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Sample records for isolation molecular identification

  1. Pythium insidiosum: morphological and molecular identification of Brazilian isolates

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    Maria Isabel de Azevedo

    2012-07-01

    Full Text Available Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identification of P. insidiosum is important in order to implement a rapid and definitive diagnosis and an effective treatment. This study reports the identification of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to confirm the identity of all Brazilian isolates as being P. insidiosum.

  2. Molecular identification of Sporothrix clinical isolates in China.

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    Liu, Ting-ting; Zhang, Ke; Zhou, Xun

    2014-01-01

    In this study, we investigated the molecular phylogeny of 64 clinical isolates which were identified as Sporothrix schenckii sensu lato by morphological identification. All of the strains were isolates from patients from several provinces in China. The phylogeny was inferred by DNA sequence analyses based on datasets of the ribosomal internal transcribed spacer (ITS) and a combined ITS and partial β-tubulin region. Reference sequences were retrieved from GenBank. Results showed that all of the isolates were clustered in a distinct clade with a type of Sporothrix globosa. Our analysis showed that S. globosa is the causal agent of the tested sporotrichosis in China, rather than S. schenckii that was generally believed to be the case. The existence of S. schenckii in China remains to be confirmed. This study improved our understanding of the distribution of the species in S. schenckii complex.

  3. Molecular identification of Candida orthopsilosis isolated from blood culture.

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    Yong, P V C; Chong, P P; Lau, L Y; Yeoh, R S C; Jamal, F

    2008-02-01

    The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.

  4. Molecular Identification of Giardia duodenalis Isolates from Fars Province, Iran.

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    Mohammad Rayani

    2014-03-01

    Full Text Available Giardia duodenalis is one of the most common human intestinal protozoan parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. The present study aimed to identify the prevalence of G. duodenalis isolates and determine the most common of its assemblages in the patients referring to health centers and hospitals in Fars province, Iran that will be subjected to further molecular investigation.We collected 1000 human fecal samples from health centers and hospitals in Shiraz, Iran in a one year period from September 2009 to August 2010. Microscopic examination for the presence of G. duodenalis cysts and trophozoites was performed by direct wet mount before and after the concentration techniques. Extraction of DNA was performed by Phenol-Chloroform-Isoamylalcohol (PCI. G. duodenalis-positive specimens were analyzed by PCR. A fragment of the SSU-rDNA (292 bp gene was amplified by PCR using the forward primer RH11 and the reverse primer RH4. Genotyping was performed using sequence analysis of G. duodenalis glutamate dehydrogenase gene using primers GDHeF, GDHiF, and GDHiR.The prevalence of Giardia infection was 10.7% (107/1000 examined based on microscopic examination. PCR identified 80% (40/50 of the samples as positive for G. duodenalis based on SSU-rDNA amplification on sucrose gradient samples. Besides, genotyping results indicated 32 isolates (80% as assemblage AII and 8 isolates (20% as assemblage BIII and BIV based on the DNA sequence analysis of the glutamate dehydrogenase locus of G. duodenalis.The findings of this study emphasize that Iran (Fars Province is a favorable area for giardiasis with an anthroponotic infection route.

  5. Molecular identification and genetic diversity among Photorhabdus and Xenorhabdus isolates.

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    Moghaieb, Reda E A; Abdelhadi, Abdelhadi A; El-Sadawy, Hanan A; Allam, Nesreen A T; Baiome, Baiome Abdelmaguid; Soliman, Mohamed H

    2017-05-01

    Five bacterial strains were isolated from the hemocoel of the greater wax moth larvae (Galleria mellonella) infected with the entomopathogenic nematodes: Heterorhabditis bacteriophora HP88, Heterorhabditis indicus RM1 and Heterorhabditis sp (S1), Steinernema abbasi and Steinernema sp. (S II). Strains were identified as Photorhabdus luminescens HRM1, P. luminescens HS1, P. luminescens HP88, Xenorhabdus indica and X. nematophila ATTC19061 using 16S rDNA sequence analysis. To reveal the genetic diversity among these strains, three molecular markers (RAPD, ISSR and SRAP) were employed. RAPD analysis showed 73.8 and 54.5 polymorphism percentages for the Photorhabdus and Xenorhabdus strains, respectively. ISSR analysis resulted in 70.1 and 75.2 polymorphism percentages among the Photorhabdus and Xenorhabdus strains, respectively. The SRAP analysis indicated that 75.6 and 61.2% genetic polymorphism was detected among Photorhabdus and Xenorhabdus strains, respectively. The cluster analysis grouped the three Photorhabdus strains together in one cluster and the two Xenorhabdus strains together in another cluster indicating the phylogenetic relationships among them. The genotype-specific markers detected from the three molecular markers (RAPD, ISSR and SRAP) were sufficient to distinguish between the different bacterial strains tested and can be used in the future IBM program that could be built on the use of these strains.

  6. Phenotypic and molecular identification of Fonsecaea pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela.

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    Carolina Rojas, O; León-Cachón, Rafael B R; Pérez-Maya, Antonio Alí; Aguirre-Garza, Marcelino; Moreno-Treviño, María G; González, Gloria M

    2015-05-01

    Chromoblastomycosis is a chronic granulomatous disease caused frequently by fungi of the Fonsecaea genus. The objective of this study was the phenotypic and molecular identification of F. pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela. Ten strains were included in this study. For phenotypic identification, we used macroscopic and microscopic morphologies, carbohydrate assimilation test, urea hydrolysis, cixcloheximide tolerance, proteolitic activity and the thermotolerance test. The antifungal activity of five drugs was evaluated against the isolates. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) ribosomal DNA regions of the isolated strains. The physiological analysis and morphological features were variable and the precise identification was not possible. All isolates were susceptible to itraconazole, terbinafine, voriconazole and posaconazole. Amphotericin B was the least effective drug. The alignment of the 559-nucleotide ITS sequences from our strains compared with sequences of GenBank revealed high homology with F. pedrosoi (EU285266.1). In this study, all patients were from rural areas, six from Mexico and four from Venezuela. Ten isolates were identified by phenotypic and molecular analysis, using ITS sequence and demonstrated that nine isolates from Mexico and Venezuela were 100% homologous and one isolate showed a small genetic distance. © 2015 Blackwell Verlag GmbH.

  7. Molecular identification of unusual Mycetoma agents isolated from patients in Venezuela.

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    Rojas, Olga C; León-Cachón, Rafael B R; Moreno-Treviño, Maria; González, Gloria M

    2017-02-01

    Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques.

  8. Molecular identification of Echinococcus granulosus isolates from ruminants in Greece.

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    Roinioti, Erifylli; Papathanassopoulou, Aegli; Theodoropoulou, Ioanna; Simsek, Sami; Theodoropoulos, Georgios

    2016-08-15

    Cystic echinococcosis is a parasitic disease caused by Echinococcus granulosus, a cestode with worldwide distribution. Data on the circulating Echinococcus granulosus genotypes in Greek livestock is scant. The aim of the present study was to conduct a genetic analysis of 82 Echinococcus granulosus isolates from ruminants in Greece, including areas which until today have not been the subject of studies. The analysis relied on a PCR assay targeting cytochrome c oxidase, subunit 1 gene (CO1), followed by bidirectional sequence analysis of the amplification product. Eighty (n=80) of the 82 (97.6%) isolates were allocated to Echinococcus granulosus sensu stricto (G1-G3) and were classified in 13 distinct haplotypes (9 common and 4 novel) with 12 polymorphic sites. The presence of the dominant haplotype EG1 as was documented in the European populations, was indicated in the country. Almost all regions shared the same common haplotype. In comparison to this predominant haplotype, the number of the nucleotide changes in all the other haplotypes ranged from 1 to 5. All nucleotide changes proved to be transitions (A↔G or C↔T). Two fertile hydatid cysts of sheep origin in different areas (Arkadia, Ilia) of the Peloponnese were identified as Echinococcus canadensis (G7 genotype).

  9. ISOLATION AND MOLECULAR IDENTIFICATION OF PATHOGENIC BACTERIA FROM URINE SAMPLE

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    Suhad A. Ibrahim

    2015-11-01

    Full Text Available Hospital environment is the major cause of variousinfections including cut and burn wounds. Urine Samples are one of the most common and devastating forms of trauma, exposing the immunosuppressed patients to early and serious infections. Faster detection of the causative Microbes and institution of proper therapy would help greatly in preventing septic complications. Recent advances in the field of Molecular Biology, including the amplification of genetic material by the Polymerase Chain Reaction (PCR technologies have led to faster and more reliable microbial detection methods. Results would help in early detection of causative organisms and treatment regimes in patients with burn wounds.

  10. Molecular identification of nocardia isolates from clinical samples and an overview of human nocardiosis in Brazil.

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    Paulo Victor Pereira Baio

    Full Text Available BACKGROUND: Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease. METHODOLOGY/PRINCIPAL FINDINGS: The majority of Brazilian human isolates in cases reported in literature were identified as Nocardia sp. Molecular characterization was used for species identification of Nocardia nova, Nocardia cyriacigeorgica, Nocardia asiatica and Nocardia exalbida/gamkensis. Data indicated that molecular analysis provided a different Nocardia speciation than the initial biochemical identification for most Brazilian isolates. All Nocardia isolates showed susceptibility to trimethoprim-sulfamethoxazole, the antimicrobial of choice in the treatment nocardiosis. N. nova isolated from different clinical specimens from one patient showed identical antimicrobial susceptibility patterns and two distinct clones. CONCLUSIONS/SIGNIFICANCE: Although Brazil is the world's fifth-largest country in terms of land mass and population, pulmonary, extrapulmonary and systemic forms of nocardiosis were reported in only 6 of the 26 Brazilian states from 1970 to 2013. A least 33.8% of these 46 cases of nocardiosis proved fatal. Interestingly, coinfection

  11. Isolation and Identification of Listeria monocytogenes in Processed Meat by a Combined Cultural-molecular Method

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    Angela Ingianni

    2007-01-01

    Full Text Available The isolation and identification of Listeria monocytogenes in processed meat samples by a combined cultural-molecular method is described. It allows the identification of Listeria strains by means of a hybridization technique with a specific DNA probe directed to the listerial internalin gene. The specificity of this method was found to be 100% and sensitivity was as low as 1 CFU/2.5 g of food sample. A total of 278 meat samples were tested in comparison with PCR and conventional cultural assays. A total of 42 (15.4% L. monocytogenes were detected. PCR analysis gave 3 false negative results and culture failed to detect the Listeria in 5 cases. With this cultural-molecular method the identification and quantitative detection of L. monocytogenes were achieved within 36 hours and no false positive or negative tests were obtained, thus fitting most food industry requirements.

  12. Molecular basis for identification of species/isolates of gastrointestinal nematode parasites

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    Ahmed M; Singh MN; Bera AK; Bandyopadhyay S; Bhattacharya D

    2011-01-01

    Gastrointestinal(GI)parasitism is the most serious constraint throughout the world in small ruminants which causes significant production loss in animals.GI parasites are major contributor to reduce productivity in terms of meat, milk and wool in animals. Control ofGI parasite is done primarily by anthelmintic treatment where choice and schedule of treatment is done after identification and quantitation of individual parasite. Identification ofGI parasites is done through microscopic method by identifying specific morphological characteristics of egg and larva (L3). Since most of parasite eggs are having similar morphological characteristics, identification up to species level through microscopy is not possible in most of cases. To address this issue, molecular techniques are the viable alternative for identification of species as well as molecular level differences within a species (isolates) of parasites. DifferentDNA based molecular techniquesviz.PCR, AFLP, RAPD, RFLP, PCR-SSCP, real timePCR, DNAmicroarrayetc. have been used for identification and to assess the genetic diversity among parasite population. For identification of species, the characteristic sequence of genomicDNAof different species should differ to allow the delineation of species, but at the same time, no/minor variation within the species should exist. In contrast, for purpose of identifying population variants (strains/isolates), a considerable degree of variation in the sequence should exist within a species. Various target regions, including nuclear ribosomal DNA (rDNA), mitochondrial DNA(mtDNA) or repetitiveDNA elements (microsatellite loci), which show considerable variation in the number of repeats within individuals have been employed to achieve the identification of parasites species or strain.

  13. Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata in northeastern Venezuela

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    Hector De Lima

    2008-06-01

    Full Text Available This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata, and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

  14. Isolation of Bacteroides from fish and human fecal samples for identification of unique molecular markers.

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    Kabiri, Leila; Alum, Absar; Rock, Channah; McLain, Jean E; Abbaszadegan, Morteza

    2013-12-01

    Bacteroides molecular markers have been used to identify human fecal contamination in natural waters, but recent work in our laboratory confirmed cross-amplification of several human-specific Bacteroides spp. assays with fecal DNA from fish. For identification of unique molecular markers, Bacteroides from human (n = 4) and fish (n = 7) fecal samples were cultured and their identities were further confirmed using Rapid ID 32A API strips. The 16S rDNA from multiple isolates from each sample was PCR amplified, cloned, and sequenced to identify unique markers for development of more stringent human-specific assays. In human feces, Bacteroides vulgatus was the dominant species (75% of isolates), whereas in tilapia feces, Bacteroides eggerthii was dominant (66%). Bacteroides from grass carp, channel catfish, and blue catfish may include Bacteroides uniformis, Bacteroides ovatus, or Bacteroides stercoris. Phylogenic analyses of the 16S rRNA gene sequences showed distinct Bacteroides groupings from each fish species, while human sequences clustered with known B. vulgatus. None of the fish isolates showed significant similarity to Bacteroides sequences currently deposited in NCBI (National Center for Biotechnology Information). This study expands the current sequence database of cultured fish Bacteroides. Such data are essential for identification of unique molecular markers in human Bacteroides that can be utilized in differentiating fish and human fecal contamination in water samples.

  15. Species identification and molecular typing of human Brucella isolates from Kuwait.

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    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  16. Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk

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    L.B. Acurcio

    2014-06-01

    Full Text Available Lactic acid bacteria species were molecularly identified in milk from Lacaune, Santa Inês and crossbred sheep breeds and their in vitro probiotic potential was evaluated. The species identified were Enterococcus faecium (56.25%, E. durans (31.25% and E. casseliflavus (12.5%. No other lactic acid bacteria species, such as lactobacilli, was identified. Most of the isolated enterococci were resistant to gastric pH (2.0 and to 0.3% oxgall. All tested enterococci were resistant to ceftazidime, oxacillin and streptomycin and sensible to clindamycin, erythromycin and penicillin. The resistance to ciprofloxacin, gentamicin, tetracycline and vancomycin varied among tested species. All tested enterococci strongly inhibited (P<0.05 Escherichia coli and Listeria monocytogenes, moderately inhibited E. faecalis and Staphylococcus aureus and did not inhibit Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium and also one E. durans sample isolated from sheep milk. Four samples of E. faecium, one of E. durans and one of E. casseliflavus presented the best probiotic potential.

  17. Molecular identification of Mycobacterium tuberculosis complex isolates from Kermanshah Province, Iran.

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    Moghaddam, Roghieh; Mosavari, Nader; Mahalati, Ardeshir Hesampoor

    2016-12-01

    Tuberculosis is one of the most important zoonotic diseases in the world. Rapid diagnosis of the disease and identification of species is extremely important for proper treatment of the disease as some species of the complex are resistant to the first-line of tuberculosis drugs. The aim of present study was molecular identification of Mycobacterium tuberculosis (MTB) complex isolates from Kermanshah Province, Iran, which were submitted to the Tuberculosis Reference Laboratory at Razi Vaccine and Serum Research Institute (Tehran, Iran). To identify the genus Mycobacterium, all isolates were subjected to 16S rRNA polymerase chain reaction (PCR), and PCR-IS6110 was subsequently used to confirm that the isolates belonged to MTB complex. Finally, region of difference (RD) typing was used to identify the species in the complex. The results of 16S rRNA and IS6110 PCR analysis showed the presence of 543-bp and 245-bp bands, respectively. Furthermore, 146bp, 172bp, 235bp, and 369bp at RD1, RD4, RD9, and RD12, respectively, were observed during RD typing. Thus, based on the results, all isolates were identified as MTB. It is worth mentioning that most tuberculosis cases are identified on the basis of acid-fast bacilli detection, and antibiotic therapy is immediately initiated subsequently. Moreover, it should be noted that some of these acid-fast positive cases might not be of genus Mycobacterium, and thus, the antibiotics prescribed might threaten the health of the patients. Additionally, if the identified bacilli are not within MTB complex, the drug therapy would differ. However, Mycobacterium bovis, which is a member of MTB complex and is resistant to pyrazinamide, requires exact strain identification. Based on the findings, individual isolates should be identified by RD typing methods, which could clearly discriminate the species from each other. Copyright © 2016.

  18. Isolation and identification of low molecular weight antioxidant compounds from fermented "chorizo" sausages.

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    Broncano, J M; Otte, J; Petrón, M J; Parra, V; Timón, M L

    2012-02-01

    This work is focused on the determination of low molecular weight compounds extracted from samples of fermented sausages. The antioxidant activity of fractions isolated from chorizo extracts was tested by their ability to quench free radicals by the DPPH-radical scavenging assay. Natural dipeptides and metabolites characteristic of meat were abundant in the fractions isolated by RP-HPLC from chorizo extracts. Due to extensive degradation during the ripening of chorizo, the extracts did not contain many peptides in a concentration that allowed identification. However, many free amino acids were identified by LC-MS/MS and HILIC-MS/MS. The fractions with the most hydrophilic compounds showed the highest antioxidant activity.

  19. Isolation and molecular identification of biodegrading Mycobacteria from water supplies of Iranian hospitals.

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    Davood Azadi

    2014-08-01

    Full Text Available Some microorganisms, mainly members of two genera including Pseudomonas and Mycobacterium, were found to be capable of transforming and degrading of polluting agents. We herein report the isolation of a few mycobacteria with the ability to biodegrade organic and inorganic compounds from water supplies of Iranian hospitals.The water samples were collected from hospital water supplies. Isolation processes were done according to standard methods. The colonies were subcultured on Löwenstein-Jensen medium to obtain a pure culture. The identification and characterization of the isolates were based on conventional and molecular methods including direct sequence analysis of almost full length of 16S rRNA gene.The almost complete 16S rRNA gene sequences of the studied strains revealed that the isolates WP16, AW18-1 and AW18-3 were identified as M. fredriksbergense, AW18-2 as M. austroafricanum, AW27-2 as M. obuenseand AW27-6 as M. phocaicum.The relationship between our isolates and standard strains of Mycobacterium was supported by a phylogenetic tree of 16S rRNA gene.In the current study we were able to isolate and characterize six mycobacteria with capability of transforming and degrading polluting agents from Iranian hospital environments. This is indeed the first report on isolation and characterization of mycobacteria with degrading capability of polluting agents from Iranian hospitals. It can be considered as a pioneer study to open up a new horizon in the study of microbial diversity in Iran with an objective-based and applied approach to tackle environmental challenges.

  20. Molecular identification of Giardia duodenalis isolates from domestic dogs and cats in Wroclaw, Poland.

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    Piekarska, Jolanta; Bajzert, Joanna; Gorczykowski, Michał; Kantyka, Magdalena; Podkowik, Magdalena

    2016-09-01

    Giardia duodenalis (G. intestinalis) is a common protozoan causing gastrointestinal disorders in many species of mammals. The genus of Giardia has high molecular diversity. Dogs and cats, in addition to their typical infection with assemblages C, D and F, may be a reservoir of zoonotic assemblages (A and B). The aim of this study was a genetic characteristic of Giardia isolates of dogs and cats from the area of Wroclaw (Poland). A total of 128 and 33 faecal samples from dogs and cats, respectively, were analyzed by routine coprological methods. The animals were diagnosed on the presence of G. duodenalis antigens in faeces soluble with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. 27 DNA isolates of Giardia were subjected to molecular identification (PCR-RFLP). The prevalence of G. duodenalis was 21.1% (27/128) in dogs and 15.1% (5/33) in cats. In dogs, C assemblage was present in 18 (81%) positive stool samples, D assemblage in 2 (9%) samples, B assemblage present in one (4.5%), and mixed assemblages (C and D) occurred in one (4.5%) sample. F assemblage was found in 4 (80%) cats' positive stool samples and A assemblage occurred in one case (20%). Confirmation of the presence of A and B zoonotic assemblages suggests that infected pets can be a threat to human health. This study describes for the first time the presence of mixed infections within host-specific C and D assemblages in dogs in Poland.

  1. Molecular identification of three Trichinella isolates from Heilongjiang Province, People's Republic of China.

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    Lu, Yixin; Han, Caixia; Yang, Jinyu; Li, Xiaoyun; Song, Mingxin

    2011-11-01

    DNAs of Trichinella dog isolate (HC), Trichinella swine isolate (HH) and Trichinella cat isolate (SW), obtained from Heilongjiang Province, were amplified by the fragments of 18S rDNA and ITS2. Two reference strains, Trichinella spiralis (ISS3) and Trichinella nativa (ISS10) were used for sequence comparison. Sequence and dendrogram analysis indicated that HC belonged to T. nativa and HH together with SW belonged to T. spiralis. This method permits rapid species identification of Trichinella isolates, although further evaluation is required before precisely identification.

  2. Molecular identification and artificial cultivation of a wild isolate of oyster mushroom in Albania

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    Jordan Merkuri

    2016-03-01

    Full Text Available Basidiomata of a wild mushroom macroscopically recognised as Pleurotus ostreatus were observed on an oak trunk in a mixed wood of northern Albania. Pure cultures of the fungus were then obtained on potato-dextrose-agar medium. Molecular analyses of genomic DNA of the fungus confirmed its identification. The rDNA ITS region nucleotide sequence of the studied Pleurotacea matched at 99% those of two P. ostreatus strains already present in NCBI GenBank database. The rDNA ITS nucelotide sequences of two pure cultures of the Albanian P. ostreatus were deposited in EMBL database under the accession numbers LN849458 and LN849459. One of the fungus isolates was subsequently cultivated under protected and semi-natural conditions. Productivity and biological efficiency of the Albanian P. ostreatus ranged from about 10% to 16% and from 33 to 53.33%, respectively. This seems to be the first report on the artificial cultivation of P. ostreatus in Albania and could have, in the next future, a high economic impact on development and diffusion of this important edible mushroom over the country.

  3. Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007-2014.

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    Lovero, Grazia; Borghi, Elisa; Balbino, Stella; Cirasola, Daniela; De Giglio, Osvalda; Perdoni, Federica; Caggiano, Giuseppina; Morace, Giulia; Montagna, Maria Teresa

    2016-01-01

    The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it

  4. Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007–2014

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    Lovero, Grazia; Borghi, Elisa; Balbino, Stella; Cirasola, Daniela; De Giglio, Osvalda; Perdoni, Federica; Caggiano, Giuseppina; Morace, Giulia; Montagna, Maria Teresa

    2016-01-01

    The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it

  5. Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007-2014.

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    Grazia Lovero

    Full Text Available The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2 and Clinical Laboratory Standards Institute (CLSI M27-A3 guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4% were identified as C. parapsilosis, and 27 (16.6% as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis to 11.1% (C. orthopsilosis, were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin

  6. Isolation, identification and molecular characterization of IBV variant from out break of visceral gout in commercial broilers

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    Amit Gaba

    Full Text Available Kidney tissue samples of field outbreak of visceral gout in commercial broilers from Anand District of Gujarat, India were processed for virus isolation, identification and molecular characterization. Isolates produced typical embryo lesions of curling and stunting suggestive of infectious bronchitis after three passages. RT-PCR and sequence analysis was carried out from infected allantoic fluid. The nucleotide sequence was found to have 99% nucleotide sequence identity with avian infectious bronchitis virus strain 4/91(pathogenic. Phylogenetic analysis using Clustal W program, the virus was found to group with 793/B and 4/91 strain of viruses. [Veterinary World 2010; 3(8.000: 375-377

  7. Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis).

    Science.gov (United States)

    Léon, Fabian; Van Boven, Maurits; de Witte, Peter; Busson, Roger; Cokelaere, Marnix

    2004-03-10

    A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.

  8. Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh,

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    Suaad S. AlWakeel

    2017-09-01

    Full Text Available This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis, alpha-hemolytic streptococci, Staphylococcus hominis, coagulase-negative staphylococci, Leuconostoc mesenteroides, Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (<60% sensitivity to the antibiotics ampicillin, erythromycin, clarithromycin and norfloxacin were observed. Ninety-seven percent similarity to the microbial bank species was noted when the isolates were compared to it. Most hematological indices recorded were within the normal range. In conclusion, exposure to toxic fumes and compounds within fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.

  9. Identification and molecular characterization of Chryseobacterium vrystaatense ST1 isolated from oligomineral water of southeast Serbia

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    Kojić M.

    2012-01-01

    Full Text Available The isolation and molecular characterization of bacterial strains isolated from water sources in the Vlasina Mountain in southeast Serbia, confirmed the presence of a new species Chryseobacterium vrystaatense ST1. This Gram- negative species showed an extremely low level of biochemical reactivity in biochemical tests. The gene for 16S rRNA was amplified by PCR using universal primers and sequenced. Comparison of 16S rRNA gene sequence and phenotypic features indicated that the isolate ST belonged to Chryseobacterium vrystaatense. A BLAST search of sequenced 1088 nucleotides of the 16S rRNA gene with all sequences deposited in the NCBI collection showed the highest similarity (98% with the strain Chryseobacterium vrystaatense sp. nov., designated as strain R-23533. The very high homology of these two strains allowed classification of our strain at the species level, but some differences indicate, and indirectly confirm, that the isolate ST is an authentic representative. On the basis of these results, we could conclude that Chryseobacterium vrystaatense ST was for first time isolated in Serbia, which is particularly important when one bears in mind that there are only three sequences of this species deposited in the NCBI collection.

  10. Microbial quality and molecular identification of cultivable microorganisms isolated from an urban drinking water distribution system (Limassol, Cyprus).

    Science.gov (United States)

    Botsaris, George; Kanetis, Loukas; Slaný, Michal; Parpouna, Christiana; Makris, Konstantinos C

    2015-12-01

    Microorganisms can survive and multiply in aged urban drinking water distribution systems, leading to potential health risks. The objective of this work was to investigate the microbial quality of tap water and molecularly identify its predominant cultivable microorganisms. Tap water samples collected from 24 different households scattered in the urban area of Limassol, Cyprus, were microbiologically tested following standard protocols for coliforms, E. coli, Pseudomonas spp., Enterococcus spp., and total viable count at 22 and 37 °C. Molecular identification was performed on isolated predominant single colonies using 16SrRNA sequencing. Approximately 85% of the household water samples were contaminated with one or more microorganisms belonging to the genera of Pseudomonas, Corynebacterium, Agrobacterium, Staphylococcus, Bacillus, Delftia, Acinetobacter, Enterococcus, Enterobacter, and Aeromonas. However, all samples tested were free from E. coli. This is the first report in Cyprus molecularly confirming specific genera of relevant microbial communities in tap water.

  11. Identification of low molecular weight proteins isolated by 2-D liquid separations.

    Science.gov (United States)

    Zhu, Kan; Miller, Fred R; Barder, Timothy J; Lubman, David M

    2004-07-01

    Proteins with molecular mass (M(r)) <20 kDa are often poorly separated in 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, low-M(r) proteins may not be readily identified using peptide mass fingerprinting (PMF) owing to the small number of peptides generated in tryptic digestion. In this work, we used a 2-D liquid separation method based on chromatofocusing and non-porous silica reversed-phase high-performance liquid chromatography to purify proteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis and protein identification. Several proteins were identified using the PMF method where the result was supported using an accurate M(r) value obtained from electrospray ionization TOFMS. However, many proteins were not identified owing to an insufficient number of peptides observed in the MALDI-TOF experiments. The small number of peptides detected in MALDI-TOFMS can result from internal fragmentation, the few arginines in its sequence and incomplete tryptic digestion. MALDI-QTOFMS/MS can be used to identify many of these proteins. The accurate experimental M(r) and pI confirm identification and aid in identifying post-translational modifications such as truncations and acetylations. In some cases, high-quality MS/MS data obtained from the MALDI-QTOF spectrometer overcome preferential cleavages and result in protein identification.

  12. Morphological and molecular identification of four Brazilian commercial isolates of Pleurotus spp. and cultivation on corncob

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    Nelson Menolli Junior

    2010-04-01

    Full Text Available The species of Pleurotus have great commercial importance and adaptability for growth and fructification within a wide variety of agro-industrial lignocellulosic wastes. In this study, two substrates prepared from ground corncobs supplemented with rice bran and charcoal were tested for mycelium growth kinetics in test tubes and for the cultivation of four Pleurotus commercial isolates in polypropylene bags. The identification of the isolates was based on the morphology of the basidiomata obtained and on sequencing of the LSU rDNA gene. Three isolates were identified as P. ostreatus, and one was identified as P. djamor. All isolates had better in-depth mycelium development in the charcoal-supplemented substrate. In the cultivation experiment, the isolates reacted differently to the two substrates. One isolate showed particularly high growth on the substrate containing charcoal.Espécies de Pleurotus têm grande importância comercial e adaptabilidade para crescimento e frutificação em uma ampla variedade de resíduos agro-industriais lignocelulósicos. Neste trabalho foram testados dois substratos à base de sabugo de milho triturado, suplementados com farelo de arroz e carvão vegetal, para avaliação da cinética de crescimento micelial em tubos de ensaio e produção em sacos de polipropileno, utilizando quatro isolados comerciais. O estudo taxonômico foi realizado com a análise da morfologia dos basidiomas obtidos em cultivo e pelo seqüenciamento do gene nLSU do DNAr, para certificar a identificação taxonômica. Os isolados tiveram melhor desenvolvimento micelial em profundidade no substrato suplementado com carvão vegetal. Em relação à produção, os isolados reagiram de formas distintas em função dos substratos, sendo significativamente melhor o substrato contendo carvão. Três isolados foram identificados como P. ostreatus e o outro foi identificado como P. djamor.

  13. Toxocara nematodes in stray cats from shiraz, southern iran: intensity of infection and molecular identification of the isolates.

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    Fattaneh Mikaeili

    2013-12-01

    Full Text Available Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection.This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites.Eight out of 30 cats (26.7% were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes.The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.

  14. Isolation, molecular identification and quinolone-susceptibility testing of Arcobacter spp. isolated from fresh vegetables in Spain.

    Science.gov (United States)

    González, Ana; Bayas Morejón, Isidro Favián; Ferrús, María Antonia

    2017-08-01

    Some species of the Arcobacter genus are considered emerging foodborne and waterborne enteropathogens. However, the presence of Arcobacter spp. in vegetables very little is known, because most studies have focused on foods of animal origin. On the other hand, quinolones are considered as first-line drugs for the treatment of infection by campylobacteria in human patients, but few data are currently available about the resistance levels to these antibiotics among Arcobacter species. Therefore, the aim of this study was to investigate the presence and diversity of arcobacters isolated from fresh vegetables such as lettuces, spinaches, chards and cabbages. Resistance to quinolones of the isolates was also investigated. One hundred fresh vegetables samples purchased from seven local retail markets in Valencia (Spain) during eight months were analysed. The study included 41 lettuces, 21 spinaches, 34 chards and 4 cabbages. Samples were analysed by culture and by molecular methods before and after enrichment. By culture, 17 out of 100 analysed samples were Arcobacter positive and twenty-five isolates were obtained from them. Direct detection by PCR was low, with only 4% Arcobacter spp. positive samples. This percentage increased considerably, up 20%, after 48 h enrichment. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 17 out of the 25 isolates were identified as A. butzleri and 8 as A. cryaerophilus. Only two A. butzleri isolates showed resistance to levofloxacin and ciprofloxacin. The sequencing of a fragment of the QRDR region of the gyrA gene from the quinolones-resistant isolates revealed the presence of a mutation in position 254 of this gene (C-T transition). This study is the first report about the presence of pathogenic species of Arcobacter spp. in chards and cabbages and confirms that fresh vegetables can act as transmission vehicle to humans. Moreover, the presence of A. butzleri quinolone resistant in vegetables could

  15. Molecular Identification and Amphotericin B Susceptibility Testing of Clinical Isolates of Aspergillus From 11 Hospitals in Korea

    Science.gov (United States)

    Heo, Min Seok; Choi, Min Ji; Park, Yeon-Joon; Lee, Hye Soo; Koo, Sun Hoe; Lee, Won Gil; Kim, Soo Hyun; Shin, Myung-Geun; Suh, Soon-Pal; Ryang, Dong-Wook

    2015-01-01

    Background We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. Methods A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and β-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. Results ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by β-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of ≥2 µg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was ≤75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). Conclusions Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance. PMID:26354348

  16. Identification and molecular epidemiology of dermatophyte isolates by repetitive-sequence-PCR-based DNA fingerprinting using the DiversiLab system in Turkey.

    Science.gov (United States)

    Koc, A Nedret; Atalay, Mustafa A; Inci, Melek; Sariguzel, Fatma M; Sav, Hafize

    2017-05-01

    Dermatophyte species, isolation and identification in clinical samples are still difficult and take a long time. The identification and molecular epidemiology of dermatophytes commonly isolated in a clinical laboratory in Turkey by repetitive sequence-based PCR (rep-PCR) were assessed by comparing the results with those of reference identification. A total of 44 dermatophytes isolated from various clinical specimens of 20 patients with superficial mycoses in Kayseri and 24 patients in Hatay were studied. The identification of dermatophyte isolates was based on the reference identification and rep-PCR using the DiversiLab System (BioMerieux). The genotyping of dermatophyte isolates from different patients was determined by rep-PCR. In the identification of dermatophyte isolates, agreement between rep-PCR and conventional methods was 87.8 % ( 36 of 41). The dermatophyte strains belonged to four clones (A -D) which were determined by the use of rep-PCR. The dermatophyte strains in Clone B, D showed identical patterns with respect to the region. In conclusion, rep-PCR appears to be useful for evaluation of the identification and clonal relationships between Trichophyton rubrum species complex and Trichophyton mentagrophytes species complex isolates. The similarity and diversity of these isolates may be assessed according to different regions by rep-PCR. © 2017 Blackwell Verlag GmbH.

  17. Morphological and molecular identification of filamentous Aspergillus flavus and Aspergillus parasiticus isolated from compound feeds in South Africa.

    Science.gov (United States)

    Iheanacho, Henry E; Njobeh, Patrick B; Dutton, Francis M; Steenkamp, Paul A; Steenkamp, Lucia; Mthombeni, Julian Q; Daru, Barnabas H; Makun, Anthony H

    2014-12-01

    Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South Africa, and subsequent extraction of their individual DNA in this study, presents a simple but rapid molecular procedure for high through-put analysis of the individual morphological forms. DNA was successfully isolated from the Aspergillus spp. from agar cultures by use of a commercial kit. Agarose gel electrophoresis fractionation of the fungi DNA, showed distinct bands. The DNA extracted by this procedure appears to be relatively pure with a ratio absorbance at 260 and 280 nm. However, the overall morphological and molecular data indicated that 67.5 and 51.1% of feed samples were found to be contaminated with Aspergillus flavus and Aspergillus parasiticus, respectively, with poultry feed having the highest contamination mean level of 5.7 × 105 CFU/g when compared to cattle (mean: 4.0 × 106 CFU/g), pig (mean: 2.7 × 104 CFU/g) and horse (1.0 × 102 CFU) feed. This technique presents a readily achievable, easy to use method in the extraction of filamentous fungal DNA and it's identification. Hence serves as an important tool towards molecular study of these organisms for routine analysis check in monitoring and improving compound feed quality against fungal contamination.

  18. Molecular Identification of Fungi Isolated from Bean Tissues with Anthracnose Symptoms

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    Katherine Maritza Vanegas Berrouet

    2014-03-01

    Full Text Available ABSTRACTIn this work, endophytic fungi from leaves and pods of bean presenting anthracnose symptoms were isolated from plantscollected at different municipalities in the province of Antioquia (Colombia. Isolates were identified by sequencing the rDNA ITS regions together with the examination of reproductive structures during sporulation in culture media. Colletotrichum lindemuthianum, the causal agent of anthracnose was isolated in all samples showing symptoms of this disease. These results were confirmed by duplex PCR using the specific primers CD1/CD2 and CY1/CY2. Additionally, 17 endophytic fungi were obtained. Fourteen isolates did not sporulate in culture media (Mycelia sterilia but were identified by phylogenetic analysis of the ITS regions as the Ascomycetes: Leptosphaerulina (3, Diaporthe (3, Gibberella (1, Plectosphaerella (1 and Biscogniauxia (1and the mitosporic genera Phoma (2, Alternaria (2 and Stemphylium(1. Three isolates were identified combining morphologica and molecular analysis as Fusarium (2 and Curvularia lunata (1. This work increases our knowledge of the mycobiota of legume plants and will serve as support of future studies aimed at determining the effect of these fungi on the development of anthracnose as well as other problems affecting the bean crop.IDENTIFICACIÓN MOLECULAR DE HONGOS AISLADOS DETEJIDOS DE FRÍJOL CON SÍNTOMAS DE ANTRACNOSISRESUMENEn este estudio se realizó el aislamiento de hongos en tejidos foliares y vainas de fríjol con síntomas de antracnosis, procedentes de cultivos de diferentes municipios del departamento de Antioquia (Colombia. La identificación de los aislamientos se realizó con base en la secuenciación de las regiones ITS del ADN ribosomal y se confirmó por observación microscópica de estructuras reproductivas en aquellos aislamientos que esporulaban en medios de cultivo. En todas las muestras sintomáticas, se logró el aislamiento del agente causal de la antracnosis

  19. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

    OpenAIRE

    Aboubaker M. Garbaj; Enas M. Awad; Salah M. Azwai; Said K. Abolghait; Naas, Hesham T.; Moawad, Ashraf A.; Fatim T. Gammoudi; Ilaria Barbieri; Ibrahim M. Eldaghayes

    2016-01-01

    Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice c...

  20. Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production.

    Science.gov (United States)

    Oelofse, A; Lonvaud-Funel, A; du Toit, M

    2009-06-01

    The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS-PCR, PCR-DGGE and REA-PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.

  1. Molecular detection and species identification of Enterocytozoon bieneusi isolated from immunocompetent Orang Asli in Malaysia.

    Science.gov (United States)

    Ashikin, Azah; Al-Mekhlafi, Hesham M; Moktar, Norhayati; Anuar, Tengku Shahrul

    2017-04-01

    Most studies of opportunistic infections focus on immunocompromised patients. However, there is a lack of information on microsporidiosis in healthy people (immunocompetent) worldwide. This study aimed to detect and identify microsporidia species in immunocompetent Orang Asli living in Pahang, Malaysia. Orang Asli is a collective term for a group of indigenous people that usually reside in the interior regions of Peninsular Malaysia. They comprise about 0.7% of the total population in Malaysia and 76% of them lived below the poverty line i.e., poor housing conditions with the lack of access to safe drinking water and adequate sanitation, contaminated environment, high illiteracy rate and unhygienic practices by these people. Stool samples were collected from 209 Orang Asli and analyzed for detecting the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis by polymerase chain reaction assay targeting small subunit ribosomal RNA gene. E. bieneusi was detected in 8 individuals (3.83%). This infection was commonly found in males than females (5.2% vs. 2.7%). All infected Orang Asli were adults, with a mean age of 44years. Diarrhea and other gastrointestinal symptoms were reported in one case (12.5%) among individuals infected with this species. These findings clearly show that exposure to E. bieneusi may actually be common than reported. The accurate detection and identification of microsporidian species by molecular technique will improve therapy, clinical manifestations and prognosis of this infection, as no antiparasitic therapy has been approved for E. bieneusi. It is hoped that these findings will allow the formulation of better health management and disease prevention advisories, and improvement in the standards of health in similar communities.

  2. Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

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    Fábio Silvestre Ataides

    2015-08-01

    Full Text Available AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7% were identified as C. parapsilosis sensu stricto , five (5.7% were identified as C. orthopsilosis , and four (4.6% were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

  3. Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China.

    Science.gov (United States)

    Li, Ling Zhao; Wang, Zhong Quan; Jiang, Peng; Zhang, Xi; Ren, Hui Jun; Cui, Jing

    2011-12-01

    The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

  4. Molecular identification of fungi isolated from bean tissues with anthracnose symptoms

    OpenAIRE

    Katherine Maritza Vanegas Berrouet; Pablo Andrés Gutiérrez Sánchez; Mauricio Alejandro Marín Montoya

    2014-01-01

    ABSTRACTIn this work, endophytic fungi from leaves and pods of bean presenting anthracnose symptoms were isolated from plantscollected at different municipalities in the province of Antioquia (Colombia). Isolates were identified by sequencing the rDNA ITS regions together with the examination of reproductive structures during sporulation in culture media. Colletotrichum lindemuthianum, the causal agent of anthracnose was isolated in all samples showing symptoms of this disease. These results ...

  5. Molecular and phenotypic identification of Candida isolates from pregnant women in Ogbomoso, Southwestern Nigeria

    Directory of Open Access Journals (Sweden)

    Akinbami Abidemi Nurat

    2016-02-01

    Conclusions: The combine uses of chromogenic agar and PCR have the advantage of efficient differentiation and identification of Candida species. [Int J Reprod Contracept Obstet Gynecol 2016; 5(2.000: 317-322

  6. Molecular identification of two strains of Cercospora rodmanii isolated from water hyacinth present in Yuriria lagoon, Guanajuato, Mexico and identification of new hosts for several other strains.

    Science.gov (United States)

    Montenegro-Calderón, José Guadalupe; Martínez-Álvarez, José Ascención; Vieyra-Hernández, Ma Teresa; Rangel-Macías, Luz Imelda; Razzo-Soria, Tannia; Chávez-Herrera, Roberto; Ponce-Noyola, Patricia; Leal-Morales, Carlos Alberto

    2011-11-01

    Water hyacinth is a beautiful monocotyledon plant that has been dispersed all over the world by humans. The plant has been present in Mexico since 1907, and many water bodies have become infested with it since then. In 2001, we initiated a survey in Yuriria lagoon in southern Guanajuato state to isolate fungi able to biocontrol the plant. We isolated 25 morphologically distinct fungal cultures, of which two were identified as members of the genus Cercospora. Cercospora species are among the most prevalent and destructive of plant pathogens and can be found on leaves, pedicels, stems, fruits, and bracts. Only two species of Cercospora, Cercospora piaropi, and Cercospora rodmanii, have been described on water hyacinth; however, the classification of these species has been controversial. Several molecular approaches have been used for Cercospora identification, and some candidate genes have been identified for use in Cercospora species determination. Although the nrRNA genes alone do not show sufficient resolution for species determination, histone H3, translation elongation factor1-α, β-tubulin, actin, and calmodulin have been shown in previous studies to have an adequate number of nucleotide changes to allow species identification. In the present study, we used partial sequences of the histone H3, actin, and calmodulin genes to identify our two isolates as C. rodmanii. Our two strains are not specific to water hyacinth, as they are also pathogenic to beet and sugar beet. Similar host ranges were found for C. rodmanii strains isolated from Tabasco in México, Zambia, and Brazil, however, the specificity for water hyacinth persists in Cercospora piaropi Tharp and C. rodmanii Conway, the latter being the most pathogenic.

  7. Morphological and molecular identification of filamentous fungi isolated from cosmetic powders

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    Flavia Cristina Jastale Pinto

    2012-12-01

    Full Text Available Seven fungi were isolated from 50 samples of cosmetic powders. Morphological analyses and ribosomal DNA Internal Transcribed Spacers sequencing were performed which allowed the discrimination of the isolated fungi as Aspergillus fumigatus, Penicillium sp., and Cladosporium sp. which could have, among their species, potentially pathogenic microorganisms.

  8. Measurement of antimicrobial activity of isolated bacteria from the Caspian sea and molecular identification of strains with antimicrobial effect

    Directory of Open Access Journals (Sweden)

    Sajad Harounabadi

    2015-12-01

    Full Text Available Introduction: Easy access and wide use of antimicrobial compounds led to the emergence of resistance among microorganisms. Therefore, screening and identifying antimicrobial compound with high effect of microorganisms in different environments is necessary and vital . Using microorganisms for biological aims change them to an important tool to control pathogens. Streptomyces griseus is one of them. The aim of this study is isolation of marine bacteria with antimicrobial effect against gram positive and negative bacteria. Finally, molecular identification of strains with antimicrobial activity. Materials and methods: In this study, 162 strains were isolated from the Caspian Sea .The strains were cultured on special medium and finally antimicrobial activity on references strains as measured. Among them four strains with remarkable antimicrobial activity were identified and selected. The strains were subjected to 16S rDNA PCR sequencing. The strains were submitted to NCBI as new Streptomyces griseus strains. Results: Among 162 strains, 4 strains had the most antimicrobial activity. The result showed, the strains were the most effective on Bacillus subtilis and Staphylococcus aureus (Gram positive bacteria and the least effect were observed on Escherichia coli and Pseudomonas aeruginosa (Gram negative bacteria. After sequencing, the strains were classified to sterptomyces griseus genu. Discussion and conclusion: In this study, 4 strains with antimicrobial activity were identified. According to the strength of these bacteria for controlling pathogenic bacteria resistant to antibiotic, we can have more pure microorganisms in optimized and controlled conditions for using in pharmaceutical industries and also for the treatment of dangerous pathogenic bacteria.

