014-06-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.

  13. Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread.

    Science.gov (United States)

    Renneberg, H; Friedetzky, A; Konrad, L; Kurek, R; Weingärtner, K; Wennemuth, G; Tunn, U W; Aumüller, G

    1999-01-01

    Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

  14. [Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

    Science.gov (United States)

    Lin, Feng; Liu, Li; Hao, Gui-Jie; Cao, Zheng; Sheng, Peng-Cheng; Wu, Ying-Lei; Shen, Jin-Yu

    2014-09-01

    White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

  15. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  16. Detection of Cardamom mosaic virus and Banana bract mosaic virus in cardamom using SYBR Green based reverse transcription-quantitative PCR.

    Science.gov (United States)

    Siljo, A; Bhat, A I; Biju, C N

    2014-01-01

    Cardamom being perennial, propagated vegetatively, detecting viruses in planting material is important to check the spread of viruses through infected material. Thus development of effective and sensitive assay for detection of viruses is need of the time. In this view, assay for the detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus (BBrMV), infecting cardamom was developed using SYBR Green one step reverse transcription-quantitative PCR (RT-qPCR). The RT-qPCR assay amplified all isolates of CdMV and BBrMV tested but no amplification was obtained with RNA of healthy plants. Recombinant plasmids carrying target virus regions corresponding to both viruses were quantified, serially diluted and used as standards in qPCR to develop standard curve to enable quantification. When tenfold serial dilutions of the total RNAs from infected plants were tested through RT-qPCR, the detection limit of the assay was estimated to be 16 copies for CdMV and 10 copies for BBrMV, which was approximately 1,000-fold higher than the conventional RT-PCR. The RT-qPCR assay was validated by testing field samples collected from different cardamom growing regions of India. This is the first report of RT-qPCR assay for the detection of CdMV and BBrMV in cardamom.

  17. Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108 cells by mefloquine

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    Dow Geoffrey S

    2003-02-01

    Full Text Available Abstract Background There is no known biochemical basis for the adverse neurological events attributed to mefloquine. Identification of genes modulated by toxic agents using microarrays may provide sufficient information to generate hypotheses regarding their mode of action. However, this utility may be compromised if sample sizes are too low or the filtering methods used to identify differentially expressed genes are inappropriate. Methods The transcriptional changes induced in rat neuroblastoma cells by a physiological dose of mefloquine (10 micro-molar were investigated using Affymetrix arrays. A large sample size was used (total of 16 arrays. Genes were ranked by P-value (t-test. RT-PCR was used to confirm (or reject the expression changes of several of the genes with the lowest P-values. Different P-value filtering methods were compared in terms of their ability to detect these differentially expressed genes. A retrospective power analysis was then performed to determine whether the use of lower sample sizes might also have detected those genes with altered transcription. Results Based on RT-PCR, mefloquine upregulated cJun, IkappaB and GADD153. Reverse Holm-Bonferroni P-value filtering was superior to other methods in terms of maximizing detection of differentially expressed genes but not those with unaltered expression. Reduction of total microarray sample size ( Conclusions Adequate sample sizes and appropriate selection of P-value filtering methods are essential for the reliable detection of differentially expressed genes. The changes in gene expression induced by mefloquine suggest that the ER might be a neuronal target of the drug.

  18. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) at ambient freshwater beaches

    Science.gov (United States)

    Fogarty, Lisa R.; Haack, Sheridan K.; Johnson, Heather E.; Brennan, Angela K.; Isaacs, Natasha M.; Spencer, Chelsea

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) are a threat to human health worldwide, and although detected at marine beaches, they have been largely unstudied at freshwater beaches. Genes indicating S. aureus (SA; femA) and methicillin resistance (mecA) were detected at 11 and 12 of 13 US Great Lakes beaches and in 18% or 27% of 287 recreational water samples, respectively. Eight beaches had mecA + femA (potential MRSA) detections. During an intensive study, higher bather numbers, staphylococci concentrations, and femA detections were found in samples collected after noon than before noon. Local population density, beach cloud cover, and beach wave height were significantly correlated with SA or MRSA detection frequency. The Panton-Valentine leukocidin gene, associated with community-acquired MRSA, was detected in 12 out of 27 potential MRSA samples. The femA gene was detected less frequently at beaches that met US enterococci criteria or EU enterococci ‘excellent’ recreational water quality, but was not related to Escherichia coli-defined criteria. Escherichia coli is often the only indicator used to determine water quality at US beaches, given the economic and healthcare burden that can be associated with infections caused by SA and MRSA, monitoring of recreational waters for non-fecal bacteria such as staphylococci and/or SA may be warranted.

  19. Influence of Host Genetics and Environment on Nasal Carriage of Staphylococcus aureus in Danish Middle-Aged and Elderly Twins

    DEFF Research Database (Denmark)

    Andersen, Paal Skytt; Pedersen, Jacob Krabbe; Fode, Peder;

    2012-01-01

    Background. Nasal carriage is a major risk factor for Staphylococcus aureus infection. Approximately, one-quarter of adults carry S. aureus. However, the role of host genetics on S. aureus nasal carriage is unknown. Methods. Nasal swabs were obtained from a national cohort of middle-aged and elde......Background. Nasal carriage is a major risk factor for Staphylococcus aureus infection. Approximately, one-quarter of adults carry S. aureus. However, the role of host genetics on S. aureus nasal carriage is unknown. Methods. Nasal swabs were obtained from a national cohort of middle.......4%-34.5%), and opposite sex (21.4%; 95% CI, 12.0%-33.4%) dizygotic twins. Despite shared childhoods, only 1 of 617 pairs was concordant with respect to lineage. Although heritability increased for S. aureus and lineage persistency, no significant heritability was detected. Conclusion. In this study, host genetic factors...

  20. 应用Vitek MS质谱检测PSM-mec对医院感染MRSA快速分型%Detection of PSM-mec by Vitek mass spectrometry for rapid identification of nosocomial-acquired methicillin-resistant Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    刘晔华; 王猛; 刘萍; 陈锦艳; 邢迎红; 张坚磊; 穆红

    2015-01-01

    Objective To evaluate the method of PSM-mec detection by Vitek MS for nosocomialacquired methicillin-resistant Staphylococcus aureus (MRSA) identification.Methods Totally 167 isolates of MRSA and 100 isolates of methicillin-sensitive Staphylococcus aureus (MSSA) used in this research were non-repetitively and prospectively collected between June 2012 and December 2013,two different SCCmec genotyping methods were applied for the MRSA strains,Vitek MS was used for identification of the isolates,the acquisition mass-spectrogram and the result mass-spectrogram at Myla system were analyzed among the different SCCmec type of MRSA.Results The 167 isolates of MRSA were classified into 5 major SCCmec types,among which SCCmec Ⅰ accounting for 3.6% (6 isolates);SCCmec Ⅱ 6.0% (10 isolates);SCCmec Ⅲ and Ⅲa 84.4% (141 isolates);SCCmec Ⅳand Ⅳ a 4.8% (8 isolates);SCCmec Ⅴ 1.2% (2 isolates),respectively.The peak adjacent to the horizontal axis of a m/z 2 500 could be visually identified between the SCCmec Ⅱ and Ⅲ MRSA,of which the delta toxin peak were presented at m/z 3 005-3 009 or m/z 3 037-3 056,while the strains without delta toxin peak and the other types of MRSA or MSSA had no characteristic peak at the same position.Conclusions Nosocomial-acquired MRSA of the drug-resistant condition could be rapidly differentiated and forecasted by Vitek MS.Vitek MS could serve as a routine clinical assistance for epidemiological investigations of nosocomial-acquired MRSA in local area.%目的 评估Vitek MS质谱快速检测PSM-mec在医院感染耐甲氧西林金黄色葡萄球菌(MRSA)分型的应用价值.方法 前瞻性研究.收集天津市第一中心医院2012年6月至2013年12月非重复分离的MRSA 167株和耐甲氧西林敏感金黄色葡萄球菌(MSSA) 100株,通过2种SCCmec分型技术检测MRSA的型别,以Vitek MS质谱对所有菌株进行常规鉴定并对菌株获取图谱和Myla结果图谱进行分析,探明不

  1. Pathogenesis of Staphylococcus aureus abscesses.

    Science.gov (United States)

    Kobayashi, Scott D; Malachowa, Natalia; DeLeo, Frank R

    2015-06-01

    Staphylococcus aureus causes many types of human infections and syndromes-most notably skin and soft tissue infections. Abscesses are a frequent manifestation of S. aureus skin and soft tissue infections and are formed, in part, to contain the nidus of infection. Polymorphonuclear leukocytes (neutrophils) are the primary cellular host defense against S. aureus infections and a major component of S. aureus abscesses. These host cells contain and produce many antimicrobial agents that are effective at killing bacteria, but can also cause non-specific damage to host tissues and contribute to the formation of abscesses. By comparison, S. aureus produces several molecules that also contribute to the formation of abscesses. Such molecules include those that recruit neutrophils, cause host cell lysis, and are involved in the formation of the fibrin capsule surrounding the abscess. Herein, we review our current knowledge of the mechanisms and processes underlying the formation of S. aureus abscesses, including the involvement of polymorphonuclear leukocytes, and provide a brief overview of therapeutic approaches.

  2. A duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups A and B.

    Science.gov (United States)

    Zhou, Gang; Cai, Wenbo; Liu, Xiaolei; Niu, Chengming; Gao, Caixia; Si, Changde; Zhang, Wei; Qu, Liandong; Han, Lingxia

    2011-05-01

    Avian leukosis is a disease that is spreading widely in the world causing large economic losses to the poultry industry. In this study, a duplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify avian leukosis virus subgroups A and B (ALVA/B). The assay was optimised to measure viral gp85 and chicken housekeeping (β-actin) genes. The result showed that the assay was specific for reference strains of ALVA/B subtype and no cross-reaction was detected with ALV subtypes E and J or with four other non-ALV viruses. The assay detected as few as 56 gp85 cDNA copies and was 100-fold more sensitive than a conventional RT-PCR. Seventy clinical blood samples were evaluated by both the qRT-PCR and the conventional RT-PCR assay, and the results show that 65 samples were positive by the qRT-PCR compared with 43 by the conventional RT-PCR. When this assay was used to quantify the viral load in ALV-inoculated embryos from three congenic chicken lines, the embryos from the B21 line showed the highest viral load, whereas the lowest load was found in the B5 line. This assay provides a powerful tool for quantitative detection of the ALVA/B and for the study of host genetic resistance to avian leukosis.

  3. Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

    Science.gov (United States)

    Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

    2015-06-01

    Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

  4. Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR 2 Primers

    Directory of Open Access Journals (Sweden)

    Abdul Rahman Siregar

    2015-11-01

    Full Text Available Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3. This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

  5. Role of Nasal Staphylococcus aureus Carriage in Transmission among Contact Athletes.

    Science.gov (United States)

    Suzuki, K; Tagami, K

    2015-12-01

    Among athletes, Staphylococcus aureus is thought to be transmitted by close physical contact with carriers. Nevertheless, evidence is limited with regard to both the tracking of individual strains and the role of S. aureus on the skin's surface. We investigated its transmission using molecular genotyping and the presence of S. aureus on the skin during exercise. In the first study, nasal samples were obtained from 172 athletes over a period of up to one year. The 200 strains of S. aureus collected from these athletes were genotyped, and transmission of S. aureus was detected by phage open reading frame typing (POT). In the second study, the presence of S. aureus on the skin's surface was compared between nasal carriers (n=9) and non-nasal carriers (n=9), who had participated in the first study. In the first study, 10 cases of transmission were confirmed. In the second study, exercise-induced sweating increased S. aureus isolates from the skin's surface (before vs. after exercise: 5.2±5.4 vs. 41.7±40.6 CFU/ml) in nasal carriers. In 5 of 9 nasal carriers, S. aureus isolates from the skin's surface were clonally identical to those from the nares. These results identify a major route of S. aureus transmission among athletes and provide insight into the role played by exercise-induced sweating in nasal carriers.

  6. Methicillin-resistant Staphylococcus aureus isolates in a hospital of Shanghai

    Science.gov (United States)

    Wang, Xiaoguang; Ouyang, Lin; Luo, Lingfei; Liu, Jiqian; Song, Chiping; Li, Cuizhen; Yan, Hongjing; Wang, Ping

    2017-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) strains are now common both in the health care setting and in the community. Active surveillance is critical for MRSA control and prevention. Specimens of patients (200 patients with 1119 specimens) as well as medical staff and hospital setting (1000 specimens) were randomly sampled in a level 2 hospital in Shanghai from September 2011 to August 2012. Isolation, cultivation and identification of S. aureus were performed. Totally, 67 S. aureus strains were isolated. 32 S. aureus strains were isolated from patient samples; 13 (13/32, 40.6%) of the 32 S. aureus isolates were MRSA; sputum sample and patients in the department of general internal medicine were the most frequent specimen and patient group for S. aureus strains isolation. Remaining 35 S. aureus strains were isolated from the medical staff and hospital setting; 20 (20/35, 57.1%) of the 35 S. aureus isolates were MRSA; specimens sampled from doctors and nurses’ hands and nose and hospital facilities were the most frequent samples to isolate S. aureus. Resistant and virulent genes detection showed that, all 33 MRSA strains were mecA positive which accounts for 49.3% of the 67 S. aureus strains; 38 isolates were Panton-Valentine leukocidin (PVL) gene positive which accounts for 56.7% of the 67 S. aureus strains; and 17 (17/67, 25.4%) isolates are mecA and PVL genes dual positive. Multidrug-resistant strains of MRSA and PVL positive S. aureus are common in patients, medical staff and hospital setting, the potential health threat is worthy of our attention. PMID:28030828

  7. Methicillin-resistant staphylococcus aureus isolates in a hospital of shanghai.

    Science.gov (United States)

    Wang, Xiaoguang; Ouyang, Lin; Luo, Lingfei; Liu, Jiqian; Song, Chiping; Li, Cuizhen; Yan, Hongjing; Wang, Ping

    2017-01-24

    Methicillin-resistant Staphylococcus aureus (MRSA) strains are now common both in the health care setting and in the community. Active surveillance is critical for MRSA control and prevention. Specimens of patients (200 patients with 1119 specimens) as well as medical staff and hospital setting (1000 specimens) were randomly sampled in a level 2 hospital in Shanghai from September 2011 to August 2012. Isolation, cultivation and identification of S. aureus were performed. Totally, 67 S. aureus strains were isolated. 32 S. aureus strains were isolated from patient samples; 13 (13/32, 40.6%) of the 32 S. aureus isolates were MRSA; sputum sample and patients in the department of general internal medicine were the most frequent specimen and patient group for S. aureus strains isolation. Remaining 35 S. aureus strains were isolated from the medical staff and hospital setting; 20 (20/35, 57.1%) of the 35 S. aureus isolates were MRSA; specimens sampled from doctors and nurses' hands and nose and hospital facilities were the most frequent samples to isolate S. aureus. Resistant and virulent genes detection showed that, all 33 MRSA strains were mecA positive which accounts for 49.3% of the 67 S. aureus strains; 38 isolates were Panton-Valentine leukocidin (PVL) gene positive which accounts for 56.7% of the 67 S. aureus strains; and 17 (17/67, 25.4%) isolates are mecA and PVL genes dual positive. Multidrug-resistant strains of MRSA and PVL positive S. aureus are common in patients, medical staff and hospital setting, the potential health threat is worthy of our attention.