  9. Molecular Identification of Candida Albicans Isolated From the Oncology Patients at Four University Hospitals in Mazandaran Province (2005-6

    Directory of Open Access Journals (Sweden)

    H. Karami, M.D.

    2007-01-01

    Full Text Available AbstractBackground and Purpose: Early detection of Candida species in body site could improve the survival of the immunosuppressed patients by allowing the initiation of specific treatment while the fungal biomass is still low. The aim of this study was the identification of Candida albicans isolated from the oncology patients by molecular methods.Materials and Methods: Sixty two of Candida albicans isolated identified by phenotypic methods (color of colony on CHROMagar medium, germ-tube formation in horse serum, chlamydospore formation on Cornmeal agar with 1% Tween 80. DNA was extracted by using a glass bead/phenol- chloroform method. The oligonucleotide primer pairs (NL1/NL4 were used to amplify a 620-bp fragment of D1/D2 region of large submit (26s ribosomal DNA gene. PCR-products were electrophoresed in a 1.5% agarose gel. Eighteen PCR-amplified products sequenced and results were evaluated by online BLAST software. Multiple sequence alignment was performed by using online CLUSTAL-W (version 1.83 software.Results: The BLAST search revealed that all of products were Candida albicans. All sequences showed >99% similarity when compared with known reference sequences at the Gene-Bank. Four different strains were obtained of albicans species, including: AA 1622b (13 samples, 24698 (3 samples, TA 62 (1samples and 551 FC (1 sample. A total of 131 nucleotide exchange sites were revealed.Conclusion: The dominant species by phenotypic approaches was Candida albicans. In addition, identification of Candida albicans by (26S rDNA sequencing was 100% concordant to the results obtained by the phenotypic metho

  10. Molecular identification of the isolated diesel degrading bacteria and optimization studies

    Directory of Open Access Journals (Sweden)

    Janani Prathiba G

    2014-08-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Diesel, a refinery product of crude oil constitutes a major source of pollution in our environment. Poor solubility in water and the higher content in sediments make diesel a potential water pollutant. Bioremediation of contaminated aquatic and soil environments has arisen as an effective technology, with a range of advantages compared to more traditional methods. A total of 9 bacteria were isolated from three petroleum contaminated soil samples and isolate 3 of sample 1 showed maximum degradation potential of diesel in both primary and secondary screening tests. Hence, it was subjected for 16srDNA study and sequence alignment by BLASTN identified the isolate as Pseudomonas aeruginosa strain KEB24. The reaction conditions for efficient diesel degradation by the isolate were optimized.

  11. Molecular identification of TEM-116 beta-lactamase gene in isolates ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research January 2017; 16 (1): 149-154 ... 1Department of Biotechnology and Genetic Engineering, 2Department of ... Keywords: P. aeruginosa, Clinical isolates, Sequencing, TEM-116, Antibiotic ...

  12. Isolation and Molecular Identification of Salmonella typhimurium from Chicken Meat in Iraq

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    Aseel A. Saeed

    2013-06-01

    Full Text Available This study was conducted to determine the prevalence of Salmonellae contamination of chicken meat imported from different origin to local markets in south of Iraq (Diwaniya. The bacteria were cultured, isolated and biochemically characterized by the analytical profiling index (API 20E system. The 16s rRNA and invA gene primers were selected specifically for the detection of Salmonella to amplify a 406 and 558 bp DNA fragments, respectively. The results of this study showed that 22 Salmonella isolates were detected by polymerase chain reaction (PCR from 100 chicken meats and only 7 isolates out of 22 were identified as S. typhimurium, the highest percent of isolates were 83.8 % for India origin and the lowest percent were 25% from Jordan origin.

  13. Isolation and Identification of Concrete Environment Bacteria

    Science.gov (United States)

    Irwan, J. M.; Anneza, L. H.; Othman, N.; Husnul, T.; Alshalif, A. F.

    2016-07-01

    This paper presents the isolation and molecular method for bacteria identification through PCR and DNA sequencing. Identification of the bacteria species is required in order to fully utilize the bacterium capability for precipitation of calcium carbonate in concrete. This process is to enable the addition of suitable catalyst according to the bacterium enzymatic pathway that is known through the bacteria species used. The objective of this study is to isolate, enriched and identify the bacteria species. The bacteria in this study was isolated from fresh urine and acid mine drainage water, Kota Tinggi, Johor. Enrichment of the isolated bacteria was conducted to ensure the bacteria survivability in concrete. The identification of bacteria species was done through polymerase chain reaction (PCR) and rRDNA sequencing. The isolation and enrichment of the bacteria was done successfully. Whereas, the results for bacteria identification showed that the isolated bacteria strains are Bacillus sp and Enterococus faecalis.

  14. Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks

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    Asma Ashraf

    Full Text Available ABSTRACT Newcastle disease (ND is a major infectious disease of the poultry caused by a virulent strain of Avian Paramyxovirus - 1, that is a single strand non-segmented negative sense RNA virus. ND virus is major threat to the poultry industry in many countries of the world. The study was aimed to isolate and identify Newcastle disease virus (NDV by using a haemagglutination inhibition (HI test and reverse transcription-polymerase chain reaction (RT-PCR assay. A total 100 samples of infected and dead birds were collected from different poultry farms. The weight of the birds was ranged 1000-1200g. The birds were divided into 3 groups. Haemagglutination assay (HA was performed to detect the presence of NDV in suspension of infected homogenized tissues and it was found that HA is not the best method to detect the virus when it is in trace amounts. RT-PCR using NDV specific primers analyzed different clinical and postmortem samples. Reverse transcriptase polymerase chain reaction and specific primers was used for determining the presence of viruses. It was found that the virus was present in most of the infected samples except the serum of infected birds. During multiple sequence alignment (MSA it was found that, our isolates have high homology (98% with other reported NDV isolates. Phylogenetic analysis revealed that our isolate was closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus.

  15. Isolation, screening and molecular identification of novel bacterial strain removing methylene blue from water solutions

    Science.gov (United States)

    Kilany, Mona

    2017-04-01

    The potentially deleterious effects of methylene blue (MB) on human health drove the interest in its removal promptly. Bioremediation is an effective and eco friendly for removing MB. Soil bacteria were isolated and examined for their potential to remove MB. The most potent bacterial candidate was characterized and identified using 16S rRNA sequence technique. The evolutionary history of the isolate was conducted by maximum likelihood method. Some physiochemical parameters were optimized for maximum decolorization. Decolorization mechanism and microbial toxicity study of MB (100 mg/l) and by-products were investigated. Participation of heat killed bacteria in color adsorption have been investigated too. The bacterial isolate was identified as Stenotrophomonas maltophilia strain Kilany_MB 16S ribosomal RNA gene with 99% sequence similarity. The sequence was submitted to NCBI (Accession number = KU533726). Phylogeny depicted the phylogenetic relationships between 16S ribosomal RNA gene, partial sequence (1442 bp), of the isolated strain and other strains related to Stenotrophomonas maltophilia in the GenBank database. The optimal conditions were investigated to be pH 5 at 30 °C, after 24 h using 5 mg/l MB showing optimum decolorization percentage (61.3%). Microbial toxicity study demonstrated relative reduction in the toxicity of MB decolorized products on test bacteria. Mechanism of color removal was proved by both biosorption and biodegradation, where heat-killed and live cells showed 43 and 52% of decolorization, respectively, as a maximum value after 24-h incubation. It was demonstrated that the mechanism of color removal is by adsorption. Therefore, good performance of S maltophilia in MB color removal reinforces the exploitation of these bacteria in environmental clean-up and restoration of the ecosystem.

  16. Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping

    DEFF Research Database (Denmark)

    Siemer, B.L.; Nielsen, Elsa; On, Stephan L.w.

    2005-01-01

    Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish...... isolates of diverse origin identified by routine phenotyping as C coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli...

  17. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

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    Aboubaker M. Garbaj

    2016-11-01

    Full Text Available Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey that include 3 isolates from cow’s milk (11%, 3 isolates from she-camel’s milk (11%, two isolates from goat’s milk (7.4% and 7 isolates from fermented raw milk samples (26%, isolates from fresh locally made soft cheeses (Maasora and Ricotta were 9 (33% and 3 (11%, respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

  18. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA.

    Science.gov (United States)

    Garbaj, Aboubaker M; Awad, Enas M; Azwai, Salah M; Abolghait, Said K; Naas, Hesham T; Moawad, Ashraf A; Gammoudi, Fatim T; Barbieri, Ilaria; Eldaghayes, Ibrahim M

    2016-11-01

    The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow's milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow's milk (11%), 3 isolates from she-camel's milk (11%), two isolates from goat's milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

  19. Morphologic and molecular identification of Naegleria dunnebackei n. sp. isolated from a water sample.

    Science.gov (United States)

    Visvesvara, Govinda S; De Jonckheere, Johan F; Marciano-Cabral, Francine; Schuster, Frederick L

    2005-01-01

    Naegleria dunnebackei n. sp., a new species of the free-living amoeboflagellate Naegleria, is described in this report. The organism was isolated from a water sample taken from drinking troughs associated with cases of primary amoebic meningoencephalitis in cattle at a ranch in southern California. The isolate grew at, but not above 37 degrees C, and did not kill young mice upon intranasal inoculation suggesting that it was not pathogenic. The new species combines morphological features of non-pathogenic Naegleria gruberi and pathogenic Naegleria fowleri. The trophic amoeba resembled other members of the genus, with a prominent vesicular nucleus and mitochondria with discoidal cristae; a Golgi apparatus was not observed by electron microscopy. The cyst stage had pores in the wall typical of those seen in pathogenic N. fowleri. Upon suspension in distilled water, amoebae transformed into temporary, non-feeding flagellates, mostly with two anterior flagella but occasionally with four. The rationale for its description as a new species was based upon sequencing of the 5.8S rDNA and internal transcribed spacers of the amoeba, which is similar to but not identical to that of Naegleria gallica, differing from that organism's DNA by six base pairs. Virus-like elements were found in the cytoplasm of trophic amoebae, often in association with crystalloids, and may be the cause of lysis of amoebae in culture.

  20. Isolation and molecular identification of actinomycetes from mycetoma patients in Sudan

    Directory of Open Access Journals (Sweden)

    Mogahid M Elhassan

    2013-01-01

    Full Text Available Introduction: In order to minimize chance of amputation due to actinomycetoma, it is important to correctly identify the causative agents. Microscopic examination of grains is not definite and further confirmatory diagnostic tests are needed. This study aims to investigate the prevalence of actinomycetoma and to explore the usefulness of strb1 gene in the diagnosis of the disease. Materials & Methods: The present study is a prospective cross-section laboratory-based study in which clinical samples (n = 100 from patients with mycetoma lesions were collected. The samples were cultured on Lowenstein Jensen and glucose yeast extract agar media. Grown colonies were initially identified using Gram′s stain, Ziehl Neelsen stain, and selected biochemical reactions. Confirmation was done by the analysis of polymerase chain reaction amplified strb1 gene. Results: Actinomycetoma was represented by a high ratio (12% among the study population. Nine out of the 12 isolates (75% were found to belong to the genus Streptomyces; whereas three isolates (25% were identified as Nocardia spp. on the basis of phenotypic and mycolic acid contents. Conclusion: It could be concluded that actinomycetoma exists with significant prevalence (12% among patients investigated in the present study. Streptomyces is the most important etiological agent of actinomycetoma compared to Nocardia.

  1. Molecular identification and potential of an isolate of white rot fungi in bioremediation of petroleum contaminated soils

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    Maryam Mohammadi-sichani

    2017-06-01

    Full Text Available Introduction:Elimination or reduction of petroleum hydrocarbons from natural resources such as water and soil is a serious problem of countries, particularly oil-rich countries of the world. Using white rotting fungi compost for bioremediation of soils contaminated by petroleum hydrocarbons is effective. The aim of this study is molecular identification and potential of anisolate of white rot fungi in bioremediation of petroleum contaminated soils. Materials and methods: Spent compost of white rotting fungi was inoculated with petroleum contaminated soil into 3%, 5% and 10% (w/w. Treatments were incubated at 25-23 °C for 3 months. Reduction of petroleum hydrocarbons in treated soil was determined by gas chromatography. Ecotoxicity of soil was evaluated by seed germination test. Results: Based on the genome sequence of 18s rRNA, it is revealed that this isolate is Ganoderma lucidum and this isolate is deposited as accession KX525204 in the Gene Bank database. Reduction of petroleum hydrocarbons in soil treated with compost (3, 5 and 10% ranged from 42% to 71%. The germination index (% in ecotoxicity tests ranged from 20.8% to 70.8%. Gas chromatography results also showed a decrease in soil Hydrocarbons compounds. Discussion and conclusion: The compost of Ganoderma lucidum, a white rot fungus, has a potential ability to remove petroleum hydrocarbons in contaminated soil. Removal of hydrocarbons was increased with increase in compost mixed with contaminated soil. Petroleum contaminated soil amended with spent compost of G.lucidum 10% during three months is appropriate to remove this pollutant.

  2. Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

    Science.gov (United States)

    Trabelsi, Houaida; Dendana, F; Neji, S; Sellami, H; Cheikhrouhou, F; Makni, F; Ayadi, A

    2016-01-01

    Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

  3. Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

    Directory of Open Access Journals (Sweden)

    Rinaldi L.

    2012-11-01

    Full Text Available In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%, precisely in 6/125 snakes (4.8% and in 3/25 lizards (12.0%; all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus.

  4. ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia coli AND Campylobacter jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU.

    Science.gov (United States)

    Caballero, Moisés; Rivera, Isabel; Jara, Luis M; Ulloa-Stanojlovic, Francisco M; Shiva, Carlos

    2015-01-01

    Feral pigeons (Columbia livia) live in close contact with humans and other animals. They can transmit potentially pathogenic and zoonotic agents. The objective of this study was to isolate and detect strains of diarrheagenic Escherichia coli and Campylobacter jejuni of urban feral pigeons from an area of Lima, Peru. Fresh dropping samples from urban parks were collected for microbiological isolation of E. coli strains in selective agar, and Campylobacter by filtration method. Molecular identification of diarrheagenic pathotypes of E.coli and Campylobacter jejuni was performed by PCR. Twenty-two parks were sampled and 16 colonies of Campylobacter spp. were isolated. The 100% of isolates were identified as Campylobacter jejuni. Furthermore, 102 colonies of E. coli were isolated and the 5.88% resulted as Enteropathogenic (EPEC) type and 0.98% as Shiga toxin-producing E. coli (STEC). The urban feral pigeons of Lima in Peru can act as a reservoir or carriers of zoonotic potentially pathogenic enteric agents.

  5. Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds.

    Science.gov (United States)

    Gehring, I; Wensing, A; Gernold, M; Wiedemann, W; Coplin, D L; Geider, K

    2014-06-01

    Assays to detect Pantoea stewartii from maize seeds should include differentiation of P. stewartii subsp. stewartii and P. stewartii subsp. indologenes. Previously published PCR primers for the identification of P. stewartii subsp. stewartii amplified signals from both subspecies using both conventional and quantitative PCR. In MALDI-TOF mass spectroscopy analysis with the Biotyper software (Bruker), subspecies stewartii and indologenes produced identical score values. Analysis against the Biotyper database produced similar score values for both subspecies. From the subtyping methods provided by the Biotyper software, only composite correlation indexing (CCI) separated both groups. By alignment of 16S rRNA sequences, no subspecies distinction was possible. To develop new techniques for the separation of these subspecies, the partial sequences of several housekeeping genes were compared. The type strains of the two subspecies showed characteristic single-nucleotide polymorphisms (SNPs) in the genes galE, glmS and recA. Other reference strains of P. stewartii subsp. stewartii followed the same nucleotide pattern, whereas known P. stewartii subsp. indologenes strains were different. Based on single-nucleotide polymorphisms in galE and recA, PCR primers were created to separate the subspecies by stepdown PCR analysis. Two putative P. stewartii strains were isolated from imported maize seeds. They were not virulent on maize seedlings, were positive in the indole assay with Kovacs reagent and identified as P. stewartii subsp. indologenes. The subspecies-specific PCR primers confirmed they were subspecies indologenes. By stepdown PCR, P. stewartii subsp. indologenes can be differentiated from P. stewartii subsp. stewartii. A possible detection of P. stewartii subsp. stewartii, the causative agent of Stewart's wilt of maize, in plant material by immunological or molecular assays must exclude contamination with P. stewartii subsp. indologenes, which would create

  6. Molecular serotyping and identification of the 85M gragment in different Colombian isolates of Listeria monocytogenes strains: A descriptive study

    Directory of Open Access Journals (Sweden)

    María Consuelo Vanegas

    2012-02-01

    Full Text Available Introduction: Listeria monocytogenes is a pathogen acquired through the consumption of contaminated foods. Thirteen serotypes have been reported, of which 1/2a, 1/2b, and 4b are responsible for 98% of human listeriosis cases. This study examines the association between serotypes and virulent clones, offering greater information and providing tools to prevent and control diseases caused by L. monocytogenes serotype 4b.Objective: To identify the serotypes from L. monocytogene strains isolated from different samples by performing the molecular subtyping technique; to determine the 85M fragment that codifies for epidemic clone I.Methods : 108 strains of L. monocytogenes were used, isolated from samples of animals, body fluids, foods, and food processing plant equipment and spaces. The samples were identified by following the Bacteriological Analytical Manual protocol described by the Food and Drug Administration (FDA. The strains were identified by PolymeraseChain Reaction (PCR using primers and a standardized protocol from a previous research project. Serotype identification was performed by multiplex PCR. The determination of the 85M fragment of the SSCS cassette was done by following the protocol by Yildrim et al.Results : Of the 108 L. monocytogenes strains analyzed, 60.2% (65 strains belonged to the 4b-4d-4e serotype, 17.6% (19 strains were identified as 1/2a-3a serotype, 14.8% (16 strains were 4a-4c serotype, 3.7% (4 strains belonged to the 1/2c-3c serotype, and (3.7% corresponded to the 1/2b-3b-7 serotype. It was determined that the L. monocytogenes strains serotype 4b-4d-4e and 1/2a-3b have the 85M fragment of the SSCS cassette.Conclusion : This study reports the predominant existence of L. monocytogenes strains serotype 4b-4d-4e in food, environmental, and clinical samples. The presence of an epidemic clone I region was also found in L. monocytogenes strains.

  7. Molecular Identification of Isolated Fungi from Unopened Containers of Greek Yogurt by DNA Sequencing of Internal Transcribed Spacer Region

    OpenAIRE

    Sulaiman, Irshad M.; Emily Jacobs; Steven Simpson; Khalil Kerdahi

    2014-01-01

    In our previous study, we described the development of an internal transcribed spacer (ITS)1 sequencing method, and used this protocol in species-identification of isolated fungi collected from the manufacturing areas of a compounding company known to have caused the multistate fungal meningitis outbreak in the United States. In this follow-up study, we have analyzed the unopened vials of Greek yogurt from the recalled batch to determine the possible cause of microbial contamination in the pr...

  8. Molecular Identification of Isolated Fungi from Unopened Containers of Greek Yogurt by DNA Sequencing of Internal Transcribed Spacer Region

    Directory of Open Access Journals (Sweden)

    Irshad M. Sulaiman

    2014-06-01

    Full Text Available In our previous study, we described the development of an internal transcribed spacer (ITS1 sequencing method, and used this protocol in species-identification of isolated fungi collected from the manufacturing areas of a compounding company known to have caused the multistate fungal meningitis outbreak in the United States. In this follow-up study, we have analyzed the unopened vials of Greek yogurt from the recalled batch to determine the possible cause of microbial contamination in the product. A total of 15 unopened vials of Greek yogurt belonging to the recalled batch were examined for the detection of fungi in these samples known to cause foodborne illness following conventional microbiological protocols. Fungi were isolated from all of the 15 Greek yogurt samples analyzed. The isolated fungi were genetically typed by DNA sequencing of PCR-amplified ITS1 region of rRNA gene. Analysis of data confirmed all of the isolated fungal isolates from the Greek yogurt to be Rhizomucor variabilis. The generated ITS1 sequences matched 100% with the published sequences available in GenBank. In addition, these yogurt samples were also tested for the presence of five types of bacteria (Salmonella, Listeria, Staphylococcus, Bacillus and Escherichia coli causing foodborne disease in humans, and found negative for all of them.

  9. Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance.

    Science.gov (United States)

    Srednik, Mariela Elizabeth; Grieben, Mario Andres; Bentancor, Adriana; Gentilini, Elida Raquel

    2015-09-27

    Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as causeof bovine intramammary infection (IMI) throughout the world in recent years. CoNShave been isolated from milk samples collected from cows with clinical andsubclinical mastitis in several countries. Identification of mastitis pathogensis important when selection appropriate antimicrobial therapy. A total of 93 strains of Staphylococcusspp isolated from bovine clinical andsubclinical mastitis in Argentina during 2010-2013 were identified by Restriction Fragment Length Polymorphism (RFLP)-PCR using the gap gene. The isolates were tested by PCR for the presence of blaZ gene and mecA gene and were tested by disk diffusion for the susceptibilityto penicillin and cefoxitin. The most common CoNS species was S.chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The blaZ gene was detected in 19 (20.4%) but only 16 (17.2%) isolates were resistant to penicillin; the mecA was detected in6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mecA-positive isolates showed oxaxillinMICs ≤ 0.5 μg/ml. CoNSare important minor mastitis pathogens and can be the cause of substantial economic losses. Despite the low resistance to PEN in Argentina, the presenceof MR isolates found in this study emphasize the importance of identificationof CoNS when an IMI is present because of the potentially risk of lateraltransfer of resistance genes between staphylococcal species.

  10. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2

    Directory of Open Access Journals (Sweden)

    M Forouzandeh-Moghadam

    2011-09-01

    Full Text Available Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and ana-lyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifi-cally of Fasciola species. Results: The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.Conclusion: The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.

  11. Identification of molecular marker and aggressiveness for different groups of Bipolaris sorokiniana isolates causing spot blotch disease in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Jaiswal, S K; Sweta; Prasad, L C; Sharma, S; Kumar, S; Prasad, R; Pandey, S P; Chand, R; Joshi, A K

    2007-08-01

    One hundred fifty-five isolates of Bipolaris sorokiniana of wheat were studied for their morphopathological characterization. These isolates were grouped in five categories--black, brown/dull black, gray cottony growth, dull white/greenish black, and white--on the basis of their growth pattern. The frequency of the black suppressed type was maximum (45.63%), whereas the white isolate displayed lowest frequency (6.96%) in the natural population. Twenty RAPD (random amplified polymorphic DNA) primers were used to observe the variability among the identified groups of B. sorokininana. From each group, eight random isolates were investigated. A total of 143 bands were amplified, out of which 107 (74.83%) were polymorphic and 36 (25.17%) were monomorphic. On an average, the total numbers of bands generated per primer were 7.15, of which 5.35 and 1.80 were polymorphic and monomorphic, respectively. Dendrograms based on molecular polymorphism unveiled a considerable amount of diversity among the isolates. Specific DNA bands were identified for selected isolates. The distinct markers appeared to be potential enough to be employed as genetic fingerprints for future strain identification and classification. The study indicated that the RAPD primers provide an easy, rapid, and simple technique for the preliminary assessment of genetic diversity among the fungal isolates.

  12. Emerging pathogen Arcobacter spp. in acute gastroenteritis: molecular identification, antibiotic susceptibilities and genotyping of the isolated arcobacters.

    Science.gov (United States)

    Kayman, Tuba; Abay, Seçil; Hizlisoy, Harun; Atabay, H Ibrahim; Diker, K Serdar; Aydin, Fuat

    2012-10-01

    The aims of this study were as follows: (i) to isolate Arcobacter spp. from the stool samples of patients with gastroenteritis; (ii) to identify them with molecular methods; (iii) to genotype them using enterobacterial repetitive intergenic consensus (ERIC)-PCR; and (iv) to determine their antibiotic susceptibilities. For the study, a total of 3287 diarrhoeal stool samples submitted to the Microbiology Laboratory of the Kayseri Training and Research Hospital, Kayseri, Turkey, between 2010 and 2011 were analysed. Campylobacter blood-free selective medium supplemented with cefoperazone, amphotericin B and teicoplanin was used for isolation. Medium inoculated with stool samples was incubated microaerobically at 37 °C for 72-96 h. Phenotypic tests, a genus-specific PCR and a multiplex PCR were used to identify the arcobacters, whilst ERIC-PCR was used for genotyping and the antibiotic susceptibilities of the isolates were detected by E-test. Arcobacter spp. were isolated from nine of the 3287 samples. These nine isolates were identified as Arcobacter butzleri and all showed different ERIC-PCR profiles. All nine isolates were resistant to ampicillin and susceptible to gentamicin, tetracycline, erythromycin and ciprofloxacin. As far as is known, this is the first study in which A. butzleri has been isolated from human acute gastrointestinal infections in Turkey. According to these results, it is recommended that, when investigating the aetiology of infections of the digestive system in humans, Arcobacter spp. be considered for inclusion. The results of this study should contribute to our knowledge related to A. butzleri infections in humans.

  13. Molecular and phylogenetic identification of an oil-producing strain of Nannochloropsis oceanica (Eustigmatophyceae) isolated from the southwestern Atlantic coast (Argentina)

    OpenAIRE

    Bongiovani, Natalia Soledad; Sanchez Puerta, Maria Virginia; Popovich, Cecilia Angelines; Leonardi, Patricia Ines

    2017-01-01

    Screening of local microalgae species with potential for oil production is essential to achieve successful commercial large-scale cultures. In this study, identification of a South American species of Nannochloropsis was carried out using molecular and phylogenetic analyses of chloroplastic and nuclear genes, rcbL and 18S rDNA, respectively. The gene sequences for the studied strain were highly similar to other strains of Nannochloropsis oceanica (100% for 18S rDNA and 99.7% for rbcL) isolate...

  14. Identification and determination of antibiotic susceptibilities of Brucella strains isolated from patients in van, Turkey by conventional and molecular methods.

    Science.gov (United States)

    Parlak, Mehmet; Güdücüoğlu, Hüseyin; Bayram, Yasemin; Çıkman, Aytekin; Aypak, Cenk; Kılıç, Selçuk; Berktaş, Mustafa

    2013-01-01

    Brucellosis is a worldwide zoonotic disease and still constitutes a major public health problem. In this study, we aimed to identify biovars of Brucella strains isolated from clinical specimens taken from brucellosis patients from the Eastern Anatolia region as well determine the susceptibility of these isolates to tigecycline and azithromycin, drugs that may serve as alternatives to the conventional drugs used in the therapy. Seventy-five Brucella spp. isolates were included in the study. All strains were identified by both conventional and molecular methods. Brucella Multiplex PCR kit (FC-Biotech, Code: 0301, Turkey) and B. melitensis biovar typing PCR kit (FC-Biotech, Code: 0302, Turkey) were used for molecular typing. Antimicrobial susceptibilities of all strains were determined by E-tests. By conventional biotyping, 73 strains were identified as B. melitensis biovar 3 and two strains as B. abortus biovar 3. Molecular typing results were compatible with conventional methods. The MIC50 and MIC90 values of doxycycline were 0.047 and 0.094; tigecycline 0.094 and 0.125; trimethoprim/sulfamethoxazole 0.064 and 0.19; ciprofloxacin 0.19 for both; streptomycin 0.75 and 1; rifampin 1 and 2 and azithromycin 4 and 8. According to the MIC values, doxycycline was found to be the most effective antibiotic, followed by tigecycline, trimethoprim-sulfamethoxazole and ciprofloxacin. Currently recommended antibiotics for the treatment of brucellosis such as doxycycline, rifampin, streptomycin, trimethoprim-sulfamethoxazole and ciprofloxacin were found to be still effective. While our results showed that tigecycline can be used an alternative agent in the treatment of brucellosis, azithromycin has not been confirmed as an appropriate agent for the treatment.

  15. Isolation and molecular identification of Leishmania ( Viannia peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae in the Peruvian Andes

    Directory of Open Access Journals (Sweden)

    J Enrique Perez

    2007-08-01

    Full Text Available Leishmania (Viannia peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru. Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia sp. was determined by polymerase chain reaction (PCR, and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V. peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

  16. Isolation and molecular identification of a UV-resistant strain of Dietzia maris and antioxidant activity of pigment

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    Narges Zamanian

    2016-09-01

    Full Text Available Introduction: The ability of radioresistant bacteria to survive high levels of UV radiation has been linked to their strong DNA repair systems and ability to produce primary and secondary metabolic products. The biosynthesis of pigments provides an opportunity for bacteria to live in radiation-rich environment. Recent radiation-responsive pigments are used commercially as food colorants, anticancer drugs, as well as antibiotics and for cosmetic purposes. Materials and methods: Soil sample of Omidiyeh city was collected during the spring of 2014 and UV-resistant strain was isolated after primary and secondary screening. Then it was identified by molecular methods (16S rRNA gene sequencing. Antioxidant activity of pigment was evaluated by 2,2 -diphenyl-1-picryl hydrazyl (DPPH and the reducing power of pigments were analyzed by ferric chloride. Results: In this present study, new UV-resistant strain NM2 was isolated and by comparison of these 16S rRNA gene sequences to public database using the BLAST, the genus and species of the isolate was identified as Dietzia maris with 99% similarity. Extraction of pigment from isolated strain was carried out by methanol and acetone as solvents. The spectrum is characterized by maximum peak at 473 nm for pigment of NM2 strain. Antioxidant activity and the reducing ability of pigments increased by increasing their concentrations. NM2 strain pigment showed EC50 concentration of 3.30 mg/ml for DPPH free radical scavenging activity, and EC50 concentration of 28.46 µg/ml for reducing power. Discussion and conclusion: Isolation of natural resources of pigment is very important with high anti-oxidant activity. In the current study, pigment of UV-resistant bacteria demonstrated a strong antioxidant activity in vitro and pigment of these bacteria could play an important role in UV tolerance. Pigment of UV-resistant bacteria may be an appropriate source for antioxidative-related functional foods and the pharmaceutical

  17. Potential use of molecular-typing methods for the identification and characterization of salmonella enterica serotypes isolated in the poultry production chain

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    CM Baratto

    2012-09-01

    Full Text Available Salmonella is widespread in nature and can be found in all links of the poultry production chain. Due to its high impact on meat processing, techniques for the rapid detection and reproducible characterization of Salmonella serotypes in foods are needed. The present study investigated the potential of molecular profiling to identify and differentiate 15 Salmonella serotypes isolated from the poultry production chain, based on 5 primers by random amplified polymorphic DNA (RAPD, enterobacterial repetitive intergenic consensus (ERIC-PCR, amplification of rDNA internal spacer analysis (RISA, and amplified ribosomal DNA restriction analysis (ARDRA of 16S-23S rRNA internal spacer region (ISR cleaved with Alu I and Hha I restriction enzymes. Three isolates of each serotype were analyzed for the identification of similar and different profiles. Dendrograms were constructed from molecular profiles using the UPGMA method (unweighted pair-group method for the arithmetic averages and the software program WinBoot. The present study indicates the usefulness of RISA and ARDRA of the 16S-23S rRNA intergenic spacer region (ISR for systematic, epidemiological, and diagnostic purposes. Since these techniques can be used for the differentiation of serotypes, they are highly promising for the characterization of Salmonella serotypes and intra-serotypes. Data indicate that these techniques may be used to produce more consistent, reliable, and reproducible results in the identification and epidemiological study (traceability of Salmonella in the poultry industry.

  18. Molecular identification and phylogenetic analysis of Lactobacillus and Bifidobacterium spp. isolated from gut of honeybees (Apis mellifera) from West Azerbaijan, Iran

    Science.gov (United States)

    Sharifpour, Mohammad Farouq; Mardani, Karim; Ownagh, Abdulghaffar

    2016-01-01

    Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees’ gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products. PMID:28144419

  19. ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia coli AND Campylobacter jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU

    Science.gov (United States)

    CABALLERO, Moisés; RIVERA, Isabel; JARA, Luis M.; ULLOA-STANOJLOVIC, Francisco M.; SHIVA, Carlos

    2015-01-01

    SUMMARY Feral pigeons (Columbia livia) live in close contact with humans and other animals. They can transmit potentially pathogenic and zoonotic agents. The objective of this study was to isolate and detect strains of diarrheagenic Escherichia coli and Campylobacter jejuni of urban feral pigeons from an area of Lima, Peru. Fresh dropping samples from urban parks were collected for microbiological isolation of E. coli strains in selective agar, and Campylobacter by filtration method. Molecular identification of diarrheagenic pathotypes of E.coli and Campylobacter jejuni was performed by PCR. Twenty-two parks were sampled and 16 colonies of Campylobacter spp. were isolated. The 100% of isolates were identified as Campylobacter jejuni. Furthermore, 102 colonies of E. coliwere isolated and the 5.88% resulted as Enteropathogenic (EPEC) type and 0.98% as Shiga toxin-producing E. coli (STEC). The urban feral pigeons of Lima in Peru can act as a reservoir or carriers of zoonotic potentially pathogenic enteric agents. PMID:26603225

  20. Molecular identification and phylogenetic analysis of Lactobacillus and Bifidobacterium spp. isolated from gut of honeybees (Apis mellifera) from West Azerbaijan, Iran.

    Science.gov (United States)

    Sharifpour, Mohammad Farouq; Mardani, Karim; Ownagh, Abdulghaffar

    2016-01-01

    Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees' gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products.

  1. Molecular Identification and Polymorphism Determination of Cutaneous and Visceral Leishmaniasis Agents Isolated from Human and Animal Hosts in Iran

    Directory of Open Access Journals (Sweden)

    Homa Hajjaran

    2013-01-01

    Full Text Available Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67% were infected with L. major and 37 (33% with L. tropica. Of the 60 rodents examined, 25 (41.6% harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT, whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

  2. Pollen gene flow and molecular identification of full-sib families in small and isolated population fragments of Gleditsia triacanthos L

    National Research Council Canada - National Science Library

    Schlarbaum, Scott E; Owusu, Sandra A; Gailing, Oliver; Carlson, John E

    2016-01-01

    To analyze the utility of isolated remnant populations for full-sibling (full-sib) identification among open-pollinated single-tree progeny in the outcrossing and insect-pollinated tree Gleditsia triacanthos L...

  3. Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of Salmonella typhi from India

    OpenAIRE

    Shanahan, Philippa M. A.; Jesudason, Mary V.; Thomson, Christopher J.; Amyes, Sebastian G. B.

    1998-01-01

    A representative sample of 21 Salmonella typhi strains isolated from cultures of blood from patients at the Christian Medical College and Hospital, Vellore, India, were tested for their susceptibilities to various antimicrobial agents. Eleven of the S. typhi strains possessed resistance to chloramphenicol (256 mg/liter), trimethoprim (64 mg/liter), and amoxicillin (>128 mg/liter), while four of the isolates were resistant to each of these agents except for amoxicillin. Six of the isolates wer...

  4. Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit▿

    OpenAIRE

    Garner, Cherilyn D.; Starr, Jennifer K.; McDonough, Patrick L.; Altier, Craig

    2010-01-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three me...

  5. Molecular Phylogenies of Blastocystis Isolates from Different Hosts: Implications for Genetic Diversity, Identification of Species, and Zoonosis

    Science.gov (United States)

    Noël, Christophe; Dufernez, Fabienne; Gerbod, Delphine; Edgcomb, Virginia P.; Delgado-Viscogliosi, Pilar; Ho, Lip-Chuen; Singh, Mulkit; Wintjens, René; Sogin, Mitchell L.; Capron, Monique; Pierce, Raymond; Zenner, Lionel; Viscogliosi, Eric

    2005-01-01

    Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1α (EF-1α) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1α-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially ≥12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans. PMID:15634993

  6. Molecular identification of phosphate-solubilizing native bacteria isolated from the rhizosphere of Prosopis glandulosa in Mexicali valley.

    Science.gov (United States)

    Moreno-Ramírez, L; González-Mendoza, D; Cecena-Duran, C; Grimaldo-Juarez, O

    2015-03-31

    One of the main limitations in intensive crop production in Northwestern Mexico is the dependence on the use of phosphate fertilizer. In this study, we isolated indigenous microorganisms with phosphate solubilization capacities from mesquite (Prosopis glandulosa) present in the Mexicali valley. In total, 4 bacteria were isolated from the rhizosphere of mesquite, including ICA01, ICA02Ba, ICA03Bs, and ICA04Ma. The bacterial isolates were identified based on their phenotypic and 16S rRNA gene sequencing data to be Acinetobacter calcoaceticus. The results showed that ICA01 was the most efficient in solubilizing phosphate, followed by ICA02Ba and ICA03Bs, while ICA04Ma showed the lowest phosphate-solubilizing activity. The pH value of the culture medium decreased with bacterial growth, suggesting that these strains produce organic acids that solubilize phosphorus. These results will be useful for biotechnological studies and A. calcoaceticus may be employed for biofertilization programs in northwest Mexico.

  7. Molecular identification of Fusarium species isolated from transgenic insect-resistant cotton plants in Mexicali valley, Baja California.

    Science.gov (United States)

    Gonzalez-Soto, T; González-Mendoza, D; Troncoso-Rojas, R; Morales-Trejo, A; Ceceña-Duran, C; Garcia-Lopez, A; Grimaldo-Juarez, O

    2015-10-02

    Cotton production in the Mexicali valley is adversely affected by wilt and root rot disease associated with Fusarium species. In the present study, we sought to isolate and identify the Fusarium species in the rhizosphere of transgenic insect-resistant cotton plants grown in the Mexicali valley. Our analyses isolated four native fungi from the rhizosphere of cotton plants, namely, T-ICA01, T-ICA03, T-ICA04, and T-ICA08. These fungal isolates were categorized as belonging to Fusarium solani using their phenotypic characteristics and ITS region sequence data. Examination of the infection index showed that T-ICA03 and T-ICA04 caused systemic colonization (90%) of seeds followed by the occurrence of radicle and coleoptile decay. In contrast, T-ICA08 strain was less pathogenic against seed tissues (40%) in comparison to the other strains isolated. Our study showed that in transgenic insect-resistant cotton the disease "Fusarium wilt" is caused by the fungus, F. solani. Future studies are necessary to characterize the F. solani populations to determine whether phenological stages might influence the genetic diversity of the fungal populations present.

  8. Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity.

    Science.gov (United States)

    Matsubayashi, Makoto; Suzuta, Fumiko; Terayama, Yoshimi; Shimojo, Kengo; Yui, Takeshi; Haritani, Makoto; Shibahara, Tomoyuki

    2014-08-01

    Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea.

  9. A low-virulence Eimeria intestinalis isolate from rabbit (Oryctolagus cuniculus) in China: molecular identification, pathogenicity, and immunogenicity.

    Science.gov (United States)

    Shi, Tuanyuan; Bao, Guolian; Fu, Yuan; Suo, Xun; Hao, Lili

    2014-03-01

    An Eimeria intestinalis isolated from a rabbit in China was first identified by amplifying the 18S small subunit (SSU) ribosomal RNA gene. The size of the amplified fragment was 1521 bp. The 18S SSU RNA gene of the E. intestinalis isolate shared 99% sequence identity with E. intestinalis isolates from France and the Czech Republic, with 100 and 96% coverage, respectively. Then, the pathogenicity and immunogenicity of the E. intestinalis isolate were evaluated in specific pathogen free (SPF) rabbits. In the pathogenicity assay, SPF rabbits in four groups were infected with 5 × 10(3), 5 × 10(4), 5 × 10(5), and 0 sporulated oocysts, respectively. Clinical signs including diarrhoea, constipation, loss of appetite, and reduction of body weight gain were observed in rabbits inoculated with 5 × 10(4) and 5 × 10(5) oocysts. And one rabbit (25 %) inoculated with 5 × 10(5) oocysts died 15 days after the inoculation. In the immunogenicity assay, SPF rabbits in five groups (named B1, B2, B3, B4, and B5) were immunised with 5 × 10(1), 5 × 10(2), 5 × 10(3), 0, and 0 sporulated oocysts, respectively. All rabbits but the B5 group were challenged with 1 × 10(6) oocysts. After the challenge, no or slight clinical signs were seen in rabbits of the B2 and B3 groups. Compared with the control, a 69.6 and 84.5% reduction of oocyst output was observed in the B2 and B3 groups, respectively. The body weight gain of the two groups was obviously higher than that of the challenge control group. All the results show that the E. intestinalis isolate has low virulence but immunogenicity in rabbit.

  10. First report of Streptococcus agalactiae isolated from Oreochromis niloticus in Piura, Peru: Molecular identification and histopathological lesions

    Directory of Open Access Journals (Sweden)

    Yessica Ortega Asencios

    2016-11-01

    Full Text Available The aim of this study was to identify the bacterium Streptococcus agalactiae isolated in farmed Nile tilapia (Oreochromis niloticus from Piura, Peru and to characterize the histopathological lesions caused by this pathogen. Sixteen tilapias were sampled with clinic signs of the disease such as erratic swimming, exophthalmia and haemorrhages on the body and fins. Qualitative PCR in real time and histopathological analysis were performed. Nine fishes positives to S. agalactiae were found. The main histopathological findings were fibrinosuppurative epicarditis, periesplenitis, meninigitis and panophtaltmitis with predominance of mononuclear infiltration in all tissues. The correlation between qPCR and histopathological findings demonstrated nine fish (prevalence of 56.25% with Cq lower than 30, associated to high degree of tissue injuries. This study reports the first isolation of S. agalactiae by PCR in real time in tilapia farmed in Peru and characterizes the major histopathological changes caused by this bacterium.

  11. Staphylococcal micrococcins. II. Isolation, purification and identification.

    Science.gov (United States)

    Breiter, J; Metz, H; Grigo, J

    1975-08-01

    Seven strains belonging to the Micrococcaceae family and excreting substances with antibiotic activity, were grown in submerged cultures on technical scale for isolation, purification and identification of biologically active compounds. Two basic substances were isolated and classified to the micrococcin antibiotics family. The naturally occurring mixture of micrococcin M1 and M3 was called micrococcin M. This antibiotic has the formula C48H50O11N12S6 and a molecular weight of about 1160, melting point 221--224 degrees C, and optical rotation [a]20/D = + 66.6. Other antibiotically active substances produced by seven investigated strains were identified as micrococcin M or as separate compounds. Comparison with previously described micrococcin and micrococcin P has been made.

  12. Isolation and molecular identification of landfill bacteria capable of growing on di-(2-ethylhexyl) phthalate and deteriorating PVC materials.

    Science.gov (United States)

    Latorre, Isomar; Hwang, Sangchul; Montalvo-Rodriguez, Rafael

    2012-01-01

    Waste materials containing Di-(2-ethylhexyl) phthalate (DEHP), a suspected endocrine disruptor and reasonably anticipated human carcinogen, are typically disposed of in landfills. Despite this, very few studies had been conducted to isolate and identify DEHP-degrading bacteria in landfill leachate. Therefore, this study was conducted to isolate and characterize bacteria in landfill leachate growing on DEHP as the sole carbon source and deteriorating PVC materials. Four strains LHM1, LHM2, LHM3 and LHM4, not previously reported as DEHP-degraders, were identified via 16S rRNA gene sequence. Gram-positive strains LHM1 and LHM2 had a greater than 97% similarity with Chryseomicrobium imtechense MW 10(T) and Lysinibacillus fusiformis NBRC 15717(T), respectively. Gram-negative strains LHM3 and LHM4 were related to Acinetobacter calcoaceticus DSM 30006(T) (90.7% similarity) and Stenotrophomonas pavanii ICB 89(T) (96.0% similarity), respectively. Phylogenetic analysis also corroborated these similarities of strains LHM1 and LHM2 to the corresponding bacteria species. Strains LHM2 and LHM4 grew faster than strains LHM1 and LHM3 in the enrichment where DEHP was the sole carbon source. When augmented to the reactors with PVC shower curtains containing DEHP, strains LHM1 and LHM2 developed greater optical densities in the solution phase and thicker biofilm on the surfaces of the shower curtains.

  13. Isolation and identification of Salmonella spp. in drinking water, streams, and swine wastewater by molecular techniques in Taiwan

    Science.gov (United States)

    Kuo, C.; Hsu, B.; Shen, T.; Tseng, S.; Tsai, J.; Huang, K.; Kao, P.; Chen, J.

    2013-12-01

    Salmonella spp. is a common water-borne pathogens and its genus comprises more than 2,500 serotypes. Major pathogenic genotypes which cause typhoid fever, enteritis and other intestinal-type diseases are S. Typhimurium, S. Enteritidis, S. Stanley, S. Agona, S.Albany, S. Schwarzengrund, S. Newport, S. Choleraesuis, and S. Derby. Hence, the identification of the serotypes of Salmonella spp. is important. In the present study, the analytical procedures include direct concentration method, non-selective pre-enrichment method and selective enrichment method of Salmonella spp.. Both selective enrichment method and cultured bacteria were detected with specific primers of Salmonella spp. by polymerase chain reaction (PCR). At last, the serotypes of Salmonella were confirmed by using MLST (multilocus sequence typing) with aroC, dnaN, hemD, hisD, purE, sucA, thrA housekeeping genes to identify the strains of positive samples. This study contains 121 samples from three different types of water sources including the drinking water (51), streams (45), and swine wastewater (25). Thirteen samples with positive invA gene are separated from culture method. The strains of these positive samples which identified from MLST method are S. Albany, S. Typhimurium, S. Newport, S. Bareilly, and S. Derby. Some of the serotypes, S. Albany, S. Typhimurium and S. Newport, are highly pathogenic which correlated to human diarrhea. In our results, MLST is a useful method to identify the strains of Salmonella spp.. Keywords: Salmonella, PCR, MLST.

  14. Molecular identification of aminoglycoside-modifying enzymes in clinical isolates of Escherichia coli resistant to amoxicillin/clavulanic acid isolated in Spain.

    Science.gov (United States)

    Fernández-Martínez, Marta; Miró, Elisenda; Ortega, Adriana; Bou, Germán; González-López, Juan José; Oliver, Antonio; Pascual, Alvaro; Cercenado, Emilia; Oteo, Jesús; Martínez-Martínez, Luis; Navarro, Ferran

    2015-08-01

    The activity of eight aminoglycosides (amikacin, apramycin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin and tobramycin) against a collection of 257 amoxicillin/clavulanic acid (AMC)-resistant Escherichia coli isolates was determined by microdilution. Aminoglycoside resistance rates, the prevalence of aminoglycoside-modifying enzyme (AME) genes, the relationship between AME gene detection and resistance phenotype to aminoglycosides, and the association of AME genes with mechanisms of AMC resistance in E. coli isolates in Spain were investigated. Aminoglycoside-resistant isolates were screened for the presence of genes encoding common AMEs [aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, ant(2″)-Ia, ant(4')-IIa and aph(3')-Ia] or 16S rRNA methylases (armA, rmtB, rmtC and npmA). In total, 105 isolates (40.9%) were resistant to at least one of the aminoglycosides tested. Amikacin, apramycin and arbekacin showed better activity, with MIC90 values of 2mg/L (arbekacin) and 8mg/L (amikacin and apramycin). Kanamycin presented the highest MIC90 (128mg/L). The most common AME gene was aac(6')-Ib (36 strains; 34.3%), followed by aph(3')-Ia (31 strains; 29.5%), ant(2″)-Ia (29 strains; 27.6%) and aac(3)-IIa (23 strains; 21.9%). aac(3)-Ia, aac(3)-IVa, ant(4')-IIa and the four methylases were not detected. The ant(2″)-Ia gene was usually associated with OXA-1 [21/30; 70%], whilst 23/25 (92%) strains producing CTX-M-15 had the aac(6')-Ib gene. The most prevalent AME gene was aac(6')-Ib (18/41; 44%) in nosocomial isolates, whilst ant(2″)-Ia and aph(3')-Ia genes (20/64; 31%) were more frequent in strains of community origin. In 64.6% isolates the phenotypic profile correlated with the presence of commonly encountered AMEs.

  15. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit.

    Science.gov (United States)

    Garner, Cherilyn D; Starr, Jennifer K; McDonough, Patrick L; Altier, Craig

    2010-06-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

  16. Isolation, Molecular Identification and Evaluation of Antifungal Effects of the Bacilli Isolated from Rhizospheric Soil of Gorgan Region against Tricophyton Mentagrophytes

    Directory of Open Access Journals (Sweden)

    Pordeli, HR. (PhD

    2013-01-01

    Full Text Available Background and Objective: Soil bacteria, particularly Bacillus genus have thepotential of producing a range of bioactive substances with antimicrobial andantifungal properties. They have the ability to produce hundreds of active andeffective biologic compound against microorganisms. Therefore, it seems to be aproper candidate in the biocontrol of fungal pathogenesis.Material and Methods: In this study, soil samples were collected from differentparts of Gorgan in order to isolate Bacillus and to determine their antifungalactivity against T.mentagrophytes. The Isolates that had the highest antifungaleffects were analyzed by PCR and 16s rRNA sequencing.Results: Of 54 strains, 14 have antifungal activity. The Isolates, S4 and S12,identified as B.cereus and B.thuringiensis respectively show the highestantidermatofit effect. These isolates based on 16s rRNA sequence analysis show97% homology with Bacillus cereusstrain KU4 and Bacillus thuringiensisstrainucsc27.Conclusion: According to the results, it seems that the soil Bacilli have biocontrolpotential against dermatophytic agents such as T.mentagrophytes.Keywords: Antifungal effects; Bacillus; Rhizospheric soil; T.mentagrophytes

  17. Coagulase-positive Staphylococcus isolated from wildlife: Identification, molecular characterization and evaluation of resistance profiles with focus on a methicillin-resistant strain.

    Science.gov (United States)

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Wojtanowicz-Markiewicz, Katarzyna; Trościańczyk, Aleksandra

    2016-02-01

    The aim of the study was molecular analysis of coagulase-positive isolates of Staphylococcus bacteria obtained from wild animals and evaluation of their resistance to antimicrobial agents. A total of 76 rectal swabs were taken from wild animals. The species of the Staphylococcus isolates was determined by MALDI TOF MS, susceptibility to antimicrobials was evaluated by phenotypic and molecular methods, epidemiological analysis (ADSRRS-fingerprinting) was also carried out. MRSA isolate was typed by MLST and spa-typing. The animals tested, were carriers (n=38) of coagulase-positive Staphylococcus (S. aureus, S. pseudintermedius and S. delphini B). Analyzed isolates were resistant to 1 or 2 antimicrobials, which was confirmed by the presence of genes (blaZ, ermA, ermB, msrA, tetK and tetM). A multi-drug resistant and methicillin-resistant isolate of S. aureus was obtained as well (MRSA, ST8, t1635, PVL-positive and ACME-negative). The ADSRRS-fingerprinting method enabled interspecific and intraspecific differentiation of coagulase-positive Staphylococcus isolates, revealing a certain degree of correlation between the species of the isolate, and the degree of similarity between the isolates. The presence of resistance genes in 13% (5/38) of the isolates obtained from wild animals, including one methicillin-resistant isolate, is relatively small in comparison to the degree of colonization by resistant strains in humans, livestock or pets. Nevertheless, due to the possibility of contact between wild animals, domestic animals and humans, transmission of resistant strains is possible, as suggested by our isolation of a MRSA strain typed as ST8 and specific spa type t1635, which had previously been isolated exclusively from humans.

  18. Molecular identification of Candida species isolated from cases of neonatal candidemia using polymerase chain reaction-restriction fragment length polymorphism in a tertiary care hospital.

    Science.gov (United States)

    Fatima, Akeela; Bashir, Gulnaz; Wani, Tehmeena; Jan, Abiroo; Kohli, Amrish; Khan, Mosin S

    2017-01-01

    Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Hospital-based cross-sectional study. Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1) and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Kappa test for agreement. The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

  19. Molecular identification of Candida species isolated from cases of neonatal candidemia using polymerase chain reaction-restriction fragment length polymorphism in a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Akeela Fatima

    2017-01-01

    Full Text Available Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1 and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

  20. Isolation, biochemical and molecular characterization of 2 ...

    African Journals Online (AJOL)

    Isolation, biochemical and molecular characterization of ... Abstract. Pure cultures of 2-chlorophenol degrading bacteria were isolated from a natural enrichment that may be adapted to ... three isolates out of six yielded positive PCR products.

  1. Site identification presentation: Basalt Waste Isolation Project

    Energy Technology Data Exchange (ETDEWEB)

    1979-11-01

    The final step in the site identification process for the Basalt Waste Isolation Project is described. The candidate sites are identified. The site identification methodology is presented. The general objectives which must be met in selecting the final site are listed. Considerations used in the screening process are also listed. Summary tables of the guidelines used are included. (DMC)

  2. Molecular identification of Aspergillus spp. isolated from coffee beans Identificação molecular de Aspergillus spp. isolados de grãos de café

    Directory of Open Access Journals (Sweden)

    Marciane Magnani

    2005-01-01

    Full Text Available Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA, a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Paraná, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2 of ribosomal DNA (rDNA. The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.Algumas espécies pertencentes ao gênero Aspergillus possuem potencial para produção de Ocratoxina A (OA, uma micotoxina de efeitos nefrotóxicos, imunossupressivos, teratogênicos e carcinogênicos. Com o objetivo de identificar as espécies de Aspergillus que contaminam o interior de grãos de café, foram coletadas amostras em diferentes estádios de maturação do produto, em 16 propriedades produtoras do norte do estado do Paraná. Um total de 108 isolados de Aspergillus spp. foram identificados ao nível de espécie, pelo sequenciamento dos espaços internos transcritos (ITS1-5,8S-ITS2 do DNA ribossomal (rDNA. Os resultados revelaram a presença de espécies potencialmente ocratoxigênicas em 82% das regiões analisadas, sendo dentre estas, Aspergillus niger a espécie mais freqüentemente detectada,seguida por A. ochraceus, e A. carbonarius. É relatada pela primeira vez a presença de A. carbonarius em frutos de café coletados na árvore.

  3. Prospective Multicenter Study of the Epidemiology, Molecular Identification, and Antifungal Susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Isolated from Patients with Candidemia ▿

    OpenAIRE

    Cantón, Emilia; Pemán, Javier; Quindós, Guillermo; Eraso, Elena; Miranda-Zapico, Ilargi; Álvarez, María; Merino, Paloma; Campos-Herrero, Isolina; Marco, Francesc; de la Pedrosa, Elia Gomez G.; Yagüe, Genoveva; Guna, Remedios; Rubio, Carmen; Miranda, Consuelo; Pazos, Carmen

    2011-01-01

    A 13-month prospective multicenter study including 44 hospitals was carried out to evaluate the epidemiology of Candida parapsilosis complex candidemia in Spain. Susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin was tested by the microdilution colorimetric method. A total of 364 C. parapsilosis complex isolates were identified by molecular methods: C. parapsilosis (90.7%), Candida orthopsilosis (8.2...

  4. Bioassay-guided isolation, identification and molecular ligand-target insight of lipoxygenase inhibitors from leaves of Anisomeles malabarica R.Br.

    Directory of Open Access Journals (Sweden)

    A Sudha

    2014-01-01

    Full Text Available Background: Anisomeles malabarica R. Br. (Lamiaceae is extensively used in traditional medicine in major parts of India for several medicinal purposes, including their use in rheumatism. Materials and Methods: The air-dried leaves of A. malabarica were extracted with ethanol, defatted with n-hexane and then successively partitioned into chloroform and n-butanol fractions. Bioassay-guided fractionation and purification of chloroform fraction from A. malabarica lead to the isolation of lipoxygenase (LOX inhibitors. The structures of isolated compounds were elucidated by ultraviolet, infrared, 1 H nuclear magnetic resonance (NMR, 13 C NMR and mass spectrometry spectroscopic techniques and assessed further by in vitro soybean lipoxygenase (sLOX assay. In addition, the enzyme type inhibition was evaluated through molecular docking technique as a part of computational study. Results: The bioactive compounds 3, 4 dihydroxy benzoic acid (1 and 4′, 5, 7-trihydroxyflavone (2 were isolated from chloroform fraction of A. malabarica, whose bioactivity was observed to be dose-dependent compared to n-butanol fraction. Among the compounds, 3, 4 dihydroxy benzoic acid showed significant sLOX inhibitory activity with 74.04% ±2.6% followed by 4′, 5, 7-trihydroxyflavone (34.68% ±1.9%. The computational analysis of compounds showed their molecular interaction with important amino acid residues and nonheme iron atom in the catalytic site of LOX by enlightening their potential binding mode at molecular level. Conclusions: The LOX inhibitory constituents were identified from A. malabarica by means of bioassay-guided fractionation process. The results derived from in vitro and computational experiments confirm the potential of the isolated compounds and provide additional evidence for its traditional use in inflammatory disorders.

  5. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    Directory of Open Access Journals (Sweden)

    S.M. Azwai

    2016-03-01

    Full Text Available The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk. Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6% yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS with culture characteristics of Vibrio spp. More than half (n=27 of processed seafood samples (n=46 yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 х104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 х104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9% were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  6. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

    Science.gov (United States)

    Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  7. Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance.

    Science.gov (United States)

    Shimuta, Ken; Unemo, Magnus; Nakayama, Shu-Ichi; Morita-Ishihara, Tomoko; Dorin, Misato; Kawahata, Takuya; Ohnishi, Makoto

    2013-11-01

    In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.

  8. Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia.

    Science.gov (United States)

    Cantón, Emilia; Pemán, Javier; Quindós, Guillermo; Eraso, Elena; Miranda-Zapico, Ilargi; Álvarez, María; Merino, Paloma; Campos-Herrero, Isolina; Marco, Francesc; de la Pedrosa, Elia Gomez G; Yagüe, Genoveva; Guna, Remedios; Rubio, Carmen; Miranda, Consuelo; Pazos, Carmen; Velasco, David

    2011-12-01

    A 13-month prospective multicenter study including 44 hospitals was carried out to evaluate the epidemiology of Candida parapsilosis complex candidemia in Spain. Susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin was tested by the microdilution colorimetric method. A total of 364 C. parapsilosis complex isolates were identified by molecular methods: C. parapsilosis (90.7%), Candida orthopsilosis (8.2%), and Candida metapsilosis (1.1%). Most candidemias (C. parapsilosis, 76.4%; C. orthopsilosis, 70.0%; C. metapsilosis, 100%) were observed in adults. No C. orthopsilosis or C. metapsilosis candidemias occurred in neonates. C. parapsilosis was most frequent in adult intensive care unit (28.8%), surgery (20.9%), and internal medicine (19.7%) departments; and C. orthopsilosis was most frequent in hematology (28.6%), pediatrics (12.0%), and neonatology (11.5%) departments. The geographic distribution of C. orthopsilosis and C. metapsilosis was not uniform. According to CLSI clinical breakpoints, all C. orthopsilosis and C. metapsilosis isolates were susceptible to the nine agents tested. Resistance (MICs > 1 mg/liter) was observed only in C. parapsilosis: amphotericin B, posaconazole, itraconazole, and caspofungin (0.3% each), anidulafungin (1.9%), and micafungin (2.5%). Applying the new species-specific fluconazole and echinocandin breakpoints, the rates of resistance to fluconazole for C. parapsilosis and C. orthopsilosis increased to 4.8% and 0.3%, respectively; conversely, for C. parapsilosis they shifted from 1.9 to 0.6% (anidulafungin) and from 2.5 to 0.6% (micafungin). Our study confirms the different prevalence of C. parapsilosis complex candidemia among age groups: neither C. orthopsilosis nor C. metapsilosis was isolated from neonates; interestingly, C. metapsilosis was isolated only from adults and the elderly. The disparity in antifungal susceptibility among species

  9. Identification of Staphylococcus saprophyticus isolated from patients with urinary tract infection using a simple set of biochemical tests correlating with 16S-23S interspace region molecular weight patterns.

    Science.gov (United States)

    Ferreira, Adriano Martison; Bonesso, Mariana Fávero; Mondelli, Alessandro Lia; da Cunha, Maria de Lourdes Ribeiro de Souza

    2012-12-01

    The emergence of Staphylococcus spp. not only as human pathogens, but also as reservoirs of antibiotic resistance determinants, requires the development of methods for their rapid and reliable identification in medically important samples. The aim of this study was to compare three phenotypic methods for the identification of Staphylococcus spp. isolated from patients with urinary tract infection using the PCR of the 16S-23S interspace region generating molecular weight patterns (ITR-PCR) as reference. All 57 S. saprophyticus studied were correctly identified using only the novobiocin disk. A rate of agreement of 98.0% was obtained for the simplified battery of biochemical tests in relation to ITR-PCR, whereas the Vitek I system and novobiocin disk showed 81.2% and 89.1% agreement, respectively. No other novobiocin-resistant non-S. saprophyticus strain was identified. Thus, the novobiocin disk is a feasible alternative for the identification of S. saprophyticus in urine samples in laboratories with limited resources. ITR-PCR and the simplified battery of biochemical tests were more reliable than the commercial systems currently available. This study confirms that automated systems are still unable to correctly differentiate CoNS species and that simple, reliable and inexpensive methods can be used for routine identification.

  10. Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

    Science.gov (United States)

    Visintin, Simonetta; Alessandria, Valentina; Valente, Antonio; Dolci, Paola; Cocolin, Luca

    2016-01-04

    Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

  11. Phenotypic and Molecular Identification of Bacteria Involved in Decubitus Ulcers

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    Mehran Dolati

    2017-07-01

    Full Text Available Background:    Bacterial secondary infection of pressure ulcers (bedsores, so called as decubitus ulcers, leads to ulcer development and it interferes with the healing process. Thus, such infections can be lethal due to the sepsis if no constructive medicinal measures regarded. Drug resistance of bacteria in pressure ulcers leads to healing inhibition. Molecular identification of bacteria involved in such infections seem necessary as culture and phenotypic approaches may result in misidentification. . The purpose of this study was to isolate and identify aerobic bacteria detected in bedsores in three Hospitals: Rasool-e-Akram, Imam Hossein and Tajrish Shohada Hospitals, Tehran, Iran.Methods:    To this end, decubitus ulcer samples of 49 patients were obtained using sterile swabs. After direct microscopic examination, the swabs were used to streak BHI agar plates supplemented with %5 defibrinated sheep blood for enrichment of probable aerobic cultures. Bacterial isolates diagnosed by biochemical tests. Antibiotic susceptibility of the isolates determined based on CLSI guideline. For molecular identification, PCR amplification of the 16S rRNA gene performed using Eubacterial universal primers. Then, the PCR products were sequenced and the nucleotide sequences of the PCR products were analyzed by BLASTN similarity search program available at NCBI. Results:   Among the isolates, Pseudomonas aeruginosa (36% had the highest frequency, followed by Staphylococcus aureus (32% and Escherichia coli (30%. The frequencies of Klebsiella pneumonia and Proteus spp. were 10% and 8%, respectively. Most of the isolated bacteria showed a widespread antibiotic resistance. Molecular identification of the bacterial isolates resulted in 6 isolates of Escherichia coli, two isolates of each of Proteus mirabilis and Shigella spp., 4 isolates of Enterobacter cloacae, and 1 isolate of each of Cronobacter sakazakii and Morganella morganii.Conclusion:

  12. Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification Estudo de bactérias do gênero Gluconobacter: isolamento, purificação, identificação fenotípica e molecular

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    André Luiz Dorini de Oliveira

    2010-03-01

    Full Text Available The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler, honey, Vitis vinifera (grape, Pyrus communis (pear, Malus sp. (apple and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone, 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid, 41 in enrichment medium (De Ley and Swings and later in the GYC medium (glucose, yeast extract and calcium carbonate. 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP in Assis, stored in malt extract at -196 ºC.A importância do estudo de bactérias acéticas, em especial as do gênero Gluconobacter, está baseada em suas aplicações industriais, pois estas possuem a capacidade de

  13. Isolation and identification of Staphylococcus sp. in powdered infant milk

    Science.gov (United States)

    Palilu, Prayolga Toban; Budiarso, Tri Yahya

    2017-05-01

    Staphylococcus sp. is one of the most dangerous bacteria that could cause food poisoning. It is a pathogenic bacterium which is able to produce enterotoxin in foods. Milk is an ideal growth medium for Staphylococcus sp., that may cause problem if it is to be consumed, especially by infant. It is the objective of this research to detect the presence of Staphylococcus sp. in powdered infant milk. As many as 14 samples obtained from market were used as samples for bacterial isolation. The isolation were done by employing enrichment step on BHI-broth, continued with Baird-Parker Agar which will produce a typical colony. It is then picked and grown on Mannitol Salt Agar, and gram staining, coagulase assay, and fermentation tests. The confirmation step was done by using API-Staph which gives the identification of Staphylococcus hemoliticus, Staphylococcus aureus and Staphylococcus epidermidis, with a percentage of identity ranging from 65.9-97.7%. Two isolates with the highest identification similarity values were then picked for molecular detection. A PCR primer pair targeting gene coding for enterotoxin A was used, and it gives positive result for the two isolates being tested. It is then concluded that the two isolates belong to Staphylococcus sp., and further research need to be done to correctly identify these isolates.

  14. Isolation and identification of a riboflavin producer yeast from Nectarine

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    Roya Daneshazari

    2014-07-01

    Full Text Available   Introduction : Many microorganisms like fungi , bacteria and yeasts, have a natural ability to produce vitamins included vitamin B2 or riboflavin. In this regard, the present study was performed to isolation and screening for riboflavin producing yeasts from various sources of soil, leaf and fruit s.   Materials and method s: samples of leaf , soil and fruits were prepared for the presence of yeasts and by its ability to produce riboflavin . After purification and enrichment of samples , in order to assay riboflavin production, spectrometry , thin layer chromatography and high performance liquid chromatography were used . Finally, the best selected isolate was identified using conventional morphological , biochemical and molecular techniques .   Results : In this study , 26 yeast strains were isolated from environmental samples, that 6 isolates showed the ability to produce riboflavin . Identification results of the best selected isolate by biochemical and phenotypic characteristics revealed that this isolate is related to Clavispora lusitaniae and considering isolation of it from nectarine, has named it Clavispora lusitaniae strain N3 (Gene accession no: JQ586258 .   Discussion and conclusion : Although only one of the six producing strains was studied and identified , observation of ability to produce among 23% of strains showed necessity for further investigation. And according to the result of absence of viewing report about production by investigated strain, it can be said that Iran has potentiality for isolation of yeasts and is capable of producing riboflavin.

  15. Visual clone identification of Penicillium commune isolates.

    Science.gov (United States)

    Hansen, Michael Edberg; Lund, Flemming; Carstensen, Jens Michael

    2003-02-01

    A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.

  16. Isolation and Identification of Yeasts from Tibet Kefir

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    Yun Li

    2015-02-01

    Full Text Available The occurrence and distribution of yeasts in Tibet kefir were investigated in this study. Five samples of Tibetan kefir from Tibet and surrounding areas were collected for yeast isolation. Based on physiological, biochemical characteristics and molecular identification results, eight species of yeast were isolated and identified from Tibet kefir, including Saccharomyces cerevisiae, Pichia fermentans, Debaryomyces hansenii, Rhodotorula mucilaginosa, Candida zeylanoide, Candida parapsilosis, Kluyveromyces marxianus and Kazachstania unispora. Among the test samples, K. marxianus, Ka. unispora and P. fermentans were the highest three species in frequency of occurrence of yeast isolates. C. zeylanoides, C. parapsilosis and R. mucilaginosa were first found the occurrence in Tibet kefir. The results provided new information of yeast composition and biodiversity of Tibet kefir.

  17. Molecular identification of Metarhizium strains isolated from soil in Anhui%安徽省土壤绿僵菌分离株的分子鉴定

    Institute of Scientific and Technical Information of China (English)

    周玉宝; 聂勇; 黄勃

    2011-01-01

    目的 进一步验证ITS序列的系统发育分析可为绿僵菌属种的鉴定提供重要的参考依据.方法 对分离自安徽土壤的13株绿僵菌菌株的内转录间隔区(ITS)片段进行PCR扩增和序列测定,采用Blast方法 将测序结果 在GenBank中进行同源搜索,依据邻接法构建获得与其相关菌株的ITS序列系统发育树.结果 供试菌株分别位于系统发育树的3个分支上,分支Ⅰ包括8个菌株和金龟子绿僵菌小孢变种,1个菌株和金龟子绿僵菌鳞鳃金龟变种形成分支Ⅲ,另外4个菌株和黄绿绿僵菌棉蚜变种聚为分支X.结论 结合同源比较的数据,将这8个、4个和1个绿僵菌菌株分别鉴定为金龟子绿僵菌小孢变种、黄绿绿僵菌棉蚜变种和金龟子绿僵菌鳞鳃金龟变种.%Objective To further manifest that the phylogenetic analysis of ITS sequence could provide an important reference basis for genus identification of Metarhizium. Method The internal transcribed spacer (ITS) sequences of 13 Metarhizium strains isolated from Anhui soil was amplified and sequenced. We searched for homology using the BLAST programs available at the web site of the National Center for Biotechnology Information, and the phylogenetic tree for the genus Metarhizium was constructed by Neighbor-Joining method. Result The tested strains were located in 3 clades of the phylogenetic tree. 8 Metarhizium strains and the Metarhizium anisopliae var. Anisopliae type were classified into clade I, 1 strain and the M. Anisopliae var. Lepidiotum type were grouped into clade Ⅲ, and 4 strains and the M. Jlavoviride var. Pemphigum type were classified into clade X. -Conclusion Combined with the homologous comparative data, the eight strains, the one strain and the four strains are identified as Metarhizium anisopliae var. Anisopliae, M. Anisopliae var. Lepidiotum and M. Jlavoviride var. Pemphigum, respectively.

  18. Identificação molecular e suscetibilidade antifúngica de Candida parapsilosis isoladas no Ceará, Brasil Molecular identification and antifungal susceptibility of Candida parapsilosis isolates in Ceará, Brazil

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    Everardo Albuquerque Menezes

    2012-12-01

    Full Text Available INTRODUÇÃO: Candida parapsilosis é a segunda ou terceira levedura mais isolada de hemoculturas em várias partes do mundo. É um patógeno comumente isolado no Brasil e no Ceará. Apresenta capacidade de formar biofilmes em cateteres e outros dispositivos médicos e esses fatores contribuem para a disseminação dessa levedura. OBJETIVOS: Identificar e avaliar a suscetibilidade aos antifúngicos de C. parapsilosis isoladas de amostras de sangue e urina de pacientes atendidos em hospitais no Ceará. MÉTODOS: Foram isoladas e identificadas 57 cepas de C. parapsilosis. As cepas foram identificadas por testes fenotípicos e moleculares. A suscetibilidade foi avaliada pelo método de microdiluição em caldo (protocolo do Clinical and Laboratory Standards Institute [CLSI] M27-A3. Foram avaliados cinco antifúngicos (anfotericina B, caspofungina, fluconazol, itraconazol e voriconazol. RESULTADOS E CONCLUSÃO: As cepas foram identificadas como C. parapsilosis por testes fenotípicos e confirmadas pelos testes moleculares. Quanto ao perfil de sensibilidade, elas se mostraram sensíveis aos antifúngicos testados, sendo a resistência ainda um fenômeno raro entre cepas de C. parapsilosis isoladas no Ceará.INTRODUCTION: C. parapsilosis is the second or third most isolated yeast from blood cultures in various parts of the world. It is a commonly isolated pathogen in Brazil and Ceará. C. parapsilosis is liable to form biofilms on catheters and other medical devices, which contributes to the spread of this yeast. OBJECTIVE: The objective of this study was to identify and assess the antifungal susceptibility of C. parapsilosis isolates from blood and urine samples collected from patients in hospitals in Ceará. METHODS: We isolated and identified 57 strains of C. parapsilosis. The strains were identified by phenotypic and molecular tests. The susceptibility to antifungals was assessed by broth microdilution (Clinical and Laboratory Standards Institute

  19. 福建省布鲁氏菌分离株的分子生物学鉴定%Molecular identification of the Brucella strains isolated in Fujian province

    Institute of Scientific and Technical Information of China (English)

    邓艳琴; 王加熊; 林代华; 陈亮; 王灵岚

    2009-01-01

    AMOS-PCR and MLVA were carried out to identify the Brucella strains isolated in Fujian province, which were classified as B. melitensis biovar 2 or 3 by conventional microbiological tests. All these 3 isolates were identified to be B. melitensis by AMOS-PCR. The genetic patterns obtained by MLVA were queried in the Brucella 2007 database and clustered with B. melitensis strains. It is evident that these two molecular assays may be used as the assistant tools in the identification of Brucella strains.%目的 利用分子生物学方法鉴定福建省布鲁氏菌分离株.方法 对布鲁氏菌分离株进行AMOS-PCR和MLVA分型.结果 3株福建省分离株经AMOS-PCR鉴定为羊种,与传统生物学分型一致;MLVA分型将其分为3个基因型,聚类分析鉴定为羊种.结论 AMOS-PCR、MLVA分型可以作为我省布鲁氏菌分离株鉴定的辅助手段.

  20. Isolation and Molecular Identification of Actinomycetes That Highly Inhibited the Activity of S. aureus%高抑制金黄色葡萄球菌活性放线菌的分离及分子鉴定

    Institute of Scientific and Technical Information of China (English)

    窦会娟; 李林珂; 刘新胜; 孔连海

    2011-01-01

    Metabolin was screened and isolated from hospital soil, which was the actinomycete with highly inhibition for S. aureus activity,and molecular identification was carried out for actinomycete. Photocopy dish was used, and Staphylococcus aureus was viewed as indicator bacteria for the isolation of actinomycetes, then carrying out identification based on 16S rDNA sequence analysis. The results indicate that A01 bacterial strain fermentation broth have better inhibition effect on S. aureus, and have good relationship with Streptomyces sp. VTTE-042674 and even reach to 99.86%, thus, it belongs to Streptomyces avermitilis. 16S rDNA sequence of the bacterial strain have already included by GenBank, moreover, the sequence registration number are HQ660584.%从医院土壤中筛选分离代谢物具有较高抑制金黄色葡萄球菌活性的放线菌,并对其进行分子鉴定.结果得出,采用影印平皿法,以金黄色葡萄球菌为指示菌进行放线菌的分离,通过16S rDNA序列分析进行鉴定.结果得出,A01菌株发酵液对金黄色葡萄球菌抑制效果较好,与Streptomyces sp.VTTE-042674亲缘关系最近,达到99.86%,应属于链霉菌属.该菌株的16S rDNA序列已被GenBank收录,序列登录号为 HQ660584.

  1. Molecular characterization of bacteria isolated from the Kingdom of ...

    African Journals Online (AJOL)

    ola

    2012-11-06

    Nov 6, 2012 ... ... bacterial isolation. Bacterial isolates were evaluated for their antimicrobial activities against pathogenic fungi which cause ... Molecular techniques based on genomic DNA have ... Isolation of total DNA from isolated strains.

  2. Molecular typing of Chinese Streptococcus pyogenes isolates.

    Science.gov (United States)

    You, Yuanhai; Wang, Haibin; Bi, Zhenwang; Walker, Mark; Peng, Xianhui; Hu, Bin; Zhou, Haijian; Song, Yanyan; Tao, Xiaoxia; Kou, Zengqiang; Meng, Fanliang; Zhang, Menghan; Bi, Zhenqiang; Luo, Fengji; Zhang, Jianzhong

    2015-06-01

    Streptococcus pyogenes causes human infections ranging from mild pharyngitis and impetigo to serious diseases including necrotizing fasciitis and streptococcal toxic shock syndrome. The objective of this study was to compare molecular emm typing and pulsed field gel electrophoresis (PFGE) with multiple-locus variable-number tandem-repeat analysis (MLVA) for genotyping of Chinese S. pyogenes isolates. Molecular emm typing and PFGE were performed using standard protocols. Seven variable number tandem repeat (VNTR) loci reported in a previous study were used to genotype 169 S. pyogenes geographically-diverse isolates from China isolated from a variety of disease syndromes. Multiple-locus variable-number tandem-repeat analysis provided greater discrimination between isolates when compared to emm typing and PFGE. Removal of a single VNTR locus (Spy2) reduced the sensitivity by only 0.7%, which suggests that Spy2 was not informative for the isolates screened. The results presented support the use of MLVA as a powerful epidemiological tool for genotyping S. pyogenes clinical isolates.

  3. Molecular characterization of distinct YMV (Yellow mosaic virus) isolates affecting pulses in India with the aid of coat protein gene as a marker for identification.

    Science.gov (United States)

    Maheshwari, Richa; Panigrahi, Gatikrushna; Angappan, K

    2014-01-01

    The present study was carried out to find out the variations present in different isolates of yellow mosaic virus (YMV) causing yellow mosaic disease of pulses in southern parts of India. The coat protein gene of YMV was amplified using gene specific and deng universal primers with DNA isolated from YMV infected samples. Further, cloning and DNA sequencing of CP gene was carried out. CP gene decrypt sequences revealed that YMV infected samples of Black gram, Cowpea and Green gram were similar to the MYMV-Tamil Nadu isolates. Whereas the YMV infected sample of Horse gram was found to be similar with HYMV. Hence, in the present study, two distinct YMV infecting pulses in Tamil Nadu (MYMV and HYMV species) were identified and it was observed that there exists considerable genetic variation among these species. In addition, Cowpea crop which was earlier supposed not to be susceptible for YMV infection also showed the presence of this virus similar to the MYMV. Overall, the findings of the present study indicate that the CP region is efficient enough to provide a simple, rapid, and reliable method for early detection of YMV infections in pulses, which would help to develop proper management strategies to control these viruses.

  4. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  5. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  6. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hana Šuranská

    2016-03-01

    Full Text Available Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

  7. Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods.

    Science.gov (United States)

    Armand, B; Motazedian, M H; Asgari, Q

    2016-01-01

    Free-living amoebae (FLA) are the most abundant and widely distributed protozoa in the environment. An investigation was conducted to determine the presence of free-living amoebae (FLA), Acanthamoeba and Vermamoeba in waterfronts of parks and squares and tap water of Shiraz City, Iran. FLA are considered pathogenic for human. These ubiquitous organisms have been isolated from different environments such as water, soil, and air. Eighty-two water samples were collected from different places of Shiraz City during the summer of 2013. All samples were processed in Dept. of Parasitology and Mycology, Shiraz University of Medical Sciences, Fars, Iran. Samples were screened for FLA and identified by morphological characters in the cultures, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Overall, 48 samples were positive for Acanthamoeba and Vermamoeba in non-nutrient agar culture based on morphological characteristics. The PCR examination was done successfully. Sequencing results were revealed T4 (62.96 %) genotypes as the most common genotype of Acanthamoeba in the Shiraz water sources. In addition, T5 (33.33 %) and T15 (3.71 %) were isolated from water supplies. Vermamoeba vermiformis was known the dominant species from this genus. The high frequency of Acanthamoeba spp. and Vermamoeba in different environmental water sources of Shiraz is an alert for the public health related to water sources. The result highlights a need for taking more attention to water supplies in order to prevent illnesses related to free-living amoebae.

  8. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

    Directory of Open Access Journals (Sweden)

    Biplab Kumar Dash

    2015-01-01

    Full Text Available A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25% as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L and sodium lauryl sulfate (0.2 g/L resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  9. Molecular Identification of D. nuttalli and D.marginatus

    Institute of Scientific and Technical Information of China (English)

    Lv; Jizhou; Wang; Zhenbao; Yuan; Xiangfen; Wu; Shaoqiang

    2014-01-01

    This study was to establish a molecular identification method to distinguish Dermacentor nuttalli(D. nuttalli) and Dermacentor marginatus(D. marginatus),and to study their phylogenetic relationship. The ticks were collected from domestic animals in Xinjiang Uygur Autonomous Region for morphological identification,then their genomic DNAs were isolated for amplifying mitochondrial 16 S rRNA and cytochrome oxidase subunit I gene(COI). The phylogenetic tree was constructed based on the sequencing results of PCR products by employing Mega 5. 0 and Mrbayes 3. 2 for homology analysis. Clustering analysis PCR product for 16 S rRNA from both D. marginatus and D. nuttalli was clustered together with their respective 16 S rRNA sequences previously accessed in GenBank,and that for COI gene as well. These are basically identical with morphological identification results. Our results indicate that morphological identification,combined with molecular markers,would be a simple and accurate method for distinguishing D. nuttalli and D. marginatus.

  10. Molecular identification of the Sporothrix schenckii complex.

    Science.gov (United States)

    Oliveira, Manoel Marques Evangelista; Almeida-Paes, Rodrigo; Gutierrez-Galhardo, Maria Clara; Zancope-Oliveira, Rosely M

    2014-01-01

    Sporothrix schenckii, an ascomycetous dimorphic organism that for over a century was recognized as the sole agent of sporotrichosis, a subcutaneous mycosis with a worldwide distribution. However, it has been proposed, based on physiologic and molecular aspects, that S. schenckii is a complex of distinct species: Sporothrix brasiliensis, Sporothrix mexicana, Sporothrix globosa, S. schenckii sensu strictu, Sporothrix luriei, and Sporothrix albicans (formerly Sporothrix pallida). Human disease has a broad range of clinical manifestations and can be classified into fixed cutaneous, lymphocutaneous, disseminated cutaneous, and extracutaneous sporotrichosis. The gold standard for the diagnosis of sporotrichosis is the culture; however, serologic, histopathologic and molecular approaches have been recently adopted for the diagnosis of this mycosis. Few molecular methods have been applied to the diagnosis of sporotrichosis to detect S. schenckii DNA from clinical specimens, and to identify Sporothrix spp. in culture. Until now, Sporothrix is the unique clinically relevant dimorphic fungus without an elucidated genome sequence, thus limiting molecular knowledge about the cryptic species of this complex, and the sexual form of all S. schenckii complex species. In this review we shall focus on the current diagnosis of the sporotrichosis, and discuss the current molecular tools applied to the diagnosis and identification of the Sporothrix complex species. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  11. Litchi Flavonoids: Isolation, Identification and Biological Activity

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    Yueming Jiang

    2007-04-01

    Full Text Available The current status of the isolation, identification, biological activity, utilization and development prospects of flavonoids found in litchi fruit pericarp (LFP tissues is reviewed. LFP tissues account for approximately 15% by weight of the whole fresh fruit and are comprised of significant amount of flavonoids. The major flavonoids in ripe LFP include flavonols and anthocyanins. The major flavanols in the LFP are reported to be procyanidin B4, procyanidin B2 and epicatechin, while cyanindin-3-rutinside, cyanidin-3-glucoside, quercetin-3-rutinosde and quercetin-3-glucoside are identified as the important anthocyanins. Litchi flavanols and anthocyanins exhibit good potential antioxidant activity. The hydroxyl radical and superoxide anion scavenging activities of procyanidin B2 are greater than those of procyanidin B4 and epicatechin, while epicatechin has the highest α,α-diphenyl-β-picrylhydrazyl radical (DPPH· scavenging activity. In addition to the antioxidant activity, LFP extract displays a dose- and time-dependent inhibitory effect on human breast cancer, which could be attributed, in part, to its inhibition of proliferation and induction of apoptosis in cancer cells through upregulation and down-regulation of multiple genes. Furthermore, various anticancer activities are observed for epicatechin, procyanidin B2, procyanidin B4 and the ethyl acetate fraction of LFP tissue extracts. Procyanidin B4 and the ethyl acetate fraction show a stronger inhibitory effect on HELF than MCF-7 proliferation, while epicatechin and procyanidin B2 have lower cytotoxicities towards MCF-7 and HELF than paclitaxel. It is therefore suggested that flavonoids from LFP might be potentially useful components for functional foods and/or anti-breast cancer drugs.

  12. Molecular characterization of Duck Plague virus isolated from Bangladesh

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    Md. Mostakin Ahamed

    2015-09-01

    Full Text Available Duck plague (DP is the most feared duck disease in the world. For isolation, identification, molecular detection and characterization of DP virus (DPV, a total of 94 samples were collected from commercial farms (n=6 and households (n=13 from Rajshahi (n=37, Netrokona (n=35 and Mymensingh (n=22 districts of Bangladesh. The samples were processed and inoculated into 11-13 days old embryonated duck eggs for virus propagation. Virus was identified using agar gel immunodiffusion test (AGIT and passive hemagglutination (PHA test, and was confirmed by polymerase chain reaction (PCR targeting DNA polymerase and gC genes, followed by sequencing. Pathogenicity tests were performed using duck embryos, ducklings and ducks. Among the 94 samples, 17 isolates were confirmed as DPV by PCR amplification of partial DNA polymerase (446-bp and gC genes (78-bp, respectively. One of the isolates (Anatid herpes 1 BAU DMH was sequenced and found to be closely related with a Chinese variant of DPV (GenBank: JQ647509.1. Thus, we assume that both Bangladeshi and Chinese isolates of DPV may have a common ancestor. [J Adv Vet Anim Res 2015; 2(3.000: 296-303

  13. Isolation and Identification of Glucoamylase Producer Fungus from Sago Hampas

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    Alfi Asben

    2013-01-01

    Full Text Available Waste of sago processing, notably hampas (ela still contains sago starch is waste that has not been utilized optimally yet  and causing pollution. Isolation and identification of glucoamylase producer fungus of sago hampas waste  were aims to obtain isolates that have gluco-amylolytic properties, and to know glucoamylase activity of selected fungus isolates after grown on artificial medium.  Indegeneous isolates that can produced glucoamylase will be use to get sugar hidrolysate from starch of sago hampas waste for bioetanol production. The study was conducted with the following stages: 1Take the sample from the tennis, 2 Isolation and Identification, 3 Characterization (clear zone, and 4 The production of glucoamylase from selected isolates, The results obtained are: 1 Isolation of fungi gluco-amylolytic from 2 sources sago hampas were produced 10 isolates. Ten isolates were divided into 4 genuses: Gliocladium (as dominant isolate, Aspergilus, Rizhopus and Geotrichum. Isolates of Gliocladium KE gaves the largest degradation of starch on PDA-Starch medium (clear zone, and followed by isolates of Aspergillus GA; 2 Production of glucoamylase on sago hampas with modificated Danial medium (1992 gave the highest activity of Gliocladium KE   on the fifth day of incubation, namely: 10.72 U / mL of crude enzyme   from the supernatant of fermentation substrate (S, and 17.16 U / mL for crude enzyme from the extract of isopropanol isolation (E.

  14. MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF Fusarium SPECIES AND THEIR PATHOGENICITY FOR WHEAT

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    Jelena Poštić

    2012-12-01

    Full Text Available From the root and lower stem parts of weeds and plant debris of maize, wheat, oat and sunflower we isolated 300 isolates of Fusarium spp. and performed morphological and molecular identification. With molecular identification using AFLP method we determined 14 Fusarium species: F. acuminatum, F. avenaceum, F. concolor, F. crookwellense, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. sporotrichioides, F. subglutinans, F. venenatum and F. verticillioides.By comparing results of morphological and molecular identification we found out that determination of 16,7% isolates was incorrect. Out of 300 isolates identified with molecular methods, 50 did not belong to the species determined with morphological determination.With pathogenicity tests of 30 chosen Fusarium isolates we determined that many of them were pathogenic to wheat and maize seedlings and to wheat heads. The most pathogenic were isolates of F. graminearum from A. retroflexus, A. theophrasti and C. album, F. venenatum from maize debris and and A. theophrasti, F. crookwellense from A. lappa. Antifungal influence of 11 essential oils on mycelia growth and sporulation of chosen Fusarium isolates determined that essential oils of T. vulgaris, P. anisum and E. caryophyllus had the strongest effect on mycelial growth. Influence of essential oils on sporulation was not statistically significant.

  15. Rapid identification of Streptomyces isolates by MALDI-TOF MS.

    Science.gov (United States)

    Loucif, Lotfi; Bendjama, Esma; Gacemi-Kirane, Djamila; Rolain, Jean-Marc

    2014-12-01

    The recent emergence of multidrug-resistant bacteria over the last decade has led to a renewal in the discovery of new antimicrobial drugs. Streptomyces members are practically unlimited sources of new antibiotics. However, the identification of Streptomyces species is difficult and time-consuming. Therefore, there is a need for alternative methods for their rapid identification. In this study, an efficient protocol of identification using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was developed and applied for the rapid identification of Streptomyces isolates from the El Kala lakes in northeastern Algeria. A collection of 48 Streptomyces isolates were used for this study. The optimized procedure allowed us to obtain specific and reproducible protein spectra for each Streptomyces isolate tested. The spectra generated were used to build a preliminary local database based on their initial 16S rRNA identification. The blind test used for the identification of 20 Streptomyces strains already available in our created database and 20 unknown Streptomyces isolates showed that all (100%) of the Streptomyces strains listed in the database were rapidly (Streptomyces isolates.