  8. Staphylococcus aureus Bacteraemia

    Directory of Open Access Journals (Sweden)

    James Price

    2010-01-01

    Full Text Available Staphylococcus aureus bacteraemia (SAB is commonly complicated by metastatic infection or relapse after treatment. Objectives. The study aim was to determine the role of bacterial, host, and management factors in development of complicated SAB. Methods. A prospectively-conducted observational study gathered data on predisposition, management and outcome of 100 consecutive SAB cases. Antibiotic susceptibilities and genetic lineage of bacterial isolates were determined. Further clinical and microbiological data were gathered on two retrospective series from 1999–2000 (n=57 and 2004 (n=116. Results. In the prospective cases, 27% met our definition of complicated disease. Expressed as RR and 95% CI, complicated disease was associated with diabetes (1.58, 1.00–2.48, injecting-drug use (5.48, 0.88–33.49, community-onset of symptoms (1.4, 1.02–1.92, and symptom duration ≥48 hours prior to starting effective antibiotic therapy (2.10, 1.22–3.61. Uncomplicated disease was associated with the presence of a central line (0.69, 0.55–0.88 and prompt removal of a primary focus (0.71, 0.57–0.90. Neither methicillin resistance nor genetic lineage was associated with complicated disease, but methicillin resistance was associated with higher mortality. Conclusions. This study demonstrates that clinical rather than microbial factors are the major determinants of SAB outcome and underscores the importance of early treatment.

  9. In vitro antimicrobial activity of honokiol against Staphylococcus aureus in biofilm mode.

    Science.gov (United States)

    Li, Wen-Li; Zhao, Xing-Chen; Zhao, Zi-Wen; Huang, Yan-Jun; Zhu, Xuan-Zhi; Meng, Ri-Zeng; Shi, Ce; Yu, Lu; Guo, Na

    2016-12-01

    Staphylococcus aureus (S. aureus) can attach to food, host tissues and the surfaces of medical implants and form a biofilm, which makes it difficult to eliminate. The purpose of this study was to evaluate the effect of honokiol on biofilm-grown S. aureus. In this report, honokiol showed effective antibacterial activity against S. aureus in biofilms. S. aureus isolates are capable of producing distinct types of biofilms mediated by polysaccharide intercellular adhesion (PIA) or extracellular DNA (eDNA). The biofilms' susceptibility to honokiol was evaluated using confocal laser scanning microscopy (CLSM) analysis. The transcript levels of the biofilm-related genes, the expression of PIA, and the amount of eDNA of biofilm-grown S. aureus exposed to honokiol were also investigated. The results of this study show that honokiol can detach existing biofilms, kill bacteria in biofilms, and simultaneously inhibit the transcript levels of sarA, cidA and icaA, eDNA release, and the expression of PIA.

  10. A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay

    DEFF Research Database (Denmark)

    Bartels, Mette Damkjaer; Boye, Kit; Rohde, Susanne Mie

    2009-01-01

    -susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte...

  11. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

    Directory of Open Access Journals (Sweden)

    Alexa J Bracht

    Full Text Available Senecavirus A (SV-A, formerly, Seneca Valley virus (SVV, has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD, a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD, swine vesicular disease (SVD, and vesicular stomatitis (VS, that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88% were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18% or without (6% vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates.

  12. 对近年来本市分离的金黄色葡萄球菌进行肠毒素检测与分析%Detection and analysis of enterotoxin in Staphylococcus aureus isolated from Suzhou city in recent years

    Institute of Scientific and Technical Information of China (English)

    邹文燕

    2012-01-01

    Objective:To survey the distribution of enterotoxin in 97 Staphylococcus aureus isolated from four types of samples. Methods: Enterotoxin was detected in 97 Staphylococcus aureus samples by mini VIDAS detector. Results: Of the 97 strains of Staphylococcus aureus , 39 strains can produce enterotoxin, the positive rate was 40.2% , among them, 17 enterotoxin strains were separated from 25 ready - to - eat food samples, the positive rate was 68. 0% . Conclusion: Higher positive rate of enterotoxigenic Staphylococcus aureus was found in food samples, which would lead to the potential hazards of acute gastroenteritis and food poisoning.%目的:了解分离于四类不同样品的97株金黄色葡萄球菌携带肠毒素的情况.方法:采用mini VIDAS酶联荧光免疫分析仪对此97株金黄色葡萄球菌进行肠毒素的检测.结果:97株菌中,有39株菌能产肠毒素,阳性率为40.2%,其中,分离自即食类食品的25株被测菌中,有17株肠毒素阳性,阳性率达68.0%.结论:食品样品中分离的金黄色葡萄球菌产肠毒素的阳性率较高,具有引发急性胃肠炎等食物中毒的潜在危害.

  13. Detection of p190BCR-ABL AND p210BCR-ABL fusion transcripts in patients with chronic myeloid leukemia (CML using qualitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Aya Bonilla, Carlos Alberto

    2014-10-01

    Full Text Available Introduction: Chronic myelogenous leukemia (CML is characterized by the presence of the Philadelphia chromosome (Ph, resulting from the balanced reciprocal translocation t(9;22(q34;q11. This marker chromosome is found less frequently in patients suffering from acute lymphoblastic leukemia. Objective: To determine the frequency of BCR-ABL gene fusions encoding the p210BCR-ABL y p190 BCR-ABL transcripts in Colombian patients diagnosed with CML in different stages of the disease and/or its treatment. Materials and methods: Cross sectional, descriptive study of thirty one CML patients (aged 15-78. Analysis was carried out through qualitative nested PCR for the isoforms P210 BCR-ABL (b3a2 e b2a2 and P190 BCR-ABL (e1a2, and based on peripheral blood samples. Results: In 29 of the 31 patients (93.6% transcript p210BCR-ABL was detected; b2a2 and b3a2 gene fusions and the coexpression b3a2 y b2a2 were identified in 55.2% (16/29, 34.5% (10/29 and 10.3% (3/29 of the cases, respectively. Conclusion: b2a2 gene fusion was the most frequent in this CML population.

  14. Rot is a key regulator of Staphylococcus aureus biofilm formation

    Science.gov (United States)

    Mootz, Joe M.; Benson, Meredith A.; Heim, Cortney E.; Crosby, Heidi A.; Kavanaugh, Jeffrey S.; Dunman, Paul M.; Kielian, Tammy; Torres, Victor J.; Horswill, Alexander R.

    2015-01-01

    AUTHOR SUMMARY Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus (CA-MRSA) strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were upregulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm. PMID:25612137

  15. Superantigen Profiling of Staphylococcus aureus Infective Endocarditis Isolates

    Science.gov (United States)

    Chung, Jin-Won; Karau, Melissa J.; Greenwood-Quaintance, Kerryl E.; Ballard, Alessandro D.; Tilahun, Ashenafi; Khaleghi, Shahryar Rostamkolaei; David, Chella S.; Patel, Robin; Rajagopalan, Govindarajan

    2014-01-01

    The frequency of superantigen production among Staphylococcus aureus isolates associated with endocarditis is not well defined. We tested 154 S. aureus isolates from definite infective endocarditis cases for the presence of staphylococcal enterotoxins A-E, H and TSST-1 by PCR, ELISA and using an HLA-DR3 transgenic mouse splenocyte proliferation assay. Sixty-three isolates (50.8%) tested positive for at least one superantigen gene, with 21 (16.9%) testing positive for more than two. tst (28.6%) was most common, followed by seb (27%), sea (22.2%), sed (20.6%), see (17.5%), and sec (11.1%). Of 41 methicillin-resistant S. aureus, 21 had superantigen genes, with sed being more frequently detected in this group compared to methicillin-susceptible S. aureus (P<0.05). Superantigen genes were not associated with mortality (P=0.81). 75% of PCR-positive isolates induced robust splenocyte proliferation. Overall, more than half of S. aureus isolates causing endocarditis carry superantigen genes of which most are functional. PMID:24745820

  16. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

  17. Application of a Real-time Reverse Transcription Loop Mediated Amplification Method to the Detection of Rabies Virus in Arctic Foxes in Greenland

    DEFF Research Database (Denmark)

    Wakeley, Philip; Johnson, Nicholas; Rasmussen, Thomas Bruun

    Reverse transcription loop mediated amplification (RT-LAMP) offers a rapid, isothermal method for amplification of virus RNA. In this study a panel of positive rabies virus samples originally prepared from arctic fox brain tissue was assessed for the presence of rabies viral RNA using a real time...... RT-LAMP. The method had previously been shown to work with samples from Ghana which clustered with cosmopolitan lineage rabies viruses but the assay had not been assessed using samples from animals infected with rabies from the arctic region. The assay is designed to amplify both cosmopolitan strains...... virus of arctic origin virus can be detected using RT-LAMP and the method reported is more rapid than the real-time RT-PCR. Further arctic fox samples are under analysis in order to confirm these findings....

  18. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Viral and Bacterial Pathogens of Infectious Diarrhea

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2014-01-01

    Full Text Available Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1, and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2. The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20–200 copies for a single virus and 102-103 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.

  19. Missense mutations in PBP2A Affecting ceftaroline susceptibility detected in epidemic hospital-acquired methicillin-resistant Staphylococcus aureus clonotypes ST228 and ST247 in Western Switzerland archived since 1998.

    Science.gov (United States)

    Kelley, William L; Jousselin, Ambre; Barras, Christine; Lelong, Emmanuelle; Renzoni, Adriana

    2015-04-01

    The development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested the in vitro activity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 μg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 μg/ml; resistant [R], >1 μg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline

  20. Chemical composition of fennel essential oil and its impact on Staphylococcus aureus exotoxin production.

    Science.gov (United States)

    Qiu, Jiazhang; Li, Hongen; Su, Hongwei; Dong, Jing; Luo, Mingjing; Wang, Jianfeng; Leng, Bingfeng; Deng, Yanhong; Liu, Juxiong; Deng, Xuming

    2012-04-01

    In this study, fennel oil was isolated by hydrodistillation, and the chemical composition was determined by gas chromatography/mass spectral analysis. The antimicrobial activity of fennel oil against Staphylococcus aureus was evaluated by broth microdilution. A haemolysis assay, tumour necrosis factor (TNF) release assay, western blot, and real-time reverse transcription (RT)-PCR were applied to investigate the influence of fennel oil on the production of S. aureus virulence-related exoproteins. The data show that fennel oil, which contains a high level of trans-anethole, was active against S. aureus, with MICs ranging from 64 to 256 μg/ml. Furthermore, fennel oil, when used at subinhibitory concentrations, could dose-dependently decrease the expression of S. aureus exotoxins, including α-toxin, Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1).

  1. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

    Science.gov (United States)

    Goodell, Christa K; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O'Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J

    2016-01-01

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.

  2. Visual detection of West Nile virus using reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip

    Directory of Open Access Journals (Sweden)

    Zengguo eCao

    2016-04-01

    Full Text Available West Nile virus (WNV causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification methodfor WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF was developed to detect the envelope (E gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl ofan WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubationof the amplification product on the visualization strip, and no cross-reaction with other closely related members of theFlavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV.The assay produced sensitivities of 101.5TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

  3. [Visual detection of H1 subtype and identification of N1, N2 subtype of avian influenza virus by reverse transcription loop-mediated isothermal amplification assay].

    Science.gov (United States)

    Peng, Yi; Xie, Zhi-Xun; Guo, Jie; Zhou, Chen-Yu; Liu, Jia-Bo; Pang, Yao-Shan; Deng, Xian-Wen; Xie, Zhi-Qin; Xie, Li-Ji; Fan, Qing; Luo, Si-Si

    2013-03-01

    In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.

  4. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    Science.gov (United States)

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

  5. Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

    Science.gov (United States)

    Fischer, Cristine Dossin Bastos; Ikuta, Nilo; Canal, Cláudio Wageck; Makiejczuk, Aline; Allgayer, Mariangela da Costa; Cardoso, Cristine Hoffmeister; Lehmann, Fernanda Kieling; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

    2013-12-01

    Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.

  6. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    Zhu Yang; Guoliang Mao; Yujun Liu; Yuan-Chuan Chen; Chengjing Liu; Jun Luo; Xihan Li

    2013-01-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N 1/2009 influenza A virus.In this study,we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus.The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers.The qRT-PCR assays with the newly designed primers are highly specific,and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human,swine,and raccoon dog origin.Furthermore,the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction,respectively.When tested with 83 clinical samples,32 were detected to be positive using the qRT-PCR assays with our designed primers,while only 25 were positive by the assays with the WHO-recommended primers.These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

  7. Prevalence of methicillin resistance and virulence determinants of Staphylococcus aureus in diabetic foot ulcers

    Directory of Open Access Journals (Sweden)

    Sumeet Sandhu

    2014-12-01

    Conclusions: DNA-based detection and characterization of bacteria in DFUs are rapid and efficient and can assist in accurate, targeted antibiotic therapy of DFU infections. The majority of S. aureus detected in this study were highly virulent and also resistant to methicillin. Further studies are required to understand the role of S. aureus in DFU trajectory. [Int J Basic Clin Pharmacol 2014; 3(6.000: 978-982

  8. Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection.