  16. Molecular characterization of lactobacilli isolated from fermented idli batter

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    Perumal Jayaprabha Agaliya

    2013-12-01

    Full Text Available Lactic acid bacteria are non pathogenic organism widely distributed in nature typically involved in a large number of spontaneous food fermentation. The purpose of this study was to characterize the bacteriocinogenic lactobacilli from fermented idli batter which can find application in biopreservation and biomedicine. Eight most promising lactobacilli were chosen from twenty two isolates based on their spectrum of activity against other lactic acid bacteria and pathogens. The eight lactobacilli were characterized based on the various classical phenotypic tests, physiological tests and biochemical tests including various carbohydrate utilization profiles. All isolates were homo fermentative, catalase, and gelatin negative. Molecular characterization was performed by RAPD, 16S rRNA analysis, 16S ARDRA, and Multiplex PCR for species identification. RAPD was carried out using the primer R2 and M13. Five different clusters were obtained based on RAPD indicating strain level variation. 16S rRNA analysis showed 99 to 100% homology towards Lactobacillus plantarum. The restriction digestion pattern was similar for all the isolates with the restriction enzyme AluI. The subspecies were identified by performing Multiplex PCR using species specific primer. Among the five clusters, three clusters were clearly identified as Lactobacillus plantarum subsp. plantarum, Lactobacillus pentosus, and Lactobacillus plantarum subsp. argentoratensis.

  17. Molecular characterization of lactobacilli isolated from fermented idli batter.

    Science.gov (United States)

    Agaliya, Perumal Jayaprabha; Jeevaratnam, Kadirvelu

    2013-12-01

    Lactic acid bacteria are non pathogenic organism widely distributed in nature typically involved in a large number of spontaneous food fermentation. The purpose of this study was to characterize the bacteriocinogenic lactobacilli from fermented idli batter which can find application in biopreservation and biomedicine. Eight most promising lactobacilli were chosen from twenty two isolates based on their spectrum of activity against other lactic acid bacteria and pathogens. The eight lactobacilli were characterized based on the various classical phenotypic tests, physiological tests and biochemical tests including various carbohydrate utilization profiles. All isolates were homo fermentative, catalase, and gelatin negative. Molecular characterization was performed by RAPD, 16S rRNA analysis, 16S ARDRA, and Multiplex PCR for species identification. RAPD was carried out using the primer R2 and M13. Five different clusters were obtained based on RAPD indicating strain level variation. 16S rRNA analysis showed 99 to 100% homology towards Lactobacillus plantarum. The restriction digestion pattern was similar for all the isolates with the restriction enzyme AluI. The subspecies were identified by performing Multiplex PCR using species specific primer. Among the five clusters, three clusters were clearly identified as Lactobacillus plantarum subsp. plantarum, Lactobacillus pentosus, and Lactobacillus plantarum subsp. argentoratensis.

  18. Isolation, identification and characterization of Bacillus amyloliquefaciens BZ-6, a bacterial isolate for enhancing oil recovery from oily sludge.

    Science.gov (United States)

    Liu, Wuxing; Wang, Xiaobing; Wu, Longhua; Chen, Mengfang; Tu, Chen; Luo, Yongming; Christie, Peter

    2012-06-01

    Over 100 biosurfactant-producing microorganisms were isolated from oily sludge and petroleum-contaminated soil from Shengli oil field in north China. Sixteen of the bacterial isolates produced biosurfactants and reduced the surface tension of the growth medium from 71 to BZ-6 was found to be the most efficient strain and the three phases (oil, water and sediment) were separated automatically after the sludge was treated with the culture medium of BZ-6. Based on morphological, physiological characteristics and molecular identification, isolate BZ-6 was identified as Bacillus amyloliquefaciens. The biosurfactant produced by isolate BZ-6 was purified and analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. There were four ion peaks representing four different fengycin A homologues.

  19. Molecular Identification and Subtype Analysis of Blastocystis.

    Science.gov (United States)

    Stensvold, C Rune; Clark, C Graham

    2016-11-18

    Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding,", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites. Recently, a publicly available online facility was developed for quick and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes) based on sequence data obtained by barcoding PCR. Moreover, a modified barcoding approach is now available using nested PCR, which enables detection of mixed subtype infections. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  20. Molecular genetic strategies for species identification

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    This paper probes into the molecular genetic mechanism of the formation of species, subspecies and variety in evolving progression, and brings forward 5 criteria of an ideal strategy in species identification: stating the specific characteristics at species, subspecies and variety level without any interference of too high polymorphism at individual or population level; keys should be distributed as 0 or 1, e. g. yes or no; satisfying re-peatability and simple operation; high veracity and reliability; adaptability to widely various specimen. Respec-tively, this paper reviews two strategies focusing on detecting the fragment length polymorphism and base re-placement and lays out some detail methods under above strategies. It demonstrates that it is not possible to solve all species problems by pursuing identification with only a single gene or DNA fragment. Only based on thorough consideration of all strategies, a method or combined several methods could bring satisfying reliability. For advanced focuses, it requires not only development and optimization of methods under above strategies, but also new originality of creative strategies.

  1. Identificación Molecular de Bacterias Productoras de Polihidroxialcanoatos en Subproductos de Lácteos y Caña de Azúcar / Molecular Identification of Polyhydroxyalkanoate-Producing Bacteria Isolated from Dairy and Sugarcane Residues

    Directory of Open Access Journals (Sweden)

    Ana Carolina Cardona Echavarría

    2013-12-01

    . In this work, the presence of PHAs-producing bacteria in whey, sugarcane molasses, cachaza and bagasse was investigated. Bacteria were isolated using a minimal medium supplemented with 2% glucoseand 1 μL mL-1 Nile red (0.1%. Colonies exhibitingfluorescence at 340 nm were further analyzed by fluorescence microscopy with Nileblue. When positive for both tests, bacterial isolates were classified as PHAs producers and their 16S rDNA sequenced. For selected isolates, the presence of the phaC gene was confirmed by PCR. A total of 38 strains, grouped into 18 different morphotypes, were identified as PHA producers. Bacteria belonging to Lactococcus, Klebsiella, Pseudomonas, Enterobacter and Enterococcus genera were isolated from whey and Bacillus, Enterobacter, Pantoea, Klebsiella and Gluconobacter from sugar cane residues. The phaC gene was detected by PCR in 16 bacteria withtype I and IV arrangements. This work opens up the possibility of using the isolated bacteria as an environmentally sustainablealternative for the disposal of agro-industrial residues as well as an additional income source.

  2. [Application of molecular biological techniques in Taenia identification].

    Science.gov (United States)

    Li, Yan; Liu, Hang; Yang, Yi-Mei

    2011-10-01

    The traditional identification of Taenia spp. based on morphological features of adult and cysticercus has difficulties in identifying the morphologically similar species. The recent development of molecular techniques provides more scientific ways for distinguishing Taenia species. This paper summarizes the application of molecular biological techniques in the identification of Taenia, such as analysis of DNA sequence, PCR-RFLP and LAMP.

  3. Testing of DNA isolation for the identification of hemp

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    Tomáš Vyhnánek

    2015-12-01

    Full Text Available Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb with nine pairs of autosomes plus XX (♀ or XY (♂ chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar of hemp with low Δ9-tetrahydrocannabinol content are used for industrial applications. Varieties with high Δ9-tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic. The DNeasy® Plant Mini Kit (Qiagen, GE was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 - 38.90 ng.µL-1, but with better purity than samples of higher weight (ratio A260nm/A280nm for low-weight samples was near 1.80. To test the applicability of DNA analysis, we used two SSR markers (CAN1347 and CAN2913. PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg. In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.

  4. Molecular and morphological identification of Colletotrichum species of clinical interest.

    Science.gov (United States)

    Cano, Josep; Guarro, Josep; Gené, Josepa

    2004-06-01

    Colletotrichum species have caused human infections in recent years. Because of the difficulties in recognizing them in vitro, we have designed a quick and unambiguous molecular test, based on the amplification of a specific fragment of the internal transcribed spacer 1 region, to distinguish any Colletotrichum isolate from other fungi, including the common pathogenic species. Analysis of the sequences of the ribosomal DNA (rDNA) fragment showed sufficient variability to clearly separate the five species of Colletotrichum that are of clinical interest, i.e., Colletotrichum coccodes, C. crassipes, C. dematium, C. gloeosporioides, and C. graminicola. Sequencing of the D1-D2 region of the large-subunit rDNA gene also supported these results. Additionally, we reviewed the most suitable morphological characteristics for the in vitro identification of these increasingly important opportunistic fungi.

  5. Isolation and identification of nondestructive desulfurizationbacterium

    Institute of Scientific and Technical Information of China (English)

    GOU; Zhongxuan; (缑仲轩); LIU; Huizhou; (罗明芳); LUO; Mingfang; (刘会洲); LI; Shan; (李珊); XING; Jianmin; (邢建民); CHEN; Jiayong; (陈家镛)

    2002-01-01

    A nondestructive desulfurization microorganism has been isolated. The metabolism product analyses show that the strain can be a kind of biocatalyst to oxidize dibenzothiophene (DBT) into 2-hydroxydiphenyl (HBP), therefore the sulfur in DBT is removed selectively. The 16SrRNA information, cell wall analysis, physical, biochemical properties and morphological properties suggest that the isolated strain is Rhodococcus erythropolis. The strain can grow in the basal salts medium (BSM) that DBT concentration is no more than 10 mmol/L, and the optimal DBT concentration for growth is 1 mmol/L, however, the optimal DBT concentration for desulfurization is 0.5 mmol/L. The further research shows that the strain can also desulfur some other organosulfur-containing compounds such as thianaphthene, phenyl sulfide and 4,6-dimethyldiben- zothiophene (4,6-DMDBT).

  6. Sporothrix schenckii complex in Iran: Molecular identification and antifungal susceptibility.

    Science.gov (United States)

    Mahmoudi, Shahram; Zaini, Farideh; Kordbacheh, Parivash; Safara, Mahin; Heidari, Mansour

    2016-08-01

    Sporotrichosis is a global subcutaneous fungal infection caused by the Sporothrix schenckii complex. Sporotrichosis is an uncommon infection in Iran, and there have been no phenotypic, molecular typing or antifungal susceptibility studies of Sporothrix species. This study aimed to identify nine Iranian isolates of the S. schenckii complex to the species level using colony morphology, carbohydrate assimilation tests, and PCR-sequencing of the calmodulin gene. The antifungal susceptibilities of these Sporothrix isolates to five antifungal agents (amphotericin B (AMB), voriconazole (VRC), itraconazole (ITC), fluconazole (FLC), and terbinafine (TRB)) were also evaluated according to the M27-A3 and M38-A2 protocols of the Clinical and Laboratory Standards Institute for yeast and mycelial phases, respectively. Five of seven clinical isolates were identified as S. schenckii, and two clinical and two environmental isolates were identified as S. globosa. This is the first report of S. globosa in Iran. There was significant agreement (73%) between the results of the phenotypic and genotypic identification methods. TRB and ITC were the most effective antifungals against the Sporothrix isolates. The minimum inhibitory concentration (MIC) values of TRB for the yeast and mycelial phases of S. schenckii differed significantly. There was also a significant difference in the minimum fungicidal concentration (MFC) values of AMB and TRB for the two phases. Considering the low efficacy of VRC and FLC and the wide MIC ranges of AMB (1-16 μg/ml and 1-8 μg/ml for yeast and mycelial forms, respectively) observed in the present study, in vitro antifungal susceptibility testing should be performed to determine appropriate therapeutic regimens.

  7. Identification and characterization of nine atypical Candida dubliniensis clinical isolates.

    Science.gov (United States)

    Albaina, Olatz; Sahand, Ismail H; Brusca, María I; Sullivan, Derek J; Fernández de Larrinoa, Iñigo; Moragues, María D

    2015-02-01

    Candida dubliniensis is a pathogenic yeast of the genus Candida closely related to Candida albicans. The phenotypic similarity of these two species often leads to misidentification of C. dubliniensis isolates in clinical samples. DNA-based methods continue to be the most effective means of discriminating accurately between the two species. Here, we report on the identification of nine unusual Candida isolates that showed ambiguous identification patterns on the basis of their phenotypic and immunological traits. The isolates were categorized into two groups. Group I isolates were unable to produce germ tubes and chlamydospores, and to agglutinate commercial latex particles coated with a mAb highly specific for C. dubliniensis. Group II isolates grew as pink and white colonies on CHROMagar Candida and ChromID Candida, respectively. Carbohydrate assimilation profiles obtained with API/ID32C together with PCR amplification with specific primers and DNA sequencing allowed reliable identification of the nine unusual clinical isolates as C. dubliniensis. © 2015 The Authors.

  8. Isolation and identification of Profenofos degrading bacteria

    Directory of Open Access Journals (Sweden)

    Saadatullah Malghani

    2009-12-01

    Full Text Available An enrichment culture technique was used to isolate bacterial strains responsible for the biodegradation of profenofos in a soil from Hubei province of central China. Two pure bacterial cultures, named W and Y, were isolated and subsequently characterized by sequencing of 16S rRNA genes and biochemical tests. Isolate W showed 96% similarity to the 16S rRNA gene of a Pseudomonas putida unlike Y which showed 99% similarity to the 16S rRNA gene of Burkholderia gladioli. Both strains grew well at pH 5.5-7.2 with a broad temperature profile ranging from 28º to 36 ºC. Bioremediation of profenofos-contaminated soil was examined using soil treated with 200 ug g-1; profenofos resulted in a higher degradation rate than control soils without inoculation. In a mineral salt medium (FTW reduction in profenofos concentration was 90% within 96 hours of incubation. A literature survey revealed that no data is available regarding the role of Burkholderia gladioli on pesticide biodegradation as well as on profenofos.

  9. Isolation and identification of Mycoplasma mycoides cluster strains from goats in Chongqing, China

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    Wang Haoju

    2014-03-01

    Full Text Available In order to evaluate the prevalence of the Mycoplasma mycoides cluster in goats in Chongqing, China, an epidemiological survey in this area was carried out. A total of 68 samples were subjected to bacteria isolation on Hartley’s medium. Four isolates (three from lung tissue and one from nasal discharges were recovered from the samples and identified as the Mycoplasma species by their morphological and biochemical characteristics. They were further confirmed by PCR using 16S rRNA specific primer pairs and by restriction enzyme analysis. In vitro antimicrobial susceptibility of the isolates indicated that some strains had developed resistance to the antibiotics tested. This is the first report on the isolation, identification, and molecular characterisation of Mycoplasma species isolated from goats in Chongqing. This study also revealed a prevalence of Mycoplasma species infection in goats in this area.

  10. Antifungal susceptibilities and identification of species of the Sporothrix schenckii complex isolated in Brazil.

    Science.gov (United States)

    Ottonelli Stopiglia, Cheila Denise; Magagnin, Cibele Massotti; Castrillón, Mauricio Ramírez; Mendes, Sandra Denise Camargo; Heidrich, Daiane; Valente, Patricia; Scroferneker, Maria Lúcia

    2014-01-01

    Sporotrichosis is a subacute or chronic mycosis caused worldwide by the dimorphic species complex, Sporothrix schenckii. We studied 85 isolates recovered in Brazil to verify their identification and evaluate their in vitro antifungal susceptibility patterns. Based on phenotypic tests (microscopic features, ability to grow at 30°C and 37°C, colony diameters, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), the strains were identified as S. schenckii, S. brasiliensis and S. globosa, with a predominance of S. schenckii isolates. There was 37.7% disagreement between the phenotypic and genotypic identification methodologies. In general, terbinafine was the most active drug, followed by ketoconazole and itraconazole, and the less active fluconazole and voriconazole. Five isolates (one S. globosa and four S. schenckii) were found to be itraconazole-resistant strains but, in general, there were no differences in the in vitro antifungal susceptibility profiles among the Sporothrix species.

  11. Molecular characterization of Danish Cryptosporidium parvum isolates

    DEFF Research Database (Denmark)

    Enemark, Heidi L.; Ahrens, Peter; Juel, Cynthia Dawn

    2002-01-01

    was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed...

  12. Isolation and identification of marine fish tumour (odontoma) associated bacteria

    Institute of Scientific and Technical Information of China (English)

    Ramalingam Vijayakumar; Kuzhanthaivel Raja; Vijayapoopathi Singaravel; Ayyaru Gopalakrishnan

    2015-01-01

    Objective: To identify fish tumour associated bacteria. Methods: The marine fish Sphyraena jello with odontoma was collected from in Tamil Nadu (Southeast India), and tumour associated bacteria were isolated. Then the isolated bacteria were identified based on molecular characters. Results: A total of 4 different bacterial species were isolated from tumour tissue. The bacterial species were Bacillus sp., Pontibacter sp., Burkholderia sp. and Macrococcus sp., and the sequences were submitted in DNA Data Bank of Japan with accession numbers of AB859240, AB859241, AB859242 and AB859243 respectively. Conclusions: Four different bacterial species were isolated from Sphyraena jello, but the role of bacteria within tumour needs to be further investigated.

  13. Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

    Science.gov (United States)

    Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia

    2016-01-01

    The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

  14. Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

    Directory of Open Access Journals (Sweden)

    Francesco Celandroni

    Full Text Available The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

  15. Hydrocarbon Degrading Bacteria: Isolation and Identification

    Directory of Open Access Journals (Sweden)

    Lies Indah Sutiknowati

    2007-11-01

    Full Text Available There is little information how to identify hydrocarbon degrading bacteria for bioremediation of marine oil spills. We have used gravel which contaminated oil mousse from Beach Simulator Tank, in Marine Biotechnology Institute, Kamaishi, Japan, and grown on enrichment culture. Biostimulation with nutrients (N and P was done to analyze biodegradation of hydrocarbon compounds: Naphthalene, Phenanthrene, Trichlorodibenzofuran and Benzo[a]pyrene. Community of bacteria from enrichment culture was determined by DGGE. Isolating and screening the bacteria on inorganic medium contain hydrocarbon compounds and determination of bacteria by DAPI (number of cells and CFU. DNA was extracted from colonies of bacteria and sequence determination of the 16S rDNA was amplified by primers U515f and U1492r. Twenty nine strains had been sequence and have similarity about 90-99% to their closest taxa by homology Blast search and few of them have suspected as new species.

  16. Molecular typing of Epidermophyton floccosum isolated from patients with dermatophytosis by RAPD-PCR.

    Science.gov (United States)

    Khosravi, Alireza; Behzad, Forough; Sabokbar, Azar; Shokri, Hojjatollah; Haddadi, Siamak; Masoudi-Nejad, Ali

    2010-12-01

    We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type Epidermophyton floccosum isolates recovered from patients with dermatophytosis originating from different regions of Iran. A total of 13 clinical isolates of E. floccosum obtained from Iranian patients were analyzed by RAPD with 7 arbitrary primers (OPN16, OPD18' OPU15, OPX19, R28, OPA04 and OPAA17). Among the applied primers, OPN16 produced banding patterns from all the isolates. In addition, some of the isolates had very close relation. The phenon line which represented the mean similarities was at the value of 73%. At this level, 4 groups were characterized. Two isolates of a patient had different molecular patterns, suggesting infection transmission from different sources in the case of a single patient. RAPD-PCR provided a rapid and practical tool for identification of E. floccosum isolates, which was independent of morphological characteristics, and enhanced laboratory diagnosis of dermatophytosis.

  17. Exopolysaccharide Production by Four Cyanobacterial Isolates and Preliminary Identification of These Isolates

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yunyi; CHI Zhenming; LU Weidong

    2007-01-01

    Four marine cyanobacterial isolates, named 104, 109, 113 and 115, from marine water off China's coast can release a large amount of exopolysaccharide (EPS) to medium. The effects of different components in medium on EPS production by the four isolates were investigated. Under the optimal condition, the EPS released by isolates 104,109, 113 and 115 reached 7.48 g L-1, 8.33 g L-1, 18.26 g L-1, and 6.78 g L-1 within 14 d,respectively. Based on the conventional identification methods for cyanobacteria,these isolates were assigned to genus Cyanothece.

  18. Molecular identification of Coccidioides spp. in soil samples from Brazil

    Directory of Open Access Journals (Sweden)

    Filho Antônio D

    2011-05-01

    Full Text Available Abstract Background Since 1991 several outbreaks of acute coccidioidomycosis (CM were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM. Results Twenty four soil samples had their DNA extracted and subsequently submitted to a semi-nested PCR technique using specific primers. While only 6 (25% soil samples were positive for C. posadasii by mice inoculation, all (100% were positive by the molecular tool. Conclusion This methodology represents a simple, sensitive and specific molecular technique to determine the environmental distribution of Coccidioides spp. in endemic areas, but cannot distinguish the species. Moreover, it may be useful to identify culture isolates. Key-words: 1. Coccidioidomycosis. 2. Coccidioides spp. 3. C. posadasii. 4. Semi-arid. 5. Semi-nested PCR

  19. Muon Identification and Isolation efficiency on full 2016 dataset

    CERN Document Server

    CMS Collaboration

    2017-01-01

    The performance of muon reconstruction and identification in CMS has been studied on data collected in pp collisions at $\\sqrt{s}$ = 13 TeV at the LHC. It has been studied on a sample of muons corresponding to an integrated luminosity of up to 36 $fb^{-1}$. We present measurements of muon reconstruction and trigger, identification and isolation efficiencies, computed with the tag-and-probe method, in different periods of the data taking. Results obtained using data are compared with Monte-Carlo predictions.

  20. Molecular characterization of mycobacteria isolated from seals.

    Science.gov (United States)

    Zumárraga, M J; Bernardelli, A; Bastida, R; Quse, V; Loureiro, J; Cataldi, A; Bigi, F; Alito, A; Castro Ramos, M; Samper, S; Otal, I; Martin, C; Romano, M I

    1999-09-01

    Tuberculosis (TB) was diagnosed in 10 seals from three species (Arctocephalus australis, Arctocephalus tropicalis and Otaria flavescens) found in South America. The mycobacteria isolated from these cases belonged to the Mycobacterium tuberculosis complex, as determined by RFLP using an IS6110 probe, spoligotyping, analysis of the 16S rRNA gene sequence and by PCR-restriction analysis of hsp65. Polymorphisms in gyrA, katG, oxyR and pncA were investigated in some of the isolates, as well as the presence of the MPB70 antigen. The insertion sequence IS6110 was present in three to seven copies in the genome of the mycobacteria isolated from seals. Using the IS6110 probe, six patterns (designated A, B, C, D, E and F) were identified from 10 different isolates. Patterns A and B were found for the mycobacteria isolated from two and four seals, respectively, indicating an epidemiological relationship between isolates grouped according to their IS6110 RFLP. The mycobacteria isolated from seals shared the majority of their IS6110 DNA-containing restriction fragments, and nine isolates had an identical spoligotype; only one isolate showed a minor difference in its spoligotype. In addition, none of these spoligotypes were found in other M. tuberculosis complex strains. These results suggest that the isolates from seals constitute a unique group of closely related strains. The mycobacteria isolated from seals showed polymorphisms at gyrA codon 95 and katG codon 463, as do group 1 M. tuberculosis, and M. bovis. Group 1 mycobacteria are associated with cluster cases. The spoligotypes found in the mycobacteria isolated from seals lack spacers 39-43, as does M. bovis, but the MPB70 antigen, which is highly expressed in M. bovis and minimally expressed in M. tuberculosis, was not detected in these mycobacteria. The mycobacteria isolated from seals also showed oxyR and pncA polymorphisms specific to M. tuberculosis. In conclusion, the mycobacteria that cause TB in seals in the South

  1. Combined morphological and molecular approach for identification of Stemphylium vesicarium inoculum in pear orchards.

    Science.gov (United States)

    Puig, Mireia; Ruz, Lídia; Montesinos, Emilio; Moragrega, Concepció; Llorente, Isidre

    2015-03-01

    Stemphylium vesicarium is the causal agent of brown spot of pear, an important disease reported in pear-growing areas of Europe. The pathogen is able to colonize pear leaf debris and dead tissues of herbaceous plants and produce abundant ascospores and conidia that are capable of infecting pear trees. Inoculum monitoring in pear orchards is mainly achieved through spore traps and species identification is based on conidial morphology, but the similarities on conidial traits among species of Stemphylium make correct identification difficult. In this work a total of thirty-seven Stemphylium isolates from pear orchards were characterized at the morphological, pathogenic, and molecular level. Correspondence among ITS and gpd sequences and morphological traits were evaluated. Species identification based exclusively on morphological data was not feasible. Combined morphological and molecular data were necessary for unambiguous identification of isolates in the S. vesicarium species group. Only isolates identified as S. vesicarium were pathogenic on pear. The study revealed that several species of Stemphylium coexist in pear orchards with S. vesicarium, the causal agent of BSP, and that combined morphological and molecular data are needed to differentiate them. Consequently, direct measurements of the airborne inoculum using volumetric spore traps may overestimate the actual pathogen population.

  2. Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood

    Directory of Open Access Journals (Sweden)

    Kee Peng Ng

    2016-12-01

    Full Text Available Background Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. Methods The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC. Results The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole, as well as amphotericin B, with MIC of less than 1 µg/ml. Discussion Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential.

  3. Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood

    Science.gov (United States)

    Ng, Kee Peng; Chan, Chai Ling; Yew, Su Mei; Yeo, Siok Koon; Toh, Yue Fen; Looi, Hong Keat; Na, Shiang Ling; Lee, Kok Wei; Yee, Wai-Yan

    2016-01-01

    Background Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. Methods The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS)-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC). Results The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole), as well as amphotericin B, with MIC of less than 1 µg/ml. Discussion Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential. PMID:28028453

  4. SIGNAL IDENTIFICATION AND ISOLATION UTILIZING RADIO FREQUENCY PHOTONICS

    Science.gov (United States)

    2017-09-01

    AFRL-RY-WP-TR-2017-0158 SIGNAL IDENTIFICATION AND ISOLATION UTILIZING RADIO FREQUENCY PHOTONICS Preetpaul S. Devgan RF/EO Subsystems Branch...MATERIEL COMMAND UNITED STATES AIR FORCE NOTICE AND SIGNATURE PAGE Using Government drawings, specifications, or other data included in this document for...ABW) Public Affairs Office (PAO) and is available to the general public, including foreign nationals. Copies may be obtained from the Defense

  5. PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF ANTIBIOTICS RESISTANCE E. CLOACAE ISOLATES

    Directory of Open Access Journals (Sweden)

    Abdalnabi J Abid

    2015-12-01

    Full Text Available The present study aimed detecting and characterizing of β-lactamases producing E.cloacae isolated from different clinical sources in Hilla hospitals using phenotypic and molecular methods. A total of 308 samples were collected from two major hospitals at Hilla Province from October 2013 to April 2014. All isolates were tested biochemically, it was found that only 15 isolates from all isolates were belonging to Enterobactercloacae. All E. cloacae isolates were primarily screened for β-lactams resistance. Antibiotic susceptibility and minimum inhibitory concentration tests were performed using disk diffusion and agar dilution methods, respectively. The molecular study documented a widespread of Amp C genes among isolates of E. cloacae isolatesrepresented by 6/15(40% positive isolates for Amp C primers. PCR assay revealed that prevalence rate of bla-TEM gene among tested isolates was 9(60%. followed by the bla-OXA gene was detected only in 3(20%.While bla-VEB gene and bla-SHV gene was not detected in any of the isolates. Some virulence factors of bacteria were also studied, and the results showed that all bacterial strains have capsule ,the results also also detected biofilm formation among isolates and the results revealed that 13(86%of the isolates are biofilm former.

  6. Isolation, identification and characterization of indigenous fungi for bioremediation of hexavalent chromium, nickel and cobalt

    Science.gov (United States)

    Hernahadini, Nelis; Suhandono, Sony; Choesin, Devi N.; Chaerun, Siti K.; Kadarusman, Ade

    2014-03-01

    Waste from nickel mining of Sorowako in South Sulawesi contains hexavalent chromium, nickel and cobalt metals in high concentration and may have a negative impact to the environment. Common waste treatment systems such as chemical treatment using a reducing reagent may still have a negative impact. Bioremediation using fungi or bacteria becomes more popular because it is an environmentally friendly alternative. The purposes of this study are to isolate and identify indigenous fungi that are resistant to heavy metals (hexavalent chromium, nickel, and cobalt) and are capable of reducing the concentration of metals in mining wastes. Ten fungal isolates were successfully isolated from the soils and pond sediments in the area of nickel mining in Sorowako. Selection of superior isolate was carried out by growing all the isolates on PDA medium, which contained all of the three metals. One superior isolate was identified to be able to grow on medium with concentrations of 6400 ppm hexavalent chromium, 200 ppm nickel and 50 ppm cobalt. Molecular identification and phylogenetic studies of the isolate using fungal PCR primers developed to amplify the ITS (internal transcribed spacer) region showed that the isolate sequence was very close to Trichoderma atroviride with 99.8% similarity. Optimum incubation time for the uptake of hexavalent chromium was 3 days, nickel and cobalt was 5 days, respectively, with an optimum pH of 4.

  7. Identification and isolation of Genotype-I Japanese Encephalitis virus from encephalitis patients

    Directory of Open Access Journals (Sweden)

    Gao Xiaoyan

    2010-11-01

    Full Text Available Abstract Historically, Japanese Encephalitis virus (JEV genotype III (GIII has been responsible for human diseases. In recent years, JEV genotype I (GI has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

  8. Isolation and Identification of Endophytic Fungi from Actinidia macrosperma and Investigation of Their Bioactivities

    Directory of Open Access Journals (Sweden)

    Yin Lu

    2012-01-01

    Full Text Available Endophytic fungi from the Chinese medicinal plant Actinidia macrosperma were isolated and identified for the first time. This was the first study to evaluate their cytotoxic and antitumour activities against brine shrimp and five types of tumour cells, respectively. In total, 17 fungal isolates were obtained. Five different taxa were represented by 11 isolates, and six isolates were grouped into the species of Ascomycete Incertae sedis with limited morphological and molecular data. Cytotoxic activity has been found in most isolates except AM05, AM06, and AM10. The isolates AM07 (4.86 μg/mL, AM11 (7.71 μg/mL, and AM17 (14.88 μg/mL exhibited significant toxicity against brine shrimp. The results of the MTT assay to assess antitumour activity revealed that 82.4% of isolate fermentation broths displayed growth inhibition (50% inhibitory concentration IC50< 100 μg/mL. Moreover, AM07, AM11, and AM17 showed strong antitumour activity in all the cell lines examined. These results suggest that endophytic fungi in A. macrosperma are valuable for the isolation and identification of novel cytotoxic and antitumour bioactive agents.

  9. Evaluation of three phenotypic identification systems for clinical isolates of Raoultella ornithinolytica.

    Science.gov (United States)

    Park, Jeong Su; Hong, Ki Ho; Lee, Hyun Jung; Choi, Soon Hee; Song, Sang Hoon; Song, Kyoung-Ho; Kim, Hong Bin; Park, Kyoung Un; Song, Junghan; Kim, Eui-Chong

    2011-04-01

    Raoultella spp. have recently been separated from the genus Klebsiella based on their molecular characteristics. It was discovered that Raoultella ornithinolytica can be misidentified as Klebsiella oxytoca by commonly used phenotypic identification systems. Therefore, this study evaluated the ability of three phenotypic systems to identify R. ornithinolytica compared with the genotypic methods sequence-specific primer PCR (SSP-PCR), 16S rRNA gene sequence analysis using the MicroSeq 500 system16S rDNA bacterial identification system or comparison with GenBank sequences using blast. The phenotypic systems examined in this study were the VITEK 2 GN ID card, the MicroScan Neg Combo 32 panel and API 20E. The SSP-PCR panel was able to distinguish the R. ornithinolytica reference strain from other Raoultella spp. and K. oxytoca. Of the 27 isolates identified as R. ornithinolytica by SSP-PCR, VITEK 2 identified all of them as R. ornithinolytica. MicroScan and API identified 25 isolates (92.6%) and 24 isolates (88.9%) as K. oxytoca, respectively. These isolates were ornithine decarboxylase (ODC) negative in all three phenotypic systems. MicroSeq 500 identified 24 isolates (88.9%) as R. ornithinolytica, whereas GenBank identification was heterogeneous. Of the 68 isolates identified as K. oxytoca by SSP-PCR, 66 isolates (97.1%) were identified as K. oxytoca by VITEK 2, MicroScan and API. MicroScan and API require additional biochemical tests to differentiate between ODC-negative R. ornithinolytica and K. oxytoca.

  10. Researches on Damage Identification in Passive Vibro-Isolation Devices

    Directory of Open Access Journals (Sweden)

    A. Leopa

    2012-01-01

    Full Text Available This paper deals with the theoretical aspects combined with experimental analysis regarding early damage identification in passive vibro-isolation devices. Basically, this research presents the relevant results obtained for a singular element. Rubber based on elements is discussed especially. This study was a naturally fall-back of the authors large analysis regarding the dynamic behaviour of the passive isolation devices against vibration, shocks and seismic waves. Main hypothesis supposed that on the exploitation time, all technical devices and systems acquire different levels of wearing because of the dynamic overloads and their derivative influences (aging, fatigue, energy dissipation, external heating or cooling, etc.. Hereby the performance characteristics changes and the system becomes working improperly. Numerical simulations were developed for simple spatial configuration of isolation device. Stochastic approach of essential results was briefly presented nearby the relevant results and discussions.

  11. Isolation and identification of marine fish tumour (odontoma associated bacteria

    Directory of Open Access Journals (Sweden)

    Ramalingam Vijayakumar

    2015-09-01

    Full Text Available Objective: To identify fish tumour associated bacteria. Methods: The marine fish Sphyraena jello with odontoma was collected from in Tamil Nadu (Southeast India, and tumour associated bacteria were isolated. Then the isolated bacteria were identified based on molecular characters. Results: A total of 4 different bacterial species were isolated from tumour tissue. The bacterial species were Bacillus sp., Pontibacter sp., Burkholderia sp. and Macrococcus sp., and the sequences were submitted in DNA Data Bank of Japan with accession numbers of AB859240, AB859241, AB859242 and AB859243 respectively. Conclusions: Four different bacterial species were isolated from Sphyraena jello, but the role of bacteria within tumour needs to be further investigated.

  12. ISOLATION AND IDENTIFICATION OF ACR YLAMIDE DEGRADING BACTERIA FROM SOIL

    Directory of Open Access Journals (Sweden)

    Nidhi Jain

    2013-02-01

    Full Text Available Acrylamide is an aliphatic amide, which is produced by industrial processes and during heating of food. It is neurotoxic and a suspected carcinogen. In the present study an attempt was made to isolate acrylamide degrading bacteria from soil. The optimum growth conditions and physiological characteristics for the isolated acrylamide degrading bacteria were investigated. The isolated bacterium was identified as Bacillus clausii strain 1779 based on full 16S rRNA molecular phylogeny. The bacteria can degrade 800 mg l-1acrylamide after eight days of incubation with concomitant cell growth. In addition to above, it also grows optimally at a concentration of acrylamide between 500-2000 mg l-1between pH 8-10 and temperature and 25 – 45 0C. Thus the isolate would be useful in the bioremediation of environment from acrylamide in alkali conditions.

  13. Molecular characterization of Shewanella and Aeromonas isolates associated with spoilage of Common carp (Cyprinus carpio).

    Science.gov (United States)

    Beaz-Hidalgo, Roxana; Agüeria, Daniela; Latif-Eugenín, Fadua; Yeannes, Maria I; Figueras, Maria J

    2015-01-01

    Storage in ice is a common way of preserving commercial fish species but some microorganisms can still contaminate and participate in the spoilage of the product; therefore, identification of potential harmful microbes is important. Thirteen colonies were isolated from common carp (Cyprinus carpio) that had been stored in ice, whose phenotypic identification revealed that they belonged to the genera Aeromonas (n = 5) and Shewanella (n = 8). Molecular genotyping with ERIC-PCR showed clonality only among two of the five Aeromonas isolates and for two groups (n = 3; n = 2) of the eight Shewanella isolates. Sequencing the rpoD gene showed that four Aeromonas isolates belonged to the species Aeromonas salmonicida and one to A. sobria. Of the eight Shewanella, seven isolates cluster with Shewanella putrefaciens and one with Shewanella profunda in the 16S rRNA phylogenetic tree. However, analysis of the gyrB gene showed that these eight isolates could constitute a new species closely related to S. baltica. The Shewanella and A. salmonicida isolates produce off-odours and reduce trimethylamine oxide, indicating that they might contribute to the spoilage of the fish. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Morphological and molecular identification of phytophthora species from maple trees in Serbia

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    Milenković Ivan

    2014-01-01

    Full Text Available The paper presents the results of the study performed with aims to determine the presence and diversity of Phytophthora species on maple trees in Serbia. Due to high aggressiveness and their multicyclic nature, presence of these pathogens is posing significant threat to forestry and biodiversity. In total, 29 samples of water, soil and tissues were taken from 10 different localities, and six different maple hosts were tested. After the isolation tests, 17 samples from five different maple hosts were positive for the presence of Phytophthora spp., and 31 isolates were obtained. After the detailed morphological and physiological classification, four distinct groups of isolates were separated. DNA was extracted from selected representative isolates and molecular identification with sequencing of ITS region was performed. Used ITS4 and ITS6 primers successfully amplified the genomic DNA of chosen isolates and morphological identification of obtained isolates was confirmed after the sequencing. Four different Phytophthora species were detected, including P. cactorum, P. gonapodyides, P. plurivora and P. lacustris. The most common isolated species was homothallic, and with very variable and semipapillate sporangia, P. plurivora with 22 obtained isolates. This is the first report of P. plurivora and P. gonapodyides on A. campestre, P. plurivora and P. lacustris on Acer heldreichii and first report of P. lacustris on A. pseudoplatanus and A. tataricum in Serbia. [Projekat Ministarstva nauke Republike Srbije, br. TR 37008

  15. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Baggesen, Dorte Lau; Porting, P.H.

    1999-01-01

    The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratorie...... (serogroup D). In conclusion, rough presumptive Salmonella isolates can be conveniently confirmed to the serogroup-revel, using the pre-mixed PCR tests. The system can be easily implemented in accredited laboratories with limited experience in molecular biology........ The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains...

  17. Identification and biotechnological potential of psychrotrophic marine isolates

    Directory of Open Access Journals (Sweden)

    Swanandi Pote

    2014-06-01

    Full Text Available Background: Importance of microbial metabolites in food, detergent, pharmaceutical, nutraceutical and cosmetic industries has now been widely established. To fulfill the requirement of these industries, psychrophilic/psychrotrophic marine microbes are being explored. These microbes help in the production of metabolites that are active and stable at extreme physiological conditions. In correlation with this scenario, the present study reports identification of 14 bacterial isolates (BRI 32- BRI 45 from marine water samples (out of which 4 are Antarctic with emphasis on their biotechnologically important characters. Materials and Methods: Bacterial isolates were identified using 16S rRNA gene sequencing. Growth of the isolates under different physiological conditions of temperature (10°C to 45°C, pH (3-10 and concentration of NaCl (0-20% was studied. Further, the isolates were examined for their ability to produce i polyunsaturated fatty acids, ii industrially important enzymes and their potential to produce bio-surfactant. Results: 16S rRNA gene sequence analysis revealed that the isolates belonged to Halomonas, Brevibacillus, Kocuria and Oceanobacillus genera. Our results indicated that the isolates could grow over a wide range of physiological conditions of pH (3-10, temperature (10-45°C and NaCl concentration. Eight out of 14 isolates showed the presence of omega-3 and omega-6 fatty acids. BRI 34 was found to produce significant amounts of eicosapentaenoic acid (39.66%. Most of the isolates exhibited the ability to produce 3 or 4 enzymes. Only BRI 35 showed potential for biosurfactant production.

  18. Molecular identification of two vegetative compatibility groups of Fusarium oxysporum f. sp. cepae.

    Science.gov (United States)

    Southwood, Michael J; Viljoen, Altus; Mostert, Glaudina; McLeod, Adéle

    2012-02-01

    Fusarium oxysporum f. sp. cepae, which causes basal rot of onion, consists of seven vegetative compatibility groups (VCGs 0420 to 0426) and several single-member VCGs (SMVs). F. oxysporum f. sp. cepae populations in South Africa and Colorado each consist of one main VCG (namely, VCG 0425 and 0421, respectively). The aim of this study was to develop sequence-characterized amplified region (SCAR) markers for the identification of VCGs 0425 and 0421, using 79 previously characterized F. oxysporum isolates. A second aim was to investigate the prevalence of VCG 0425 among 88 uncharacterized South African onion F. oxysporum isolates using (i) the developed SCAR markers and (ii) inter-retrotransposon (IR)- and random amplified polymorphic DNA (RAPD) fingerprinting. Only two RAPD primers provided informative fingerprints for VCG 0425 isolates but these could not be developed into SCAR markers, although they provided diagnostic fragments for differentiation of VCG 0425 from VCG 0421. IR fingerprinting data were used to develop a multiplex IR-SCAR polymerase chain reaction method for the identification of VCG 0421, VCG 0425, and SMV 4 isolates as a group. Molecular identification of the uncharacterized collection of 88 F. oxysporum isolates (65 F. oxysporum f. sp. cepae and 23 F. oxysporum isolates nonpathogenic to onion) confirmed that VCG 0425 is the main VCG in South Africa, with all but 3 of the 65 F. oxysporum f. sp. cepae isolates having the molecular characteristics of this VCG. Genotyping and VCG testing showed that two of the three aforementioned isolates were new SMVs (SMV 6 and SMV 7), whereas the third (previously known as SMV 3) now belongs to VGC 0247.

  19. Molecular and antimicrobial susceptibility characterization of Globicatella sulfidifaciens isolated from sow's urinary tract infection.

    Science.gov (United States)

    Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Amigo, Cristina R; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

    2017-12-01

    The Globicatella genus comprises Gram-positive, facultative anaerobic, α-hemolytic and catalase negative cocci morphologically and phenotypically very similar to Streptococcus and Aerococcus genus which can lead to misidentification and underestimation of this pathogen. Globicatella species have already been isolated from human and animals with heart and brain disorders. Their clinical relevance in animals, and its zoonotic potential, remains unknown due to the difficulty in their identification. To present the isolation, phenotypic and molecular characterization of G. sulfidifaciens from urinary tract infection in sows. Urine samples from 140 sows of two swine herds located in São Paulo State (Brazil) yielded the isolation of three presumptive G. sulfidifaciens strains. Identification and species confirmation were done by MALDI-TOF MS and 16S rRNA sequencing. Strains were further characterized by single enzyme amplified fragments length polymorphism (SE-AFLP) and broth microdilution techniques. All three isolates were confirmed as G. sulfidifaciens. The SE-AFLP genotyping resulted in distinct fingerprint patterns for each strain. All isolates presented high MIC values to tetracycline, sulphonamides, aminoglycosides and tylosin tartrate, which present high usage in human and animal medicine. Globicatella sulfidifaciens could be related to sporadic urinary tract infections in swine and appear to present alarming antimicrobial susceptibility profile. It is necessary to differentiate Streptococcus-like microorganisms in routine laboratory diagnostics for the correct identification of underestimated species potentially pathogenic to animals.

  20. Molecular sidebands of refractory elements for ISOL

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, A. [Oak Ridge Associated Universities, Oak Ridge (United States)], E-mail: kronenberga@ornl.gov; Spejewski, E.H.; Carter, H.K.; Mervin, B.; Jost, C. [Oak Ridge Associated Universities, Oak Ridge (United States); Stracener, D.W. [Oak Ridge National Laboratory, TN 37831 (United States); Lapi, S. [TRIUMF (Canada); Bray, T. [Auburn University (United States)

    2008-10-15

    The formation of molecular sidebands of refractory elements, such as V, Re, Zr, Mo, Tc, is discussed. The focus is on in situ sideband formation and its advantage for the release process. An atomic {sup 48}V beam has been produced in a two step process, forming the oxide in situ, transporting it through the target-ion source as a chloride and destroying the chlorine sideband in the ion source. The sideband formation of Re, Zr, Mo, Tc is discussed.

  1. Molecular Sidebands of Refractory Elements for ISOL

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, Andreas [Oak Ridge Associated Universities (ORAU); Spejewski, Eugene H. [Oak Ridge Associated Universities (ORAU); Carter, H Kennon [Oak Ridge Associated Universities (ORAU); Mervin, Brenden T. [Oak Ridge Associated Universities (ORAU); Jost, Cara [Oak Ridge Associated Universities (ORAU); Stracener, Daniel W [ORNL; Lapi, Suzanne [TRIUMF, Canada; Bray, T. H. [Auburn University, Auburn, Alabama

    2008-01-01

    The formation of molecular sidebands of refractory elements, such as V, Re, Zr, Mo, Tc, is discussed. The focus is on in situ sideband formation and its advantage for the release process. An atomic 48V beam has been produced in a two step process, forming the oxide in situ, transporting it through the target-ion source as a chloride and destroying the chlorine sideband in the ion source. The sideband formation of Re, Zr, Mo, Tc is discussed.