    Directory of Open Access Journals (Sweden)

    Ryan A Waters

    Full Text Available Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD. Current approaches involve either; 1 Detection of FMD virus (FMDV with immuochromatographic antigen lateral flow devices (LFD, which have relatively low analytical sensitivity, or 2 portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription-LAMP (RT-LAMP assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing "proof of concept" for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.

  9. Establishment and application of multiplex PCR detection method for Staphylococcus aureus,Streptococcus,Salmonella spp and Escherichia coli%链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌多重PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    许信刚; 杨丽花; 童德文

    2012-01-01

    根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。%A multiplex PCR assay was developed and evaluated for the clinical detection of Staphylococcus aureus,Streptococcus,Salmonella spp and Escherichia coli.Four pairs of primers were designed and synthesized according to highly conserved regions of the Streptococcus ef-tu gene,Staphylococcus aureus nuc gene,Salmonella spp hut gene and Escherichia coli 23SrRNA gene of NCBI.The multiplex PCR reaction condition was optimized and the multiplex PCR method for detecting the co-infection of Staphylococcus aureus,Streptococcus,Salmonella spp and Escherichia coli was established.The specificity test showed that a fragment of 197,278,495 and 652 bp was amplified from genomic DNA of Streptococcus,Staphylococcus aureus,Salmonella spp and Escherichia coli,respectively.No amplification was achieved from control groups of other bacteria.The sensitivity test showed that the multiplex PCR could detect genome DNA of 25.6 pg for Streptococcus,33.2 pg for Staphylococcus aureus,35.7 pg for Salmonella spp

  10. Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus.

    Science.gov (United States)

    Liu, Pei; Han, Lei; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2016-08-15

    Staphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLTTS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By functionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19CFUmL(-1)S. aureus within 30min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments.

  11. Prevalence of enterotoxigenic Staphylococcus aureus in organic milk and cheese in Tabriz, Iran.

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    Yalda Rahbar Saadat

    2014-10-01

    Full Text Available Staphylococcal food poisoning is a gastrointestinal disease, which is caused by consumption of contaminated food with enterotoxins produced by Staphylococcus aureus (SEs. Milk and its products are known sources of food borne diseases. This study was carried out to evaluate the prevalence of enterotoxigenic S. aureus strains in organic milk and cheese in Tabriz - Iran.A total of 200 samples (100 milk samples and 100 cheese samples were collected from farms and milk collection points in Tabriz - Iran. The samples were cultured and identified by standard bacteriological methods, then PCR was performed to detect sea gene.Staphylococcus aureus was found in 27% of all samples (milk and cheese. Results of PCR showed that 12.96% of S. aureus isolates possessed sea gene. It suggested the potential public health threat of S. aureus resulting from contamination of dairy products. So, efforts are required to improve safety standards for preventing staphylococcal food poisoning.

  12. Mild Staphylococcus aureus skin infection improves the course of subsequent endogenous S. aureus bacteremia in mice

    NARCIS (Netherlands)

    S. van den Berg (Sanne); C.P. de Vogel (Corné); A.F. van Belkum (Alex); I.A.J.M. Bakker-Woudenberg (Irma)

    2015-01-01

    textabstractStaphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and ev

  13. [Protein toxins of Staphylococcus aureus].

    Science.gov (United States)

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

  14. Rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Pan, Chu-Hsiang; Jong, Ming-Hwa; Huang, Yu-Liang; Huang, Tien-Shine; Chao, Parn-Hwa; Lai, Shiow-Suey

    2008-07-01

    A simple one-step reverse transcription polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild type and vaccine strains of Classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.

  15. One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification

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    Wei Qi-Wei

    2012-09-01

    Full Text Available Abstract Background Bean pod mottle virus (BPMV is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. Methods A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. Results The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn’t be used for detection of BPMV using crude RNA extract from soybean seeds. Conclusion A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.

  16. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    Science.gov (United States)

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

  17. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

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    Cui Shang-jin

    2010-05-01

    Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

  18. Detection of different Staphylococcus aureus strains in bovine milk from subclinical mastitis using PCR and routine techniques Detecção de diferentes cepas de Staphylococcus aureus de mastite bovina subclínica através da técnica de PCR e técnicas tradicionais

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    Olney Vieira-da-Motta

    2001-03-01

    Full Text Available Contamination of fresh milk with Staphylococcus aureus was assessed comparatively through routine phenotypic (coagulase tube test and coagulase slide test and genotypic (PCR screening of 128 S. aureus strains isolated from 555 milk samples. These samples were collected from 362 cows with subclinical mastitis, hosted in different dairy herds at various locations of the Northern and Northeastern rural areas of the State of Rio de Janeiro, 39.7% of which were CMT-positive. All S. aureus isolates tested positive for the presence of the coagulase gene by PCR and the isolates could be grouped into four distinct classes according to the size of the PCR product. The strains also yielded variable results when assayed with coagulase test. Taken together, these data indicate the existence of extensive polymorphism at the coagulase gene locus in the genus Staphylococcus and exemplifies the extent of molecular and phenotypic heterogeneity associated with the strains circulating in rural herds.Quinhentas e cinqüenta e cinco amostras de leite, provenientes de 362 vacas com mastite subclínica em diferentes propriedades rurais do Estado do Rio de Janeiro, Brasil, de 1995 a 1997, foram submetidas ao teste "Califórnia Mastitis Test" (CMT. 39,7% das amostras foram positivas, das quais foram isoladas 128 cepas de Staphylococcus aureus. Todas as cepas isoladas foram positivas para o gene da coagulase utilizando a técnica de PCR, todavia, resultados de coagulase através das técnicas em tubo e "coagulase slide test" foram variáveis. Após a amplificação do gen de coagulase através da técnica de PCR utilizando iniciadores específicos para o referido gen, fragmentos com diferentes pesos moleculares foram vistos através de análise em gel de agarose, sugerindo a ocorrência de polimorfismo genético. O estudo também sugere a ocorrência de diferentes cepas da bactéria atuando nos rebanhos leiteiros causando mastite bovina.

  19. Determination of enterotoxigenic and methicillin resistant Staphylococcus aureus in ice cream.

    Science.gov (United States)

    Gücükoğlu, Ali; Çadirci, Özgür; Terzi, Göknur; Kevenk, T Onur; Alişarli, Mustafa

    2013-05-01

    The aim of this study was to determine the prevalence of enterotoxigenic and methicillin-resistant Staphylococcus aureus in ice creams. After culture-based identification of isolates, the presence of 16S rRNA and nuc was confirmed by mPCR. S. aureus was identified in 18 of 56 fruity (32.1%), 4 of 32 vanilla (12.5%), and 1 of 12 chocolate (8.3%) ice creams. S. aureus was identified as 38 isolates in 23 ice cream samples by culture-based techniques, but only 35 isolates were confirmed by PCR as S. aureus. To determine the enterotoxigenic properties of PCR-confirmed S. aureus isolates, a toxin detection kit was used (SET RPLA®). Of the 12 enterotoxigenic S. aureus isolates, 9 SEB (75%), 1 SED (8.3%), 1 SEB+SED (8.3%), and 1 SEA+SEB+SED (8.3%) expressing isolates were found. The presence of enterotoxin genes (sea, seb, sed) was identified in 13 (37.1%) out of 35 isolates by the mPCR technique. In the ice cream isolates, the sea, seb, and sed genes were detected: 1 sea (7.6%), 9 seb (69.2%), 1 sed (7.6%), 1 seb+sed (7.6%), and 1 sea+seb+sed (7.6%), respectively. The sec gene was not detected in any of these isolates. One of the 35 (2.8%) S. aureus strain was mecA positive.

  20. Biofilm formation by Staphylococcus aureus isolates from a dental clinic in Konya, Turkey.

    Science.gov (United States)

    Torlak, Emrah; Korkut, Emre; Uncu, Ali T; Şener, Yağmur

    2017-02-14

    The ability of Staphylococcus aureus to form biofilm is considered to be a major virulence factor influencing its survival and persistence in both the environment and the host. Biofilm formation in S. aureus is most frequently associated with production of polysaccharide intercellular adhesion by ica operon-encoded enzymes. The present work aimed at evaluating the in vitro biofilm production and presence of the icaA and icaD genes in S. aureus isolates from a dental clinic in Konya, Turkey. The surfaces of inanimate objects were sampled over a period of six months. S. aureus isolates were subjected to Congo Red Agar (CRA) and crystal violet (CV) staining assays to evaluate their ability of biofilm production, while the presence of the icaA and icaD genes was determined by polymerase chain reaction. S. aureus contamination was detected in 13.2% of the environmental samples. All the 32 isolates were observed to be positive for both the icaA and icaD genes. Phenotypic evaluations revealed that CV staining assay is a more reliable alternative to CRA assay to determine biofilm formation ability. A high percentage of agreement (91%) was observed between the results from CV staining and ica genes' detection assays. Phenotypic and genotypic evaluations should be combined to detect biofilm formation in S. aureus. Our findings indicate that dental clinic environments should be considered as potential reservoir for biofilm-producing S. aureus and thus cross contamination.

  1. Staphylococcus aureus Transcriptome Architecture : From Laboratory to Infection-Mimicking Conditions

    NARCIS (Netherlands)

    Mäder, Ulrike; Nicolas, Pierre; Depke, Maren; Pané-Farré, Jan; Debarbouille, Michel; van der Kooi-Pol, Magdalena M; Guérin, Cyprien; Dérozier, Sandra; Hiron, Aurelia; Jarmer, Hanne; Leduc, Aurélie; Michalik, Stephan; Reilman, Ewoud; Schaffer, Marc; Schmidt, Frank; Bessières, Philippe; Noirot, Philippe; Hecker, Michael; Msadek, Tarek; Völker, Uwe; van Dijl, Jan Maarten

    2016-01-01

    Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S.

  2. Atlas(®) Listeria monocytogenes LmG2 Detection Assay Using Transcription Mediated Amplification to Detect Listeria monocytogenes in Selected Foods and Stainless Steel Surface.

    Science.gov (United States)

    Bres, Vanessa; Yang, Hua; Hsu, Ernie; Ren, Yan; Cheng, Ying; Wisniewski, Michele; Hanhan, Maesa; Zaslavsky, Polina; Noll, Nathan; Weaver, Brett; Campbell, Paul; Reshatoff, Michael; Becker, Michael

    2014-01-01

    The Atlas Listeria monocytogenes LmG2 Detection Assay, developed by Roka Bioscience Inc., was compared to a reference culture method for seven food types (hot dogs, cured ham, deli turkey, chicken salad, vanilla ice cream, frozen chocolate cream pie, and frozen cheese pizza) and one surface (stainless steel, grade 316). A 125 g portion of deli turkey was tested using a 1:4 food:media dilution ratio, and a 25 g portion for all other foods was tested using 1:9 food:media dilution ratio. The enrichment time and media for Roka's method was 24 to 28 h for 25 g food samples and environmental surfaces, and 44 to 48 h for 125 g at 35 ± 2°C in PALCAM broth containing 0.02 g/L nalidixic acid. Comparison of the Atlas Listeria monocytogenes LmG2 Detection Assay to the reference method required an unpaired approach. For each matrix, 20 samples inoculated at a fractional level and five samples inoculated at a high level with a different strain of Listeria monocytogenes were tested by each method. The Atlas Listeria monocytogenes LmG2 Detection Assay was compared to the Official Methods of Analysis of AOAC INTERNATIONAL 993.12 method for dairy products, the U.S. Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 8.08 method for ready-to-eat meat and environmental samples, and the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 10 method for frozen foods. In the method developer studies, Roka's method, at 24 h (or 44 h for 125 g food samples), had 126 positives out of 200 total inoculated samples, compared to 102 positives for the reference methods at 48 h. In the independent laboratory studies, vanilla ice cream, deli turkey and stainless steel grade 316 were evaluated. Roka's method, at 24 h (or 44 h for 125 g food samples), had 64 positives out of 75 total inoculated samples compared to 54 positives for the reference methods at 48 h. The Atlas Listeria monocytogenes LmG2 Detection Assay detected all 50

  3. Detection of staphylococcus aureus enterotoxin genes in chronic rhinosinusitis%慢性鼻-鼻窦炎中金黄色葡萄球菌肠毒素基因研究

    Institute of Scientific and Technical Information of China (English)

    申迹; 项锦银; 寇巍; 洪苏玲

    2012-01-01

    目的 检测国人慢性鼻-鼻窦炎患者鼻腔金黄色葡萄球菌肠毒素基因谱,探讨与国人慢性鼻-鼻窦炎发病相关的金黄色葡萄球菌肠毒素基因类型.方法 本实验取国人慢性鼻-鼻窦炎不伴鼻息肉组、慢性鼻-鼻窦炎伴鼻息肉组及鼻中隔偏曲组患者中鼻道黏膜拭子进行金黄色葡萄球菌分离培养鉴定,通过PCR技术检测实验组每例患者金黄色葡萄球菌样本18种肠毒素基因的携带情况,并进行统计学分析.结果 3组患者金黄色葡萄球菌肠毒素基因均有较高的阳性检出率,但差异无统计学意义;3组患者阳性检出例数均较高的几种肠毒素基因为:seg、sem、sen、sei、seo、seu;金黄色葡萄球菌肠毒素基因seq在慢性鼻-鼻窦炎伴鼻息肉患者中有16例阳性检出,而对照组无阳性检出,差异有统计学意义.结论 金黄色葡萄球菌肠毒素seg、sem、sen、sei、seo、seu可能在慢性鼻-鼻窦炎的发病中起主要作用,肠毒素seq与慢性鼻-鼻窦炎伴鼻息肉发病的关系还有待进一步深入研究.%Objective This study was to investigate whether the presence of any of the 18 currently known staphylococcal enterotoxin genes could be correlated with chronic rhinosinusitis. Methods The Staphylococcus strains were taken from the middle mea-tus of patients suffering from chronic rhinosinusitis, with or without nasal polyposis,and controls were systematically tested for the presence of the 18 currently known enterotoxin genes by PCR. Results Higher positive rates of staphylococcus aureus enterotoxin genes were detected in three groups. There was no statistically significant difference between these rates;The most frequently detected enterotoxin genes were:seg,sem,sen,sei,seo,seu in three groups;the enterotoxin gene seq was frequently observed in CRSwNP group for the first time, but absent in the control group(P = 0. 03). Conclusion The enterotoxins: seg, sem, sen, sei, seo, seu may play roles in

  4. Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

    Directory of Open Access Journals (Sweden)

    Emily J Johnson

    Full Text Available Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion.87 samples [42 oropharyngeal (OP and 45 expectorated sputum (ES] were analyzed. 59 samples (68% cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%, followed by FemA qPCR (52% and 16SrRNA qPCR (34%. For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs.Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.

  5. Tracing and inhibiting growth of Staphylococcus aureus in barbecue cheese production after product recall.

    Science.gov (United States)

    Johler, S; Zurfluh, K; Stephan, R

    2016-05-01

    Staphylococcal food poisoning is one of the most prevalent causes of foodborne intoxication worldwide. It is caused by ingestion of enterotoxins formed by Staphylococcus aureus during growth in the food matrix. Following a recall of barbecue cheese due to the detection of staphylococcal enterotoxins in Switzerland in July 2015, we analyzed the production process of the respective dairy. Although most cheese-making processes involve acidification to inhibit the growth of pathogenic bacteria, barbecue cheese has to maintain a pH >6.0 to prevent undesired melting of the cheese. In addition, the dairy decided to retain the traditional manual production process of the barbecue cheese. In this study, therefore, we aimed to (1) trace Staph. aureus along the barbecue cheese production process, and (2) develop a sustainable strategy to inhibit growth of Staph. aureus and decrease the risk of staphylococcal food poisoning without changing the traditional production process. To this end, we traced Staph. aureus in a step-wise blinded process analysis on 4 different production days using spa (Staphylococcus protein A gene) typing, DNA microarray profiling, and pulsed-field gel electrophoresis analysis. We subsequently selected a new starter culture and used a model cheese production including a challenge test assay to assess its antagonistic effect on Staph. aureus growth, as well as its sensory and technological implications. We detected Staph. aureus in 30% (37/124) of the collected samples taken from the barbecue cheese production at the dairy. This included detection of Staph. aureus in the final product on all 4 production days, either after enrichment or using quantitative detection. We traced 2 enterotoxigenic Staph. aureus strains (t073/CC45 and t282/CC45) colonizing the nasal cavity and the forearms of the cheesemakers to the final product. In the challenge test assay, we were able to show that the new starter culture inhibited growth of Staph. aureus while meeting

  6. Prevention of Healthcare Associated Staphylococcus aureus Infections

    NARCIS (Netherlands)

    L.G.M. Bode (Lonneke)

    2014-01-01

    markdownabstract__Abstract__ S. aureus colonizes the skin and mucosae of a proportion of the human population. Carriers of S. aureus are at increased risk of developing infections with this pathogen. The aim of this thesis was to add to the prevention of healthcare associated S. aureus infections.

  7. Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

    Science.gov (United States)

    Wehner, Stefanie; Mannala, Gopala K; Qing, Xiaoxing; Madhugiri, Ramakanth; Chakraborty, Trinad; Mraheil, Mobarak A; Hain, Torsten; Marz, Manja

    2014-01-01

    The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

  8. Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

    Directory of Open Access Journals (Sweden)

    Stefanie Wehner

    Full Text Available The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30% of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs. We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected

  9. Detection of Very Long Antisense Transcripts by Whole Transcriptome RNA-Seq Analysis of Listeria monocytogenes by Semiconductor Sequencing Technology

    Science.gov (United States)

    Wehner, Stefanie; Mannala, Gopala K.; Qing, Xiaoxing; Madhugiri, Ramakanth; Chakraborty, Trinad; Mraheil, Mobarak A.; Hain, Torsten; Marz, Manja

    2014-01-01

    The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host. PMID

  10. Dermatology case: segmental lichen aureus

    OpenAIRE

    Fernandes, I.; S. Carvalho; Machado, S.; Alves,R.; Selores, M.

    2012-01-01

    ABSTRACT The authors describe a clinical case of a six-year-old boy with history of a segmental brownish maculopapular skin eruption on his left thoracic and lumbar wall, since the last four months. Based on clinical and histological findings he was diagnosed with segmental lichen aureus.

  11. A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus.

    Science.gov (United States)

    Ambagala, A; Pahari, S; Fisher, M; Lee, P-Y A; Pasick, J; Ostlund, E N; Johnson, D J; Lung, O

    2017-04-01

    Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory.

  12. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

    Science.gov (United States)

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab

    2016-01-01

    Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments. PMID:27904520

  13. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

    Science.gov (United States)

    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  14. Very quick reverse transcription polymerase chain reaction for detecting 2009 H1N1 influenza A using wire-guide droplet manipulationst.

    Science.gov (United States)

    You, David J; Tran, Phat L; Kwon, Hyuck-Jin; Patel, Deepa; Yoon, Jeong-Yeol

    2011-01-01

    Reverse transcription polymerase chain reaction (RT-PCR) is currently a gold standard in identifying influenza A virus, especially H1N1 flu. Typical RT-PCR assays take about 1-2 h for thermocycling, and there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point-of-care detection tool. There have been attempts to further speed up the PCR assays by reducing its volume. There have also been attempts to use droplet microfluidics technology to PCR, primarily to automate the PCR enrichment processes and take advantage of its small volume. In all these attempts, heating and cooling is made by conduction heat transfer. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convection heat transfer, whose heat transfer coefficients are much higher than that of conduction. We used our newly-invented method of wire-guide droplet manipulations towards very quick RT-PCR. Computational fluid dynamics (CFD) simulation of our system revealed that heating/cooling for each temperature change takes 1-4 s for a 10 microL droplet, as compared to >30 s in the other quick PCRs. Theoretically a 30-cycle process can take as short as 13 s x 30 cycles = 6 min 30 s. The entire system was made as a single instrument, with the components made by a milling machine and a rapid prototyping device. No additional equipment and external computers are required. With this newly developed system, 160 bp gene sequence was amplified from 2009 H1N1 influenza A (human origin). The 30-cycle process took as short as 6 min 50 s for a 10 microL droplet (with additional 4 min for reverse transcription). Its product was confirmed by traditional gel electrophoresis, subsequent imaging as well as gene sequencing, which has been very difficult with the other stationary droplet/nanodrop approaches. The proposed system has a

  15. The Frequency of Staphylococcus aureus Isolated from Endocervix of Infertile Women in Northwest Iran

    Directory of Open Access Journals (Sweden)

    Akhi Mohammad Taghi

    2017-01-01

    Full Text Available Background Infertility is one of the major social issues. Due to the asymptomatic cervical infection associated with Staphylococcus aureus (S. aureus, the majority of patients remain undiagnosed. The present study intended to assess the frequency of S. aureus isolated from infertile women’s endocervix in northwest Iran. Materials and Methods In a descriptive cross sectional study, specimens were randomly collected during vagina examination using a sterile speculum and swabbing. After performance of antibiotic susceptibility testing, polymerase chain reaction (PCR was used to identify methicillin-resistance S. aureus (MRSA and toxic shock syndrome toxin-1 (TSST-1. Results About 26 (26% and 9 (9% women’s urogenital tracts were colonized by S. aureus and Candida spp., respectively, of which three (11.5% patients were infected with fungi and S. aureus, simultaneously. Antibiotic susceptibility results showed high activity of vancomycin and co-trimoxazole on isolates. Regarding PCR results, mecA sequences were detected in 7 (26.9% strains, whilst the tst gene encoding TSST-1 was not detected in any of clinical strains. Conclusion The prevalence of S. aureus was very high in infertile women. Therefore, it demands all patients undergoing infertility treatment to be investigated thoroughly for this type of infection.

  16. Phage sensitivity and prophage carriage in Staphylococcus aureus isolated from foods in Spain and New Zealand.

    Science.gov (United States)

    Gutiérrez, Diana; Rodríguez-Rubio, Lorena; García, Pilar; Billington, Craig; Premarante, Aruni; Rodríguez, Ana; Martínez, Beatriz

    2016-08-02

    Bacteriophages (phages) are a promising tool for the biocontrol of pathogenic bacteria, including those contaminating food products and causing infectious diseases. However, the success of phage preparations is limited by the host ranges of their constituent phages. The phage resistance/sensitivity profile of eighty seven Staphylococcus aureus strains isolated in Spain and New Zealand from dairy, meat and seafood sources was determined for six phages (Φ11, K, ΦH5, ΦA72, CAPSa1 and CAPSa3). Most of the S. aureus strains were sensitive to phage K (Myoviridae) and CAPSa1 (Siphoviridae) regardless of their origin. There was a higher sensitivity of New Zealand S. aureus strains to phages isolated from both Spain (ΦH5 and ΦA72) and New Zealand (CAPSa1 and CAPSa3). Spanish phages had a higher infectivity on S. aureus strains of Spanish dairy origin, while Spanish strains isolated from other environments were more sensitive to New Zealand phages. Lysogeny was more prevalent in Spanish S. aureus compared to New Zealand strains. A multiplex PCR reaction, which detected ΦH5 and ΦA72 sequences, indicated a high prevalence of these prophages in Spanish S. aureus strains, but were infrequently detected in New Zealand strains. Overall, the correlation between phage resistance and lysogeny in S. aureus strains was found to be weak.

  17. Synthesis and function of phospholipids in Staphylococcus aureus.

    Science.gov (United States)

    Kuhn, Sebastian; Slavetinsky, Christoph J; Peschel, Andreas

    2015-02-01

    Phospholipids are the major components of bacterial membranes, and changes in phospholipid composition affect important cellular processes such as metabolism, stress response, antimicrobial resistance, and virulence. The most prominent phospholipids in Staphylococcus aureus are phosphatidylglycerol, lysyl-phosphatidylglycerol, and cardiolipin, whose biosynthesis is mediated by a complex protein machinery. Phospholipid composition of the staphylococcal membrane has to be continuously adjusted to changing external conditions, which is achieved by a series of transcriptional and biochemical regulatory mechanisms. This mini-review outlines the current state of knowledge concerning synthesis, regulation, and function of the major staphylococcal phospholipids.

  18. Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

    Directory of Open Access Journals (Sweden)

    Benjamin P Howden

    2011-11-01

    Full Text Available Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA and vancomycin-intermediate ST239 S. aureus (VISA; each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

  19. Evaluation of non-invasive biological samples to monitor Staphylococcus aureus colonization in great apes and lemurs.

    Directory of Open Access Journals (Sweden)

    Frieder Schaumburg

    Full Text Available INTRODUCTION: Reintroduction of endangered animals as part of conservational programs bears the risk of importing human pathogens from the sanctuary to the natural habitat. One bacterial pathogen that serves as a model organism to analyze this transmission is Staphylococcus aureus as it can colonize and infect both humans and animals. The aim of this study was to evaluate the utility of various biological samples to monitor S. aureus colonization in great apes and lemurs. METHODS: Mucosal swabs from wild lemurs (n=25, Kirindy, Madagascar, feces, oral and genital swabs from captive chimpanzees (n=58, Ngamba and Entebbe, Uganda and fruit wadges and feces from wild chimpanzees (n=21, Taï National Parc, Côte d'Ivoire were screened for S. aureus. Antimicrobial resistance and selected virulence factors were tested for each isolate. Sequence based genotyping (spa typing, multilocus sequence typing was applied to assess the population structure of S. aureus. RESULTS: Oro-pharyngeal carriage of S. aureus was high in lemurs (72%, n=18 and captive chimpanzees (69.2%, n=27 and 100%, n=6, respectively. Wild chimpanzees shed S. aureus through feces (43.8, n=7 and fruit wadges (54.5, n=12. Analysis of multiple sampling revealed that two samples are sufficient to detect those animals which shed S. aureus through feces or fruit wadges. Genotyping showed that captive animals are more frequently colonized with human-associated S. aureus lineages. CONCLUSION: Oro-pharyngeal swabs are useful to screen for S. aureus colonization in apes and lemurs before reintroduction. Duplicates of stool and fruit wadges reliably detect S. aureus shedding in wild chimpanzees. We propose to apply these sampling strategies in future reintroduction programs to screen for S. aureus colonization. They may also be useful to monitor S. aureus in wild populations.

  20. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Science.gov (United States)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  1. Multiplex Reverse Transcription-PCR for Simultaneous Detection of Beet Necrotic Yellow Vein Virus, Beet Soilborne Virus, and Beet Virus Q and Their Vector Polymyxa betae KESKIN on Sugar Beet

    OpenAIRE

    Meunier, Alexandre; Schmit, Jean-François; Stas, Arnaud; Kutluk, Nazli; Bragard, Claude

    2003-01-01

    Three soilborne viruses transmitted by Polymyxa betae KESKIN in sugar beet have been described: Beet necrotic yellow vein virus (BNYVV), the agent of rhizomania, Beet soilborne virus (BSBV), and Beet virus Q (BVQ). A multiplex reverse transcription-PCR technique was developed to simultaneously detect BNYVV, BSBV, and BVQ, together with their vector, P. betae. The detection threshold of the test was up to 128 times greater than that of an enzyme-linked immunosorbent assay. Systematic associati...