  2. Isolation and Molecular Characterization of Acanthamoeba Strains from Dental Units in Costa Rica.

    Science.gov (United States)

    Retana-Moreira, Lissette; Abrahams-Sandí, Elizabeth; Castro-Artavia, Esteban; Fernández-Sánchez, Ana; Castro-Castillo, Alfredo; Reyes-Batlle, María; Lorenzo-Morales, Jacob

    2015-01-01

    Free-living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin-America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway.

  3. Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates.

    Science.gov (United States)

    Brisse, Sylvain; Duijkeren, Engeline van

    2005-02-25

    The objectives of this study were to determine the distribution of Klebsiella species and phylogenetic groups in animal clinical samples and to determine the levels of antimicrobial resistance of animal Klebsiella clinical isolates. One hundred Klebsiella veterinary clinical isolates were identified using gyrA PCR-RFLP and rpoB gene sequencing as a confirmatory method. Klebsiella pneumoniae phylogenetic group KpI was dominant (78 isolates), but KpII, KpIII (K. variicola), K. oxytoca, K. planticola and K. terrigena were also represented. The relative frequencies in animal infections of Klebsiella species and phylogenetic groups were similar to those observed in human nosocomial infections, suggesting that similar ecological and molecular factors cause Klebsiella infections in both situations. Resistance was common against ampicillin (99%) and cephalexin (43%) but not against ceftazidime, ceftiofur, tetracycline, enrofloxacin, gentamicin and trimethoprim-sulfamethoxazole. Thirteen isolates resistant to three or more antimicrobials or combinations thereof were found, but acquired antimicrobial resistance remains lower among animal isolates than among human nosocomial isolates.

  4. Identification of Colletotrichum isolates from Capsicum chinense in Amazon.

    Science.gov (United States)

    de Oliveira, C V S; Matos, K S; de Albuquerque, D M C; Hanada, R E; da Silva, G F

    2017-06-29

    Chili pepper (Capsicum chinense) is a great economic important culture on the State of Amazonas, and it represents, approximately, a production of 1.9 thousand tons per year. It is one of the hosts of Colletotrichum genus in the North region of Brazil. The aim of the study was to differentiate and to identify isolates of Colletotrichum collected from C. chinense in Amazon. Molecular characterization, using RFLP-PCR, ERIC-PCR and ISSR, was carried out initially for screening of morphologically similar isolates. Furthermore, phylogenetic analyses were performed using combined regions: Actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the three isolates, INPA 2066, INPA 2286 and INPA 1858, plus superoxide dismutase (SOD2) for INPA 2066. We showed that the molecular markers were able to distinguish the isolates of Colletotrichum studied and these results were confirmed with the phylogenetic analyses, three different occurrences of Colletotrichum species (C. siamense, C. scovillei and C. brevisporum) causing anthracnose in C. chinense in the State of Amazonas. This study represents the first report of the species C. siamense and C. scovillei in this host in Brazil.

  5. 基于TPI基因的城市污水中蓝氏贾第鞭毛虫的分子鉴定%Molecular identification of Giardia lamblia isolates from raw urban wastewater using the TPI gene

    Institute of Scientific and Technical Information of China (English)

    张唯哲; 凌虹; 张晓丽; 李懿宏; 姬红; 杨凤坤; 沈玉娟; 刘爱芹

    2012-01-01

    目的 对哈尔滨市污水处理厂原水中的蓝氏贾第鞭毛虫进行分子鉴定.方法 收集哈尔滨市污水处理厂原水样,提取基因组DNA,巢式PCR扩增蓝氏贾第鞭毛虫TPI基因,通过序列分析确定蓝氏贾第鞭毛虫的集聚体类型和亚型,并进行同源性分析.结果 从哈尔滨市污水处理厂原水DNA提取物中扩增出蓝氏贾第鞭毛虫TPI基因,经分子鉴定为蓝氏贾第鞭毛虫集聚体AⅡ,其序列与文献报道人源蓝氏贾第鞭毛虫集聚体AII序列(AB516351,FJ560570,EF688021)的同源性为100%.结论 哈尔滨市污水处理厂原水中存在蓝氏贾第鞭毛虫集聚体AII,威胁当地居民健康.%Objective To molecularly identify Giardia lamblia isolates from raw urban wastewater at a wastewater treatment plant in Haerbin. Methods Genomic DNA of G. lamblia was extracted and the TPI gene was amplified by nested PCRj the assemblage/genotype and sub-assemblage/subtype of C lamblia were identified and sequenced. The ho-mology of the gene was analyzed with reference nucleotide sequences downloaded from GenBank. Results The TPI gene of G. lamblia was amplified from DNA extracts prepared from raw wastewater from a wastewater treatment plant in Harbin, and the isolate was identified as assemblage AH. The obtained sequence based on TPI had 100% homology with the previously reported sequences of human-derived G. lamblia assemblage All (AB516351 ;FJ560570;EF688021). Conclusion The presence of G. lamblia assemblage All in raw urban wastewater in Harbin poses a threat to the health of local inhabitants.

  6. Isolation and identification of iron ore-solubilising fungus

    Directory of Open Access Journals (Sweden)

    Damase Khasa

    2010-09-01

    Full Text Available Potential mineral-solubilising fungi were successfully isolated from the surfaces of iron ore minerals. Four isolates were obtained and identified by molecular and phylogenetic methods as close relatives of three different genera, namely Penicillium (for isolate FO, Alternaria (for isolates SFC2 and KFC1 and Epicoccum (for isolate SFC2B. The use of tricalcium phosphate (Ca3(PO42in phosphate-solubilising experiments confirmed isolate FO as the only phosphate solubiliser among the isolated fungi. The bioleaching capabilities of both the fungus and its spent liquid medium were tested and compared using two types of iron ore materials, conglomerate and shale, from the Sishen Iron Ore Mine as sources of potassium (K and phosphorus (P. The spent liquid medium removed more K (a maximum of 32.94% removal, from conglomerate, than the fungus (a maximum of 21.36% removal, from shale. However, the fungus removed more P (a maximum of 58.33% removal, from conglomerate than the spent liquid medium (a maximum of 29.25% removal, from conglomerate. The results also indicated a potential relationship between the removal of K or P and the production of organic acids by the fungus. A high production of gluconic acid could be related to the ability of the fungus to reduce K and P. Acetic, citric and maleic acids were also produced by the fungus, but in lower quantities. In addition, particle size and iron ore type were also shown to have significant effects on the removal of potassium and phosphorus from the iron ore minerals. We therefore conclude that the spent liquid medium from the fungal isolate FO can potentially be used for biobeneficiation of iron ore minerals.

  7. Contemporary nucleic acid-based molecular techniques for detection, identification, and characterization of Bifidobacterium.

    Science.gov (United States)

    Mianzhi, Yao; Shah, Nagendra P

    2017-03-24

    Bifidobacteria are one of the most important bacterial groups found in the gastrointestinal tract of humans. Medical and food industry researchers have focused on bifidobacteria because of their health-promoting properties. Researchers have historically relied on classic phenotypic approaches (culture and biochemical tests) for detection and identification of bifidobacteria. Those approaches still have values for the identification and detection of some bifidobacterial species, but they are often labor-intensive and time-consuming and can be problematic in differentiating closely related species. Rapid, accurate, and reliable methods for detection, identification, and characterization of bifidobacteria in a mixed bacterial population have become a major challenge. The advent of nucleic acid-based molecular techniques has significantly advanced isolation and detection of bifidobacteria. Diverse nucleic acid-based molecular techniques have been employed, including hybridization, target amplification, and fingerprinting. Certain techniques enable the detection, characterization, and identification at genus-, species-, and strains-levels, whereas others allow typing of species or strains of bifidobacteria. In this review, an overview of methodological principle, technique complexity, and application of various nucleic acid-based molecular techniques for detection, identification, and characterization of bifidobacteria is presented. Advantages and limitations of each technique are discussed, and significant findings based on particular techniques are also highlighted.

  8. Systematic internal transcribed spacer sequence analysis for identification of clinical mold isolates in diagnostic mycology: a 5-year study.

    Science.gov (United States)

    Ciardo, Diana E; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V; Böttger, Erik C

    2010-08-01

    The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

  9. Molecular, chemical and biological screening of soil actinomycete isolates in seeking bioactive peptide metabolites

    Directory of Open Access Journals (Sweden)

    Javad Hamedi

    2015-10-01

    Full Text Available Background and Objective: Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the interests of researchers. In this study, molecular, chemical and biological screening of soil actinomycetes was carried out in order to search for peptide-producing actinomycetes.Materials and Methods: 60 actinomycetes were isolated from soils of Iran. The isolates were subjected to molecular screening for detection NRPS (non-ribosomal peptide synthetases gene. Phylogenic identification of NRPS containing isolates was performed. Chemical screening of the crude extracts was performed using chlorine o-dianisidine as peptide detector reagent and bioactivity of peptide producing strains was determined by antimicrobial bioassay. High pressure liquid chromatography- mass spectrometry (HPLC-MS with UV-visible spectroscopy was performed for detection of the metabolite diversity in selected strain.Results: Amplified NRPS adenylation gene (700 bp was detected among 30 strains. Phylogenic identification of these isolates showed presence of rare actinomycetes genera among the isolates and 10 out of 30 strains were subjected to chemical screening. Nocardia sp. UTMC 751 showed antimicrobial activity against bacterial and fungal test pathogens. HPLC-MSand UV-visible spectroscopy results from the crude extract showed that this strain has probably the ability to produce new metabolites.Conclusion: By application of a combined approach, including molecular, chemical and bioactivity analysis, a promising strain of Nocardia sp. UTMC 751 was obtained. This strain had significant activity against Staphylococcus aureus and Pseudomonas aeruginosa. Strain Nocardia sp. UTMC 751 produce five unknown and most probably new metabolites with molecular weights of 274.2, 390.3, 415.3, 598.4 and 772.5. This strain had showed 99% similarity to Nocardia ignorata DSM 44496 T.

  10. Identification of lactic acid bacteria isolated from corn stovers.

    Science.gov (United States)

    Pang, Huili; Zhang, Meng; Qin, Guangyong; Tan, Zhongfang; Li, Zongwei; Wang, Yanping; Cai, Yimin

    2011-10-01

    One hundred and twenty-six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram-positive, catalase-negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover-associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

  11. Molecular typing of clinical isolates of Cryptococcus neoformans/Cryptococcus gattii species complex from Northeast Mexico.

    Science.gov (United States)

    González, Gloria M; Casillas-Vega, Néstor; Garza-González, Elvira; Hernández-Bello, Romel; Rivera, Gildardo; Rodríguez, Jesús Ancer; Bocanegra-Garcia, Virgilio

    2016-01-01

    Cryptococcosis is caused by members of the Cryptococcus neoformans/Cryptococcus gattii species complex. Based on molecular identification, these two species have been further differentiated into molecular types. The aim of this work was to characterize clinical cryptococcal isolates recovered from six hospitals in Northeast Mexico from 1995 to 2011. One hundred and sixty-six isolates, which were characterized by biochemical tests and in vitro susceptibility to amphotericin B, fluconazole, and voriconazole, and M13 PCR fingerprinting, were included in this study. Utilizing phenotypic tests, 153 isolates (92.16 %) were identified as C. neoformans and 13 (7.83 %) as C. gattii. All isolates were susceptible to all antifungals tested. Employing M13 PCR fingerprinting, eight molecular types were detected. VNI was the most common genotype (124 cases; 74.6 %), followed by VNII (15 cases; 9 %), VNIII (8 cases; 4.8 %), VNIV (6 cases; 3.6 %), VGI (6 cases; 3.6 %), VGII (3 cases; 1.8 %), and VGIII and VGIV (2 cases, 1.2 % each). We confirm the presence of C. gattii in clinical isolates in Northeast Mexico, and a high clonal diversity in the studied strains of C. neoformans/C. gattii species complex.

  12. Molecular Microbial Analysis of Lactobacillus Strains Isolated from the Gut of Calves for Potential Probiotic Use

    Directory of Open Access Journals (Sweden)

    Lorena P. Soto

    2010-01-01

    Full Text Available The intestinal microbiota has an influence on the growth and health status of the hosts. This is of particular interest in animals reared using intensive farming practices. Hence, it is necessary to know more about complexity of the beneficial intestinal microbiota. The use of molecular methods has revolutionized microbial identification by improving its quality and effectiveness. The specific aim of the study was to analyze predominant species of Lactobacillus in intestinal microbial ecosystem of young calves. Forty-two lactic acid bacteria (LAB isolated from intestinal tract of young calves were characterized by: Amplified Ribosomal DNA Restriction Analysis (ARDRA, by using Hae III, Msp I, and Hinf I restriction enzymes, and 16S rDNA gene sequencing. ARDRA screening revealed nine unique patterns among 42 isolates, with the same pattern for 29 of the isolates. Gene fragments of 16S rDNA of 19 strains representing different patterns were sequenced to confirm the identification of these species. These results confirmed that ARDRA is a good tool for identification and discrimination of bacterial species isolated from complex ecosystem and between closely related groups. This paper provides information about the LAB species predominant in intestinal tract of young calves that could provide beneficial effects when administered as probiotic.

  13. Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells

    Directory of Open Access Journals (Sweden)

    Bo-jiang Li

    2015-08-01

    Full Text Available The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

  14. Isolation, molecular characterization and phylogenetic analysis of canine parvovirus.

    Science.gov (United States)

    Mohan Raj, J; Mukhopadhyay, H K; Thanislass, J; Antony, P X; Pillai, R M

    2010-12-01

    Canine parvovirus type 2 (CPV-2) causes acute haemorrhagic enteritis in dogs. Canine parvovirus is prone to genetic evolution and has undergone several mutations that produced different strains like CPV-2a, CPV-2b, New CPV-2a, New CPV-2b and CPV-2c in the past three decades. Mutations affecting the VP2 gene of CPV have been responsible for evolution of different antigenic variants. Sequence analysis of VP2 gene of the virus and subsequent characterization is important for molecular epidemiology. The present study was conducted to isolate and to characterize the virus by amplifying partial VP2 gene and further sequence analysis and also to estimate phylogenetic relationship of field virus with the reference strains. Out of 77 samples, 51 samples were found to be positive by PCR and all the 51 samples were subjected for virus isolation in CRFK cell line. Sixteen viruses could be isolated and 10 randomly selected isolates were subjected to sequence analysis along with four random clinical samples. All the 10 isolates and 4 clinical samples were characterized as New CPV-2a (CPV2a with 297-Ser→Ala). One of the field isolates was found to be phylogenetically closely related to New CPV-2a strains of Japan and India; another field isolates was found to share ancestral origins with New CPV-2a strains of Korea, USA, Italy, Brazil, Germany, Taiwan and Vietnam; rest other sequences had distinct lineage but shared molecular relationship with New CPV-2a reference strains.

  15. Molecular characterisation of Aspergillus flavus isolates from peanut fields in India using AFLP

    Directory of Open Access Journals (Sweden)

    Diwakar Singh

    2015-09-01

    Full Text Available Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84% was more than that between two populations (16%. The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.

  16. Metabolite identification and molecular fingerprint prediction through machine learning.

    Science.gov (United States)

    Heinonen, Markus; Shen, Huibin; Zamboni, Nicola; Rousu, Juho

    2012-09-15

    Metabolite identification from tandem mass spectra is an important problem in metabolomics, underpinning subsequent metabolic modelling and network analysis. Yet, currently this task requires matching the observed spectrum against a database of reference spectra originating from similar equipment and closely matching operating parameters, a condition that is rarely satisfied in public repositories. Furthermore, the computational support for identification of molecules not present in reference databases is lacking. Recent efforts in assembling large public mass spectral databases such as MassBank have opened the door for the development of a new genre of metabolite identification methods. We introduce a novel framework for prediction of molecular characteristics and identification of metabolites from tandem mass spectra using machine learning with the support vector machine. Our approach is to first predict a large set of molecular properties of the unknown metabolite from salient tandem mass spectral signals, and in the second step to use the predicted properties for matching against large molecule databases, such as PubChem. We demonstrate that several molecular properties can be predicted to high accuracy and that they are useful in de novo metabolite identification, where the reference database does not contain any spectra of the same molecule. An Matlab/Python package of the FingerID tool is freely available on the web at http://www.sourceforge.net/p/fingerid. markus.heinonen@cs.helsinki.fi.

  17. Molecular Identification of Human Fungal Pathogens

    Science.gov (United States)

    2014-05-01

    military environment are urgently needed. The objective of this proposal is to develop a rapid molecular diagnostic assay using a new PCR technology...called LATE- PCR (Linear-After-The-Exponential Polymerase Chain Reaction). This study has three aims: 1) selection of targets suitable for a multiplex PCR ...reaction, 2) design of primers and probes for each class of fungus, all of which will be compatible in a single reaction, and 3) pilot testing on

  18. Improving time in molecular identification of Mycosphaerella fijiensis ...

    African Journals Online (AJOL)

    cantocanche

    2012-04-24

    Apr 24, 2012 ... Direct colony polymerase chain reaction (PCR): An ... DC-PCR may also be applicable to prepare DNA templates for basic PCR- ... Abbreviations: ITS, Internal transcribed spacer; PCR, ... however, these structures are difficult to produce in .... Figure 2. DC-PCR for diagnostic molecular identification of M.

  19. Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus.

    Directory of Open Access Journals (Sweden)

    Jimei Du

    Full Text Available Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935 S. aureus isolates, including 28 (3.0%, 28/935 MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%, ampicillin/sulbactam (83.3% and trimethoprim/sulfamethoxazole (93.1%. 82%, (23/28 of the MRSA isolates and 66% (77/116 of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin--an agent commonly used for nasal decolonization. 16 different sequence types (STs, as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779 and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.

  20. Isolation and identification of sulfate reducing bacteria (SRB) from the sediment pond after a coal mine in Samarinda, East Kalimantan

    Science.gov (United States)

    Kusumawati, Eko; Sudrajat, Putri, Junita Susilaning

    2017-02-01

    Title isolation and identification of sulfate reducing bacteria (SRB) of sediment pond former coal mine in Samarinda, East Kalimantan. Sulfate reducing bacteria (SRB) is a group of microbes that can be used to improve the quality of sediment former coal mine. In the metabolic activities, the SRB can reduce sulfate to H2S which immediately binds to metals that are widely available on mined lands and precipitated in the form of metal sulfides reductive. Isolation and identification of sulfate reducing bacteria carried out in the Laboratory of Microbiology and Molecular Genetics, Faculty of Mathematics and Natural Sciences, University of Mulawarman, Samarinda. Postgate B is a liquid medium used for isolation through serial dilution. Physiological and biochemical characterization was done by Bergey's Manual of Determinative Bacteriology. Six isolates of sulfate reducing bacteria were isolated from the sediment pond former coal mine in Samarinda. Several groups of bacteria can grow at 14 days of incubation, however, another group of bacteria which takes 21 days to grow. The identification results showed that two isolates belong to the genus Desulfotomaculum sp., and each of the other isolates belong to the genus Desulfococcus sp., Desulfobacter sp., Desulfobulbus sp. and Desulfobacterium sp.

  1. Identification of trypanosomes isolated from reduviidae from north Chile.

    Science.gov (United States)

    Schaub, G A; Schottelius, J

    1984-01-01

    Examination of 54 Triatoma infestans from a village near the European Southern Observatory La Silla in Chile and of 9 Triatoma spinolai from the territory of the observatory showed that 10 T. infestans were infected with trypanosomatids. Mice were infected with in vitro cultures initiated with five different trypanosomatid isolates and treated with the immunosuppressive drug cyclophosphamide to increase the parasitemia of the flagellates. Evidence of the presence of T. cruzi was provided by a comparative biometrical analysis of blood trypomastigotes and the occurrence of intracellular amastigotes. Three methods for further identification were used: examination of kDNA ultrastructure, disc electrophoresis of soluble proteins and the Aaptos papillata II lectin induced agglutination. We obtained the following results for all isolates: (1) presence of a central band of the kDNA; (2) T. cruzi specific double bands of the protein patterns; (3) positive reaction with Aaptos papillata II. No differences between the five isolates from Chile and T. cruzi or T. cruzi-like strains from other countries could be observed. Based on these results an infection of the bugs with T. rangeli and T. conorhini could be excluded.

  2. Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates

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    Meng Dong-Ya

    2014-01-01

    Full Text Available To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs of DNA gyrase (gyrA and gyrB and topoisomerase IV (parC and parE in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX, intermediate resistant to Levofloxacin (LVX and Sparfloxacin (SFX, and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.

  3. Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates.

    Science.gov (United States)

    Meng, Dong-Ya; Sun, Chang-Jian; Yu, Jing-Bo; Ma, Jun; Xue, Wen-Cheng

    2014-01-01

    To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.

  4. Molecular characterization of Rhodococcus equi isolates in equines

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    Rabyia Javed

    2017-01-01

    Full Text Available Aim: The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra, molecular characterization and to determine the antibiotic resistance pattern of R. equi. Materials and Methods: A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. Conclusion: From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi.

  5. Molecular characterization of Rhodococcus equi isolates in equines.

    Science.gov (United States)

    Javed, Rabyia; Taku, A K; Sharma, R K; Badroo, Gulzaar Ahmed

    2017-01-01

    The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi.

  6. Molecular characterization of Rhodococcus equi isolates in equines

    Science.gov (United States)

    Javed, Rabyia; Taku, A. K.; Sharma, R. K.; Badroo, Gulzaar Ahmed

    2017-01-01

    Aim: The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. Materials and Methods: A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. Conclusion: From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi. PMID:28246441

  7. Selection, isolation, and identification of fungi for bioherbicide production.

    Science.gov (United States)

    Souza, Angélica Rossana Castro de; Baldoni, Daiana Bortoluzzi; Lima, Jessica; Porto, Vitória; Marcuz, Camila; Machado, Carolina; Ferraz, Rafael Camargo; Kuhn, Raquel C; Jacques, Rodrigo J S; Guedes, Jerson V C; Mazutti, Marcio A

    Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  8. Selection, isolation, and identification of fungi for bioherbicide production

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    Angélica Rossana Castro de Souza

    Full Text Available Abstract Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.

  9. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Science.gov (United States)

    2016-01-01

    Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate. PMID:27597865

  10. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

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    Faruku Bande

    2016-01-01

    Full Text Available Avian leukosis virus (ALV belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

  11. Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

    Science.gov (United States)

    Knolhoff, Ann M.; Zheng, Jie; McFarland, Melinda A.; Luo, Yan; Callahan, John H.; Brown, Eric W.; Croley, Timothy R.

    2015-08-01

    The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MSn spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

  12. Biochemical and Molecular Analysis of Staphylococcus aureus Clinical Isolates from Hospitalized Patients

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    Amit Karmakar

    2016-01-01

    Full Text Available Staphylococcus aureus is opportunistic human as well as animal pathogen that causes a variety of diseases. A total of 100 Staphylococcus aureus isolates were obtained from clinical samples derived from hospitalized patients. The presumptive Staphylococcus aureus clinical isolates were identified phenotypically by different biochemical tests. Molecular identification was done by PCR using species specific 16S rRNA primer pairs and finally 100 isolates were found to be positive as Staphylococcus aureus. Screened isolates were further analyzed by several microbiological diagnostics tests including gelatin hydrolysis, protease, and lipase tests. It was found that 78%, 81%, and 51% isolates were positive for gelatin hydrolysis, protease, and lipase activities, respectively. Antibiogram analysis of isolated Staphylococcus aureus strains with respect to different antimicrobial agents revealed resistance pattern ranging from 57 to 96%. Our study also shows 70% strains to be MRSA, 54.3% as VRSA, and 54.3% as both MRSA and VRSA. All the identified isolates were subjected to detection of mecA, nuc, and hlb genes and 70%, 84%, and 40% were found to harbour mecA, nuc, and hlb genes, respectively. The current investigation is highly important and informative for the high level multidrug resistant Staphylococcus aureus infections inclusive also of methicillin and vancomycin.

  13. Molecular identification of entomopathogenic Fusarium species associated with Tribolium species in stored grains.

    Science.gov (United States)

    Chehri, Khosrow

    2017-03-01

    Fusarium species are common pathogens of plants, animals and insects worldwide, including Iran. The occurrence of entomopathogenic Fusarium species isolated from Tribolium species as one of the most important insect pests of stored grains were sampled from various provinces in western Iran. In total, 15 Tribolium species belonging to T. castaneum (Herbst) and T. confusum (Du Val) (Col: Tenebrionidae) were detected and 8 isolates from Fusarium spp. were collected from them. Based on morphological features, the Fusarium isolates were classified into F. keratoplasticum and F. proliferatum. The phylogenetic trees based on tef1 dataset clearly separated all morphological taxa. DNA sequences of ITS regions and β-tubulin gene were also confirmed morphological taxa. All of the Fusarium isolates were evaluated for their pathogenicity on T. confusum. Maximum mortality rate was observed for F. keratoplasticum (isolate FSSCker2) and this isolate may be considered as a good candidate for biological control in the ecosystem of stored grains. This is the first report on molecular identification of Fusarium species isolated from insects in Iran and F. keratoplasticum and F. proliferatum were isolated for the first time from Tribolium species as two entomopathogenic fungi. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. PREVALENCE AND IDENTIFICATION OF VIBRIO SPP. ISOLATED ON AQUACULTURED GILTHEAD SEA BREAM

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    C. Scarano

    2011-01-01

    Full Text Available The aim of the study was to investigate the prevalence of Vibrio spp isolated from gilthead sea bream (Sparus aurata farmed on sea cages and to identify and characterize the pathogen by molecular techniques. Eighty fish were collected from two hatcheries located on the North-Est Sardinian Mediterranean coast, and microbiological analysis were performed on different body parts such as skin, gills, muscle and intestinal tract. Subsequently 100 pure colonies with typical morphology and phenotypic characteristics were selected and submitted to the molecular identification. The analysis on the prevalence of Vibrio spp showed the effect of the hatchery rearing system (P<0.001, of the date of sampling (P<0.001, and of the body part (P<0.001. All the strains selected were confirmed to be members of the genus Vibrio spp by the molecular method/techinique/identification, whereas the rpoA gene sequence analyses allowed to identify 89 strains belonging to the species Vibrio harveyi, 6 to V. diabolicus, 2 to V. parahaemolyticus and 1 to V. mediterranei.

  15. An evaluation of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Staphylococcus pseudintermedius isolates from canine infections.

    Science.gov (United States)

    Silva, Marcella Braga; Ferreira, Fabienne Antunes; Garcia, Luize Neli Nunes; Silva-Carvalho, Maria Cícera; Botelho, Larissa Alvarenga Batista; Figueiredo, Agnes Marie Sá; Vieira-da-Motta, Olney

    2015-03-01

    It has been proposed, based on taxonomic and molecular studies, that all canine isolates belonging to Staphylococcus intermedius group (SIG) should be renamed Staphylococcus pseudintermedius. However, isolates of SIG and other coagulase-positive staphylococci share many phenotypic characteristics, which could lead to misidentification. The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying S. pseudintermedius isolates obtained from canine infections was evaluated, using a polymerase chain reaction (PCR)-based identification as the gold standard. In addition, MALDI-TOF MS was compared with conventional biochemical tests. A central problem was the incorrect identification of S. pseudintermedius isolates as S. intermedius by either MALDI-TOF MS or biochemical identification. From the 49 S. pseudintermedius isolates identified by the molecular method, only 21 could be assigned to this species by the biochemical approach and only 12 by MALDI-TOF MS. The 6 S. aureus isolates were correctly identified by all 3 techniques. However, using biochemical tests, 9 S. pseudintermedius were mistakenly classified as S. aureus, indicating a reduced specificity relative to the MALDI-TOF MS system. Analysis with the MALDI-TOF MS platform allowed rapid and accurate identification of the 49 isolates to the S. intermedius group but the approach was very limited in identifying S. pseudintermedius isolates, as only 12 of 49 isolates were correctly identified, a sensitivity of 0.24 (95% confidence interval: 0.13-0.39).

  16. Sequence-identification of Candida species isolated from candidemia.

    Science.gov (United States)

    Fathi, Naeimeh; Mohammadi, Rasoul; Tabatabaiefar, Mohammad Amin; Ghahri, Mohammad; Sadrossadati, Seyedeh Zahra

    2016-01-01

    Candida species are the most prevalent cause of invasive fungal infections such as candidemia. Candidemia is a lethal fungal infection among immunocompromised patients worldwide. Main pathogen is Candida albicans but a global shift in epidemiology toward non-albicans species have reported. Species identification is imperative for good management of candidemia as a fatal infection. The aim of the study is to identify Candida spp. obtained from candidemia and determination of mortality rate among this population. The study was performed during February 2014 to March 2015 in Tehran, Iran. Two-hundred and four blood cultures were evaluated for fungal bloodstream infection. Identification of isolates was carried out using phenotypic tests and polymerase chain reaction sequencing technique. Twenty-two out of 204 patients (10.8%) had candidemia. Candida parapsilosis was the most prevalent species (45.4%), followed by C. albicans (31.8%) and Candida glabrata (22.7%). Male to female sex ratio was 8/14. The emergence of resistant strains of Candida species should be considered by physicians to decrease the mortality of this fatal fungal infection by appropriate treatment.

  17. Sequence-identification of Candida species isolated from candidemia

    Science.gov (United States)

    Fathi, Naeimeh; Mohammadi, Rasoul; Tabatabaiefar, Mohammad Amin; Ghahri, Mohammad; Sadrossadati, Seyedeh Zahra

    2016-01-01

    Background: Candida species are the most prevalent cause of invasive fungal infections such as candidemia. Candidemia is a lethal fungal infection among immunocompromised patients worldwide. Main pathogen is Candida albicans but a global shift in epidemiology toward non-albicans species have reported. Species identification is imperative for good management of candidemia as a fatal infection. The aim of the study is to identify Candida spp. obtained from candidemia and determination of mortality rate among this population. Materials and Methods: The study was performed during February 2014 to March 2015 in Tehran, Iran. Two-hundred and four blood cultures were evaluated for fungal bloodstream infection. Identification of isolates was carried out using phenotypic tests and polymerase chain reaction sequencing technique. Results: Twenty-two out of 204 patients (10.8%) had candidemia. Candida parapsilosis was the most prevalent species (45.4%), followed by C. albicans (31.8%) and Candida glabrata (22.7%). Male to female sex ratio was 8/14. Conclusions: The emergence of resistant strains of Candida species should be considered by physicians to decrease the mortality of this fatal fungal infection by appropriate treatment. PMID:27713871

  18. Reduced order observer based identification of base isolated buildings

    Institute of Scientific and Technical Information of China (English)

    Satish Nagarajaiah; Prasad Dharap

    2003-01-01

    The objective of this study is to identify system parameters from the recorded response of base isolated buildings, such as USC hospital building, during the 1994 Northridge earthquake. Full state measurements are not available for identification. Additionally, the response is nonlinear due to the yielding of the lead-rubber bearings. Two new approaches are presented in this paper to solve the aforementioned problems. First, a reduced order observer is used to estimate the unmeasured states. Second, a least squares technique with time segments is developed to identify the piece-wise linear system properties. The observer is used to estimate the initial conditions needed for the time segmented identification. A series of equivalent linear system parameters are identified in different time segments. It is shown that the change in system parameters, such as frequencies and damping ratios, due to nonlinear behavior of the lead-rubber bearings, are reliably estimated using the presented technique. It is shown that the response was reduced due to yielding of the lead-rubber bearings and period lengthening.

  19. Rapid molecular identification and characteristics of Lactobacillus strains.

    Science.gov (United States)

    Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

    2010-09-01

    Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

  20. Isolation, identification and antibiotic resistance of Campylobacter strains isolated from domestic and free-living pigeons.

    Science.gov (United States)

    Dudzic, A; Urban-Chmiel, R; Stępień-Pyśniak, D; Dec, M; Puchalski, A; Wernicki, A

    2016-04-01

    1. The aim of this study was to evaluate the occurrence of Campylobacter spp. in domestic and free-living pigeons and to evaluate the antibiotic resistance profiles. 2. The material consisted of cloacal swabs obtained from 108 homing pigeons and fresh faeces from 72 wild birds from Lublin and its vicinity. The identification of strains isolated on differential/selective media for Campylobacter spp. was carried out by MALDI-TOF and PCR. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in Mueller-Hinton broth. 3. A total of 35 strains of Campylobacter spp. were isolated; 27 were identified as Campylobacter jejuni and 8 as Campylobacter coli. Over half of the isolates were resistant to erythromycin and streptomycin, 40% of strains were resistant to tetracycline and ampicillin and 37% isolates were resistant to amoxicillin. Resistance to two or more antibiotics was observed in all strains tested. 4. The results indicate that both domestic and free-living pigeons are reservoirs for bacteria of the genus Campylobacter, which are characterised by varied and growing resistance to commonly used antibiotics.

  1. Identification of lead- resistant endophytic bacteria isolated from rice.

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    Alexander Pérez-Cordero

    2015-06-01

    Full Text Available   The objective of this study was to evaluate in vitro the endophytic bacteria resistance to different lead concentrations. The sampling was undertaken in the first half of 2013, when tissue samples of commercial varieties of rice at tillering stage were collected in Montería, Cordoba, Colombia. Each tissue was subjected to surface cleaning. Endophytic bacteria in agar R2A medium were isolated. Population density (CFU/g tissue was determined from each tissue, by direct counting of R2A medium surface. morphotypes were classified by shape, color, size, and appearance. A total of 168 morphotypes were isolated from root, tillers, and leaf of different commercial varieties of rice. The lead resistance test was performed in vitro, to do that, suspensions of endophytic bacteria in log phase were prepared and inoculated in minimal medium with five concentrations of lead as Pb(NO32. The experiment was incubated at 32 °C and agitated at 150 rpm, for five days. Every hour afterstarting the test, turbidimetry measuring at 600 nm was conducted. Results showed the ability of endophytic bacteria to grow at concentrations of 100% of Pb as Pb(NO32. The results of the identification with kit API20E confirmed the presence of Burkholderia cepacia and Pseudomonas putida, which showed resistance to different lead concentrations.

  2. Study of the Mechanical Properties and Vibration Isolation Performance of a Molecular Spring Isolator

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    Muchun Yu

    2016-01-01

    Full Text Available Molecular Spring Isolator (MSI is a novel passive vibration isolation technique, providing High-Static-Low-Dynamic (HSLD stiffness based on the use of molecular spring material. The molecular spring material is a solid-liquid mixture consisting of water and hydrophobic nanoporous materials. Under a certain level of external pressure, water molecules can intrude into the hydrophobic pores of nanoporous materials, developing an additional solid-liquid interface. Such interfaces are able to store, release, and transform mechanical energy, providing properties like mechanical spring. Having been only recently developed, the basic mechanic properties of a MSI have not been studied in depth. This paper focuses on the stiffness influence factors, the dynamic frequency response, and the vibration isolation performance of a MSI; these properties help engineers to design MSIs for different engineering applications. First, the working mechanism of a MSI is introduced from a three-dimensional general view of the water infiltration massive hydrophobic nanoporous pores. Next, a wide range of influence factors on the stiffness properties of MSI are studied. In addition, the frequency response functions (FRFs of the MSI vibration isolation system are studied utilizing the matching method based on equivalent piecewise linear (EPL system. Finally, the vibration isolation properties of MSI are evaluated by force transmissibility.

  3. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    Science.gov (United States)

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  4. Molecular characterization of Bovine herpesvirus type 1 Indonesian isolates

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    Muharam Saepulloh

    2009-03-01

    Full Text Available Different subtypes of bovine herpesvirus 1 (BHV-1 have been associated with different clinical conditions of cattle. For that reason subtypes differentiation has become an essential tool for understanding the pathogenesis and epidemiology of BHV infections. In search for a genomic region that would allow a clear distinction between BHV-1.1 and BHV-1.2 of glycoprotein D (gD genes of 8 Indonesian isolates were amplified and sequenced. The amino acid sequence alignments revealed that the levels of genomic similarity ranging from 98.8 to 100% among BHV-1 Indonesian isolates and its results were also similar between BHV-1 Indonesia isolates and BHV-1.1 reference, and 98.4 to 98.8% between BHV-1 Indonesian isolates and BHV-1.2 reference. The isolates could be clearly separated into BHV-1.1 and BHV-1.2 after phylogenetic analysis. The results showed that the Indonesian isolates were characterized as BHV-1.1 as agent caused respiratory tract infections in cattle or infectious bovine rhinotracheitis (IBR disease. The results suggest that the phylogenetic analysis performed here can be used as a potential molecular epidemiological tool for herpesviruses.

  5. Molecular analysis of Korean isolate of barley yellow mosaic virus.

    Science.gov (United States)

    Lee, Kui Jae; Choi, Min Kyung; Lee, Wang Hyu; Rajkumar, Mani

    2006-04-01

    The complete sequences of both RNAs of an isolate of barley yellow mosaic virus (BaYMV) from Haenam, Korea, were determined. RNA1 is 7639 nucleotides long [excluding the 3'-poly(A)], and codes for a 270 kDa polyprotein of 2411 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3582 nucleotides long and codes for a polyprotein of 890 amino acids, which contains a 28 kDa putative proteinase (P1) and a 73 kDa polypeptide (P2). The whole sequences of Korean isolate (BaYMV-K) closely resembled those of an isolate from Japan (BaYMV-J) (99.6 identical nucleotides for RNA1; 99.4 for RNA2) and china (BaYMV-C) (96.7 and 96.2%, respectively) than from Germany (BaYMV-G) (93.6 and 90.4%, respectively). The greatest differences between the BaYMV-K and BaYMV-J isolates were in the 3'-NCRs of RNA1 and 5' NCRs of RNA2 and there were also some other regions of difference in Nib Pro (RNA1) and P1 (RNA2). Further, the phylogenetic analysis of CP region showed that Asian and European isolates formed distinct clusters. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.

  6. Molecular Identification of SHV,TEM, CTX-M β lactamases Genes and Antibiotics Resistance Pattern of k.pneumoniae Isolates Collected from ICU Patients of Namazi Hospital, Shiraz, Iran

    Directory of Open Access Journals (Sweden)

    T Archin

    2014-01-01

    Full Text Available Abstract Background and aim: β-lactamase enzymes producing bacteria ESBL have spread widely throughout the world. The production of enzymes induces bacterial resistance to a wide range of antibiotics which is leading to the limitation of infection control and correct treatment. The aim of the present study was to investigate patterns of antibiotic susceptibility to antibiotics and the presence of β-lactamase genes SHV, TEM, CTX-M, in Klebsiella pneumoniae isolates from clinical specimens of intensive care. Methods: Susceptibility of isolated bacteria against 10 antibiotics was determined by agar disk diffusion method according to the CLSI guidelines. The strains (DDST were examined for the presence of the spectrum β-lactamase enzymes. Using E-test, the minimum inhibitory concentration (MIC of the antibiotic was determined to cefotaxime. Moreover, SHV, TEM, CTX-M genes were identified by, Multiplex PCR method, and some of them were sequenced. Results: The antibiotic resistance against 10 antibiotics was determined. The highest percentage of isolates was resistant to ampicillin (100% and sensitivity to imipenem was 1.66%. In this study, the majority of strains produced ESBL (60%. TEM gene in 34.38% and all three genes (TEM and SHV and CTX at 33.13% of isolates were observed. Conclusion: The present study showed that the K. pneumoniae producing ESBL in patients in ICU are common. Therefore, the use of procedures and policies for infection control in hospitals and especially ICU is necessary. Key words: Klebsiella pneumoniae, ESBL, Multiplex PCR, antibiotic sensitivity

  7. Viridans Group Streptococci clinical isolates: MALDI-TOF mass spectrometry versus gene sequence-based identification.

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    Silvia Angeletti

    Full Text Available Viridans Group Streptococci (VGS species-level identification is fundamental for patients management. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS has been used for VGS identification but discrimination within the Mitis group resulted difficult. In this study, VGS identifications with two MALDI-TOF instruments, the Biotyper (Bruker and the VITEK MS (bioMérieux have been compared to those derived from tuf, soda and rpoB genes sequencing. VGS isolates were clustered and a dendrogram constructed using the Biotyper 3.0 software (Bruker. RpoB gene sequencing resulted the most sensitive and specific molecular method for S. pneumonia identification and was used as reference method. The sensitivity and the specificity of the VITEK MS in S. pneumonia identification were 100%, while the Biotyper resulted less specific (92.4%. In non pneumococcal VGS strains, the group-level correlation between rpoB and the Biotyper was 100%, while the species-level correlation was 61% after database upgrading (than 37% before upgrading. The group-level correlation between rpoB and the VITEK MS was 100%, while the species-level correlation was 36% and increases at 69% if isolates identified as S. mitis/S. oralis are included. The less accurate performance of the VITEK MS in VGS identification within the Mitis group was due to the inability to discriminate between S. mitis and S. oralis. Conversely, the Biotyper, after the release of the upgraded database, was able to discriminate between the two species. In the dendrogram, VGS strains from the same group were grouped into the same cluster and had a good correspondence with the gene-based clustering reported by other authors, thus confirming the validity of the upgraded version of the database. Data from this study demonstrated that MALDI-TOF technique can represent a rapid and cost saving method for VGS identification even within the Mitis group but improvements of spectra

  8. Identification of Molecular Laser Transitions Using the Finite Element Method

    Science.gov (United States)

    1995-12-01

    Vibration Levels," Physical Review, Vol. 34, 1929 12 Arfken , George Mathematical Methods for Physicists. San Diego: Academic Press, Inc. 1985 13...unsolvable. Mathematical techniques such as the classical Rayleigh-Ritz method , variational calculus, and Galerkin’s weighted residuals method , much...AFIT/GAP/ENP/95D-14 IDENTIFICATION OF MOLECULAR LASER TRANSITIONS USING THE FINITE ELEMENT METHOD THESIS Matthew C. Smitham, Captain, USAF AFIT/GAP

  9. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  10. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

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    Tongjit Thanchomnang

    Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  11. Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

    Science.gov (United States)

    Kano, Rui; Okubo, Miki; Siew, Han Hock; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2015-04-01

    Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia.

  12. Molecular identification of four phenotypes of human Demodex in China.

    Science.gov (United States)

    Hu, Li; Zhao, Ya-E; Cheng, Juan; Ma, Jun-Xian

    2014-07-01

    Traditional classification of Demodex mites by hosts and phenotypic characteristics is defective because of environmental influences. In this study, we proposed molecular identification of four phenotypes of two human Demodex species based on mitochondrial cox1 fragments for the first time. Mites collected from sufferers' facial skin were classified into four phenotypes: phenotype A-C with finger-like terminus, and phenotype D with cone-like terminus. The results of molecular data showed that cox1 sequences were all 429 bp. Divergences, genetic distances and transition/transversion ratios among the three phenotypes with finger-like terminus were 0.0-3.0%, 0.000-0.031, and 6/3-5/0, respectively, in line with intraspecific differences. However, those measures between the phenotype with cone-like terminus and phenotypes with finger-like terminus were 19.6-20.5%, 0.256-0.271, and 0.58 (31/53)-0.66 (35/53), respectively, reaching interspecific level. Phylogenetic trees also showed that the three phenotypes with finger-like terminus clustered as one clade, and the phenotype with cone-like terminus formed another one. Therefore, we conclude that mitochondrial cox1 sequence is a good marker for identification of two human Demodex species. Molecular data indicate no subspecies differentiation. Terminus is an effective character for species identification. Mites with finger-like terminus are Demodex folliculorum, and those with cone-like terminus are Demodex brevis.

  13. MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

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    Patricia de Souza Bonfim-Mendonca

    2013-12-01

    Full Text Available SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

  14. An insight into the isolation, enumeration and molecular detection of Listeria monocytogenes in food

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    Jodi Woan-Fei Law

    2015-11-01

    Full Text Available Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria Enrichment Broth (BLEB, Fraser broth and University of Vermont Medium (UVM Listeria enrichment broth are recommended by regulatory agencies such as FDA-BAM, USDA-FSIS and ISO. Many plating media are available for the isolation of L. monocytogenes, for instance, PALCAM, Oxford and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. MPN technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction (PCR, multiplex polymerase chain reaction (mPCR, real-time/quantitative polymerase chain reaction (qPCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP, DNA microarray and Next Generation Sequencing (NGS technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labour-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features.

  15. An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are

  16. Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

    Directory of Open Access Journals (Sweden)

    Andréia Henzel

    2012-06-01

    Full Text Available Feline calicivirus (FCV and feline herpesvirus type 1 (FHV-1 are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55 of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55 of the animals that tested positively, and co-infection were detected in 9.1% (5/55 of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.