  2. Changes in the Staphylococcus aureus transcriptome during early adaptation to the lung.

    Directory of Open Access Journals (Sweden)

    Donald O Chaffin

    Full Text Available Staphylococcus aureus is a common inhabitant of the human nasopharynx. It is also a cause of life-threatening illness, producing a potent array of virulence factors that enable survival in normally sterile sites. The transformation of S. aureus from commensal to pathogen is poorly understood. We analyzed S. aureus gene expression during adaptation to the lung using a mouse model of S. aureus pneumonia. Bacteria were isolated by bronchoalveolar lavage after residence in vivo for up to 6 hours. S. aureus in vivo RNA transcription was compared by microarray to that of shake flask grown stationary phase and early exponential phase cells. Compared to in vitro conditions, the in vivo transcriptome was dramatically altered within 30 minutes. Expression of central metabolic pathways changed significantly in response to the lung environment. Gluconeogenesis (fbs, pckA was down regulated, as was TCA cycle and fermentation pathway gene expression. Genes associated with amino acid synthesis, RNA translation and nitrate respiration were upregulated, indicative of a highly active metabolic state during the first 6 hours in the lung. Virulence factors regulated by agr were down regulated in vivo and in early exponential phase compared to stationary phase cells. Over time in vivo, expression of ahpCF, involved in H(2O(2 scavenging, and uspA, which encodes a universal stress regulator, increased. Transcription of leukotoxic α and β-type phenol-soluble modulins psmα1-4 and psmβ1-2 increased 13 and 8-fold respectively; hld mRNA, encoding δ-hemolysin, was increased 9-fold. These were the only toxins to be significantly upregulated in vivo. These data provide the first complete survey of the S. aureus transcriptome response to the mammalian airway. The results present intriguing contrasts with previous work in other in vitro and in vivo models and provide novel insights into the adaptive and temporal response of S. aureus early in the pathogenesis of pneumonia.

  3. Changes in the Staphylococcus aureus transcriptome during early adaptation to the lung.

    Science.gov (United States)

    Chaffin, Donald O; Taylor, Destry; Skerrett, Shawn J; Rubens, Craig E

    2012-01-01

    Staphylococcus aureus is a common inhabitant of the human nasopharynx. It is also a cause of life-threatening illness, producing a potent array of virulence factors that enable survival in normally sterile sites. The transformation of S. aureus from commensal to pathogen is poorly understood. We analyzed S. aureus gene expression during adaptation to the lung using a mouse model of S. aureus pneumonia. Bacteria were isolated by bronchoalveolar lavage after residence in vivo for up to 6 hours. S. aureus in vivo RNA transcription was compared by microarray to that of shake flask grown stationary phase and early exponential phase cells. Compared to in vitro conditions, the in vivo transcriptome was dramatically altered within 30 minutes. Expression of central metabolic pathways changed significantly in response to the lung environment. Gluconeogenesis (fbs, pckA) was down regulated, as was TCA cycle and fermentation pathway gene expression. Genes associated with amino acid synthesis, RNA translation and nitrate respiration were upregulated, indicative of a highly active metabolic state during the first 6 hours in the lung. Virulence factors regulated by agr were down regulated in vivo and in early exponential phase compared to stationary phase cells. Over time in vivo, expression of ahpCF, involved in H(2)O(2) scavenging, and uspA, which encodes a universal stress regulator, increased. Transcription of leukotoxic α and β-type phenol-soluble modulins psmα1-4 and psmβ1-2 increased 13 and 8-fold respectively; hld mRNA, encoding δ-hemolysin, was increased 9-fold. These were the only toxins to be significantly upregulated in vivo. These data provide the first complete survey of the S. aureus transcriptome response to the mammalian airway. The results present intriguing contrasts with previous work in other in vitro and in vivo models and provide novel insights into the adaptive and temporal response of S. aureus early in the pathogenesis of pneumonia.

  4. Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus.

    LENUS (Irish Health Repository)

    Shore, Anna C

    2011-08-01

    Methicillin resistance in staphylococci is mediated by penicillin binding protein 2a (PBP 2a), encoded by mecA on mobile staphylococcal cassette chromosome mec (SCCmec) elements. In this study, two clonal complex 130 (CC130) methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients in Irish hospitals were identified that were phenotypically PBP 2a positive but lacked mecA by conventional PCR and by DNA microarray screening. The isolates were identified as methicillin-susceptible S. aureus using the GeneXpert real-time PCR assay. Whole-genome sequencing of one isolate (M10\\/0061) revealed a 30-kb SCCmec element encoding a class E mec complex with highly divergent blaZ-mecA-mecR1-mecI, a type 8 cassette chromosome recombinase (ccr) complex consisting of ccrA1-ccrB3, an arsenic resistance operon, and flanking direct repeats (DRs). The SCCmec element was almost identical to that of SCCmec type XI (SCCmec XI) identified by the Sanger Institute in sequence type 425 bovine MRSA strain LGA251 listed on the website of the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. The open reading frames (ORFs) identified within SCCmec XI of M10\\/0061 exhibited 21 to 93% amino acid identity to ORFs in GenBank. A third DR was identified ca. 3 kb downstream of SCCmec XI, indicating the presence of a possible SCC remnant. SCCmec XI was also identified in the second CC130 MRSA isolate by PCR and sequencing. The CC130 MRSA isolates may be of animal origin as previously reported CC130 S. aureus strains were predominantly from bovine sources. The highly divergent nature of SCCmec XI relative to other SCCmec elements indicates that it may have originated in another taxon.

  5. Population structure of Staphylococcus aureus from remote African Babongo Pygmies.

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    Frieder Schaumburg

    Full Text Available BACKGROUND: Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA predominantly encode the Panton-Valentine leukocidin (PVL, which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive. METHODOLOGY/PRINCIPAL FINDINGS: Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins, S. aureus protein A (spa types and multi-locus sequence types (MLST were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%, enterotoxin B (20.6%, exfoliative toxin D (11.7% and the epidermal cell differentiation inhibitor B (11.7% were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88. CONCLUSIONS: The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.

  6. Occurence and antimicrobial resistance of Staphylococcus aureus and Salmonella spp. in retail fish samples in Turkey.

    Science.gov (United States)

    Ertas Onmaz, Nurhan; Abay, Secil; Karadal, Fulden; Hizlisoy, Harun; Telli, Nihat; Al, Serhat

    2015-01-15

    The aims of this study were to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxins, as well as Salmonella spp. and to determine the antimicrobial susceptibilities of the isolates from fish samples. A total of 100 fish samples were analysed consisting of 30 anchovy, 35 trout and 35 sea bream. The presence of SEs was detected using ELISA and its genes confirmed by mPCR. Also, S. aureus and Salmonella spp. were detected in 9 (9%) and 5 (5%) samples, respectively. None of the S. aureus isolates had SEs and SEs genes. The resistance rates of the S. aureus isolates to erythromycin, tetracycline, and penicillin G were found to be 33% while Salmonella spp. isolates were resistant to trimethoprim-sulfamethoxazole, gentamicin and neomycine in 20%, 20% and 80%, respectively of the samples. It is of utmost important for public health that retail fish markets need to use hygienic practices in handling and processing operations.

  7. Reverse transcription PCR-based detection of Crimean-Congo hemorrhagic fever virus isolated from ticks of domestic ruminants in Kurdistan province of Iran.

    Science.gov (United States)

    Fakoorziba, Mohammad Reza; Golmohammadi, Parvaneh; Moradzadeh, Rahmatollah; Moemenbellah-Fard, Mohammad Djaefar; Azizi, Kourosh; Davari, Behrooz; Alipour, Hamzeh; Ahmadnia, Sara; Chinikar, Sadegh

    2012-09-01

    Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal viral vector-borne zoonosis which has a mortality rate of up to 30% without treatment in humans. CCHF virus is transmitted to humans by ticks, predominantly from the Hyalomma genus. Following the report of two confirmed and one suspected death due to CCHF virus in Kurdistan province of Iran in 2007, this study was undertaken to determine the fauna of hard ticks on domestic ruminants (cattle, sheep, and goats) and their possible infection with CCHF virus using reverse transcription PCR technique. This is the first detection of CCHF virus in ticks from the Kurdistan province of Iran. Overall, 414 ixodid ticks were collected from two districts in this province. They represented four genera from which 10 separate species were identified. The Hyalomma genus was the most abundant tick genus (70%). It was the only genus shown to be infected with the CCHF virus using RT-PCR technique. The number of ticks positive for CCHF virus was 5 out of 90 (5.6%) adult ticks. The three remaining genera (Haemaphysalis, Rhipicephalus, and Dermacentor) were all negative following molecular survey. Four of the five virally-infected ticks were from cattle mainly in the Sanandaj district. We concluded that CCHF virus is present in the Hyalomma ticks on domestic ruminants (cattle) in Kurdistan province of Iran.

  8. Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Kang, Xiaoping; Wu, Weili; Zhang, Chuntao; Liu, Licheng; Feng, Huahua; Xu, Lizhi; Zheng, Xin; Yang, Honglei; Jiang, Yongqiang; Xu, Bianli; Xu, Jin; Yang, Yinhui; Chen, Weijun

    2014-09-01

    The first case of avian influenza A/H7N9 infection was reported in Shanghai in mid-February, 2013; by May 1, 2013, it had infected 127 people and caused 26 deaths in 10 provinces in China. Therefore, it is important to obtain reliable epidemiological data on the spread of this new infectious agent, a need that may be best met by the development of novel molecular methods. Here, a new method was described for the detection of avian influenza A/H7N9 using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Using serial dilutions of avian influenza A H7N9 cultures, the detection limit of the assay was determined to be approximately 3.2×10(-4) HAUs (hemagglutination units) for the H7 gene and 6.4×10(-4) HAUs for N9 gene. In tests of serial dilutions of in vitro-transcribed avian influenza A H7 and N9 gene RNA, positive results were obtained for target RNA containing at least three copies of the H7 gene and six copies of the N9 gene. Thirteen throat swabs from H7N9 patients were tested; all tested positive in the assay. Specificity was evaluated by testing 18 other subtypes of influenza viruses; all tested negative. A total of 180 throat swabs from patients infected with influenza virus, including 60 from patients infected with seasonal influenza A/H1N1 virus, 60 from patients infected with pandemic influenza A/H1N1/2009 virus, 30 from patients infected with seasonal influenza A/H3N2 virus and 30 from patients infected with influenza B virus, were also tested; all tested negative.

  9. Detection of Viral Hemorrhagic Septicemia Virus by Quantitative Reverse Transcription Polymerase Chain Reaction from Two Fish Species at Two Sites in Lake Superior

    Science.gov (United States)

    Cornwell, Emily R.; Eckerlin, Geofrey E.; Getchell, Rodman G.; Groocock, Geoffrey H.; Thompson, Tarin M.; Batts, William N.; Casey, Rufina N.; Kurath, Gael; Winton, James R.; Bowser, Paul R.; Bain, Mark B.; Casey, James W.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  10. Detection of feline coronavirus in cheetah (Acinonyx jubatus) feces by reverse transcription-nested polymerase chain reaction in cheetahs with variable frequency of viral shedding.

    Science.gov (United States)

    Gaffney, Patricia M; Kennedy, Melissa; Terio, Karen; Gardner, Ian; Lothamer, Chad; Coleman, Kathleen; Munson, Linda

    2012-12-01

    Cheetahs (Acinonyx jubatus) are a highly threatened species because of habitat loss, human conflict, and high prevalence of disease in captivity. An epidemic of feline infectious peritonitis and concern for spread of infectious disease resulted in decreased movement of cheetahs between U.S. zoological facilities for managed captive breeding. Identifying the true feline coronavirus (FCoV) infection status of cheetahs is challenging because of inconsistent correlation between seropositivity and fecal viral shedding. Because the pattern of fecal shedding of FCoV is unknown in cheetahs, this study aimed to assess the frequency of detectable fecal viral shedding in a 30-day period and to determine the most efficient fecal sampling strategy to identify cheetahs shedding FCoV. Fecal samples were collected from 16 cheetahs housed at seven zoological facilities for 30 to 46 consecutive days; the samples were evaluated for the presence of FCoV by reverse transcription-nested polymerase chain reaction (RT-nPCR). Forty-four percent (7/16) of cheetahs had detectable FCoV in feces, and the proportion of positive samples for individual animals ranged from 13 to 93%. Cheetahs shed virus persistently, intermittently, or rarely over 30-46 days. Fecal RT-nPCR results were used to calculate the probability of correctly identifying a cheetah known to shed virus given multiple hypothetical fecal collection schedules. The most efficient hypothetical fecal sample collection schedule was evaluation of five individual consecutive fecal samples, resulting in a 90% probability of identifying a known shedder. Demographic and management risk factors were not significantly associated (P cheetahs shed virus intermittently to rarely, fecal sampling schedules meant to identify all known shedders would be impractical with current tests and eradication of virus from the population unreasonable. Managing the captive population as endemically infected with FCoV may be a more feasible approach.

  11. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    Science.gov (United States)

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB.

  12. Superantigens in Staphylococcus aureus isolated from prosthetic joint infection

    Science.gov (United States)

    Kim, Choon K.; Karau, Melissa J.; Greenwood-Quaintance, Kerryl E.; Tilahun, Ashenafi Y.; David, Chella S.; Mandrekar, Jayawant N.; Patel, Robin; Rajagopalan, Govindarajan

    2014-01-01

    Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea, seb, sec, sed, see, seg, seh, sei and tst, were assayed by PCR. Seventy-eight (92.9%) isolates carried at least one SAg gene studied, with 61 (72.6%) harboring more than one. seg was most commonly (70.2%) and seh was least frequently (4.8%) detected. tst-positive isolates were associated with early infection and increased ESR at diagnosis (P = 0.006 and P = 0.021, respectively). seg and sei were associated with methicillin resistance (P = 0.008 and 0.002, respectively). SAg genes are prevalent in S. aureus causing PJI; a majority of PJI-associated isolates produce biologically active SAgs in both planktonic and biofilm growth modes. PMID:25619753

  13. Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

    Directory of Open Access Journals (Sweden)

    Lamb JoAnn FS

    2011-04-01

    -Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

  14. Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Rasigade Jean

    2011-12-01

    Full Text Available Abstract Background Staphylococcus aureus is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by fnbA/B, are major pathogenesis determinants in these infections through their involvement in S. aureus adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs of antibiotics, frequently occurring in vivo because of impaired drug diffusion at the infection site, can alter S. aureus phenotype. We therefore investigated their impact on S. aureus fibronectin-mediated adhesiveness and invasiveness. Methods After in vitro challenge of S. aureus 8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored fnbA/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts. Results Oxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in fnbA/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting fnbA/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or fnbA/B transcription levels. Only oxacillin-treated S. aureus displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls. Conclusion Our findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in S. aureus in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled in vitro conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during in vivo infections is thus still uncertain and requires further investigations.

  15. Detection and serological type identification of enterotoxins in dairy-derived Staphylococcus aureus%乳源性金黄色葡萄球菌肠毒素的检测与基因分型

    Institute of Scientific and Technical Information of China (English)

    杨军; 张驰

    2011-01-01

    In the present study, we collected dairy-derived S. aureus strains in the inspection of various dairy products, and then examined the enterotoxin production of the isolates using enzyme-linked fluorescent assay (mini-VIDAS). Further, Real-time PCR analysis was performed to identify 11 of enterotoxin serological types in these strains. The frequency of enterotoxin-positive isolates presented unique characteristics, as well as the distribution of enterotoxin serological types. In addition, there was no significant difference between the test results of enterotoxin in dairy-derived S. aureus using VIDAS assay and Real-time PCR.%通过检验各类乳制品收集了大量乳源性金葡菌菌株,应用酶联荧光免疫分析法对菌株的肠毒素进行检测分析,并进一步使用荧光定量PCR法对肠毒素基因进行分型.结果表明,乳源性金葡菌肠毒素的检出率与基因型分布均呈现一定显著特征,应用酶联荧光免疫分析法与荧光定量PCR法对肠毒素的检测结果总体无显著性差异.

  16. Reversal of methicillin resistance in Staphylococcus aureus by thioridazine

    DEFF Research Database (Denmark)

    Klitgaard, Janne K; Skov, Marianne N; Kallipolitis, Birgitte H;

    2008-01-01

    Objectives Thioridazine has been shown to reverse oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) in vitro. The aim of this study was to investigate whether thioridazine alone or in combination with oxacillin affects the transcription of the methicillin resistance gene...... mecA and the protein level of the encoded protein PBP2a. Methods Viability of MRSA was determined in liquid media in the presence of oxacillin or thioridazine alone or in combination. Transcription of mecA was analysed by primer extension, and the protein level of PBP2a was analysed by western...... of thioridazine in the presence of a fixed amount of oxacillin. Furthermore, the protein level of PBP2a was reduced when bacteria were treated with the combination of oxacillin and thioridazine. The two drugs also affected the mRNA level of the beta-lactamase gene, blaZ. Conclusions The present study indicates...

  17. Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

    Science.gov (United States)

    Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

    2015-07-01

    Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients.

  18. Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

    Directory of Open Access Journals (Sweden)

    Ulrich Andergassen

    2013-01-01

    Full Text Available It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19. B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.

  19. Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd.

    Science.gov (United States)

    Guimarães, F F; Manzi, M P; Joaquim, S F; Richini-Pereira, V B; Langoni, H

    2017-01-01

    Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals' health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.

  20. Drugs resistance and penicillinase activity in skin isolated Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Bhat K

    1990-01-01

    Full Text Available A study was carried out to evaluate the drug resistance pattern and penicillinase production in skin isolated Staphylococcus aurpus. The disk diffusion method showed prevalence of: multidrug resistance among S. aureus, strains, isolated from locafised skin abscesses. method for detection of penicilfinase could detect this enzyme m 98.60/o of the isolates all fo which were resistant to penicillin and ampicillin. C16xacillin resistance as detected by the agar dilution method was found in 1.4% of the isolates. On the whole cloxacillin and gentamy′cin were found to be the most effective ′antistaphylococcal antibotics.

  1. Characterization of Staphylococcus aureus in Goose Feces from State Parks in Northeast Ohio.

    Science.gov (United States)

    Thapaliya, Dipendra; Dalman, Mark; Kadariya, Jhalka; Little, Katie; Mansell, Victoria; Taha, Mohammed Y; Grenier, Dylan; Smith, Tara C

    2017-03-10

    Staphylococcus aureus can colonize a range of species. Although numerous studies have isolated pathogenic bacteria from wild birds, very little is known regarding S. aureus and their potential to spread methicillin-resistant (MRSA) strains. The objective of this study was to determine the presence and molecular characteristics of S. aureus in geese fecal samples collected from ten state parks across Northeast Ohio (NEO). A total of 182 fecal samples from Canada geese (Branta canadensis) were collected in April 2015. Isolates were characterized using multi-locus sequence (MLST) and spa typing, as well as PCR to detect the presence of Panton-Valentine leukocidin (PVL), mecA, and scn genes. Antibiotic susceptibility testing was done via Vitek-2 system. The overall contamination by S. aureus in fecal samples was 7.1% (13/182); 7/182 (3.8%) were MRSA and 6/182 (3.3%) were methicillin-susceptible S. aureus (MSSA). One isolate was positive for PVL. A total of eight different spa types were observed. MLST included ST5, ST8, ST291, ST298, and ST2111. One (7.7%) MSSA isolate was multi-drug resistant. The S. aureus contamination in NEO state parks ranged from 0% (park 1, 4, 8, 9) to 35% (7/20) (park 5). Parks 2, 3, 6, and 7 had 5% (1/20) positive. The results of this study indicate that the feces of geese collected at various state parks in NEO may harbor S. aureus.

  2. Sclareol protects Staphylococcus aureus-induced lung cell injury via inhibiting alpha-hemolysin expression.

    Science.gov (United States)

    Ouyang, Ping; Sun, Mao; He, Xuewen; Wang, Kaiyu; Yin, Zhongqiong; Fu, Hualin; Li, Yinglun; Geng, Yi; Shu, Gang; He, Changliang; Liang, Xiaoxia; Lai, Weiming; Li, Lixia; Zou, Yuanfeng; Song, Xu; Yin, Lizi

    2016-09-23

    Staphylococcus aureus (S. aureus) is a common Gram-positive bacterium that causes serious infections in human and animals. With the continuous emergence of the methicillin-resistant S. aureus (MRSA) strains, antibiotics have limited efficacy in treating MRSA infections. Accordingly, novel agents that act on new targets are desperately needed to combat these infections. S. aureus alpha-hemolysin plays an indispensable role in its pathogenicity. In this study, we demonstrate that sclareol, a fragrant chemical compound found in clary sage, can prominently decrease alpha-hemolysin secretion in S. aureus strain USA300 at sub-inhibitory concentrations. Hemolysis assays, western-blotting and RT-PCR were used to detect the production of alpha-hemolysin in the culture supernatant. When USA300 was co-cultured with and A549 epithelial cells, sclareol could protect A549 cells at a final concentration of 8 µg/ml. The protective capability of sclareol against the USA300-mediated injury of A549 cells was further shown by cytotoxicity assays and live/dead analysis. In conclusion, sclareol was shown to inhibit the production of S. aureus alpha-hemolysin. Sclareol has potential for development as a new agent to treat S. aureus infections.

  3. High-resolution subtyping of Staphylococcus aureus strains by means of Fourier-transform infrared spectroscopy.

    Science.gov (United States)

    Johler, Sophia; Stephan, Roger; Althaus, Denise; Ehling-Schulz, Monika; Grunert, Tom

    2016-05-01

    Staphylococcus aureus causes a variety of serious illnesses in humans and animals. Subtyping of S. aureus isolates plays a crucial role in epidemiological investigations. Metabolic fingerprinting by Fourier-transform infrared (FTIR) spectroscopy is commonly used to identify microbes at species as well as subspecies level. In this study, we aimed to assess the suitability of FTIR spectroscopy as a tool for S. aureus subtyping. To this end, we compared the subtyping performance of FTIR spectroscopy to other subtyping methods such as pulsed field gel electrophoresis (PFGE) and spa typing in a blinded experimental setup and investigated the ability of FTIR spectroscopy for identifying S. aureus clonal complexes (CC). A total of 70 S. aureus strains from human, animal, and food sources were selected, for which clonal complexes and a unique virulence and resistance gene pattern had been determined by DNA microarray analysis. FTIR spectral analysis resulted in high discriminatory power similar as obtained by spa typing and PFGE. High directional concordance was found between FTIR spectroscopy based subtypes and capsular polysaccharide expression detected by FTIR spectroscopy and the cap specific locus, reflecting strain specific expression of capsular polysaccharides and/or other surface glycopolymers, such as wall teichoic acid, peptidoglycane, and lipoteichoic acid. Supervised chemometrics showed only limited possibilities for differentiation of S. aureus CC by FTIR spectroscopy with the exception of CC45 and CC705. In conclusion, FTIR spectroscopy represents a valuable tool for S. aureus subtyping, which complements current molecular and proteomic strain typing.

  4. Evasion of Neutrophil Killing by Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Will A. McGuinness

    2016-03-01

    Full Text Available Staphylococcus aureus causes many types of infections, ranging from self-resolving skin infections to severe or fatal pneumonia. Human innate immune cells, called polymorphonuclear leukocytes (PMNs or neutrophils, are essential for defense against S. aureus infections. Neutrophils are the most prominent cell type of the innate immune system and are capable of producing non-specific antimicrobial molecules that are effective at eliminating bacteria. Although significant progress has been made over the past few decades, our knowledge of S. aureus-host innate immune system interactions is incomplete. Most notably, S. aureus has the capacity to produce numerous molecules that are directed to protect the bacterium from neutrophils. Here we review in brief the role played by neutrophils in defense against S. aureus infection, and correspondingly, highlight selected S. aureus molecules that target key neutrophil functions.

  5. Postoperative Staphylococcus aureus infections in Medicare beneficiaries.

    Directory of Open Access Journals (Sweden)

    Moaven Razavi

    Full Text Available Staphylococcus aureus (S. aureus infections are important because of their increasing frequency, resistance to antibiotics, and high associated rates of disabilities and deaths. We examined the incidence and correlates of S. aureus infections following 219,958 major surgical procedures in a 5% random sample of fee-for-service Medicare beneficiaries from 2004-2007. Of these surgical patients, 0.3% had S. aureus infections during the hospitalizations when index surgical procedures were performed; and 1.7% and 2.3%, respectively, were hospitalized with infections within 60 days or 180 days following admissions for index surgeries. S. aureus infections occurred within 180 days in 1.9% of patients following coronary artery bypass graft surgery, 2.3% following hip surgery, and 5.9% following gastric or esophageal surgery. Of patients first hospitalized with any major infection reported during the first 180 days after index surgery, 15% of infections were due to S. aureus, 18% to other documented organisms, and no specific organism was reported on claim forms in 67%. Patient-level predictors of S. aureus infections included transfer from skilled nursing facilities or chronic hospitals and comorbid conditions (e.g., diabetes, congestive heart failure, chronic obstructive pulmonary disease, and chronic renal disease. In a logarithmic regression, elective index admissions with S. aureus infection stayed 130% longer than comparable patients without that infection. Within 180 days of the index surgery, 23.9% of patients with S. aureus infection and 10.6% of patients without this infection had died. In a multivariate logistic regression of death within 180 days of admission for the index surgery with adjustment for demographics, co-morbidities, and other risks, S. aureus was associated with a 42% excess risk of death. Due to incomplete documentation of organisms in Medicare claims, these statistics may underestimate the magnitude of S. aureus infection

  6. AKTIVITAS ANTIBAKTERI GETAH PEPAYA KERING TERHADAP Staphylococcus aureus PADA DANGKE [Antibacterial Activity of Dried Papaya Latex toward Staphylococcus aureus in Dangke

    Directory of Open Access Journals (Sweden)

    Rifah Hestyani Arum

    2014-06-01

    Full Text Available Dangke is a traditional milk curd product, made by coagulation of milk using fresh papaya latex. This product is usually kept at room temperature (27-30ºC until consumption. Dried papaya latex was used in this study to produce dangke, and its effect to S. aureus was determined by direct contact in TSB and dangke. Fresh papaya latex was dried using vacuum oven at 50-55ºC for 22 hours. Dried papaya latex at a concentration of 2.7x10-3 g/100 mL could reduce S. aureus approximately 1 log CFU/mL in TSB after 24 hours. Dried papaya latex and papain could maintain the S. aureus number in dangke within 24 hours storage at room temperature. The antibacterial activity of non-proteolytic compound of papaya latex, i.e ethanolic extract of papaya latex was determined by macrodilution method, resulted an the MIC90 of 8 mg/mL. The cell membrane leakage after exposure was detected by measuring the optical density of bacterial supernatant at 260 nm. The result showed that exposure to increasing antibacterial concentration resulted in increasing of optical density of S. aureus supernatant, indicating that the antibacterial caused the S. aureus membrane leakage. Fluorescence microscopy imaging showed that S. aureus exposure to antibacterial caused membrane leakage thus gave Propidium Iodide (PI chance to penetrate into the cell, as indicated by changing of fluorescence color from green to red.

  7. Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus clonal complexes in bulk tank milk from dairy cattle herds in Lombardy Region (Northern Italy).

    Science.gov (United States)

    Cortimiglia, C; Luini, M; Bianchini, V; Marzagalli, L; Vezzoli, F; Avisani, D; Bertoletti, M; Ianzano, A; Franco, A; Battisti, A

    2016-10-01

    Staphylococcus aureus is the most important causative agent of subclinical mastitis in cattle resulting in reduced milk production and quality. Methicillin-resistant S. aureus (MRSA) strains has a clear zoonotic relevance, especially in the case of occupational exposure. The aim of the study was to evaluate the prevalence of S. aureus and MRSA in bulk tank milk (BTM) from dairy cattle herds in the Lombardy Region (Northern Italy) and to identify the main MRSA circulating genotypes. MRSA strains were characterized by susceptibility testing, multi-locus sequence typing (MLST), spa typing and SCCmec typing. A total 844 BTM samples were analysed and S. aureus and MRSA were detected in 47·2% and 3·8% of dairy herds, respectively. MLST showed that the majority (28/32) of isolates belonged to the typical livestock-associated lineages: ST398, ST97 and ST1. Interestingly, in this study we report for the first time the new ST3211, a single locus variant of ST(CC)22, with the newly described 462 aroE allele. Our study indicates high diffusion of S. aureus mastitis and low, but not negligible, prevalence of MRSA in the considered area, suggesting the need for planning specific control programmes for bovine mastitis caused by S. aureus, especially when MRSA is implicated.