  17. Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

    Science.gov (United States)

    Henzel, Andréia; Brum, Mário Celso Sperotto; Lautert, Cláudia; Martins, Mathias; Lovato, Luciane Teresinha; Weiblen, Rudi

    2012-01-01

    Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary. PMID:24031864

  18. Principal methods for isolation and identification of soil microbial communities.

    Science.gov (United States)

    Stefanis, Christos; Alexopoulos, Athanasios; Voidarou, Chrissa; Vavias, Stavros; Bezirtzoglou, Eugenia

    2013-01-01

    Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes.

  19. 山东地区新城疫病毒分离株的分离鉴定及其F基因的分子特性分析%Isolation,Identification and Molecular Characterization for F Gene of Newcastle Disease Virus Isolated in Shandong

    Institute of Scientific and Technical Information of China (English)

    杨金兴; 朱瑞良

    2013-01-01

      2011年从山东不同地区的发病鸡体内分离到4株新城疫病毒,分别命名为SD1~SD4。4株毒株经病毒蚀斑克隆纯化后动物回归试验可产生典型的NDV病变;为了解其遗传起源和分子特性对其融合蛋白(Fusion,F)进行序列和分子特性的分析。研究结果发现,生物学毒力测定显示4株毒株均为强毒。 F蛋白多肽裂解位点的序列为RRQKRF 117,符合NDV强毒氨基酸基序。 F基因分型显示,SD01、SD02、SD03和SD04与另外一株鸡源SG01以及广东鹅分离株等属于基因Ⅶ型。同源性比较显示:SD1-SD4与同期分离株的同源性明显高于其他的传统毒株,且与国内分离株的同源性高于国外的分离株。%Four Newcastle Disease Virus isolates named SD 01-SD04 were collected from chicken in Shandong in 2011.The four strains were purified by plaque assay can form typical cytopathic .The F genes of the isolates were cloned and sequenced to study its molecular characterization and hereditary variation .The deduced amino acid se-quence of F gene showed that these viruses shared a velogenic motif 112 RRQKRF 117 at the cleavage site which were coincident with biological index .Phylogenetic analysis showed that SD 01-SD04 and the viruses derived from chicken and goose in China belonged to genotype Ⅶ.For F gene of the isolates, the homologies of the deduced amino acid se-quences of SD01-SD04 with the same time isolates in China were higher compared with traditional trains .There was a closer genetic relationship between the four isolates and epidemic strains in China than foreign epidemic strains .

  20. Isolation, identification and molecular docking as cyclooxygenase (COX) inhibitors of the main constituents of Matricaria chamomilla L. extract and its synergistic interaction with diclofenac on nociception and gastric damage in rats.

    Science.gov (United States)

    Ortiz, Mario I; Fernández-Martínez, Eduardo; Soria-Jasso, Luis Enrique; Lucas-Gómez, Isaac; Villagómez-Ibarra, Roberto; González-García, Martha P; Castañeda-Hernández, Gilberto; Salinas-Caballero, Mireya

    2016-03-01

    Chamomile (Matricaria chamomilla L., Asteraceae) is a medicinal plant widely used as remedy for pain and gastric disorders. The association of non-steroidal anti-inflammatory drugs (NSAIDs) with medicinal plant extracts may increase its antinociceptive activity, permit the use of lower doses and limit side effects. The aim was to isolate and identify the main chemical constituents of Matricaria chamomilla ethanolic extract (MCE) as well as to explore their activity as cyclooxygenase (COX) inhibitors in silico; besides, to examine the interaction between MCE and diclofenac on nociception in the formalin test by isobolographic analysis, and to determine the level of gastric injury in rats. Three terpenoids, α-bisabolol, bisabolol oxide A, and guaiazulene, were isolated and identified by (1)H NMR. Docking simulation predicted COX inhibitory activity for those terpenoids. Diclofenac, MCE, or their combinations produced an antinociceptive effect. The sole administration of diclofenac and the highest combined dose diclofenac-MCE produced significant a gastric damage, but that effect was not seen with MCE alone. An isobologram was constructed and the derived theoretical ED35 for the antinociceptive effect was significantly different from the experimental ED35; hence, the interaction between diclofenac and MCE that mediates the antinociceptive effect is synergist. The MCE contains three major terpenoids with plausible COX inhibitory activity in silico, but α-bisabolol showed the highest affinity. Data suggest that the diclofenac-MCE combination can interact at the systemic level in a synergic manner and may have therapeutic advantages for the clinical treatment of inflammatory pain.

  1. Molecular identification of indigenous manganese solubilising bacterial biodiversity from manganese mining deposits.

    Science.gov (United States)

    Ghosh, Shreya; Mohanty, Sansuta; Nayak, Sanghamitra; Sukla, Lala B; Das, Alok P

    2016-03-01

    Manganese (Mn) ranks twelfth among the most exuberant metal present in the earth's crust and finds its imperative application in the manufacturing steel, chemical, tannery, glass, and battery industries. Solubilisation of Mn can be performed by several bacterial strains which are useful in developing environmental friendly solutions for mining activities. The present investigation aims to isolate and characterize Mn solubilising bacteria from low grade ores from Sanindipur Manganese mine of Sundargh district in Odisha state of India. Four morphologically distinct bacterial strains showing visible growth on Mn supplemented plates were isolated. Mn solubilising ability of the bacterial strains was assessed by visualizing the lightening of the medium appearing around the growing colonies. Three isolates were gram negative and rod shaped while the remaining one was gram positive, coccobacilli. Molecular identification of the isolates was carried out by 16S rRNA sequencing and the bacterial isolates were taxonomically classified as Bacillus anthrasis MSB 2, Acinetobacter sp. MSB 5, Lysinibacillus sp. MSB 11, and Bacillus sp. MMR-1 using BLAST algorithm. The sequences were deposited in NCBI GenBank with the accession number KP635223, KP635224, KP635225 and JQ936966, respectively. Manganese solubilisation efficiency of 40, 96, 97.5 and 48.5% were achieved by MMR-1, MSB 2, MSB 5 and MSB 11 respectively. The efficiency of Mn solubilisation is suggested with the help of a pH variation study. The results are discussed in relation to the possible mechanisms involved in Manganese solubilisation efficiency of bacterial isolates.

  2. Molecular detection of an atypical, highly resistant, clonal Pseudomonas aeruginosa isolate in cystic fibrosis patients.

    LENUS (Irish Health Repository)

    Keating, Deirdre

    2013-03-01

    The identification of Pseudomonas aeruginosa (P. aeruginosa) isolates in sputum from cystic fibrosis (CF) patients can be challenging due to the multitude of phenotypic changes isolates undergo during adaptation to the microenvironment of the CF lung.

  3. Classical and Molecular Identification of Thermotolerant Campylobacters from Poultry Meat

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    Tina Zorman

    2002-01-01

    Full Text Available Poultry meat samples from Slovenian retail market were examined for the presence of thermotolerant campylobacters. The isolates were identified by phenotypic and genotypic methods. ISO 10272 recommendations were followed for phenotypic identification. Different PCR assays, targeting species specific DNA regions in C. jejuni and C. coli, were checked for their applicability in identification. High degree of tested samples was positive (27/33, with significant proportion of C. coli (32 % among identified strains. High percentage of C. jejuni strains (54 % were hippurate negative. Phenotypic identification was therefore found to be inconvenient because of the presence of the strains with atypical phenotype and possible misinterpretation of test results. Multiplex PCR, targeting hippuricase gene in C. jejuni and species specific region in C. coli, was found to be an efficient method that allowed fast, simple and accurate identification of C. jejuni and C. coli. FlaA PCR is a reliable method to identify the group C. jejuni/C. coli, but it does not differentiate between the two species. CdtB PCR is inconvenient because of many false negative and some false positive results.

  4. [Morphological and molecular characterization of isolates of Macrophomina phaseolina associated with sugarcane in Mexico].

    Science.gov (United States)

    Leyva-Mir, Santos G; Velázquez-Martínez, Guadalupe C; Tlapal-Bolaños, Bertha; Tovar-Pedraza, Juan M; Rosas-Saito, Greta H; Alvarado-Gómez, Omar G

    2015-01-01

    Charcoal rot caused by Macrophomina phaseolina is an important disease of sugarcane in Mexico. This study was carried out to characterize isolates of M. phaseolina obtained from sugarcane by the combination of morphological and molecular analyses. The morphological characterization of 10 isolates was performed using scanning electron microscopy and light microscopy. To confirm the morphological identification, rDNA from two representative isolates was extracted, and the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction and sequenced using specific primers MpKF1 and MpKR1. Based on their morphological characteristics, all isolates were identified as M. phaseolina. Moreover, the analysis of two ITS sequences showed 100% similarity with the M. phaseolina sequences deposited in the GenBank. To our knowledge, this is the first study in the world aimed at characterizing isolates of M. phaseolina obtained from sugarcane. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. Improvement in Isolation and Identification of Mouse Oogonial Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhiyong Lu

    2016-01-01

    Full Text Available Female germline stem cells (FGSCs or oogonial stem cells (OSCs have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS. The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse.

  6. Isolation and Identification of L-asparaginase producing Erwinia strains which isolated from Potato Farms

    Directory of Open Access Journals (Sweden)

    Arastoo Badoei-Dalfard

    2016-09-01

    Full Text Available Introduction: L-Asparaginase can be effectively used for the treatment of lymphoblastic leukemia. The rapid growth of cancer cells are needed for L-asparagine abundant storage. L-asparaginase catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. The purpose of this study was to isolate and identify the L-asparaginase producing Erwinia strains from the potato farms of Jiroft. Materials and methods: Pectolytic Erwinia species isolated from crumbling potato in M9 medium. The desired L-asparaginase producing bacteria were isolated based on the color changes. Biochemical-microbial and the plant pathogenicity tests of these strains were also investigated with potato and geranium. The L-asparaginase production and molecular detection of these Erwinia strains were also investigated. Results: In this study, L-asparaginase producing Erwinia was isolated on the CVP and M9 mediums. The inoculation of Erwinia strains on the potato and geranium plants showed that Er8 and Er11 species have the ability to cause plant pathogenicity. Results showed that the maximum pathogenicity of Er8 and Er11 was observed after 48 and 15 h of inoculation in potato and geranium plants, respectively. 16S rDNA sequencing and phylogenetic analyses exhibited that Er8 and Er11 strains were similar to Erwinia chrysanthemi with 98% homology. Discussion and conclusion: Because of several applications of the Erwinia L-asparaginase in various fields, isolated Erwinia and their L-asparaginase can be suitable for applied utilization.

  7. Isolation and identification of Bacillus spp. from compost material, compost and mushroom casing soil active against Trichoderma spp.

    Directory of Open Access Journals (Sweden)

    Stanojević Olja

    2016-01-01

    Full Text Available The isolation of bacteria was carried out from samples of straw and chicken manure, compost at various stages of the composting process and casing soil used for growing button mushrooms. A preliminary screening of 108 bacterial isolates for antagonistic activity against Trichoderma aggressivum f. europaeum showed that 23 tested isolates inhibited mycelial growth of the pathogenic fungus. Further screening with four indicator isolates of fungi revealed that all 23 bacterial isolates inhibited the growth of T. aggressivum f. europaeum, T. harzianum and T. koningii, while only 13 isolates inhibited the growth of T. atroviride. T. aggressivum f. europaeum proved to be the most sensitive, with many bacterial isolates generating a high percentage of growth inhibition. Only two bacterial isolates (B-129 and B-268 were successful in inhibiting the growth of all 4 tested pathogens. All 23 bacterial isolates were characterized as Gram-positive and catalase-positive and were subjected to molecular identification based on the partial sequence, the hypervariant region of the 16S rDNA. It was shown that the obtained bacterial strains belong to Bacillus subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus species. [Projekat Ministarstva nauke Republike Srbije, br. 31043 i br. 173026

  8. Molecular identification of Leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of Iran.

    Science.gov (United States)

    Eslami, Gilda; Hajimohammadi, Bahador; Jafari, Abbas Ali; Mirzaei, Farzaneh; Gholamrezai, Mostafa; Anvari, Hossein; Khamesipour, Ali

    2014-12-01

    The most common form of the disease is cutaneous leishmaniasis (CL) which is a public health and social problem in many countries especially Iran. In endemic areas where other diseases with similar clinical symptoms occur, definitive diagnosis of CL is very important. The detection and identification of Leishmania in infected patients is crucial for achieving a correct treatment and prognosis. To our knowledge, this is the first comprehensive study in terms of geographical distribution and molecular identification of Leishmania tropica isolates in central of Iran. This study was performed between 2010 and 2011, during which 218 CL suspected patients referred to Shahid Sadoughi University of Medical Sciences in Yazd, Iran for confirmation were examined. After microscopic analysis, DNA extraction was performed for identification. The molecular target region was ITS1 gene. Results showed that out of 218 isolates, 102 (46.8%) samples were positive for Leishman body using molecular assay. After PCR-RFLP, analysis identified 50 (49.01%) samples as L. major and 52 (50.98%) as L. tropica. Two samples showed a different pattern that were reported as unknown. Among L. tropica, six different isolates were identified in this endemic area. Finally, this study showed heterozygosity among L. tropica isolates in this endemic area such as some other studies from the world. This heterozygosity among the strains may suggest a sexual recombination or genetic exchange between strains.

  9. Identification and Characterization of Imipenem-Resistant Klebsiella pneumoniae and Susceptible Klebsiella variicola Isolates Obtained from the Same Patient.

    Science.gov (United States)

    Garza-Ramos, Ulises; Moreno-Dominguez, Stephania; Hernández-Castro, Rigoberto; Silva-Sanchez, Jesús; Barrios, Humberto; Reyna-Flores, Fernando; Sanchez-Perez, Alejandro; Carrillo-Casas, Erika M; Sanchez-León, María Carmen; Moncada-Barron, David

    2016-04-01

    Klebsiella variicola, a bacterium closely genetically related to Klebsiella pneumoniae, is commonly misidentified as K. pneumoniae by biochemical tests. To distinguish between the two bacteria, phylogenetic analysis of the rpoB gene and the identification of unique genes in both bacterial species by multiplex-polymerase chain reaction (PCR) provide the means to reliably identify and genotype K. variicola. In recent years, K. variicola has been described both as the cause of an intrahospital outbreak in a pediatric hospital, which resulted in sepsis in inpatients, and as a frequent cause of bloodstream infections. In the present study, K. pneumoniae and K. variicola were isolated from a unique patient displaying different antimicrobial susceptibility phenotypes and different genotypes of virulence determinants. Eight clinical isolates were obtained at different time intervals; all during a 5-month period. The isolates were identified as K. pneumoniae by an automated identification system. The clinical (biochemical test) and molecular (multiplex-PCR and rpoB gene) characterization identified imipenem resistance in the first six K. pneumoniae ST258 isolates, which encode the SHV-12 cephalosporinase and KPC-3 carbapenemase genes. The two last remaining isolates corresponded to susceptible K. variicola. The bacterial species showed a specific profile of virulence-associated determinants, specifically the fimA, fimH, and ecpRAB fimbrial-encoding genes identified only in K. pneumoniae isolates. However, the entb (enterobactin), mrkD (fimbrial adhesin), uge (epimerase), ureA (urease), and wabG (transferase) genes were shared between both bacterial species. Recent studies attribute a higher mortality rate to K. variicola than to K. pneumonia. This work highlights the identification of K. pneumoniae and the closely related K. variicola isolated from the same patient. The value of distinguishing between these two bacterial species is in their clinical significance, their

  10. Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk.

    Science.gov (United States)

    Pilla, Rachel; Snel, Gustavo G M; Malvisi, Michela; Piccinini, Renata

    2013-05-01

    Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase-or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or-negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it

  11. Molecular epidemiology of clinical isolates of Pseudomonas aeruginosa isolated from horses in Ireland

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    Tazumi A

    2009-07-01

    Full Text Available Abstract Clinical isolates (n = 63 of Pseudomonas aeruginosa obtained from various sites in 63 horses were compared using ERIC2 RAPD PCR to determine their genetic relatedness. Resulting banding patterns (n = 24 genotypes showed a high degree of genetic heterogeneity amongst all isolates examined, indicating a relative non-clonal relationship between isolates from these patients, employing this genotyping technique. This study characterised 63 clinical isolates into 24 distinct genotypes, with the largest cluster (genotype E accounting for 10/63 (15.9% of the isolates. ERIC2 RAPD PCR proved to be a highly discriminatory molecular typing tool of P. aeruginosa in isolates recovered from horses. With the adoption of several controls to aid reproducibility, this technique may be useful as an alternative to PFGE, particularly in epidemiological investigations of outbreaks where speed may be a significant parameter. This is the first report of clonal heterogeneity amongst P. aeruginosa from horses and demonstrated that ERIC RAPD PCR is a rapid method for the examination of this species in horses, which may be useful in outbreak analysis.

  12. Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum.

    Science.gov (United States)

    Malandrakis, Anastasios A; Markoglou, Anastasios N; Konstantinou, Sotiris; Doukas, Eleftherios G; Kalampokis, John F; Karaoglanidis, George S

    2013-04-01

    Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the β-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the

  13. Isolation and Identification of Microorganisms in JSC Mars-1 Simulant Soil

    Science.gov (United States)

    Mendez, Claudia; Garza, Elizabeth; Gulati, Poonam; Morris, Penny A.; Allen, Carlton C.

    2005-01-01

    Microorganisms were isolated and identified in samples of JSC Mars-1, a Mars simulant soil. JSC Mars-1 is an altered volcanic ash from a cinder cone south of Mauna Kea, Hawaii. This material was chosen because of its similarity to the Martian soil in physical and chemical composition. The soil was obtained by excavating 40 cm deep in a vegetated area to prevent contamination. In previous studies, bacteria from this soil has been isolated by culturing on different types of media, including minimal media, and using biochemical techniques for identification. Isolation by culturing is successful only for a small percentage of the population. As a result, molecular techniques are being employed to identify microorganisms directly from the soil without culturing. In this study, bacteria were identified by purifying and sequencing the DNA encoding the 16s ribosomal RNA (16s rDNA). This gene is well conserved in species and demonstrates species specificity. In addition, biofilm formation, an indicator of microbial life, was studied with this soil. Biofilms are microbial communities consisting of microbes and exopolysaccharides secreted by them. This is a protective way of life for the microbes as they are more resistant to environmental pressures.

  14. Rapid Isolation and Molecular Detection of Streptomycin-Producing Streptomycetes

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    M Motovali-bashi

    2006-07-01

    Full Text Available Introduction: Streptomyces species are mycelial, aerobic gram-positive bacteria that are isolated from soil and produce a diverse range of antibiotics. Streptomyces griseus produces the antibiotic, streptomycin and forms spores even in a liquid culture. The gene cluster for the production of Streptomycin antibiotic contains strR gene that encodes StrR, a pathway-specific regulator. Then, this pathway-specific regulator induces transcription of other streptomycin production genes in the gene cluster. The overall aim of this work was rapid isolation and molecular detection of streptomycin-producing Streptomycetes, especially S. griseus, from Iranian soils in order to manipulate them for increased production of streptomycin. Methods: This research used new initiative half-specific medium for isolation of Streptomycetes from natural environments, called FZmsn. The fifty colonies of Streptomyces strains grown on the surface of FZmsn medium isolated from environmental samples were defined on the basis of their morphological characteristics and light microscope studies. A set of primers was designed to detect strR by OLIGO software. Results: In colony-PCR reactions followed by gel electrophoresis, 6 colonies from Streptomyces strains colonies were detected as S. griseus colonies. Conclusion: These native Streptomyces strains will be used for genetic manipulation of S. griseus in order to increase production levels of streptomycin.

  15. Identification and characterisation of oil sludge degrading bacteria isolated from compost

    Directory of Open Access Journals (Sweden)

    Ubani Onyedikachi

    2016-06-01

    Full Text Available Compounds present in oil sludge such as polycyclic aromatic hydrocarbons (PAHs are known to be cytotoxic, mutagenic and potentially carcinogenic. Microorganisms including bacteria and fungi have been reported to degrade oil sludge components to innocuous compounds such as carbon dioxide, water and salts. In the present study, we isolated different bacteria with PAH-degrading capabilities from compost prepared from oil sludge and animal manures. These bacteria were isolated on a mineral base medium and mineral salt agar plates. A total of 31 morphologically distinct isolates were carefully selected from 5 different compost treatments for identification using polymerase chain reaction (PCR of the 16S rRNA gene with specific primers (universal forward 16S-P1 PCR and reverse 16S-P2 PCR. The amplicons were sequenced and sequences were compared with the known nucleotides from the GenBank. The phylogenetic analyses of the isolates showed that they belong to 3 different clades; Firmicutes, Proteobacteria and Actinobacteria. These bacteria identified were closely related to the genera Bacillus, Arthrobacter, Staphylococcus, Brevibacterium, Variovorax, Paenibacillus, Ralstonia and Geobacillus. The results showed that Bacillus species were predominant in all composts. Based on the results of the degradation of the PAHs in the composts and results of previous studies on bacterial degradation of hydrocarbons in oil, the characteristics of these bacterial isolates suggests that they may be responsible for the breakdown of PAHs of different molecular weights in the composts. Thus, they may be potentially useful for bioremediation of oil sludge during compost bioremediation.

  16. Soy protein isolate molecular level contributions to bulk adhesive properties

    Science.gov (United States)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  17. Identification of Malassezia Species Isolated from Patients with Pityriasis Versicolor Using PCR-RFLP Method in Markazi Province, Central Iran.

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    Mojtaba Didehdar

    2014-05-01

    Full Text Available The lipophilic yeasts of Malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor. Pityriasis versicolor is a common superficial fungal infection with world-wide distribution. The phenotypic methods for identification of Malassezia species usually are time consuming and unreliable to differentiate newly identified species. But DNA-based techniques rapidly and accurately identified Malassezia species. The purpose of this study was isolation and identification of Malassezia Species from patients with pityriasis versicolor by molecular methods in Markazi Province, Central Iran in 2012.Mycologic examinations including direct microscopy and culture were performed on clinical samples. DNA extraction was performed from colonies. The ITS1 region of rDNA from isolates of Malassezia species were amplified by PCR reaction. The PCR were digested by Cfo I enzyme.From 70 skin samples, were microscopically positive for Malassezia elements, 60 samples were grown on culture medium (85.7%. Using PCR-RFLP method, that was performed on 60 isolates, 37(61.6% M. globosa, 14(23.3% M. furfur, 5(8.4% M. sympodialis and 4(6.7% M. restrictawere identified. In one case was isolated M. globosa along with M. restricta.The PCR-RFLP method is a useful and reliable technique for identification of differentiation of Malas-sezia species.

  18. 乌江网箱养殖丁(魚歲)细菌的分离与分子鉴定%Isolation and Molecular Identification of Bacteria from Tinca tinca Linnaeus of Net Cage Culture in the Wujiang River

    Institute of Scientific and Technical Information of China (English)

    李顺; 王庆容

    2009-01-01

    利用微生物学常用的细菌分离、纯化方法从丁(魚歲)中分离出9株菌株,编号分别为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅷ、Ⅸ.分别扩增出9株菌株的16S rRNA基因约600 bp的片段,并对其测序,将测序结果输入到NCBI(http://www.ncbi.nlm.nih.gov/)中,用BLAST工具对序列进行同源性比对分析,并利用MEGA4绘制系统进化树,结果表明:9株菌株的16S rRNA基因测序结果分别与不动杆菌(Acinetobacter)、嗜水气单胞菌(Aeromonas hydrophi-la)、希瓦氏菌(Shewanella baltica)、不动杆菌(Acinetobacter)、希瓦氏菌(Shewanella baltica)、霍乱弧菌(Vibriocholerae)、枯草芽孢杆菌(Bacillus subtilis)、杀鲑气单胞菌(Aeromonas salmonicida)、肺炎克雷伯氏菌(Klebsiellapneumoniae)的16S rRNA基因的核苷酸序列同源,同源性分别为96%,99%,99%,97%,98%,98%,99%,98%,98%.9株菌株的系统进化树明显分为3支,Ⅰ和Ⅳ聚为一支,Ⅶ独聚一支,Ⅱ、Ⅲ、Ⅴ、Ⅵ、Ⅷ、Ⅸ聚为一支,其中Ⅱ和Ⅷ相聚,Ⅲ和Ⅴ相聚后再与Ⅵ相聚,最后与Ⅸ相聚.%The present experiment was undertaken with the tranditional microbiology method to isolate the bacteria from Tinca tinca Linnaeus of net cage culture in Wujiang River and 9 strains were isolated. The numbers of 9 strains were Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ, Ⅸ respectively and the 600bp fragments of 16S rRNA gene of nine strains were amplificated by PCR methold. These 600bp sequences were successfully sequenced and were entered into the NCBI (http://www. ncbi. nlm. nih. gov/). Objective to analyse the homology, BLAST tool was used and the Phylogenetic tree of the nine strains was constructed by the soft-ware of MEGA4. The results indicated that: the sequencing results of 16S rRNA gene of 9 strains have homology with the 16S rRNA gene nucleotide sequence of Acinetobacter, Aeromonas hydrophila, Shewanella baltica, Acinetobacter, Shewanella bahica, Vibrio cholerae, Bacillus subtilis, Aeromonas

  19. Molecular Characterization of Fusarium oxysporum and Fusarium commune Isolates from a Conifer Nursery.

    Science.gov (United States)

    Stewart, Jane E; Kim, Mee-Sook; James, Robert L; Dumroese, R Kasten; Klopfenstein, Ned B

    2006-10-01

    ABSTRACT Fusarium species can cause severe root disease and damping-off in conifer nurseries. Fusarium inoculum is commonly found in most container and bareroot nurseries on healthy and diseased seedlings, in nursery soils, and on conifer seeds. Isolates of Fusarium spp. can differ in virulence; however, virulence and colony morphology are not correlated. Forty-one isolates of Fusarium spp., morphologically indistinguishable from F. oxysporum, were collected from nursery samples (soils, healthy seedlings, and diseased seedlings). These isolates were characterized by amplified fragment length polymorphism (AFLP) and DNA sequencing of nuclear rDNA (internal transcribed spacer including 5.8S rDNA), mitochon-drial rDNA (small subunit [mtSSU]), and nuclear translation elongation factor 1-alpha. Each isolate had a unique AFLP phenotype. Out of 121 loci, 111 (92%) were polymorphic; 30 alleles were unique to only highly virulent isolates and 33 alleles were unique to only isolates nonpathogenic on conifers. Maximum parsimony and Bayesian analyses of DNA sequences from all three regions and the combined data set showed that all highly virulent isolates clearly separated into a common clade that contained F. commune, which was recently distinguished from its sister taxon, F. oxysporum. Interestingly, all but one of the nonpathogenic isolates grouped into a common clade and were genetically similar to F. oxysporum. The AFLP cladograms had similar topologies when compared with the DNA-based phylograms. Although all tested isolates were morphologically indistinguishable from F. oxysporum based on currently available monographs, some morphological traits can be plastic and unreliable for identification of Fusarium spp. We consider the highly virulent isolates to be F. commune based on strong genetic evidence. To our knowledge, this is the first reported evidence that shows F. commune is a cause of Fusarium disease (root rot and dampingoff) on Douglas-fir seedlings. Furthermore

  20. Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

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    Meltem Yalinay Cirak

    2003-12-01

    Full Text Available The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction following by RFLP (restriction fragment length polymorphism analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction. The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS. Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

  1. Isolation, Identification, and Characterization of a Cellulolytic Bacillus amyloliquefaciens Strain SS35 from Rhinoceros Dung

    OpenAIRE

    2013-01-01

    Cellulose hydrolyzing bacteria were isolated from rhinoceros dung and tested for clear zone formation around the colonies on the agar plates containing the medium amended with carboxymethylcellulose as a sole carbon source. Isolates were further screened on the basis of carboxymethylcellulase production in liquid medium. Out of 36 isolates, isolate no. 35 exhibited maximum enzyme activity of 0.079 U/mL and was selected for further identification by using conventional biochemical tests and phy...

  2. A molecular method for S-allele identification in apple based on allele-specific PCR.

    Science.gov (United States)

    Janssens, G A; Goderis, I J; Broekaert, W F; Broothaerts, W

    1995-09-01

    cDNA sequences corresponding to two self-incompatibility alleles (S-alleles) of the apple cv 'Golden Delicious' have previously been described, and now we report the identification of three additional S-allele cDNAs of apple, one of which was isolated from a pistil cDNA library of cv 'Idared' and two of which were obtained by reverse transcription-PCR (RT-PCR) on pistil RNA of cv 'Queen's Cox'. A comparison of the deduced amino acid sequences of these five S-allele cDNAs revealed an average homology of 69%. Based on the nucleotide sequences of these S-allele cDNAs, we developed a molecular technique for the diagnostic identification of the five different S-alleles in apple cultivars. The method used consists of allele-specific PCR amplification of genomic DNA followed by digestion of the amplification product with an allele-specific restriction endonuclease. Analysis of a number of apple cultivars with known S-phenotype consistently showed coincidence of phenotypic and direct molecular data of the S-allele constitution of the cultivars. It is concluded that the S-allele identification approach reported here provides a rapid and useful method to determine the S-genotype of apple cultivars.

  3. Molecular identification of Taenia specimens after long-term preservation in formalin.

    Science.gov (United States)

    Jeon, Hyeong-Kyu; Kim, Kyu-Heon; Eom, Keeseon S

    2011-06-01

    The majority of Taenia tapeworm specimens in the museum collections are usually kept in a formalin fixative for permanent preservation mainly for use in morphological examinations. This study aims to improve Taenia tapeworm identification even of one preserved in formalin for a maximum of 81 years. Taenia tapeworms were collected by the parasite collection unit of the Swiss Natural History Museum and from units in Indonesia, Japan and Korea. A small amount of formalin-fixed tissue (100 mg) was crushed in liquid nitrogen and then soaked in a Tris-EDTA buffer for 3-5h. The sample was then digested in SDS and proteinase K (20 mg/ml) for 3-5h at 56 °C. After the addition of proteinase K (20mg/ml), SDS and hexadecyl-trimethyl-ammonium bromide (CTAB), incubation was continued for another 3h at 65 °C. A maximum yield of genomic DNA was obtained from this additional step and the quality of genomic DNA obtained with this extraction method seemed to be independent of the duration of storage time in the formalin fixative. The molecular identification of Taenia tapeworms was performed by using PCR and DNA sequences corresponding to position 80-428 of cox1 gene. T. asiatica was detected in the isolates of Indonesia, Japan and Korea. Improvements in the genomic DNA extraction method from formalin fixed museum collections will help in the molecular identification of parasites.

  4. Molecular Characterisation of Salmonella enterica Serovar Typhi Isolated from Typhoidial Humans

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    Arunava Das

    2012-09-01

    Full Text Available Aims: Salmonella enterica serovar Typhi is the major causative agent for typhoidial fever around the globe among human population reported till date. Present research work was carried out for detection and molecular characterisation of Salmonella enterica serovar Typhi isolated from humans with Typhoidial fever by biochemical, phenotypical and virulence gene based polymerase chain reaction (PCR techniques. The isolated strains were also investigated for antibiotic susceptibility patterns as a control measure. Methodology and Results: A total of 16 clinical samples were collected from the same numbers of patients (7 males and 9 females from Coimbatore, Erode and Salem districts of Tamil Nadu and were processed via broth enrichment methods for isolation and identification of the causative agent S. enterica serovar Typhi. Microbiological and biochemical investigations revealed the presence of S. Typhi from 16 samples. The biotyping of the isolates showed that all the isolates belonged to biotype IV. The PCR analysis confirmed the presence of invA (Invasion gene, 244bp, tyv (Tyveloseepimerase gene, 615 bp, fliC-d (Phage-1 flagellin gene for d-antigen, 750 bp and viaB (Vi antigen gene, 439bp in all 16 clinical samples. The antibiotic susceptibility test that was carried out among the isolates against 12 antimicrobial agents, showed 100 % resistance to only ampicillin and 100 % sensitivity to carbenicillin, chloramphenicol, clindamycin, gentamycin, kanamycin and tetracycline.Conclusion, significance and impact of study: This study confirmed the association of virulent strains of S. enterica serovar Typhi from Typhoidial fever among human population and suggested that PCR based diagnostic could be very useful for the rapid detection of S. Typhi isolates. Present study emphasized the use of antibiotic like chloramphenicol or in combination with other antibiotics for the effective control of S. Typhi.

  5. Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence.

    Science.gov (United States)

    Talarmin, A; Chandler, L J; Kazanji, M; de Thoisy, B; Debon, P; Lelarge, J; Labeau, B; Bourreau, E; Vié, J C; Shope, R E; Sarthou, J L

    1998-09-01

    This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest.

  6. Isolation and identification of an allelopathic phenylethylamine in rice.

    Science.gov (United States)

    Le Thi, Ho; Lin, Chung-Ho; Smeda, Reid J; Leigh, Nathan D; Wycoff, Wei G; Fritschi, Felix B

    2014-12-01

    Allelopathy is the process whereby an organic chemical (allelochemical) released from one plant influences the growth and development of other plants. Allelochemicals produced by specific rice (Oryza sativa L.) cultivars have potential to manage barnyard grass (Echinochloa crus-galli L.), a major yield-limiting weed species in rice production systems in Asia and North America. In this study, isolation and identification of an allelopathic compound, N-trans-cinnamoyltyramine (NTCT), in a Vietnamese rice cultivar 'OM 5930' was accomplished through bioassay-guided purification using reversed-phase liquid chromatography coupled with spectroscopic techniques, including tandem mass spectrometry, high resolution mass spectrometry, as well as one-dimensional and two-dimensional (1)H NMR and (13)C NMR spectroscopy. The identified compound, NTCT is considered a β-phenylethylamine. NTCT inhibited root and hypocotyl growth of cress (Lepidium sativum L.), barnyard grass and red sprangletop (Leptochloa chinensis L. Nees) at concentrations as low as 0.24 μM. The ED50 (concentration required for 50% inhibition) of NTCT on barnyard grass root and hypocotyl elongation were 1.35 and 1.85 μM, respectively. Results further demonstrated that mortality of barnyard grass and red sprangletop seedlings was >80% at a concentration of 2.4 μM of NTCT. By 20 days after transplanting, 0.425 nmol of NTCT per OM 5930 rice seedling was released into the culture solution. With concentrations of 42 μg g(-1) fresh weight, production of NTCT in intact rice plants can be considered high. These findings suggest that developing plants of Vietnamese rice cultivar OM 5930 release NTCT and may be utilized to suppress barnyard grass in rice fields. The potency of NTCT may encourage development of this compound as a bio-herbicide. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Theory and Application of Dissociative Electron Capture in Molecular Identification

    CERN Document Server

    Havey, C D; Jones, T; Voorhees, K J; Laramee, J A; Cody, R B; Clougherty, D P; Eberhart, Mark; Voorhees, Kent J.; Laramee, James A.; Cody, Robert B.; Clougherty, Dennis P.

    2006-01-01

    The coupling of an electron monochromator (EM) to a mass spectrometer (MS) has created a new analytical technique, EM-MS, for the investigation of electrophilic compounds. This method provides a powerful tool for molecular identification of compounds contained in complex matrices, such as environmental samples. EM-MS expands the application and selectivity of traditional MS through the inclusion of a new dimension in the space of molecular characteristics--the electron resonance energy spectrum. However, before this tool can realize its full potential, it will be necessary to create a library of resonance energy scans from standards of the molecules for which EM-MS offers a practical means of detection. Here, an approach supplementing direct measurement with chemical inference and quantum scattering theory is presented to demonstrate the feasibility of directly calculating resonance energy spectra. This approach makes use of the symmetry of the transition-matrix element of the captured electron to discriminat...

  8. Caracterização morfológica e identificação molecular de isolados de Fusarium graminearum associados à giberela do trigo e triticale no sul do Brasil Morphological characterization and molecular identification of Fusarium graminearum isolates associated with fusarium head blight in wheat and triticale in Southern Brazil

    Directory of Open Access Journals (Sweden)

    Francislene Angelotti

    2006-06-01

    Full Text Available A giberela ou fusariose da espiga é uma das principais doenças do trigo e triticale no sul do Brasil. A espécie de fungo Fusarium graminearum é citada como agente causal da doença, muito embora, em outros países, outras espécies de Fusarium também estejam associadas à doença. No País, não existem relatos de levantamentos de espécies associadas à doença. O objetivo deste trabalho foi identificar espécies de Fusarium associados à giberela do trigo e triticale procedentes do sul do Brasil, com base na morfologia e no emprego da reação da polimerase em cadeia (PCR baseada em oligonucletídeos específicos para espécies de Fusarium. A patogenicidade dos isolados em trigo foi avaliada em espigas de plantas cultivadas em casa de vegetação. Os 20 isolados monospóricos analisados, obtidos de espigas doentes e sementes, foram identificados como F. graminearum.Fusarium head blight (FHB or scab is an important disease of wheat and triticale in Southern Brazil. The fungus Fusarium graminearum has been reported as the causal agent of this disease in Brazil even though other Fusarium species are also associated to FHB in other regions of the world. The aim of this study was to identify species of Fusarium associated with FHB on wheat and triticale in Southern Brazil. The identification was based on morphological features, and uppon polymerase chain reaction using species specific primers. The pathogenicity of the isolates in wheat was evaluated after inoculation of ears under greenhouse conditions. The twenty single spores isolates obtained from diseased ears and seeds were classified as F. graminearum.

  9. Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius.

    Science.gov (United States)

    Khoodoo, M H R; Sahin, F; Donmez, M F; Fakim, Y Jaufeerally

    2005-06-01

    Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.

  10. Isolation and Molecular Characterization of Free-Living Amoebae from Different Water Sources in Italy

    Directory of Open Access Journals (Sweden)

    Margherita Montalbano Di Filippo

    2015-03-01

    Full Text Available Free-living amoebae (FLA are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7% of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively. Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1% Acanthamoeba isolates (genotypes T4 and T15 and 21 (45.6% Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.

  11. Isolation and molecular characterization of free-living amoebae from different water sources in Italy.

    Science.gov (United States)

    Montalbano Di Filippo, Margherita; Santoro, Maristella; Lovreglio, Piero; Monno, Rosa; Capolongo, Carmen; Calia, Carla; Fumarola, Luciana; D'Alfonso, Rossella; Berrilli, Federica; Di Cave, David

    2015-03-24

    Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.

  12. Morphological and molecular characterisation of Echinococcus granulosus from goat isolates in Iran.

    Science.gov (United States)

    Rajabloo, Mohammad; Hosseini, Seyed Hossein; Jalousian, Fatemeh

    2012-08-01

    Hydatidosis is considered to be an important economic and human public health problem in Iran, where a variety of animals act as intermediate hosts. There is limited information about the genotypes of Echinococcus granulosus in goats. In this study, 20 isolates of E. granulosus obtained from goats were characterised by mitochondrial DNA sequencing and morphology of the metacestode. The mitochondrial cytochrome oxidase 1 sequences were evaluated, and the sequence analysis indicated two genotypes within the isolates. 17 samples were identified as G1 strain, and 3 isolates were identified as G6 strain. The results of the morphological studies support the findings of the molecular studies. Two types of rostellar hooks were observed in the goat isolates, in agreement with the strain identification. Type 1 hooks were morphologically similar to those of the common sheep strains, whereas the dimensions of the hooks in type 2 were similar to those normally found in the camel strain. The morphological results suggest that Echinococcus of goat origin is phenotypically similar to either the sheep (G1) or the camel (G6) strains. Further, these results suggest that the transmission of the G1 genotype between sheep and goats seems to be the predominant mode of transmission, but further work is required to verify this.

  13. Molecular identification of 4 clinical Aeromonas isolates by gyrB and rpoB gene sequencing%4株气单胞菌临床分离株基于gyrB和rpoB基因序列的分子鉴定

    Institute of Scientific and Technical Information of China (English)

    韦小瑜; 游旅; 田克诚; 朱琳; 张仁俊; 吉光辉; 唐光鹏; 王定明

    2016-01-01

    目的:对4株气单胞菌临床分离株进行菌种的分子鉴定,为气单胞菌的种型鉴定提供病原学依据。方法对4株系统生化鉴定为气单胞菌的菌株,提取核酸作为模板,分别使用gyrB、rpoB基因进行扩增及产物测序,将所得序列在GenBank中进行同源性搜索,与GenBank中已经公布的同源性较高的气单胞菌序列比较构建系统发育树并进行分析。结果4株气单胞菌gyrB基因序列经同源性搜索,与多株嗜水气单胞菌和豚鼠气单胞菌同源性为99%,rpoB基因序列则与多株豚鼠气胞菌同源性为99%~100%,对gyrB基因和rpoB基因序列分别进行系统发育树分析显示,4株气单胞菌菌株在gyrB基因系统发育树中与多株嗜水气单胞菌和豚鼠气单胞菌聚为一簇,而在rpoB基因系统发育树中与多株豚鼠气单胞菌聚在同一分枝内。结论4株气单胞菌经rpoB基因测序分析明确鉴定为豚鼠气单胞菌,gyrB基因测序分析不能有效鉴定其种型。%Objective To identify the gene species of 4 clinical Aeromonas isolates and to provide the etiologic basis for the identification of Aeromonas species. Methods The isolates were identified as Aeromonas by conventional biochemical methods. The DNA of Aeromonas isolates was extracted as the template of PCR. The gyrB and rpoB gene were amplified and sequenced. Two sequence homology searches were conducted on GenBank and selected closely homology strains. Two molecular phylogenetic trees were constructed for analyze. Results The sequence homology of gyrB gene was 99% among Aeromonas isolates、 Aeromonas hydrophila and Aeromonas caviae strains,99%~100%(rpoB) among Aeromonas isolates and Aeromonas caviae strains. The phylogenetic tree of gyrB gene showed that 4 Aeromonas isolates、Aeromonas caviae strains and Aeromonas hydrophila strains were clustered in the same group. While 4 Aeromonas isolates fell into the same branch with Aeromonas caviae strains

  14. Rapid identification of mycobacteria and rapid detection of drug resistance in Mycobacterium tuberculosis in cultured isolates and in respiratory specimens.

    Science.gov (United States)

    Yam, Wing-Cheong; Siu, Kit-Hang Gilman

    2013-01-01

    Recent advances in molecular biology and better understanding of the genetic basis of drug resistance have allowed rapid identification of mycobacteria and rapid detection of drug resistance of Mycobacterium tuberculosis present in cultured isolates or in respiratory specimens. In this chapter, several simple nucleic acid amplification-based techniques are introduced as molecular approach for clinical diagnosis of tuberculosis. A one-tube nested IS6110-based polymerase chain reaction (PCR) is used for M. tuberculosis complex identification; the use of a multiplex allele-specific PCR is demonstrated to detect the isoniazid resistance; PCR-sequencing assays are applied for rifampicin and ofloxacin resistance detection and 16S rDNA sequencing is utilized for identification of mycobacterial species from cultures of acid fast bacilli (AFB). Despite the high specificity and sensitivity of the molecular techniques, mycobacterial culture remains the "Gold Standard" for tuberculosis diagnosis. Negative results of molecular tests never preclude the infection or the presence of drug resistance. These technological advancements are, therefore, not intended to replace the conventional tests, but rather have major complementary roles in tuberculosis diagnosis.

  15. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    LENUS (Irish Health Repository)

    Ojo, Olabisi O

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (\\'U\\' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  16. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    Science.gov (United States)

    Ojo, Olabisi O; Sheehan, Stella; Corcoran, Daniel G; Nikolayevsky, Vladyslav; Brown, Timothy; O'Sullivan, Margaret; O'Sullivan, Kathleen; Gordon, Stephen V; Drobniewski, Francis; Prentice, Michael B

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) ('U' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  17. Rapid molecular identification of pathogenic yeasts by pyrosequencing analysis of 35 nucleotides of internal transcribed spacer 2.

    Science.gov (United States)

    Borman, Andrew M; Linton, Christopher J; Oliver, Debra; Palmer, Michael D; Szekely, Adrien; Johnson, Elizabeth M

    2010-10-01

    Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures.