  8. Diversity of Staphylococcus aureus Isolates in European Wildlife

    Science.gov (United States)

    Monecke, Stefan; Gavier-Widén, Dolores; Hotzel, Helmut; Peters, Martin; Guenther, Sebastian; Lazaris, Alexandros; Loncaric, Igor; Müller, Elke; Reissig, Annett; Ruppelt-Lorz, Antje; Shore, Anna C.; Walter, Birgit; Coleman, David C.; Ehricht, Ralf

    2016-01-01

    Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation. PMID:27992523

  9. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    Science.gov (United States)

    Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Morrison, John M.; Dunman, Paul M.

    2009-01-01

    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript. PMID:18992285

  10. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

    Science.gov (United States)

    Figueiredo, L T; Batista, W C; Igarashi, A

    1997-01-01

    We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.

  11. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

    Directory of Open Access Journals (Sweden)

    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  12. A novel HMM-based method for detecting enriched transcription factor binding sites reveals RUNX3 as a potential target in pancreatic cancer biology.

    Directory of Open Access Journals (Sweden)

    Liron Levkovitz

    Full Text Available BACKGROUND: Pancreatic adenocarcinoma (PAC is one of the most intractable malignancies. In order to search for potential new therapeutic targets, we relied on computational methods aimed at identifying transcription factor binding sites (TFBSs over-represented in the promoter regions of genes differentially expressed in PAC. Though many computational methods have been implemented to accomplish this, none has gained overall acceptance or produced proven novel targets in PAC. To this end we have developed DEMON, a novel method for motif detection. METHODOLOGY: DEMON relies on a hidden Markov model to score the appearance of sequence motifs, taking into account all potential sites in a promoter of potentially varying binding affinities. We demonstrate DEMON's accuracy on simulated and real data sets. Applying DEMON to PAC-related data sets identifies the RUNX family as highly enriched in PAC-related genes. Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs exhibits time-dependent increases in normal but not in PAC cells. These increases are accompanied by changes in mRNA levels of putative RUNX gene targets. CONCLUSIONS: The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

  13. Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species

    Directory of Open Access Journals (Sweden)

    Pueyo Carmen

    2007-09-01

    -stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

  14. CHROMOSOMAL MAPPING IN STRAINS OF STAPHYLOCOCCUS AUREUS,

    Science.gov (United States)

    STAPHYLOCOCCUS AUREUS , CHROMOSOMES), (*CHROMOSOMES, MAPPING), NITROSO COMPOUNDS, GUANIDINES, GENETICS, MUTATIONS, DRUGS, TOLERANCES(PHYSIOLOGY), TEST METHODS, DEOXYRIBONUCLEIC ACIDS, INHIBITION, RESISTANCE(BIOLOGY).

  15. Mastite com lesões sistêmicas por Staphylococus aureus subesp. aureus em coelhos Mastitis with systemic lesions due to Staphylococus aureus subesp. aureus in rabbits

    OpenAIRE

    Sandra Davi Traverso; Leonardo da Cunha; Joaquim César Teixeira Fernandes; Alexandre Paulino Loretti; Adriana Rhoden; Elsio Wunder Jr.; David Driemeier

    2003-01-01

    Em uma criação composta por 1800 coelhos, 33% das matrizes apresentaram mastite e lesões cutâneas crostosas e purulentas. Estes animais apresentavam-se entre 10 a- 12 meses de idade e em segunda parição. Quinze coelhos afetados foram sacrificados e necropsiados. Na necropsia, além das lesões cutâneas haviam microabscessos em diversos órgãos. Das amostras coletadas isolou-se Staphylococcus aureus subesp. aureus. S. aureus subesp. aureus também foi isolado de "swab" nasal coletado do tratador e...

  16. Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice.

    Directory of Open Access Journals (Sweden)

    Sanne van den Berg

    Full Text Available Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.

  17. Assessment of Interpersonal Motivation in Transcripts (AIMIT): an inter- and intra-rater reliability study of a new method of detection of interpersonal motivational systems in psychotherapy.

    Science.gov (United States)

    Fassone, G; Valcella, F; Pallini, S; Scarcella, F; Tombolini, L; Ivaldi, A; Prunetti, E; Manaresi, F; Liotti, G

    2012-01-01

    Assessing Interpersonal Motivations in Transcripts (AIMIT) is a coding system aiming to systematically detect the activity of interpersonal motivational systems (IMS) in the therapeutic dialogue. An inter- and intra-rater reliability study has been conducted. Sixteen video-recorded psychotherapy sessions were selected and transcribed according to the AIMIT criteria. Sessions relate to 16 patients with an Axis II diagnosis, with a mean Global Assessment of Functioning of 51. For the intra-rater reliability evaluation, five sessions have been selected and assigned to five independent coders who where asked to make a first evaluation, and then a second independent one 14 days later. For the inter-rater reliability study, the sessions coded by the therapist-coder were jointly revised with another coder and finally classified as gold standard. The 16 standard sessions were sent to other evaluators for the independent coding. The agreement (κ) was estimated according to the following parameters for each coding unit: evaluation units supported by the 'codable' activation of one or more IMS; motivational interaction with reference to the ongoing relation between patient and therapist; an interaction between the patient and another person reported/narrated by the patient; detection of specific IMS: attachment (At), caregiving (CG), rank (Ra), sexuality (Se), peer cooperation (PC); and transitions from one IMS to another were also scored. The intra-rater agreement was evaluated through the parameters 'cod', 'At', 'CG', 'Ra', 'Se' and 'PC' described above. A total of 2443 coding units were analysed. For the nine parameters on which the agreement was calculated, eight ['coded (Cod)', 'ongoing relation (Rel)', 'narrated relation (Nar)', 'At', 'CG', 'Ra', 'Se' and 'PC'] have κ values comprised between 0.62 (CG) and 0.81 (Cod) and were therefore satisfactory. The scoring of 'transitions' showed agreement values slightly below desired cut-off (0.56). Intra-rater reliability was

  18. Quantitative microbial risk assessment for Staphylococcus aureus in natural and processed cheese in Korea.

    Science.gov (United States)

    Lee, Heeyoung; Kim, Kyunga; Choi, Kyoung-Hee; Yoon, Yohan

    2015-09-01

    This study quantitatively assessed the microbial risk of Staphylococcus aureus in cheese in Korea. The quantitative microbial risk assessment was carried out for natural and processed cheese from factory to consumption. Hazards for S. aureus in cheese were identified through the literature. For exposure assessment, the levels of S. aureus contamination in cheeses were evaluated, and the growth of S. aureus was predicted by predictive models at the surveyed temperatures, and at the time of cheese processing and distribution. For hazard characterization, a dose-response model for S. aureus was found, and the model was used to estimate the risk of illness. With these data, simulation models were prepared with @RISK (Palisade Corp., Ithaca, NY) to estimate the risk of illness per person per day in risk characterization. Staphylococcus aureus cell counts on cheese samples from factories and markets were below detection limits (0.30-0.45 log cfu/g), and pert distribution showed that the mean temperature at markets was 6.63°C. Exponential model [P=1 - exp(7.64×10(-8) × N), where N=dose] for dose-response was deemed appropriate for hazard characterization. Mean temperature of home storage was 4.02°C (log-logistic distribution). The results of risk characterization for S. aureus in natural and processed cheese showed that the mean values for the probability of illness per person per day were higher in processed cheese (mean: 2.24×10(-9); maximum: 7.97×10(-6)) than in natural cheese (mean: 7.84×10(-10); maximum: 2.32×10(-6)). These results indicate that the risk of S. aureus-related foodborne illness due to cheese consumption can be considered low under the present conditions in Korea. In addition, the developed stochastic risk assessment model in this study can be useful in establishing microbial criteria for S. aureus in cheese.

  19. Application of Real-time PCR technology in Staphylococcus aureus detection%实时荧光定量PCR技术在金黄色葡萄球菌检测中的应用

    Institute of Scientific and Technical Information of China (English)

    韩齐; 孔保华; 李沛军; 李暮春

    2013-01-01

    金黄色葡萄球菌(Staphylococcus aureus)是主要的食源性致病菌之一.实时荧光定量PCR(Real-time PCR)技术是近年来广泛应用于食源性致病菌的快速检测方法,具有特异性强、灵敏度高等特点.本文综述了Real-time PCR 技术原理、分类及其在检测食品中金黄色葡萄球菌的应用,并对其应用前景进行了展望.

  20. Characterisation of Staphylococcus aureus bacteraemia at Tygerberg hospital

    NARCIS (Netherlands)

    Orth, H.; Dreyer, Z.S.; Makgotlho, E.; Oosthuysen, W.; Sinha, B.; Wasserman, E.

    2013-01-01

    To elucidate the local epidemiology of Staphylococcus aureus bacteraemia, we characterised blood culture isolates using molecular methods and prospectively collected clinical data to determine the occurrence of community-acquired, methicillinresistant S. aureus (MRSA). Consecutive S. aureus blood cu

  1. Staphylococcus aureus and Escherichia coli in nham (Thai-style fermented pork sausage).

    Science.gov (United States)

    Petchsing, U; Woodburn, M J

    1990-05-01

    The fate of Staphylococcus aureus and Escherichia coli was determined when they were introduced into ground pork made into nham (Thai-style fermented pork sausage) with or without 0.75 or 1.5% added starter culture. Without starter culture, the numbers of E. coli remained little changed but there was slow multiplication of S. aureus. With 0.75% starter culture, S. aureus was no longer detectable after 48 h and E. coli numbers decreased by 1 log after 96 h. No viable S. aureus or E. coli were recovered after 36 h and 96 h, respectively, when 1.5% starter culture was added. The addition of a starter culture is recommended when making nham.

  2. Cross reactivity of S. aureus to murine cytokine assays: A source of discrepancy.

    Science.gov (United States)

    Javed, Numan; Xue, Guang; Lu, Ailing; Xing, Yue; Iwakura, Yoichiro; Xiao, Hui; Lecoeur, Hervé; Späth, Gerald F; Meng, Guangxun

    2016-05-01

    Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1β and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1β and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.

  3. Contact petri dish in detection of Staphylococcus aureus and Escherichia coli on hands of blood collection staff%采用接触式培养皿法对采血人员手部金黄色葡萄球菌与大肠埃希菌的检测效果分析

    Institute of Scientific and Technical Information of China (English)

    赵安成

    2012-01-01

    OBJECTIVE To evaluate the effect of contact petri dish on the detection of Staphylococcus aureus and Escherichia coli in the hands of the blood collection staff. METHODS A total of 28 cases of blood collection staff were divided into two groups according to the disinfection methods before the puncture for the blood sampling. The contact petri dish group was taken with contact petri dish method, and the swab group was taken with the swab method. The number of the species of pathogens were determined by sampling, culture, colony counting, and identification culture medium for S. aureus and E. coli. RESULTS The blood collection staff in the contact petri dish group before the disinfection, with the total bacterial colony counts more than 30 CFU/30 cm2 , accounted for 50. 00% , significantly higher than 7. 13% of the swap group. The blood collection staff in the contact petri dish group after the disinfection, with the bacterial colony counts 21 - 30 CFU/30 cm2 , accounted for 14. 29% , there were no strains of pathogens detected in the swab group (P<0. 05) ; before and after disinfection, the volume of S. aureus and E. coli detected by the contact petri dish before and after the disinfections was significantly higher than that by the swab (P<0. 05). CONCLUSION The contact petri dish method is effective and simple in the detection of the total bacterial colonies counts of S. aureus and E. coli.%目的 评价接触式培养皿法对采血人员手部金黄色葡萄球菌与大肠埃希菌的检测效果.方法 将28名采血人员在穿刺采血之前的手部消毒前后,根据采取的消毒方式不同分为两组,其中接触式培养皿组采用接触式培养皿法,棉签擦拭组采用棉签擦拭法进行,分别取样、培养、菌落计数、用金黄色葡萄球菌和大肠埃希菌鉴别培养基鉴定该种类菌株数.结果 接触式培养皿法消毒前细菌总数>30 CFU/30 cm2的人数占50.00%,显著高于棉签擦拭法的7.13

  4. Staphylococcus aureus autoinducer-2 quorum sensing decreases biofilm formation in an icaR-dependent manner

    Directory of Open Access Journals (Sweden)

    Yu Dan

    2012-12-01

    Full Text Available Abstract Background Staphylococcus aureus is an important pathogen that causes biofilm-associated infection in humans. Autoinducer 2 (AI-2, a quorum-sensing (QS signal for interspecies communication, has a wide range of regulatory functions in both Gram-positive and Gram-negative bacteria, but its exact role in biofilm formation in S. aureus remains unclear. Results Here we demonstrate that mutation of the AI-2 synthase gene luxS in S. aureus RN6390B results in increased biofilm formation compared with the wild-type (WT strain under static, flowing and anaerobic conditions and in a mouse model. Addition of the chemically synthesized AI-2 precursor in the luxS mutation strain (ΔluxS restored the WT phenotype. Real-time RT-PCR analysis showed that AI-2 activated the transcription of icaR, a repressor of the ica operon, and subsequently a decreased level of icaA transcription, which was presumably the main reason why luxS mutation influences biofilm formation. Furthermore, we compared the roles of the agr-mediated QS system and the LuxS/AI-2 QS system in the regulation of biofilm formation using the ΔluxS strain, RN6911 and the Δagr ΔluxS strain. Our data indicate a cumulative effect of the two QS systems on the regulation of biofilm formation in S. aureus. Conclusion These findings demonstrate that AI-2 can decrease biofilm formation in S. aureus via an icaR-activation pathway. This study may provide clues for therapy in S. aureus biofilm-associated infection.

  5. Evaluación de estrategias de control frente a biopelículas de Staphylococcus aureus potencialmente presnetes en la industria pesquera

    OpenAIRE

    Vázquez-Sánchez, Daniel

    2014-01-01

    Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing ...

  6. Human-associated Staphylococcus aureus strains within great ape populations in Central Africa (Gabon).

    Science.gov (United States)

    Nagel, M; Dischinger, J; Türck, M; Verrier, D; Oedenkoven, M; Ngoubangoye, B; Le Flohic, G; Drexler, J F; Bierbaum, G; Gonzalez, J-P

    2013-11-01

    The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa-type t148. It was isolated from three different individuals of the western lowland gorilla (Gorilla gorilla gorilla), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees (Pan troglodytes) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.