  18. Reliable molecular identification of nine tropical whitefly species

    Science.gov (United States)

    Ovalle, Tatiana M; Parsa, Soroush; Hernández, Maria P; Becerra Lopez-Lavalle, Luis A

    2014-01-01

    The identification of whitefly species in adult stage is problematic. Morphological differentiation of pupae is one of the better methods for determining identity of species, but it may vary depending on the host plant on which they develop which can lead to misidentifications and erroneous naming of new species. Polymerase chain reaction (PCR) fragment amplified from the mitochondrial cytochrome oxidase I (COI) gene is often used for mitochondrial haplotype identification that can be associated with specific species. Our objective was to compare morphometric traits against DNA barcode sequences to develop and implement a diagnostic molecular kit based on a RFLP-PCR method using the COI gene for the rapid identification of whiteflies. This study will allow for the rapid diagnosis of the diverse community of whiteflies attacking plants of economic interest in Colombia. It also provides access to the COI sequence that can be used to develop predator conservation techniques by establishing which predators have a trophic linkage with the focal whitefly pest species. PMID:25614792

  19. Reliable molecular identification of nine tropical whitefly species.

    Science.gov (United States)

    Ovalle, Tatiana M; Parsa, Soroush; Hernández, Maria P; Becerra Lopez-Lavalle, Luis A

    2014-10-01

    The identification of whitefly species in adult stage is problematic. Morphological differentiation of pupae is one of the better methods for determining identity of species, but it may vary depending on the host plant on which they develop which can lead to misidentifications and erroneous naming of new species. Polymerase chain reaction (PCR) fragment amplified from the mitochondrial cytochrome oxidase I (COI) gene is often used for mitochondrial haplotype identification that can be associated with specific species. Our objective was to compare morphometric traits against DNA barcode sequences to develop and implement a diagnostic molecular kit based on a RFLP-PCR method using the COI gene for the rapid identification of whiteflies. This study will allow for the rapid diagnosis of the diverse community of whiteflies attacking plants of economic interest in Colombia. It also provides access to the COI sequence that can be used to develop predator conservation techniques by establishing which predators have a trophic linkage with the focal whitefly pest species.

  20. Use of Fourier transform infrared spectroscopy (FTIR spectroscopy for rapid and accurate identification of Yeasts isolated from human and animals

    Directory of Open Access Journals (Sweden)

    M. Taha

    2013-06-01

    Full Text Available Rapid and accurate identification of yeast is increasingly important to stipulate the appropriate therapy thus reducing morbidity and mortality related to yeast infections. Vibrational spectroscopic techniques (infrared (IR and Raman could provide potential alternatives to conventional typing methods, because they constitute a rapid, inexpensive and highly specific spectroscopic fingerprint through-which microorganism can be identified. The present study evaluate (FTIR spectroscopy as a sensitive and effective assay for the identification of the most frequent yeast species isolated from human and animals. One hundred and twenty-eight yeasts isolated from infected human mouths/vaginas, chronic diseased cows, crop mycosis in chicken and soil contaminated with pigeon droppings were phenotypically identified. Using universal primers, ITS1/ITS4, we have amplified ITS1-5.8S-ITS2 rDNA regions for 39 yeast isolates as representative samples. The PCR products were digested with restriction enzyme MspI and examined by PCR-RFLP, which was an efficient technique for identification of Candida spp., Cryptococcus neoformans and Trichosporon asahii. Further, identification of the same 39 isolates were done by FTIR spectroscopy and considered as reference for other strains by comparison of their FTIR spectra. The current study has sharply demonstrated the significant spectral differences between the various examined species of Candida, Cryptococcus, Trichosporon, Rhodotorula and Geotrichum isolated from different sources. Decisively, our research has confirmed that FTIR spectroscopy is a promising diagnostic tool, because of its sensitivity, rapidity, high differentiation capacity and simplicity compared to conventional/molecular techniques.

  1. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    OpenAIRE

    Wang Nan; Sun Changjiang; Wang Quankai; Du Rui; Wang Shijie; Gao Yugang; Zhang Pengju; Zhang Lianxue

    2011-01-01

    Abstract Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical...

  2. Identification of lactic acid bacteria isolated from corn stovers

    National Research Council Canada - National Science Library

    PANG, Huili; ZHANG, Meng; QIN, Guangyong; TAN, Zhongfang; LI, Zongwei; WANG, Yanping; CAI, Yimin

    2011-01-01

    One hundred and twenty‐six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram‐positive, catalase...

  3. Molecular identification and phylogenetic study of Demodex caprae.

    Science.gov (United States)

    Zhao, Ya-E; Cheng, Juan; Hu, Li; Ma, Jun-Xian

    2014-10-01

    The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

  4. Molecular toolbox for the identification of unknown genetically modified organisms.

    Science.gov (United States)

    Ruttink, Tom; Demeyer, Rolinde; Van Gulck, Elke; Van Droogenbroeck, Bart; Querci, Maddalena; Taverniers, Isabel; De Loose, Marc

    2010-03-01

    Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization (unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events. Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition, we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO, which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification of a novel unknown event without prior knowledge of its transgene

  5. Identification of Pediococcus Strains Isolated from Feces of Indonesian Infants With in vitro Capability to Consume Prebiotic Inulin and to Adhere on Mucus

    Directory of Open Access Journals (Sweden)

    Widodo .

    2015-11-01

    Full Text Available The aim of this experiment was to identify isolates obtained from feces of Indonesian infants and to evaluate their capability as probiotics. Identification of isolates was carried out based on morphology, physiology and biochemical identifications, and molecular identification based on 16S rRNA sequence. Morphological and physiological identification was carried out based on Gram staining, shape, motility, spore formation and catalase production. Biochemical identifications based on production of CO2 and NH3 from glucose, the ability to grow on different temperature (10 and 45°C and pH (4.4 and 9.6, and different salt concentration (6.5 and 18%. Probiotics capability of isolates was assayed on the ability to grow on low pH (pH 2.0, on different bile salts concentration (0.3; 0.5; 1.0 and 1.5%, the capacity to grow on media with inulin as the only carbon source, and in vitro adhesion ability on porcine mucin. Morphological, physiological and biochemical identification suggest that all of isolates belong to lactic acid bacteria. Further molecular identification of five isolates showedthat isolates AA, BE and BK were strains of Pediococcus acidilactici (similarity 99%, while isolate AP and AG were strains of Lactobacillus casei (similarity 99-100%. Probiotic assays showed that more than 80% of cells of Pediococcus acidilactici isolates AA, BE and BK were viable after grown on pH 2.0 for 90 min, and around 80% of cells from the same isolates were survived on media supplemented with bile salt 1.5% for 2 h. All of isolates had high adhesion capacity as seen by more than 75% of cells attached on pig gastric mucin. Investigationof isolates to grow on inulin showed Pediococcus acidilactici isolate BE was able to consume inulin as the only carbon source. It is concluded that Pediococcus acidilactici isolate BE was a candidate probiotics and subject to further in vivo evaluation using animal models to examine their beneficial health effects.Key word

  6. Molecular identification of Malaysian Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) using life stage specific mitochondrial DNA.

    Science.gov (United States)

    Kavitha, R; Tan, T C; Lee, H L; Nazni, W A; Sofian, A M

    2013-06-01

    DNA identification of blow fly species can be a very useful tool in forensic entomology. One of the potential benefits that mitochondrial DNA (mtDNA) has offered in the field of forensic entomology is species determination. Conventional identification methods have limitations for sibling and closely related species of blow fly and stage and quality of the specimen used. This could be overcome by DNA-based identification methods using mitochondrial DNA which does not demand intact or undamaged specimens. Mitochondrial DNA is usually isolated from whole blow fly and legs. Alternate sources for mitochondrial DNA isolation namely, egg, larva, puparium and empty puparium were explored in this study. The sequence of DNA obtained for each sample for every life cycle stage was 100% identical for a particular species, indicating that the egg, 1st instar, 2nd instar, 3rd instar, pupa, empty puparium and adult from the same species and obtained from same generation will exhibit similar DNA sequences. The present study also highlighted the usefulness of collecting all life cycle stages of blow fly during crime scene investigation with proper preservation and subsequent molecular analysis. Molecular identification provides a strong basis for species identification and will prove an invaluable contribution to forensic entomology as an investigative tool in Malaysia.

  7. Molecular identification and culture trials of Eutreptiella gymnastica (Eutreptiales, Euglenophyceae)

    Institute of Scientific and Technical Information of China (English)

    XU Na; PANG Shaojun; SHAN Tifeng; LIU Feng; ZHAO Xiaobo; GAO Suqin

    2012-01-01

    During a survey for marine microalgal resources,we isolated a rare marine euglenoid from the coastal waters of Qingdao,China in 2009,and established a pure culture.Electron microscopic and molecular phylogenetic (18S rDNA and 16S rDNA sequences) analyses revealed a close affinity with Eutreptiella gymnastica,a bloom-forming species.Different culture conditions were monitored to understand optimal E.gymnastica growth characteristics.The optimal growth conditions in a batch culture of this isolate were 20℃,160 μmol photons/(m2·s) of white light,and a salinity of 10-31.Nutrient experiments demonstrated that growth increased dramatically with a phosphorus concentration greater than 72 μmol/L.Understanding the effect of culture conditions on E.gymnastica may help understanding the blooming mechanism of this alga in its natural environment.

  8. MOLECULAR IDENTIFICATION AND GENOTYPING OF CIAUSCOLO AUTOCHTHONOUS MICROFLORA: PRELIMINARY STUDY

    Directory of Open Access Journals (Sweden)

    A. Petruzzelli

    2011-01-01

    Full Text Available The present study reports the results of a preliminary characterization of the bacterial population of Ciauscolo, a typical Italian fermented sausage, traditionally manufactured in Marche region. The bacterial community involved in Ciauscolo fermentation was investigated using both molecular and culturebased methods. The estimation of genotypic intra-species variation of the autochthonous bacteria isolated was also evaluated by using randomly amplified polymorphic DNA (RAPD analysis and unweighted pairgroup method with arithmetic averages (UPGMA cluster analysis. Our findings revealed an high diversity of the autochthonous bacterial population investigated, both at species and strain level.

  9. Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda

    Directory of Open Access Journals (Sweden)

    Denis Rwabiita Mugizi

    2015-01-01

    Full Text Available Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

  10. MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates

    Directory of Open Access Journals (Sweden)

    Ashutosh Panda

    2014-01-01

    Full Text Available Background & objectives: Pathogenic bacteria often cause life threatening infections especially in immunocompromised individuals. Therefore, rapid and reliable species identification is essential for a successful treatment and disease management. We evaluated a rapid, proteomic based technique for identification of clinical bacterial isolates by protein profiling using matrix-assisted laser desorption-ionization time - of - flight mass spectrometry (MALDI-TOF MS. Methods: Freshly grown bacterial isolates were selected from culture plates. Ethanol/formic acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with α-cyano-4 hydroxy-cinnamic acid matrix solution. Identification was performed using the MALDI BioTyper 1.1, software for microbial identification (Bruker Daltonik GmbH, Bremen, Germany. Results: A comparative analysis of 82 clinical bacterial isolates using MALDI -TOF MS and conventional techniques was carried out. Amongst the clinical isolates, the accuracy at the species level for clinical isolates was 98.78%. One out of 82 isolates was not in accordance with the conventional assays because MALDI-TOF MS established it as Streptococcus pneumoniae and conventional methods as Streptococcus viridans. Interpretation & conclusions: MALDI - TOF MS was found to be an accurate, rapid, cost-effective and robust system for identification of clinical bacterial isolates. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care.

  11. Isolation and identification mould micoflora inhabiting plant leaf litter from Mount Lawu, Surakarta, Central Java

    Directory of Open Access Journals (Sweden)

    MUHAMMAD ILYAS

    2007-04-01

    Full Text Available A study on isolation and identification mould inhabiting plant leaf litter had been conducted. The objective of the study was to isolate and identify mould inhabiting plant leaf litter from Mount Lawu, Surakarta, Central Java. The mould isolation was based on washing and filtering with membrane isolation method. The result showed that 39 moulds generas with 55 species varians, one group identified in class level, and three groups of unidentified mould isolates had been isolated. Taxas distributions showed that there were endophyte and phytopatogen mould isolates had been isolated such as Fusarium, Pestalotiopsis, Phoma, and Coelomycetes. However, typical soil taxa and common saprobic fungi such as Aspergillus, Cunninghamella, Mucor, Paecilomyces, Penicillium, Rhizopus, and Trichoderma remain dominated the resulted isolates.

  12. Identification of Bifidobacterium Strains Isolated from Kashk-e Zard: A Traditional Iranian Fermented Cereal-Dairy Based Food

    Directory of Open Access Journals (Sweden)

    Mashak

    2016-09-01

    Full Text Available Objectives The genus Bifidobactrium enjoys considerable significance among the probiotic bacteria for having appropriately adapted to the human gastrointestinal tract. As the properties of Bifidobacteria are strain-oriented and niche-dependent, there is growing interest in studying the different sources of these probiotics. Kashk-e Zard, a traditional fermented food produced from wheat and yogurt through a two-week, two-step fermentation process, is rich in probiotics and is worthy of study in this regard. The present study aimed to identify Bifidobacterium spp. in Kashk-e Zard. Methods Twenty-three samples of Kashk-e Zard were collected and subjected to Bifidobacterium identification experiments. Polymerase chain reaction (PCR and sequencing methods were applied for bacterial identification. Results Twelve of the isolates obtained were G +, rod-shaped, and catalase-, whereas only three of them identified positive for fructose 6-phosphate phosphoketolase (F6PPK a Bifidobacterium specific test and mupirocin resistance. These three isolates were then considered for further identification using the 16SrDNA sequencing technique. Conclusions Although carbohydrate fermentation patterns specified these three isolates as B. infantis, B. bifidum, and B. longum, the molecular results did not confirm B. longum, which is still also controversial in the literature. Overall, our results demonstrated that Kashk-e Zard is a rich potential source of probiotic bacteria and further investigations should be undertaken.

  13. SEROLOGICAL DETECTION AND MOLECULAR CHARACTERIZATION OF A BEGOMOVIRUS ISOLATE OBTAINED FROM Macroptilium lathyroides

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    JOSÉ ALBERSIO DE ARAUJO LIMA

    2012-01-01

    Full Text Available The viruses from the genus Begomovirus, family Geminiviridae are considered emergent pathogens, mainly because of the population explosion of their insect vectors. For this reason, more attention needs to be directed to the correct virus species identification inside the genus. The present paper had the objectives of serologically detecting a begomovirus in Macroptilium lathyroides plants in the State of Ceará, and developing biological, serological and molecular studies with a virus isolate obtained from M. lathyroides. Indirect ELISA with antiserum for Macroptilium golden mosaic virus (MaGMV demonstrated that the samples collected from M. lathyroides showing golden mosaic in the field were infected with a begomovirus. The virus isolate obtained was transmitted by grafting to eight species of the family Leguminosae, four species of Solonaceae, and one species in the family Amaranthaceae. The virus also was transmitted from M. lathyroides to M. lathyroides by the whitefly Bemisia tabaci biotype B. A DNA fragment of 1.2 kb was obtained by PCR with the primers PAL1v 1978 and PAR1c 496 for component A, and a DNA fragment of 0.5 kb was obtained with the primers PBL1v 2040 and PCR cl for component B, confirming the presence of a begomovirus infecting M. lathyroides. Molecular studies indicated that the begomovirus isolate showed 77% genomic similarity with Bean golden mosaic virus and 75% with Cowpea golden mosaic virus for their cp and rep genes, indicating the possibility that the isolate is a distinct virus species of the Begomovirus genus.

  14. GC-MS Quantitation and Identification of Bisphenol-A Isolated from Water

    Science.gov (United States)

    Mead, Ralph N.; Seaton, Pamela J.

    2011-01-01

    Isolation and identification of organic compounds is a necessary skill chemistry students must be able to do with proficiency. In this upper-level undergraduate laboratory, students isolate bisphenol-A (BPA; 4-4'-isopropylidenediphenol) from water using solid-phase extraction (SPE) followed by derivatization with analysis by GC-MS. The students…

  15. Isolation and Molecular Characterization of Circulating Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Xi Luo

    2014-05-01

    Full Text Available Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.

  16. Development of a novel, simple and rapid molecular identification system for clinical Candida species

    NARCIS (Netherlands)

    Deak, R.; Bodia, L.; Aarts, H.J.M.; Maraz, A.

    2004-01-01

    Identification of clinical yeast isolates causing candidiasis is routinely performed by commercial yeast identification systems based on biochemical, morphological and physiological tests. These systems require 3-5 days and the proportion of identifications that are incorrect is high. Our novel and

  17. Isolation, characterization, and molecular regulation of muscle stem cells

    Directory of Open Access Journals (Sweden)

    So-ichiro eFukada

    2013-11-01

    Full Text Available keletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse’s genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background mdx mice that has a much more severe phenotype than the frequently used C57BL/10-mdx mice. The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders.

  18. Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

    Science.gov (United States)

    Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

    2006-09-01

    This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

  19. Isolation, identification and molecular characterization of Inclusion body hepatitis virus

    Directory of Open Access Journals (Sweden)

    Amit Gaba

    Full Text Available Inclusion body hepatitis caused by avian adenovirus with highly pathogenic strain lead to heavy mortality and unusual lesion like hydropericardium in commercial broilers. Samples were collected from an outbreak in 12000 broiler flock from Andhra Pradesh with sudden high mortality initiated from 32 days of age. Liver was enlarged and showed petechae. Hydropericardium was extensive. 10% liver suspension inoculated to SPF chicks produced mortality with hydropericardium and showed presence of intranuclear inclusions in the hepatocytes. Viral DNA extracted from infected liver processed for PCR produced an amplicon of 890 bp. Nucleotide sequence analysis using blast programme of NCBI showed 99% identity with fowl adenovirus. [Veterinary World 2010; 3(9.000: 414-416

  20. Isolation and molecular identification of a Serratia strain from ...

    African Journals Online (AJOL)

    ONOS

    2010-04-05

    Apr 5, 2010 ... The tree shrews are small animals belonging to the family Tupaiidae, mainly ... shrews holds significant promise as research models and great use could be .... Science and Technology project of Yunan Province. “Project for ...

  1. Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

    Science.gov (United States)

    Fadda, M E; Pisano, M B; Scaccabarozzi, L; Mossa, V; Deplano, M; Moroni, P; Liciardi, M; Cosentino, S

    2013-01-01

    This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of

  2. Isolation, Separation, and Identification of Synthetic Food Colors.

    Science.gov (United States)

    Dixon, E. A.; Renyk, G.

    1982-01-01

    Describes a simple, inexpensive experiment for extraction of synthetic dyes permitted in foodstuffs, and their separation and identification using thin-layer chromatography and ultraviolet/visible spectroscopy. (Author/SK)

  3. Accuracy of conventional identification methods used for Enterobacteriaceae isolates in three Nigerian hospitals

    Science.gov (United States)

    Ogunlowo, Peter Oladejo; Olley, Mitsan; Springer, Burkhard; Allerberger, Franz; Ruppitsch, Werner

    2016-01-01

    Background Enterobacteriaceae are ubiquitously present in nature and can be found in the intestinal tract of humans and animals as commensal flora. Multidrug-resistant Enterobacteriaceae are increasingly reported and are a threat to public health implicating a need for accurate identification of the isolates to species level. In developing countries, identification of bacteria basically depends on conventional methods: culture and phenotypic methods that hamper the accurate identification of bacteria. In this study, matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique was compared to conventional identification techniques. Materials and Methods In total, 147 Enterobacteriaceae isolates were collected from March to May 2015 from three medical microbiology laboratories of hospitals in Edo state, Nigeria, after being tested according to the individual laboratories standard operating procedures. All isolates were stored at −20°C until tested centrally by MALDI-TOF MS. Results One hundred and forty five (98.6%) isolates had a MALDI Biotyper best score > or =2.0, indicating a secure genus and probable species identification; and 2(1.36%) isolates had a best score or =2.0 comprised nine genera and 10 species, respectively. A total of 57.2% and 33.1% of isolates identified had agreement between MALDI-TOF MS and conventional techniques for identification at genus and species level, respectively, when analyzing bacteria with MALDI Biotyper best scores > or =2.0. Conclusion The results of our study show that the applied conventional identification techniques for Enterobacteriaceae in the investigated Nigerian hospitals are not very accurate. Use of state-of-the-art identification technologies for microorganisms is necessary to guarantee comparability of bacteriological results. PMID:27703855

  4. Molecular identification and prevalence of malassezia species in pityriasis versicolor patients from kashan, iran.

    Science.gov (United States)

    Talaee, Rezvan; Katiraee, Farzad; Ghaderi, Maryam; Erami, Mahzad; Kazemi Alavi, Azam; Nazeri, Mehdi

    2014-08-01

    Malassezia species are lipophilic yeasts found on the skin surface of humans and other warm-blooded vertebrates. It is associated with various human diseases, especially pityriasis versicolor, which is a chronic superficial skin disorder. The aim of the present study was to identify Malassezia species isolated from patients' samples affected by pityriasis versicolor, using molecular methods in Kashan, Iran. A total of 140 subjects, suspected of having pityriasis versicolor from Kashan, were clinically diagnosed and then confirmed by direct microscopic examination. The scraped skin specimens were inoculated in modified Dixon's medium. DNA was extracted from the colonies and PCR amplification was carried out for the 26s rDNA region. PCR products were used to further restriction fragment length polymorphism by CfoI enzyme. Direct examination was positive in 93.3% of suspected pityriasis versicolor lesions. No statistically significant difference was observed in the frequency of Malassezia species between women and men. The highest prevalence of tinea versicolor was seen in patients 21-30 years-of-age. No difference could be seen in the frequency of Malassezia species depending on the age of the patients. In total, 65% of patients with pityriasis versicolor had hyperhidrosis. The most commonly isolated Malassezia species in the pityriasis versicolor lesions were; Malassezia globosa (66%), M. furfur (26%), M. restricta (3%), M. sympodialis (3%), and M. slooffiae (2%). Malassezia species were mainly isolated from the neck and chest. This study showed M. globosa to be the most common Malassezia species isolated from Malassezia skin disorders in Kashan, Iran. The PCR-RFLP method was useful in the rapid identification of the Malassezia species. By using these methods, the detection and identification of individual Malassezia species from clinical samples was substantially easier.

  5. First molecular identification of Entamoeba moshkovskii in Malaysia.

    Science.gov (United States)

    Anuar, Tengku Shahrul; Al-Mekhlafi, Hesham M; Ghani, Mohamed Kamel Abdul; Azreen, Siti Nor; Salleh, Fatmah Md; Ghazali, Nuraffini; Bernadus, Mekadina; Moktar, Norhayati

    2012-10-01

    Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. Although sporadic cases of human infection with E. moshkovskii have been reported, the amoeba is still considered primarily as a free-living amoeba. A cross-sectional study was carried out among Orang Asli communities in 3 different states of Peninsular Malaysia. Fecal samples were examined by formalin-ether sedimentation and trichrome staining techniques and then single-round PCR assay was used to detect E. moshkovskii. Out of 500 fecal samples examined microscopically, 93 (18·6%) samples were positive for E. histolytica/E. dispar/E. moshkovskii complex cysts and/or trophozoites. PCR products were detected in 106 fecal samples. E. moshkovskii isolates were detected in 13 (12·3%) fecal samples. Of the 13 E. moshkovskii-positive samples, 5 were of single isolation of E. moshkovskii, 6 were also positive for E. dispar, and only 2 samples were positive for E. dispar and E. histolytica. Moreover, 3 E. moshkovskii-positive samples were collected from symptomatic individuals while the remaining 10 samples were from asymptomatic subjects. This is the first report on the identification of E. moshkovskii in Malaysia. Further studies are needed to confirm the pathogenicity of E. moshkovskii infection and determine the epidemiology among Orang Asli communities in Malaysia.

  6. Identification study on base isolation systems by full-scale buildings

    Energy Technology Data Exchange (ETDEWEB)

    Loh, C.; Lee, C. (National Taiwan University, Taipei (Taiwan, Province of China))

    1992-10-15

    In this paper, seismic response characteristics of two types of base-isolated buildings are investigated by using identification techniques. Equivalent linear system was first assumed in connection with the dynamic behavior of the structural system, then the nonlinear Bouc-Wen's model was assumed as second stage to model the isolation system and evaluate the hysteretic damping of the system. For structural identification, both ' model minimization method ' and ' extended Kalman filtering techniques ' were used. It was illustrated that the natural frequency of the isolation system of ' Law and Justice Center Building ' in California, the USA was designed greater than the natural frequency of the building while the natural frequency of the isolation system of the test building of Tohoku University was designed smaller than the natural frequency of the building. These results could be obtained by the system identification techniques. 20 refs., 12 figs., 2 tabs.

  7. Molecular identification and culture observation on Acrochaete leptochaete (Chaetophoraceae, Chlorophyta) from China

    Science.gov (United States)

    Deng, Yunyan; Tang, Xiaorong; Huang, Bingxin; Teng, Linhong; Ding, Lanping

    2012-05-01

    Acrochaete leptochaete (Huber) Nielsen (Chaetophoraceae, Chlorophyta) was isolated from the macroalgae Chaetomorpha collected from intertidal pools in Rongcheng, Shandong, China. 18S rDNA combined with ITS regions were used to ascertain the morphological identification of the isolated material. Based on the unialgal culture, asexual reproduction and growth characteristics of A. leptochaete were investigated over wide ranges of temperature and irradiance. Results revealed that asexual reproduction of A. leptochaete could be realized by biflagellate zoospores. The zoospores germinated directly to give self-replicating generations. Zoospore germination was bipolar. A temperature range from 13-21°C and a lower irradiance of 36 μmol/(m2·s) were most favorable for the growth of A. leptochaete. Thallus organization, an important taxonomic criterion for the genus Acrochaete, was affected markedly by temperature and irradiance. Our results extend the knowledge about the species' general biology and its morphological plasticity. For classification and identification of a simple microphytic algae like A. leptochaete, which are traditionally placed in the class Chaetophoraceae, we propose that molecular tools associated with culture observations are applied.

  8. What causes canine sino-nasal aspergillosis? A molecular approach to species identification.

    Science.gov (United States)

    Talbot, Jessica J; Johnson, Lynelle R; Martin, Patricia; Beatty, Julia A; Sutton, Deanna A; Billen, Frédéric; Halliday, Catriona L; Gibson, Justine S; Kidd, Sarah; Steiner, Jörg M; Ujvari, Beata; Barrs, Vanessa R

    2014-04-01

    On the basis of phenotypic identification methods, Aspergillus fumigatus is reported as the most commonly identified aetiological agent of canine sino-nasal aspergillosis (SNA). However, definitive identification of Aspergillus spp. using phenotypic features alone is unreliable. The aim of this study was to determine the molecular identities of fungal species causing SNA in dogs. Genomic DNA was extracted from 91 fungal isolates from 90 dogs diagnosed with SNA in Australia, the USA and Belgium, and the ITS1-5.8S-ITS2 ribosomal DNA and partial β-tubulin regions were sequenced. Eighty-eight of 91 (96.7%) isolates were identified as A. fumigatus and 3/91 (3.3%) belonged to Aspergillus section Nigri spp. (Aspergillus tubingensis: 2/91; Aspergillus uvarum: 1/91). These findings confirm that A. fumigatus is the most common aetiological agent of canine SNA. This is the first report to document a pathogenic role for A. tubingensis and A. uvarum in dogs. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  9. Molecular identification and culture observation on Acrochaete leptochaete (Chaetophoraceae, Chlorophyta)from China

    Institute of Scientific and Technical Information of China (English)

    DENG Yunyan; TANG Xiaorong; HUANG Bingxin; TENG Linhong; DING Lanping

    2012-01-01

    Acrochaete leptochaete (Huber) Nielsen (Chaetophoraceae,Chlorophyta) was isolated from the macroalgae Chaetomorpha collected from intertidal pools in Rongcheng,Shandong,China.18S rDNA combined with ITS regions were used to ascertain the morphological identification of the isolated material.Based on the unialgal culture,asexual reproduction and growth characteristics of A.leptochaete were investigated over wide ranges of temperature and irradiance.Results revealed that asexual reproduction of A.leptochaete could be realized by biflagellate zoospores.The zoospores germinated directly to give selfreplicating generations.Zoospore germination was bipolar.A temperature range from 1 3-21 ℃ and a lower irradiance of 36 μmol/(m2·s) were most favorable for the growth ofA.leptochaete.Thallus organization,an important taxonomic criterion for the genus Acrochaete,was affected markedly by temperature and irradiance.Our results extend the knowledge about the species' general biology and its morphological plasticity.For classification and identification of a simple microphytic algae like A.leptochaete,which are traditionally placed in the class Chaetophoraceae,we propose that molecular tools associated with culture observations are applied.

  10. Significance of molecular identification and antifungal susceptibility of clinically significant yeasts and moulds in a global antifungal surveillance programme.

    Science.gov (United States)

    Pfaller, Michael A; Woosley, Leah N; Messer, Shawn A; Jones, Ronald N; Castanheira, Mariana

    2012-10-01

    The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45 °C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7 %): Aspergillus spp. (64.1 % correct); Candida spp. (60.1 % correct); non-Candida yeasts (58.9 % correct); non-Aspergillus moulds (30.4 % correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0  %), C. lusitaniae (100 %), C. dubliniensis (92.3 %), C. kefyr (100 %), and C. neoformans (90.2 %). Only 50.0 % of isolates of C. albicans and 59.1 % of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of

  11. Isolation and molecular characterization of polyvinyl chloride (PVC) plastic degrading fungal isolates.

    Science.gov (United States)

    Ali, Muhammad Ishtiaq; Ahmed, Safia; Robson, Geoff; Javed, Imran; Ali, Naeem; Atiq, Naima; Hameed, Abdul

    2014-01-01

    The recalcitrant nature of polyvinyl chloride creates serious environmental concerns during manufacturing and waste disposal. The present study was aimed to isolate and screen different soil fungi having potential to biodegrade PVC films. After 10 months of soil burial experiment, it was observed that a number of fungal strains were flourishing on PVC films. On morphological as well as on 18rRNA gene sequence and phylogenetic basis they were identified as Phanerochaete chrysosporium PV1, Lentinus tigrinus PV2, Aspergillus niger PV3, and Aspergillus sydowii PV4. The biodegradation ability of these fungal isolates was further checked in shake flask experiments by taking thin films of PVC (C source) in mineral salt medium. A significant change in color and surface deterioration of PVC films was confirmed through visual observation and Scanning electron microscopy. During shake flask experiments, P. chrysosporium PV1 produced maximum biomass of about 2.57 mg ml(-1) followed by A. niger PV3. P. chrysosporium PV1 showed significant reduction (178,292 Da(-1)) in Molecular weight of the PVC film than control (200,000 Da(-1)) by gel permeation chromatography. Furthermore more Fourier transform infrared spectroscopy and nuclear magnetic resonance also revealed structural changes in the PVC. It was concluded that isolated fungal strains have significant potential for biodegradation of PVC plastics.

  12. Identification of possible virulence marker from Campylobacter jejuni isolates.

    Science.gov (United States)

    Harrison, James W; Dung, Tran Thi Ngoc; Siddiqui, Fariha; Korbrisate, Sunee; Bukhari, Habib; Tra, My Phan Vu; Hoang, Nguyen Van Minh; Carrique-Mas, Juan; Bryant, Juliet; Campbell, James I; Studholme, David J; Wren, Brendan W; Baker, Stephen; Titball, Richard W; Champion, Olivia L

    2014-06-01

    A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.

  13. Isolation, identification and screening of potential cellulase-free ...

    African Journals Online (AJOL)

    compaq

    2012-09-05

    Sep 5, 2012 ... tation has gained interest from researchers in recent ... Eight fungal isolates were screened by xylan-agar diffusion method ... Xylanase activity was determined by mixing 0.9 ml of 1% (w/v) birch .... The qualitative and quantitative assay of xylanases by isolated eight fungal strains in solid state fermentation.

  14. The isolation and identification of predatory bacteria from a ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-02-01

    Feb 1, 2010 ... ISSN 1684–5315 © 2010 Academic Journals. Full Length .... Water samples were collected from surface waters of Hartbees- poort dam where ... Culture of bacterial isolates: An inoculum of the isolate was cultured in a 250 mℓ ...

  15. Identification of bifidobacteria isolated from Asian elephant (Elephas maximus)

    Indian Academy of Sciences (India)

    Vera Bunesova; Eva Vlkova; Vojtech Rada; Jiri Killer; Vladimir Kmet

    2013-06-01

    Bifidobacteria are considered as one of the key genera in intestinal tracts of animals, and their species composition vary depending on the host. The aim of this study was to identify faecal bifidobacteria from Asian elephants (Elephas maximus), housed in Zoological gardens (Ostrava, Czech Republic). Using culturing, bifidobacteria were found in counts 7.60±0.56 log CFU/g. Twenty-six pure strains were isolated from faeces of Asian elephant. The isolates were clustered into two groups according to fingerprinting profiles and fermentation characteristic. Bacteria were identified by a combination of MALDI-TOF MS, PCR methods and sequencing as B. boum (12 isolates) and B. adolescentis (14 isolates). Elephant strains showed different fingerprinting profiles than type and collection strains. Since these two species are frequently isolated from gastrointestinal tract of herbivores, they seem to be typical of animals fed plant diets.

  16. Genetic identification of rickettsiae isolated from ticks in Japan.

    Science.gov (United States)

    Fournier, Pierre-Edouard; Fujita, Hiromi; Takada, Nobuhiro; Raoult, Didier

    2002-06-01

    Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.

  17. Taxonomic identification of algae (morphological and molecular): species concepts, methodologies, and their implications for ecological bioassessment.

    Science.gov (United States)

    Manoylov, Kalina M

    2014-06-01

    Algal taxonomy is a key discipline in phycology and is critical for algal genetics, physiology, ecology, applied phycology, and particularly bioassessment. Taxonomic identification is the most common analysis and hypothesis-testing endeavor in science. Errors of identification are often related to the inherent problem of small organisms with morphologies that are difficult to distinguish without research-grade microscopes and taxonomic expertise in phycology. Proposed molecular approaches for taxonomic identification from environmental samples promise rapid, potentially inexpensive, and more thorough culture-independent identification of all algal species present in a sample of interest. Molecular identification has been used in biodiversity and conservation, but it also has great potential for applications in bioassessment. Comparisons of morphological and molecular identification of benthic algal communities are improved by the identification of more taxa; however, automated identification technology does not allow for the simultaneous analysis of thousands of samples. Currently, morphological identification is used to verify molecular taxonomic identities, but with the increased number of taxa verified in algal gene libraries, molecular identification will become a universal tool in biological studies. Thus, in this report, successful application of molecular techniques related to algal bioassessment is discussed.

  18. Molecular epidemiology of microorganisms isolated from food workers and enteral feeding of public hospitals.

    Science.gov (United States)

    Borges, Liana J; Campos, Maria Raquel H; Cardoso, Juliana L; André, Maria Cláudia D P B; Serafini, Álvaro B

    2010-09-01

    This study aimed to compare strains of Staphylococcus aureus and E. coli isolated from food workers and enteral diet samples obtained from 2 public hospitals (H1/H2) in Goiania, Goias, Brazil, by the means of antibiogram and pulsed field gel electrophoresis (PFGE). In the H1, strains of S. aureus were present in 2 enteral diet samples and in 13 food worker swabs. Strains of E. coli were found in an enteral diet sample from H1 and in 2 enteral diet samples from H2 and in 6 food worker swabs in the H1 and in 12 food worker swabs from H2. According to the antibiogram, the 6 susceptibility profiles (A to F) of 15 S. aureus strains colonizing personnel and enteral feeding did not allow the identification of the probable source of diet contamination. All 20 E. coli strains isolated from the H1 and H2 were grouped in 4 phenotypic profiles (A to D). The phenotypes A (H1) and C (H2) showed the same profile for microorganisms isolated from handlers and diets, suggesting more phenotypic similarity among these samples. PFGE genotyping showed that S. aureus isolates from diets were related to a single strain isolated from a food worker suggesting that in this case the reason for the diet contamination may be a result of food handling. The food worker appears to be the most probable source of E. coli contamination for enteral feeding from H2. This fact emphasizes on the food workers as a risk of bacterial transmission for the diets and that the diet chain production must be controlled. The study emphasizes the importance of monitoring the enteral diet microbiological quality and the factors associated to its contamination. The study highlights the use of molecular biology as an instrument to correlate strains to determine the origin of the final product contamination.

  19. Application and validation of PFGE for serovar identification of Leptospira clinical isolates.

    Directory of Open Access Journals (Sweden)

    Renee L Galloway

    Full Text Available Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT, the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78% were identified to the serovar level by the database, and an additional 27 isolates (15% have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.

  20. Identification of new IS711 insertion sites in Brucella abortus field isolates

    Directory of Open Access Journals (Sweden)

    Moriyón Ignacio

    2011-08-01

    Full Text Available Abstract Background Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated. Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. Results We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Conclusions Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  1. Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods

    Directory of Open Access Journals (Sweden)

    Kavita Diddi

    2013-01-01

    Full Text Available Background & objectives: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly. Methods: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP. Results: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae. Interpretation & conclusions: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

  2. Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes.

    Science.gov (United States)

    Alvarez, Julio; García, Ignacio Gómez; Aranaz, Alicia; Bezos, Javier; Romero, Beatriz; de Juan, Lucía; Mateos, Ana; Gómez-Mampaso, Enrique; Domínguez, Lucas

    2008-04-01

    Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

  3. An integrated microfluidic platform for rapid tumor cell isolation, counting and molecular diagnosis.

    Science.gov (United States)

    Hung, Lien-Yu; Chuang, Ying-Hsin; Kuo, Hsin-Tzu; Wang, Chih-Hung; Hsu, Keng-Fu; Chou, Cheng-Yang; Lee, Gwo-Bin

    2013-04-01

    Ovarian cancer is the second most common of the gynecological cancers in Taiwan. It is challenging to diagnose at an early stage when proper treatment is the most effective. It is well recognized that the detection of tumor cells (TCs) is critical for determining cancer growth stages and may provide important information for accurate diagnosis and even prognosis. In this study, a new microfluidic platform integrated with a moving-wall micro-incubator, a micro flow cytometer and a molecular diagnosis module performed automated identification of ovarian cancer cells. By efficiently mixing the cells and immunomagnetic beads coated with specific antibodies, the target TCs were successfully isolated from the clinical samples. Then counting of the target cells was achieved by a combination of the micro flow cytometer and an optical detection module and showed a counting accuracy as high as 92.5 %. Finally, cancer-associated genes were amplified and detected by the downstream molecular diagnosis module. The fluorescence intensity of specific genes (CD24 and HE4) associated with ovarian cancer was amplified by the molecular diagnosis module and the results were comparable to traditional slab-gel electrophoresis analysis, with a limit of detection around 10 TCs. This integrated microfluidic platform realized the concept of a "lab-on-a-chip" and had advantages which included automation, disposability, lower cost and rapid diagnosis and, therefore, may provide a promising approach for the fast and accurate detection of cancer cells.

  4. Molecular Identification of Microorganisms Associated to the Rhizosphere of Vanilla Plants in Colombia

    Directory of Open Access Journals (Sweden)

    Claudia Lucía Álvarez López

    2013-06-01

    Full Text Available The cultivation of vanilla (Vanilla planifolia is highly promising in Colombia, but more research is needed on its agronomical management and beneficial microorganisms that grow associated to its rhizosphere, on which the plant depends for its nutrition and growth. This study involved the identification of microorganisms associated to the rhizosphere of vanilla plants in a crop located in Sopetrán, Colombia. The microbes were isolated in selective media for functional groups such as cellulolytic, proteolytic, inorganic and organic phosphate (phytate solubilizers, and asymbiotic nitrogen fixing bacteria. After isolation and purification, 109 microbial isolates were obtained. DNA was extracted from 52 selected isolates for molecular identification based on ITS and 16S rDNA sequencing, for fungi and bacteria, respectively. The diversity of rhizosphere microorganismsfound was significant. Bacteria such as Bacillus megaterium, Pseudomonas koreensis and Acinetobacter sp., and the fungus Plectosphaerella sp., may have a high potential to be used as biofertilizers to improve vanilla plant nutrition and growth.IDENTIFICACIÓN MOLECULAR DE MICROORGANISMOS ASOCIADOS A LA RIZOSFERA DE PLANTAS DE VAINILLA EN COLOMBIAEl cultivo de vainilla es altamente promisorio en Colombia, pero se requiere mayor conocimiento de su manejo agronómico y de los microorganismos que crecen asociados a su rizosfera, de los cuales depende esta planta para su nutrición y crecimiento. En este trabajo se realizaron aislamientos de microorganismos de la rizosfera de plantas de vainilla en un cultivo piloto ubicado en el municipio de Sopetrán (Antioquia, Colombia. Los microorganismos se aislaron en medios selectivos de crecimiento para evaluar su capacidad para descomponer celulosa, proteínas, solubilizar fosfato inorgánico y orgánico (fitato y fijar nitrógeno en forma asimbiótica. Una vez aislados y purificados, se obtuvieron un total de 109 aislamientos, de los

  5. [Isolation, screening and identification of yeast for aquaculture water purification].

    Science.gov (United States)

    Xie, Fengxing; Zhang, Fengfeng; Zhou, Ke; Zhao, Yujie; Sun, Haibo; Wang, Yun

    2015-05-04

    In order to get excellent yeast strains for aquiculture water purification, we isolated, screened and identified yeasts from the aquacultural environment and intestinal tract of shrimp. The potential water purification ability of yeasts, isolated from the activated sludge of aquacultural environment and intestinal tract of white shrimp and mantis shrimp under normal and low temperature, was evaluated in the simulated wastewater. Morphological physio-biochemical characteristics, 5.8S rDNA ITS gene sequence analysis were used to identify the strains. Thirty-seven yeast strains were isolated from 3 samples, among them 16 strains were isolated under normal temperature (25 °C) while 21 strains were isolated under low temperature (15° C). Water purification test suggested 5 strains isolated under 25 °C and 6 strains isolated under 15 °C had higher removal ability of nitrite and ammonia from water. After 48 hours treatment with DN9 and CN6, 10.64 mg/L nitrite in the water was completely removed. After 96 hours treatment, CODcr degradation rates of the 2 strains were 52% and 67%, respectively. According to morphological, physio-biochemical characteristics and 5.8S rDNA ITS gene sequence analysis, the strain DN9 was identified as Rhodotorula mucilaginosa and CN6 as Rhodosporidium paludigenum. Strains DN9 and CN6 would be promising for water purification in aquiculture.

  6. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex.

    Directory of Open Access Journals (Sweden)

    Carolina Firacative

    Full Text Available BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this

  7. A grass molecular identification system for forensic botany: a critical evaluation of the strengths and limitations.

    Science.gov (United States)

    Ward, Jodie; Gilmore, Simon R; Robertson, James; Peakall, Rod

    2009-11-01

    Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA-based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single-nucleotide polymorphisms were utilized as molecular markers for subfamily to species-level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated "proof of concept" of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.

  8. Isolation and identification of bacteria causing arthritis in chickens

    Directory of Open Access Journals (Sweden)

    B. Y. Rasheed

    2011-01-01

    Full Text Available Sixty chickens 30-55 days old with arthritis symptoms, were collected from different broiler chickens farms, all samples were examined clinically, post mortem and bacterial isolation were done. The results revealed isolation of 26 (50.98% of Staphylococcus aureus, which were found highly sensitive to amoxycillin. The experimental infection of 10 chickens was carried out on 35 days old by intravenous inoculated with 107 cfu/ml of isolated Staphylococcus aureus. Arthritis occurred in 8 (80% chickens. Clinical signs and post mortem findings confined to depression, swollen joints, inability to stand.

  9. Isolation and identification of indigenous lactic acid bacteria from North Sumatra river buffalo milk

    Directory of Open Access Journals (Sweden)

    Heni Rizqiati

    2015-06-01

    Full Text Available Buffalo milk is a source of various lactic acid bacteria (LAB which is potential as culture starter as well as the probiotic. This study was conducted to isolate and identify LAB from indigenous North Sumatra river buffalo milk. Lactic acid bacteria was isolated and grown in medium De Man Rogosa Sharpe Agar (MRSA. The isolation was conducted to obtain pure isolate. The identification of LAB was studied in terms of morphology, physiology, biochemistry and survival on low pH. Morphology tests were conducted by Gram staining and cell forming; physiology tests were conducted for growing viability at pH 4.5 and temperature at 45oC; whereas biochemistry tests were conducted for CO2, dextran and NH3 productions. Determination of LAB species was conducted using Analytical Profile Index (API test CHL 50. Results of identification showed that 41 isolates were identified as LAB with Gram-positive, catalase-negative, rod and round shaped characteristics. Resistance test done to low pH (pH 2 for the lactic acid bacteria showed decrease of bacteria viability up to1.24±0.68 log cfu/ml. The resistant isolates at low pH were L12, L16, L17, L19, L20, M10, P8, S3, S19 and S20. Identification with API test CHL 50 for 10 isolates showed that four isolates were identified as Lactobacillus plantarum, L. brevis, L. pentosus and Lactococuslactis.