  7. Prevalence, toxin gene profiles, and antimicrobial resistance of Staphylococcus aureus isolated from quick-frozen dumplings.

    Science.gov (United States)

    Hao, Dan; Xing, Xiaonan; Li, Guanghui; Wang, Xin; Zhang, Min; Zhang, Weisong; Xia, Xiaodong; Meng, Jianghong

    2015-01-01

    The aim of this study was to investigate the prevalence of Staphylococcus aureus in quick-frozen dumplings and to characterize these strains. A total of 120 dumpling samples, including lamb (n = 13), vegetarian (n = 14), seafood (n = 12), and pork (n = 81) stuffing, were collected in Shaanxi province in China and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, and detection of genes encoding staphylococcal enterotoxins, exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin 1 (tsst-1), and resistance to methicillin-oxacillin (mecA). In all, 60.0% of all samples were positive for S. aureus, and 117 S. aureus isolates, including seven mecA-positive strains, were recovered from these positive samples. In addition, all mecA-positive S. aureus isolates were recovered from products of animal origin. In these S. aureus isolates, resistance was observed most frequently to ampicillin (92.3%) and penicillin (86.3%), followed by clarithromycin, erythromycin, midecamycin, tetracycline, and kanahemycin (from 53.8 to 28.2%). All isolates were sensitive to cefoperazone, minocycline, vancomycin, and ofloxacin. The predominant toxin gene was sec (38.5%), followed by seg (19.7%), sej (16.2%), see (12.8%), sea (11.1%), and seb (10.3%), whereas eta, etb, and tsst-1 genes were not detected. These findings indicate that S. aureus was present commonly in quick-frozen dumplings, accompanied by multiple antimicrobial resistance and toxin genes. Our findings highlight the urgency for stricter hygiene strategies in food production and the prudent use of antibiotics in the breeding industry.

  8. Haemin represses the haemolytic activity of Staphylococcus aureus in an Sae-dependent manner.

    Science.gov (United States)

    Schmitt, Julia; Joost, Insa; Skaar, Eric P; Herrmann, Mathias; Bischoff, Markus

    2012-10-01

    Staphylococcus aureus is a major human pathogen and a common cause of nosocomial infections. This facultative pathogen produces a large arsenal of virulence factors, including the haemolysins, which allow the bacterium to lyse erythrocytes and thereby release large amounts of the haem-containing haemoglobin. The released haem is thought to be the main iron source of this organism during the course of infection, and is considered to be crucial for bacterial proliferation in vivo. High concentrations of haem and its degradation products, on the other hand, are known to be toxic for S. aureus, making it essential for the pathogen to tightly control haem release from red blood cells. Here we show that S. aureus responds to haemin by downregulating the expression of haemolysins. Subinhibitory concentrations of haemin were found to significantly reduce transcription of the haemolysin genes hlb (encoding β-haemolysin) and hlgA (encoding the S-class component of γ-haemolysin), while hla (encoding α-haemolysin) and RNAIII (encoding δ-haemolysin) transcription did not appear to be affected. The presence of haemin also reduced the haemolytic potential of the supernatants of S. aureus LS1 cultures. Inactivation of the sae locus in LS1 abolished the haemin effect on the transcription of haemolysin genes, indicating that the two-component regulatory system is required for this regulatory effect. Iron limitation, on the other hand, was found to induce the expression of haemolysins, and this effect was again abolished in the sae mutant, indicating that S. aureus modulates its haemolysin production in response to iron and haem availability in an Sae-dependent manner.

  9. Fluorescent reporters for markerless genomic integration in Staphylococcus aureus

    Science.gov (United States)

    de Jong, Nienke W. M.; van der Horst, Thijs; van Strijp, Jos A. G.; Nijland, Reindert

    2017-01-01

    We present integration vectors for Staphylococcus aureus encoding the fluorescent reporters mAmetrine, CFP, sGFP, YFP, mCherry and mKate. The expression is driven either from the sarA-P1 promoter or from any other promoter of choice. The reporter can be inserted markerless in the chromosome of a wide range of S. aureus strains. The integration site chosen does not disrupt any open reading frame, provides good expression, and has no detectable effect on the strains physiology. As an intermediate construct, we present a set of replicating plasmids containing the same fluorescent reporters. Also in these reporter plasmids the sarA-P1 promoter can be replaced by any other promoter of interest for expression studies. Cassettes from the replication plasmids can be readily swapped with the integration vector. With these constructs it becomes possible to monitor reporters of separate fluorescent wavelengths simultaneously. PMID:28266573

  10. A pig model of acute Staphylococcus aureus induced pyemia

    DEFF Research Database (Denmark)

    Nielsen, O. L.; Iburg, T.; Aalbæk, B.;

    2009-01-01

    Background: Sepsis caused by Staphylococcus aureus constitutes an important cause of morbidity and mortality in humans, and the incidence of this disease-entity is increasing. In this paper we describe the initial microbial dynamics and lesions in pigs experimentally infected with S. aureus......, with the aim of mimicking human sepsis and pyemia. Methods: The study was conducted in anaesthetized and intravenously inoculated pigs, and was based on bacteriological examination of blood and testing of blood for IL-6 and C-reactive protein. Following killing of the animals and necropsy bacteriological...... was not detected in the blood and C-reactive protein did not increase, probably because of the short time course of the study. Conclusion: This study demonstrates the successful induction of acute pyemia (microabscesses), and forms a basis for future experiments that should include inoculation with strains of S...

  11. Development of multiplex PCR for detection of pathogenic genes in the Staphylococcus aureus strains isolated from skin and soft tissue infections%皮肤软组织及创伤感染金黄色葡萄球菌分离株的毒素基因检测

    Institute of Scientific and Technical Information of China (English)

    邓家德; 陈惠玲; 叶惠芬; 凌艳英; 李焕庭; 杨银梅; 周小棉

    2012-01-01

    Objective To examine the prevalence of the genes encoding Panton-Valentine leukocidin (PVL), exfoliative toxin (ETs) , toxic shock syndrome toxin 1 (TSST-1) , and some antibiotic resistant genes in the Staphylococcus aureus strains isolated from skin and soft tissue infections. Methods A total of 90 strains of S. aureus were isolated from skin and soft tissue infections. Antibiotic susceptibility testing was done by Kirby-Bauer method. The results were interpreted according to CLSI 2009 breakpoints. Multiplex PCR was designed to detect S. aureus specific 16S rRNA gene, mecA, and PVL gene. The genes encoding, TSST-1 and EtsA and EtsB were amplified by PCR. Results The prevalence of mecA gene was 52. 2% (47/90). Positive mecA defines the strain as methicillin-resistant (MRSA). EtsB gene was positive in 1 of the MRSA strains. TSST-1 gene was also identified in only 1 MRSA strain. PVL gene was positive in 7 of the S. aureus strains, 3 of which carried mecA gene. Conclusions S. aureus may release various toxins. PVL-positive strains usually cause severe infection. Attention must be paid to the toxin-producing MRSA strains.%目的 了解皮肤软组织及创伤感染的金葡菌携带的杀白细胞素(Panton-Valentine Leukocidin,PVL)基因、表皮剥脱性毒素(ETs)基因、中毒性休克综合征毒素-1(TSST-1)的tst基因的特点.方法 对连续收集的皮肤软组织感染中分离的90株金葡菌,采用多重PCR同时检测金葡菌特异性16SrRNA基因、mecA基因、PVL基因,采用PCR法检测TSST-1及EtsA、B基因.结果 金葡菌mecA基因阳性47株(占金葡菌52.2%)为耐甲氧西林金葡菌(MRSA).有1株MRSA的EtsB基因阳性,1株MRSA的TSST-1阳性,有7株金葡菌携带PVL基因,其中3株为mecA基因阳性株(MRSA).结论 金葡菌可分泌多种毒素,携带PVL毒素的金葡菌常可以引起严重的侵袭性感染,尤其对产毒的MRSA感染引起足够的重视,是防控的重点.

  12. ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

    Science.gov (United States)

    Daly, Seth M.; Elmore, Bradley O.; Kavanaugh, Jeffrey S.; Triplett, Kathleen D.; Figueroa, Mario; Raja, Huzefa A.; El-Elimat, Tamam; Crosby, Heidi A.; Femling, Jon K.; Cech, Nadja B.; Horswill, Alexander R.; Oberlies, Nicholas H.

    2015-01-01

    Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation. PMID:25645827

  13. Chromones from an ascomycete,Chaetomium aureus

    Institute of Scientific and Technical Information of China (English)

    Li Mei Li; Qiang Zou; Guo You Li

    2010-01-01

    A novel chromone,named chaetoaurin (1),along with six known chromone derivatives (2-7),was isolated from the ethyl acetate extract of a solid-state fermented culture of Chaetomium aureus.Their structures were elucidated by extensive spectral analysis.All of these compounds were reported from C.aureus for the first time.

  14. Immunogenicity of toxins during Staphylococcus aureus infection

    NARCIS (Netherlands)

    N.J. Verkaik (Nelianne); O. Dauwalder (Olivier); K. Antri (Kenza); I. Boubekri (Ilhem); C.P. de Vogel (Corné); C. Badiou (Cédric); M. Bes (Michèle); F. Vandenesch (François); M. Tazir (Mohammed); H. Hooijkaas (Herbert); H.A. Verbrugh (Henri); A.F. van Belkum (Alex); J. Etienne (Jerome); G. Lina (Gérard); N. Ramdani-Bouguessa (Nadjia); W.J.B. van Wamel (Willem)

    2010-01-01

    textabstractAB - BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects we

  15. Distribution of food-borne Staphylococcus aureus enterotoxin genes.

    Science.gov (United States)

    Hu, W D

    2016-01-29

    We identified and analyzed 5 new-type enterotoxin genes, including SEj, SEl, SEq, SEm, and SEr, to explore the distribution of 5 enterotoxin genes in Staphylococcus aureus of different origins as well as their correlations and differences. We examined the distribution of the S. aureus enterotoxin genes and their pathogenic mechanisms. A total of 660 specimens were collected from January 2011 to December 2014, and 217 strains of S. aureus were isolated. The template DNA of S. aureus was extracted. The Primer6.0 and Oligo7 software were used to design and synthesize polymerase chain reaction primers. Amplification results were analyzed by electrophoresis, and the amplification products were recovered and sequenced. Thirty-six bacterial strains contained the SEj gene (16.6%), including 15, 8, 8, 4, and 1 strains in fresh meat, quick-frozen food, raw milk, human purulent tissue, and living environment, respectively. Thirty-one bacterial strains contained the SEr gene (14.3%), including 16, 9, and 6 strains in fresh meat, quick-frozen food, and raw milk, respectively. Twenty-one bacterial strains contained the enterotoxin SEq gene (9.7%), including 8, 6, 6, and 1 strains in fresh meat, quick-frozen food, raw milk, and human purulent tissue, respectively. No SEm and SEl genes were detected. Different types of foods carry different types of enterotoxins, providing a basis for quick tracing for food poisoning. Three enterotoxin genes, SEj, SEr, and SEq, showed the highest carrier rate in quick-frozen food. It is imperative to improve their detection in quick-frozen food.

  16. Staphylococcus aureus and hand eczema severity

    DEFF Research Database (Denmark)

    Haslund, P; Bangsgaard, N; Jarløv, J O

    2009-01-01

    BACKGROUND: The role of bacterial infections in hand eczema (HE) remains to be assessed. OBJECTIVES: To determine the prevalence of Staphylococcus aureus in patients with HE compared with controls, and to relate presence of S. aureus, subtypes and toxin production to severity of HE. METHODS......: Bacterial swabs were taken at three different visits from the hand and nose in 50 patients with HE and 50 controls. Staphylococcus aureus was subtyped by spa typing and assigned to clonal complexes (CCs), and isolates were tested for exotoxin-producing S. aureus strains. The Hand Eczema Severity Index...... was used for severity assessment. RESULTS: Staphylococcus aureus was found on the hands in 24 patients with HE and four controls (P hands...

  17. Methicillin-resistant Staphylococcus aureus containing mecC in Swedish dairy cows

    Directory of Open Access Journals (Sweden)

    Unnerstad Helle Ericsson

    2013-01-01

    Full Text Available Abstract Background Hitherto, methicillin-resistant Staphylococcus aureus (MRSA has not been detected in Swedish cattle. However, due to the report of mecC, a novel homologue to the mecA gene, there was reason to re-evaluate susceptibility results from strain collections of Staphylococcus aureus and test suspected isolates for the presence of mecC. Findings Bovine isolates of S. aureus with elevated minimum inhibitory concentrations of beta-lactams were retrospectively tested for presence of mecC. In four of the isolates mecC was detected. Conclusion In Sweden, this is the first finding of MRSA in cattle and the first detection of MRSA harbouring mecC of domestic animal origin. MRSA in animal populations has implications as a potential reservoir with risk for spread to humans. Occurrence of MRSA among Swedish cattle appears still very limited.

  18. 离子液体保护聚苯胺纳米金复合膜传感器的制备及其在检测乳制品中金黄色葡萄球菌毒素B的应用%An Electrochemical Immunosensor Modified with Polyaniline-Gold Composite Film for Detecting Staphylococcus Aureus Enterotoxin B in Dairy Products

    Institute of Scientific and Technical Information of China (English)

    孙秀兰; 高博; 张银志

    2012-01-01

    采用原位合成法制得聚苯胺修饰纳米金的复合膜,并采用紫外、红外及透射电镜对其进行表征,以离子液体1,3- 二丁基-咪唑六氟磷酸盐为保护复合膜的溶剂,成功制得离子液体保护的聚苯胺纳米金复合膜免疫传感器,并应用于乳品中金黄色葡萄球菌肠毒素B(SEB)的检测.在Fe(CN)63-/4-溶液中,采用循环伏安法(CV)及交流阻抗法(EIS)对传感器进行表征与测定,建立了SEB检测标准曲线,Y=933.46x+ 1399.8,线性范围为0.1~8.0 μg/L,相关系数R2=0.9932,检出限明显降低(0.033μg/L,S/N=3).结果表明,本传感器特异