  10. Isolation, Characterization and Identification of Microalgae from the Red Sea

    KAUST Repository

    Luque Alanís, Patricio

    2013-05-01

    Eukaryotic microalgae from the Red Sea were isolated, characterized and identified with the purpose of building a culture collection that will serve future research activities in the area of industrial microbiology. Seven sampling locations were surveyed using an in-house designed isolation protocol. Microalgae enrichment was carried out in vitro using the streak plate method and fluorescence activated cell sorting approaches. Colonial and cellular microscopy, growth media preference assays, as well as temperature, pH and salinity tolerance tests were carried out to describe the isolates. DNA extraction, PCR amplification, template sequencing and in silico analyses were carried out to identify the isolates and arrange them in a proper phylogenetic description. In total, 129 isolates were obtained. From these, only 39 were selected for characterization given their increased ability of accumulating large amounts of biomass in solid and liquid media in relatively short periods of time. All of these have a green color, are unicellular, non-motile, photosynthetic organisms and have a cell size ranging from 5 to 8 µm. More than half of them showed growth preference in Walne media, followed by F/2, MN and BG-11 SW. Maximum temperature tolerance of all organisms was around 38 ºC, while optimum growth was observed close to 25 ºC. pH preference was diverse and three groups were identified: acidic (6), intermediate (8 - 9) and alkaline (> 10) growing isolates. Salinity tests showed an overall growth preference at 25 PSU, approximately 10 units lower than that found at the sampling stations. Most isolates showed diminished growth at high salinity and high pH, except for OS3S1b which grew well in both cases, and could be an interesting strain to study further. Twenty four isolates were related to Ulvophyceae sp. MBIC10591 by BLAST approaches with a maximum identity of 96 - 97%. A maximum likelihood phylogenetic tree was created for these isolates, relative to the BLAST hits

  11. Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing.

    Science.gov (United States)

    Lv, Xu-Cong; Huang, Zhi-Qing; Zhang, Wen; Rao, Ping-Fan; Ni, Li

    2012-01-01

    Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.

  12. Isolation and identification of an ester from a crude oil

    Science.gov (United States)

    Phillips, H.F.; Breger, I.A.

    1958-01-01

    A dioctylphthalate has been isolated from a crude oil by means of adsorption column chromatography. The ester was identified by means of elemental analysis, refractive index, and its infra-red absorption spectrum. Saponification of the isolate and examination of the resultant alcohol by means of infrared absorption spectra led to the conclusion that the ester is a branched chain dioctylphthalate. This is the first reported occurrence of an ester in crude petroleum. ?? 1958.

  13. [Isolation and purification and toxigenic characteristic identification of microcystis].

    Science.gov (United States)

    Ban, Haiqun; Cheng, Xuemin; Zhang, Huizhen; Zhu, Jingyuan

    2007-07-01

    To establish methods of isolation and purification of microcystis, and to identify it's biological property. By the means of 96 well microplates and utmost dilution, microcystis was isolated and purified from Xiliu Lake of Zhengzhou. Absorbency of isolated microcystis was determined by visble light spectrophotometric method, and it's growth curve was ploted, calculated growth rate constant and double time. Toxigenicity of isolated microcystis was identified by means of the Whole cell PCR and ELISA, and the concentration of microcystin in crude extractive solution from freeze-dried microcystis cells was determined by ELISA. Two strains of microcystis, Microcystis XLH6 and Microcystis XLH10 were isolated successfully from Xiliu Lake. Their growth curves were "S" style, their growth rate constants rapidly ascended firstly, and then slowly descended. Average growth rate constants of the two strains were 0.294 and 0.345, average double times of the two strains were 82h and 70h, respectively. The results of the Whole cell PCR and ELISA of the two strains were positive, and both of them were toxigenic. Every milligram of freeze-dried Microcystis XLH6 and Microcystis XLH10 produced 1.4microg and 2.4microg microcystin. Xiliu Lake was polluted by toxigenic microcystis. 96 well microplates can be used to isolate microcystis.

  14. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    Science.gov (United States)

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  15. Isolation, identification and optimization of ethanol producing bacteria from Saccharomyces-based fermentation process of alcohol industries in Iran

    Directory of Open Access Journals (Sweden)

    Hoda Ebrahimi

    2013-01-01

    Full Text Available Introduction: Due to the vast growth of world population, consumption of a lot of energy, limited energy supply and rising prices of fuel oil in the future, other alternative energy source is essential. Ethanol is renewable and a safe fuel and it is mainly based on microbial fermentation. The purpose of this study was isolation of high ethanol producing bacteria from the fermentation process of alcohol industries and optimization of growth conditions to be introduced to the industries. Materials and methods: The samples that were collected from fermentation tanks of alcohol industries were enriched in ZSM medium. To isolate the ethanol producing bacteria, the enriched culture was transferred on RMA agar. Bacterial growth conditions and their effects on ethanol production were optimized based on pH, growth temperature, agitation, fermentation time, initial substrate concentration and carbon and nitrogen sources. In addition, the morphological, physiological and molecular characterizations were investigated for identification of the isolates.Results: Three bacterial isolates ZYM7, ZYM8 and ZYM9 were isolated from fermentation tank. All isolates were able to produce ethanol 5.00, 7.60 and 4.00 gL-1 after 48 hours, respectively. The results demonstrated that all isolates were able to consume most sugars sources specially pentose carbon xylose. The isolate ZYM7 produced 13.00 gL-1 ethanol by consumption of xylose. The results of morphological and physiological characteristics showed that ZYM7 belonged to Lactobacillus sp. and ZYM8 and ZYM9 belonged to Acetobacter sp. Moreover, 16S rRNA sequencing and phylogenetic analyses exhibited that ZYM7 was similar to Lactobacillus rhamnosus with 99% homology and ZYM8 and ZYM9 were similar to Acetobacter pasteurianus with 99 and 98% homology, respectively.Discussion and conclusion: The results showed that that the isolated bacteria were suitable candidates to produce ethanol from raw material enriched with

  16. Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

    Science.gov (United States)

    Mulet, M; Gomila, M; Ramírez, A; Cardew, S; Moore, E R B; Lalucat, J; García-Valdés, E

    2017-02-01

    Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

  17. Molecular Characterization of Vibrio cholerae Isolated From Clinical Samples in Kurdistan Province, Iran.

    Science.gov (United States)

    Ramazanzadeh, Rashid; Rouhi, Samaneh; Shakib, Pegah; Shahbazi, Babak; Bidarpour, Farzam; Karimi, Mohammad

    2015-05-01

    Vibrio cholerae causes diarrhoeal disease that afflicts thousands of people annually. V. cholerae is classified on the basis of somatic antigens into serovars or serogroups and there are at least 200 known serogroup. Two serogroups, O1 and O139 have been associated with epidemic diseases. Virulence genes of these bacteria are OmpW, ctxA and tcpA. Due to the importance of V. cholerae infection and developing molecular diagnostics of this organism in medical and microbiology sciences, this study aimed to describe molecular characterization of V. cholerae isolated from clinical samples using a molecular method. In this study, 48 samples were provided during summer 2013 (late August and early September) by reference laboratory. Samples were assessed using biochemical tests initially. The primer of OmpW, ctxA and tcpA genes was used in Polymerase Chain Reaction (PCR) protocols. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and Repetitive Extragenic Palindromic (REP)-PCR methods were used to subtype V. cholerae. In this study, from a total of 48 clinical stool samples 39 (81.2 %) were positive for V. cholerae in biochemical tests and bacteria culture tests. The PCR results showed that of 39 positive isolates 35 (89.7%), 34 (87.1%) and 37 (94.8%) were positive for ctxA, tcpA and OmpW gene, respectively. Also, in the REP-PCR method with ERIC primer strains were divided into 10 groups. In the REP-PCR method with REP primer, strains were divided into 13 groups. Polymerase chain reaction has specificity and accuracy for identification of the organism and is able to differentiate biotypes. Enterobacterial repetitive intergenic consensus sequence is one of the informative and discriminative methods for the analysis of V. cholerae diversity. The REP-PCR is a less informative and discriminative method compared to other methods for the analysis of V. cholerae diversity.

  18. Molecular Identification, Phylogenic and Virulence Analysis of a Legionella Strain Isolated from Aquatic Environment%一株水源分离军团菌的分子生物学鉴定和系统发育学及毒力分析

    Institute of Scientific and Technical Information of China (English)

    关望; 陈建平; 徐佳楠; 许颖

    2012-01-01

    Objective CD-1 was isolated from aquatic environment, which grew with strict L-cysteine dependence. In this study, we applied molecular methods to identify CD-1, and animal test to understand its virulence. Methods To identify CD-1 strain, CD-1 strain was tested for genus-specific 16S rRNA of Legionella via PCR amplification, then its rpoB gene was sequenced for phylogenic analysis. To understand the virulence, CD-1 was detected for mip gene, which was an indispensable virulent gene of Legionella. Then, BABL/c mice were infected by CD-1 in different dosages. Results For identification, CD-1 was positive for genus-specific 16S rRNA of Legionella, while in the phylogenic tree CD-1 was a sister to Legionella longbeachae with high posterior probability (PP=1. 00). For the virulence analysis, CD-1 was positive for mip gene detection. In the animal test, all mice tested died when the infection dose of CD-1 strain reached 107 cfu/mL. Conclusion CD-1 strain was identified to be Legionella longbeachae with strong virulence to BALB/c mice. It may be a potential virulent strain to human. This is the first strain of Legionella longbeachae isolated in Sichuan province, and this is the first virulence analysis of Legionella strain isolated from aquatic environment in China.%目的 对我室2009年分离获得的一株疑似军团菌菌株(编号为CD-1)进行分子生物学鉴定、系统发育学及毒力分析.方法 对CD-1株进行PCR扩增军团菌属特异性16S rRNA作为分子生物学鉴定,扩增其rpoB 基因片段并测序进行系统发育学分析.对CD-1株进行PCR扩增以检测其毒力基因mip,并用CD-1株感染BABL/c小鼠以研究其毒力.结果 PCR扩增军团菌属特异性16S rRNA显示CD-1株在650 bp处有特异条带,说明CD-1株为一株军团菌,系统发育学分析显示CD-1株与长滩军团菌(Legionella longbeachae)聚为一枝,其后验概率为1.00.对CD-1株进行军团菌毒力基因mip扩增,扩增产物在650 bp

  19. Molecular identification of nematode larvae different from those of the Trichinella genus detected by muscle digestion.

    Science.gov (United States)

    Marucci, Gianluca; Interisano, Maria; La Rosa, Giuseppe; Pozio, Edoardo

    2013-05-20

    Although larvae of the genus Trichinella are the most common parasite species detected in vertebrate muscles using artificial digestion, nematode larvae belonging to other genera are sometimes detected and incorrectly identified as Trichinella. However, it is often very difficult to identify these larvae at the species, genus or family level using microscopy because of the absence of specific morphological characters or cuticle damage, and the only means of identification is PCR and sequencing of specific molecular markers (12S mtDNA; COI; 18S rDNA; and ITS1). From 2008 to 2011, 18 nematode isolates not belonging to the genus Trichinella were collected from different host species. Eleven of these isolates were successfully identified at the species, genus or superfamily level: larvae from two common kestrels, three hooded crows, a hen harrier and a domestic pig were identified as Toxocara cati; larvae from a badger were identified as Toxocara canis; larvae from a domestic pig were identified as a free-living nematode of the genus Panagrolaimus; larvae from a wild boar were identified as belonging to the Metastrongylus genus; and larvae from a rough-legged buzzard were identified as belonging to the superfamily Filarioidea. The recovery of nematodes belonging to genera other than Trichinella during routine meat inspection suggests that the persons performing the analyses need to be informed of the possibility of false positives and that a molecular-based identification system that allows for a rapid and reliable response must be adopted (i.e., a DNA barcoding-like system).

  20. Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must.

    Science.gov (United States)

    Tofalo, Rosanna; Chaves-López, Clemencia; Di Fabio, Federico; Schirone, Maria; Felis, Giovanna E; Torriani, Sandra; Paparella, Antonello; Suzzi, Giovanna

    2009-04-15

    The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.

  1. Identification and classification of Cronobacter spp. isolated from powdered food in Korea.

    Science.gov (United States)

    Park, Jong-Hyun; Lee, Young-Duck; Ryu, Tae-Wha; Chang, Hyo-Ihl

    2010-04-01

    Cronobacter is a major food-borne pathogen in powdered infant formula and can lead to serious developmental aftereffect and death to infants. The contamination of Cronobacter may be a high risk for the powdered foods. To isolate and identify Korean Cronobacter from the powdered foods such as powdered infant formula and Saengsik, conventional culture method, rapid identification system, PCR, 16S rDNA sequencing were performed. As the results of isolation, seven Cronobacter were isolated from seven out of 102 powdered infant formulas and 41 Cronobacter were isolated from 41 out of 86 Saengsiks. Forty eight Cronobacter isolates were identified into C.sakazakii and C.dublenisis by 16s rDNA sequence analysis. Almost the isolates were C.sakazakii and 13% of the isolates were C. dublinesis. One fourth of the C.sakazakii showed different biochemical characteristics of negative nitrate reduction and non-motility activities with the other strains reported previously.

  2. Molecular identification of the chitinase genes in Plasmodium relictum.

    Science.gov (United States)

    Garcia-Longoria, Luz; Hellgren, Olof; Bensch, Staffan

    2014-06-18

    Malaria parasites need to synthesize chitinase in order to go through the peritrophic membrane, which is created around the mosquito midgut, to complete its life cycle. In mammalian malaria species, the chitinase gene comprises either a large or a short copy. In the avian malaria parasites Plasmodium gallinaceum both copies are present, suggesting that a gene duplication in the ancestor to these extant species preceded the loss of either the long or the short copy in Plasmodium parasites of mammals. Plasmodium gallinaceum is not the most widespread and harmful parasite of birds. This study is the first to search for and identify the chitinase gene in one of the most prevalent avian malaria parasites, Plasmodium relictum. Both copies of P. gallinaceum chitinase were used as reference sequences for primer design. Different sequences of Plasmodium spp. were used to build the phylogenetic tree of chitinase gene. The gene encoding for chitinase was identified in isolates of two mitochondrial lineages of P. relictum (SGS1 and GRW4). The chitinase found in these two lineages consists both of the long (PrCHT1) and the short (PrCHT2) copy. The genetic differences found in the long copy of the chitinase gene between SGS1 and GRW4 were higher than the difference observed for the cytochrome b gene. The identification of both copies in P. relictum sheds light on the phylogenetic relationship of the chitinase gene in the genus Plasmodium. Due to its high variability, the chitinase gene could be used to study the genetic population structure in isolates from different host species and geographic regions.

  3. Evaluation of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in clinical isolates.

    Science.gov (United States)

    Marzouk, Manel; Kahla, Imen Ben; Hannachi, Naila; Ferjeni, Asma; Salma, Walid Ben; Ghezal, Samira; Boukadida, Jalel

    2011-04-01

    Identification of Mycobacterium tuberculosis complex (MTC) remains slow. Over the years, several new technologies have been proposed to accelerate and simplify the detection of MTC. In this context, we evaluated an immunochromatographic assay (ICA) (BIO-LINE SD Ag MPT64 TB) for rapid identification of MTC, based on detection of a specific MPT64 antigen of MTC. We have tested it on i) mycobacterial cultures: 210 MTC strains and 28 nontuberculous mycobacteria; ii) M. bovis bacille Calmette-Guérin strain SSI (Statens Serum Institut, Denmark); and iii) 22 microorganisms other than mycobacteria, isolated from cultures. We concluded that this kit has an excellent specificity (100%) and sensitivity (99%) from isolated cultures. The ICA (BIO-LINE SD Ag MPT64 TB) allows excellent MTC identification from clinical isolates. It is a rapid, simple, and inexpensive test, and has a definite contribution in the rapid laboratory diagnosis of tuberculosis.

  4. Monolithic molecularly imprinted polymeric capillary columns for isolation of aflatoxins.

    Science.gov (United States)

    Szumski, Michał; Grzywiński, Damian; Prus, Wojciech; Buszewski, Bogusław

    2014-10-17

    Monolithic molecularly imprinted polymers extraction columns have been prepared in fused-silica capillaries by UV or thermal polymerization in a two-step process. First, a poly-(trimethylolpropane trimethacrylate) (polyTRIM) core monolith was synthesized either by UV or thermal polymerization. Then it was grafted with the mixture of methacrylic acid (MAA) as a functional monomer, ethylene dimethacrylate (EDMA) as a cross-linking agent, 5,7-dimethoxycoumarin (DMC) as an aflatoxin-mimicking template, toluene as a porogen solvent and 2,2-azobis-(2-methylpropionitrile) (AIBN) as an initiator of the polymerization reaction. Different thermal condition of the photografting and different concentrations of the grafting mixture were tested during polymerization. The extraction capillary columns were evaluated in the terms of their hydrodynamic and chromatographic properties. Retention coefficients for aflatoxin B1 and DMC were used for assessment of the selectivity and imprinting factor. The obtained results indicate that the temperature of photografting and concentration of the grafting mixture are key parameters that determine the quality of the prepared MIPs. From the MIP columns characterized by the highest permeability the column of the highest imprinting factor was applied for isolation of aflatoxins B1, B2, G1 and G2 from the model aqueous sample followed by on-line chromatographic separation. The process was performed using a micro-MISPE-microLC-LIF system of a novel design, which allowed for detection of the eluates from the sample preparation part as well as from the chromatographic separation.

  5. Isolation and Molecular Identification of Bacteria from Xiaoqing River of Jinan and Bioremediation of Polluted Water%济南小清河水样细菌的分离、分子鉴定及对污染河水的修复作用

    Institute of Scientific and Technical Information of China (English)

    岳寿松; 边斐; 邹晓凤; 王翠萍; 孙林波; 田顺; 孙凯; 陈文娟

    2014-01-01

    从济南小清河水样中共获得不同菌落特征的细菌微生物13种,利用分子生物学方法对培养得到的菌株进行分子鉴定及系统发育分析,结果表明:13株微生物分属于8个属,分别为肠杆菌属( Enterobacter)、气单胞菌属(Aeromonas)、克雷伯氏菌属(Klebsiella)、微杆菌属(Microbacterium)、希瓦氏菌属(Shewanella)、金黄杆菌属(Chryseobacterium)、芽孢杆菌属(Bacillus)、枸橼酸杆菌属(Citrobacter)和寡养单胞菌属(Stenotroph-omonas)。研究了地衣芽孢杆菌QH11和嗜麦芽寡养(窄食)单胞菌QH13对水体化学需氧量( CODCr )和氨氮(NH3-N)的去除作用,结果表明,处理5天后,QH11对 CODCr和NH3-N的去除率分别为70.6%和84.8%, QH13的去除率分别为58.5%和52.3%。%Total 13 kinds of bacteria were isolated from Xiaoqing River and made genetic identification and phylogenetic analysis by molecular biology method .The 13 strains of bacteria belonged to eight genera , which were Enterobacter, Aeromonas, Klebsiella, Microbacterium, Shewanella, Chryseobacterium, Bacillus, Citrobacter and Stenotrophomonas.The removal effects of Bacillus licheniformis QH11 and Stenotrophomonas maltophilia QH13 on chemical oxygen demand ( CODCr ) and NH3 -N were measured.After treating for 5 days, the CODCr and NH3 -N removal rates of Bacillus licheniformis QH11 were 70.6% and 84.8%, and those of Stenotrophomonas maltophilia QH13 were 58 .5%and 52 .3%respectively .

  6. Molecular variability among isolates of Fusarium oxysporum associated with root rot disease of Agave tequilana.

    Science.gov (United States)

    Vega-Ramos, Karla L; Uvalle-Bueno, J Xavier; Gómez-Leyva, Juan F

    2013-04-01

    In this study, 115 isolates of Fusarium oxysporum from roots of Agave tequilana Weber cv azul plants and soil in commercial plantations in western Mexico were characterized using morphological and molecular methods. Genetic analyses of monosporic isolates included restriction enzyme analysis of rDNA (ARDRA) using HaeIII and HinfI, and genetic diversity was determined using Box-PCR molecular markers. Box-PCR analysis generated 14 groups. The groups correlated highly with the geographic location of the isolate and sample type. These results demonstrate the usefulness of ARDRA and Box-PCR techniques in the molecular characterization of the Fusarium genus for the discrimination of pathogenic isolates.

  7. Identification of potential local isolated for biosurfactant production

    Science.gov (United States)

    Shafiei, Zahra; Yusoff, Wan Mohtar Wan; Hamid, Aidil Abdul; Moazami, Nasrin; Hamzah, Ainon; Fooladi, Taybeh

    2013-11-01

    Biosurfactant are amphiphilic molecule that have received increasing attention in recent years because of their role in the growth of microorganisms on water-insoluble hydrophobic materials such as hydrocarbons as well as their commercial potential in the cosmetics, food, oil recovery and agricultural industries. In this study a potential biosurfactant producing strain was isolated from several soil samples of Terengganu oil refinery, Malaysia and selected during preliminary screening using hemolytic activity, oil spreading and drop collapsed technique. Isolates with at least more than one positive response to these three methods were subjected to complementary screening by measuring surface tension reduction as well as emulsification capacity. The biosurfactant produced by isolated 5M was able to reduced surface tension of culture medium from 60 mN/m to30mN/m. The biochemical and morphological characterization, 16SrRNA gene sequencing showed that the isolated 5M belongs to bacillus groups. The maximum production of biosurfactant by Bacillus 5M was observed after 48 h of incubation.

  8. Isolation, identification and antimicrobial suscep- tibility profiles of ...

    African Journals Online (AJOL)

    examined for the presence of Salmonella following standard techniques and procedures outlined by the International Organization for Standardization. ... of the 28 Salmonella isolates, 18 (64.3%), 3 (10.7%) and 7(25%) were from lac- .... Different studies conducted in Ethiopia revealed fragmented substantial prev-.

  9. Identification of Leishmania isolates from Varzaneh city, Isfahan province, Iran using nested polymerase chain reaction method

    Directory of Open Access Journals (Sweden)

    Reza Arjmand

    2014-01-01

    Full Text Available Background: Leishmaniasis, a parasitic disease, is caused by the Leishmania genus, a protozoan parasite transmitted by sand fly arthropods. Cutaneous leishmaniasis (CL in old world is usually caused by L. major, L. tropica, and L. aethiopica complexes. One of the most important hyper endemic areas of CL in Iran is Isfahan province. Varzaneh is a city in the eastern part of Isfahan province. Due to different biological patterns of parasite strains which are distributed in the region, this study was design to identify Leishmania species from human victims using Kinetoplastid DNA as templates in a molecular PCR method. Materials and Methods: Among 186 suspected cases, 50 cases were confirmed positive by direct microscopy after Giemsa staining. Species characterization of the isolates was done using Nested- PCR as a very effective and sensitive tool to reproduce mini circle strands. Results: After Nested-PCR from all 50 cases, 560 bp bands were produced which according to products of reference strains indicate that the infection etiologic agent has been L. major. 22 (44% of patients were females and 28 (56% of them were males. Their age ranges were between 7 months and 60 years. Conclusion: According to the results of the study and the particular pattern of infection prevalent in the region, genetic studies and identification of Leishmania parasites are very important in the disease control and improvement of regional strategy of therapy protocols.

  10. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  11. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells.

    Science.gov (United States)

    Haqqani, Arsalan S; Delaney, Christie E; Tremblay, Tammy-Lynn; Sodja, Caroline; Sandhu, Jagdeep K; Stanimirovic, Danica B

    2013-01-10

    In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood-brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in 'externalizing' brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons

  12. Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil.

    Science.gov (United States)

    de Oliveira Rocha, Liliana; Reis, Gabriela Martins; da Silva, Valéria Nascimento; Braghini, Raquel; Teixeira, Marta Maria Geraldes; Corrêa, Benedito

    2011-01-31

    Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B(1) and B(2) production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB(1) and FB(2), respectively, P methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Astronomical identification of CN-, the smallest observed molecular anion

    CERN Document Server

    Agundez, M; Guelin, M; Kahane, C; Roueff, E; Klos, J; Aoiz, F J; Lique, F; Marcelino, N; Goicoechea, J R; Garcia, M Gonzalez; Gottlieb, C A; McCarthy, M C; Thaddeus, P

    2010-01-01

    We present the first astronomical detection of a diatomic negative ion, the cyanide anion CN-, as well as quantum mechanical calculations of the excitation of this anion through collisions with para-H2. CN- is identified through the observation of the J = 2-1 and J = 3-2 rotational transitions in the C-star envelope IRC +10216 with the IRAM 30-m telescope. The U-shaped line profiles indicate that CN-, like the large anion C6H-, is formed in the outer regions of the envelope. Chemical and excitation model calculations suggest that this species forms from the reaction of large carbon anions with N atoms, rather than from the radiative attachment of an electron to CN, as is the case for large molecular anions. The unexpectedly large abundance derived for CN-, 0.25 % relative to CN, makes likely its detection in other astronomical sources. A parallel search for the small anion C2H- remains so far unconclusive, despite the previous tentative identification of the J = 1-0 rotational transition. The abundance of C2H...

  14. Characterization and identification of newly isolated Acinetobacter baumannii strain serdang 1 for phenol removal

    Science.gov (United States)

    Yadzir, Z. H. M.; Shukor, M. Y.; Nazir, M. S.; Abdullah, M. A.

    2012-09-01

    A new indigenous bacterial strain from Malaysian soil contaminated with petroleum waste had been successfully isolated, characterized and identified for phenol removal. The gram negative bacteria showed 98% identity with Acinetobacter baumannii based on Biolog{trade mark, serif} Identification System and the determination of a partial 16S ribosomal RNA (rRNA) sequence. The isolate clustered with species belonging to Acinetobacter clade in a 16S rDNA-based neighbour-joining phylogenetic tree.

  15. Identification at Biovar Level of Brucella Isolates Causing Abortion in Small Ruminants of Iran

    OpenAIRE

    Ali Mohammad Behroozikhah; Ramin Bagheri Nejad; Karim Amiri; Ali Reza Bahonar

    2012-01-01

    To determine the most prevalent biovar responsible for brucellosis in sheep and goat populations of Iran, a cross-sectional study was carried out over 2 years in six provinces selected based on geography and disease prevalence. Specimens obtained from referred aborted sheep and goat fetuses were cultured on Brucella selective media for microbiological isolation. Brucellae were isolated from 265 fetuses and examined for biovar identification using standard microbiological methods. Results show...

  16. Identification and diversity of Fusarium species isolated from tomato fruits

    Directory of Open Access Journals (Sweden)

    Murad Nur Baiti Abd

    2016-07-01

    Full Text Available Fruit rot of tomato is a serious disease caused by Fusarium species. Sampling was conducted throughout Selangor, Malaysia and fungal species identification was conducted based on morphological and gene encoding translation elongation factor 1-α (tef1-α sequence analysis. Five species of Fusarium were discovered namely F. oxysporum (including F. oxysporum f. sp. lycopersici, F. solani, F. equiseti, F. proliferatum and F. verticillioides. Our results provide additional information regarding the diversity of Fusarium species associated with fruit rot disease of tomato.

  17. Isolation and identification of fungal communities in compost and vermicompost.

    Science.gov (United States)

    Anastasi, Antonella; Varese, Giovanna Cristina; Marchisio, Valeria Filipello

    2005-01-01

    This research illustrates the qualitative and quantitative composition of the mycoflora of both a green compost (thermophilically produced from plant debris) and a vermicompost (mesophilically produced by the action of earthworms on plant and animal wastes after thermophilic preconditioning). Fungi were isolated using three media (PDA, CMC, PDA plus cycloheximide), incubated at three temperatures (24, 37 and 45 C). Substantial qualiquantitative differences in the species composition of the two composts were observed. The total fungal load was up to 8.2 X 10(5) CFU/g dwt in compost and 4.0 x 10(5) CFU/g dwt in vermicompost. A total of 194 entities were isolated: 118 from green compost, 142 from vermicompost; 66 were common to both. Structural characterization of this kind is necessary to determine the most appropriate application of a compost and its hygienic quality.

  18. Isolation and identification of Gluconacetobacter azotocaptans from corn rhizosphere.

    Science.gov (United States)

    Mehnaz, Samina; Weselowski, Brian; Lazarovits, George

    2006-09-01

    Six acetic acid producing, diazotrophic bacteria were isolated from soil adhering to corn roots. These isolates were shown to be Gluconacetobacter azotocaptans and they shared some features with G. johannae and G. diazotrophicus but differed on the basis of colony morphology on different media, use of carbon sources and use of l-amino acids as a nitrogen source. The species identity was confirmed using 16S rDNA sequence analysis, PCR amplification of 16S rRNA gene with species-specific primers and amplified rDNA restriction analysis. This is the first report of the presence of this bacteria on corn plants. Scope of the paper: This is the first report of the occurrence and association of Gluconacetobacter azotocaptans with corn.

  19. Isolation, identification and antimicrobial activity of propolis-associated fungi.

    Science.gov (United States)

    de Souza, Giovanni Gontijo; Pfenning, Ludwig Heinrich; de Moura, Fabiana; Salgado, Mírian; Takahashi, Jacqueline Aparecida

    2013-01-01

    Propolis is a natural product widely known for its medicinal properties. In this work, fungi present on propolis samples were isolated, identified and tested for the production of antimicrobial metabolites. Twenty-two fungal isolates were obtained, some of which were identified as Alternaria alternata, Aspergillus flavus, Bipolaris hawaiiensis, Fusarium merismoides, Lasiodiplodia theobromae, Penicillium citrinum, Penicillium crustosum, Penicillium janthinellum, Penicillium purpurogenum, Pestalotiopsis palustris, Tetracoccosporium paxianum and Trichoderma koningii. These fungi were grown in liquid media to obtain crude extracts that were evaluated for their antibiotic activity against pathogenic bacteria, yeast and Cladosporium cladosporioides and A. flavus. The most active extract was obtained from L. theobromae (minimum inhibitory concentration = 64 μg/mL against Listeria monocitogenes). Some extracts showed to be more active than the positive control in the inhibition of Staphylococcus aureus and L. monocitogenes. Therefore, propolis is a promising source of fungi, which produces active agents against relevant food poisoning bacteria and crop-associated fungi.

  20. Isolation and Identification of Burkholderia glumae from Symptomless Rice Seeds

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A survey on isolation and detection of the casual organism of bacterial grain rot of rice was conducted during 1997-2006.In 2006,six pathogenic bacterial strains were isolated from two symptomiess seed samples of rice (Oryza sativa L.) originally produced in Hainan Province and then planted in Zhejiang Province,China.They were identified as Burkholderia glumae which is the causal organism of bacterial grain rot of rice by physiological characteristics,colony morphology,pathogenicity test,Biolog,fatty acid methyl ester (FAME) analysis and RAPD-PCR compared with the four standard reference strains.It is confirmed that there is the infection of B.glumae in so-called 'health looking seeds'.

  1. ISOLATION AND IDENTIFICATION OF FLAVONOIDS FROM PROSOPIS JULIFLORA

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    KHANDELWAL PREETI

    2017-01-01

    Full Text Available Objective: The Flavonoids are remarkable and important secondary metabolites, found in plants. The present study was carried out to investigate the flavonoid content present in the leaves, stem, and pods of Prosopis juliflora. Methods: The established protocol of Subramanian & Nagarajan, 1969 was used for isolation of flavonoids. The structure of the isolated compounds was established on the basis of physical and chemical test and spectroscopic evidences (TLC, IR and GC- MS. Results: The study was revealed that the three types of flavonoids Quercetin, Kaempferol and Luteolin were found in Prosopis juliflora. Out of these three flavonoids, Quercetin was found as a major flavonoid. Kaempferol and luteolin were found as minor flavonoids. Conclusion: The present flavonoids may be responsible for various activities found in this plant, like- Antioxidant activity, anti-microbial activity etc.

  2. Isolation and Identification of Prenylflavonoids from Humulus lupulus

    Institute of Scientific and Technical Information of China (English)

    WANG Wen-Shu; ZHOU Ya-Wei; YE Yun-Hua; LI Mei-Lan

    2003-01-01

    @@ The hop plant (Humulus lupulus L. Cannabinaceae ) is cultivated widely through the temperate zones of the world. The female inflorescences of the hop plant (hops) are used in the brewing industry to give bitterness and aroma of beer. In China, hops are used as stomachics, diuretic and tranquilizer. [1] A number of prenylflavonoids have been isolated from hops, [2,3] which caused attention because of their potential anti-cancer properties, [4] endocrine activities, [5] and diacylglycerol acyltransferase inhibition. [6

  3. Isolation and identification of phenolic compounds from Gynura divaricata leaves

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    Chunpeng Wan

    2011-01-01

    Full Text Available Background: Phenolic constituents were the principle bioactivity compounds exist in Gynura divaricata, little phenolic compounds were reported from the plant previously. Materials and Methods: 60% ethanol extract from the leaves of Gynura divaricata were isolated and purified by column chromatography of Silica gel, ODS and Sephadex LH-20, the structures of the isolated compounds were identified by UV, 1H-NMR, 13C-NMR and MS spectroscopic techniques. Additionally, a high-performance liquid chromatography-diode array detector-electrospray ionization-mass (HPLC-DAD-ESI-MS analytical method was developed to identify some minor constituents in the n-butanol fraction of the ethanol extract of Gynura divaricata. Results: Six flavonols and one Dicaffeoylquinic acid were isolated from the leaves of Gynura divaricata, and these compounds were identified as follows: quercetin (1, kaempferol (2, kaempferol-3-O-β-D-glucopyranoside (3, quercetin-3-O-rutinoside (4, kaempferol-3,7-di-O-β-D-glucopyranoside (5, kaempferol-3-O-rutinoside-7-O-β-D-glucopyranoside (6, and 3,5-dicaffeoylquinic acid (7. A total of 13 compounds, including 9 flavonol glycosides and 4 phenolic acids, were tentatively identified by comparing their retention time (RT, UV, and MS spectrum values with those that had been identified and the published data. Conclusion: This was the first time to use the HPLC-DAD-ESI-MS method to identify the phytochemicals of the genera Gynura. Moreover, compounds (6 and (7 have been isolated for the first time from the genus Gynura.

  4. Isolation and Identification of Listeria monocytogenes from Retail

    Directory of Open Access Journals (Sweden)

    A.D.I. Alsheikh

    2013-02-01

    Full Text Available Listeria species are widely distributed in environment and L. monocytogenes are the causal agent of Listeriosis, the disease that can be serious and fatal to human and animals. The objectives of this study were to detect, isolate and identify Listeria monocytogenes from retail broiler chicken ready to eat meat products in restaurants-Khartoum state, Sudan. A total of 250 retail broiler chicken ready to eat meat products were collected from restaurants in Khartoum State, 50 sample from frozen chicken burger, 50 sample from frozen chicken sausages, 50 sample from frozen chicken meat balls (kofta, 50 sample from chicken shawerma and 50 sample from chicken mortedella, Listeria spp. were isolated by the conventional International Organization for Standardization method and L. monocytogenes identified by biochemical test. The results showed that out of total 250 samples, 95 (38% were found to be contaminated with Listeria spp. the isolation rate was as follows: L. monocytogenes (13.6%, L. ivanovi (20.8%, L. grayi (1.6%, L. seeligeri (0.8% and L. welshimeri (1.2%. The results presented in this study indicate the contamination of retail broiler chicken ready to eat meat products with L. monocytogenes. This study reported the occurrence and distribution of L. monocytogenes and other Listeria species in retail meat products (frozen chicken burger, frozen chicken sausages, frozen chicken meat balls (kofta, chicken shawerma and chicken mortedella, purchased from restaurants in Khartoum state Sudan.

  5. Molecular characterization of pneumococcal isolates from pets and laboratory animals.

    Directory of Open Access Journals (Sweden)

    Mark van der Linden

    Full Text Available BACKGROUND: Between 1986 and 2008 Streptococcus pneumoniae was isolated from 41 pets/zoo animals (guinea pigs (n = 17, cats (n = 12, horses (n = 4, dogs (n = 3, dolphins (n = 2, rat (n = 2, gorilla (n = 1 treated in medical veterinary laboratories and zoos, and 44 laboratory animals (mastomys (multimammate mice; n = 32, mice (n = 6, rats (n = 4, guinea pigs (n = 2 during routine health monitoring in an animal facility. S. pneumoniae was isolated from nose, lung and respiratory tract, eye, ear and other sites. METHODOLOGY/PRINCIPAL FINDINGS: Carriage of the same isolate of S. pneumoniae over a period of up to 22 weeks was shown for four mastomys. Forty-one animals showed disease symptoms. Pneumococcal isolates were characterized by optochin sensitivity, bile solubility, DNA hybridization, pneumolysin PCR, serotyping and multilocus sequence typing. Eighteen of the 32 mastomys isolates (56% were optochin resistant, all other isolates were optochin susceptible. All mastomys isolates were serotype 14, all guinea pig isolates serotype 19F, all horse isolates serotype 3. Rats had serotypes 14 or 19A, mice 33A or 33F. Dolphins had serotype 23F, the gorilla serotype 14. Cats and dogs had many different serotypes. Four isolates were resistant to macrolides, three isolates also to clindamycin and tetracycline. Mastomys isolates were sequence type (ST 15 (serotype 14, an ST/serotype combination commonly found in human isolates. Cats, dogs, pet rats, gorilla and dolphins showed various human ST/serotype combinations. Lab rats and lab mice showed single locus variants (SLV of human STs, in human ST/serotype combinations. All guinea pig isolates showed the same completely new combination of known alleles. The horse isolates showed an unknown allele combination and three new alleles. CONCLUSIONS/SIGNIFICANCE: The isolates found in mastomys, mice, rats, cats, dogs, gorilla and dolphins are most likely identical to human pneumococcal isolates. Isolates from

  6. Isolation and identification of the acetonitrile-degrading Pseudomonas from the Ab-anbar-e Ganjali Khan in Kerman

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    Narjes Ramezani–pour

    2016-06-01

    Full Text Available Introduction: Nitriles are toxic and hazardous compounds for all organisms which are produced enormously by human being and cause environment pollution. Biodegradation is the best method for Nitrile elimination in sewage. The aim of this study was isolation and identification of native bacteria Acetonitrile-degrading from the sewage of the city of Kerman. Materials and methods: The enrichment and screening of Acetonitrile degrading bacteria was performed in a specific medium containing 1 % Acetonitrile as sole carbon and nitrogen source. Enzyme activity and ammonium production was determined in growth medium using a modification of the phenol/hypochlorite method. Identification of the isolates was undertaken using microbial - biochemical and molecular tests. The optimization of enzyme production was examined. The amount of nitrile and acid were also determined by gas chromatography. Results: Among three isolated nitrile hydrolyzing producing species (FA3, FA8 and AB19, FA8 isolate showed maximum enzyme productivity. The results of characteristic tests showed that these bacteria belonged to the Pseudomonas sp. Moreover, 16S rRNA sequencing and phylogenetic analyses exhibited that FA11, FA8 and AB19 strains were similar to Pseudomonas Otitidis and Pseudomonas geniculate with 99, 100% homology, respectively. Results of medium optimization of Pseudomonas FA8 strain showed that glucose (10 gL-1 and yeast extract (5 gL-1 in neutral medium strongly supported enzyme production. Gas chromatography results showed that FA8 produced 58% acetic acid at 48 h of incubation. Discussion and conclusion: The results demonstrated that Pseudomonas FA8 isolated bacterium is a suitable candidate for degradation of Acetonitrile. This bacterium could be used for treatment of industrial wastewater and sites that contaminated with Acetonitrile.

  7. Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus)

    Science.gov (United States)

    Laith, AA; Ambak, Mohd Azmi; Hassan, Marina; Sheriff, Shahreza Md.; Nadirah, Musa; Draman, Ahmad Shuhaimi; Wahab, Wahidah; Ibrahim, Wan Nurhafizah Wan; Aznan, Alia Syafiqah; Jabar, Amina; Najiah, Musa

    2017-01-01

    Aim: The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia (Oreochromis niloticus) in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents. Materials and Methods: The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection. Results: The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, β-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity), reconfirmed by 16S rRNA gene sequencing (99% similarity) and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule, hyperemic and

  8. Isolation and identification of the three rice monotelosomics

    Institute of Scientific and Technical Information of China (English)

    YU Hengxiu; GONG Zhiyun; SU Yan; GU Minghong

    2005-01-01

    In order to obtain rice monotelosomic, the progeny of 24 telotrisomics, derived from an indica rice variety, Zhongxian 3037, were screened. The variants that differed morphologically from the diploids and the original primary trisomics as well as the telotrisomics were collected for cytological identification. The variants with 24 chromosomes were selected according to the prometaphase chromosomes. From these variants, three monotelosomics with one chromosome arm deletion in each were verified by fluorescence in situ hybridization (FISH) using a rice centromeric BAC clone of 17p22 as a marker probe. The three monotelosomics were derived from telotrisomic 1S, 4L and 11L, respectively. Further identification was conducted on the prometaphase or pachytene chromosomes of the three variants, which were probed with the same centromeric BAC clone together with the corresponding chromosome arm specific makers, a0059H02 (on the short arm of chromosome 1), a0034E24 (on the long arm of chromosome 4), and a0071H11 (on the long arm of chromosome 11). The results indicated that the telocentric chromosomes in the three monotelosomics were derived from their respective corresponding telotrisomics. According to the telocentric chromosomes of the variants, they were monotelosomic 1S (one long arm of chromosome 1 was lost), monotelosomic 4L (one short arm of chromosome 4 was lost) and monotelosomic 11L (one short arm of chromosome 11 was lost), respectively.

  9. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    Directory of Open Access Journals (Sweden)

    Wang Nan

    2011-02-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE, indirect immunoperoxidase test (IPX and electron microscopy(EM. The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV and 3 strains of border disease virus(BDV in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

  10. Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community.

    Science.gov (United States)

    Raja, Huzefa A; Miller, Andrew N; Pearce, Cedric J; Oberlies, Nicholas H

    2017-03-24

    Fungi are morphologically, ecologically, metabolically, and phylogenetically diverse. They are known to produce numerous bioactive molecules, which makes them very useful for natural products researchers in their pursuit of discovering new chemical diversity with agricultural, industrial, and pharmaceutical applications. Despite their importance in natural products chemistry, identification of fungi remains a daunting task for chemists, especially those who do not work with a trained mycologist. The purpose of this review is to update natural products researchers about the tools available for molecular identification of fungi. In particular, we discuss (1) problems of using morphology alone in the identification of fungi to the species level; (2) the three nuclear ribosomal genes most commonly used in fungal identification and the potential advantages and limitations of the ITS region, which is the official DNA barcoding marker for species-level identification of fungi; (3) how to use NCBI-BLAST search for DNA barcoding, with a cautionary note regarding its limitations; (4) the numerous curated molecular databases containing fungal sequences; (5) the various protein-coding genes used to augment or supplant ITS in species-level identification of certain fungal groups; and (6) methods used in the construction of phylogenetic trees from DNA sequences to facilitate fungal species identification. We recommend that, whenever possible, both morphology and molecular data be used for fungal identification. Our goal is that this review will provide a set of standardized procedures for the molecular identification of fungi that can be utilized by the natural products research community.

  11. The isolation and identification of steroidal sapogenins in Kleingrass.

    Science.gov (United States)

    Patamalai, B; Hejtmancik, E; Bridges, C H; Hill, D W; Camp, B J

    1990-08-01

    Kleingrass (Panicum coloratum L) has been reported to cause hepatogenous photosensitization in sheep and goats in West Texas since 1973. The toxin in Kleingrass has been suspected of being a steroidal saponin as Kleingrass produced characteristic birefringent crystals similar to those produced by saponin-containing plants such as Agave lecheguilla and Tribulus terrestris. In this present study, steroidal saponis were isolated from Kleingrass and their sapogenins were identified as diosgenin and yamogenin by means of thin-layer chromatography and infrared spectrophotometric analysis.

  12. Isolation and identification of novel geosmin-degrading bacteria.

    Science.gov (United States)

    Xue, Qiang; Chen, Gang; Shimizu, Kazuya; Sakharkar, Meena Kishore; Utsumi, Motoo; Chen, Honghan; Li, Miao; Zhang, Zhenya; Sugiura, Norio

    2011-06-01

    Three novel geosmin-degrading bacteria were isolated from the sediments of Lake Kasumigaura, Japan. All strains were identified as Acinetobacter spp. by 16S rRNA gene sequence analysis and can biodegrade geosmin at an initial geosmin concentration of 2 mg/L after 2 days. Furthermore, at an initial geosmin concentration of 40 microg/L, geosmin removal was more than 68per cent by GSM-2 strain, and the degradation mechanism followed a pseudo-first-order mode. A rate constant of 0.026 reveals rapid geosmin degradation. This is the first report on geosmin degradation by by Acinetobacter spp.

  13. Chemicals from Agave sisalana Biomass: Isolation and Identification

    Directory of Open Access Journals (Sweden)

    Jener David Gonçalves Santos

    2015-04-01

    Full Text Available Agave sisalana (sisal is known worldwide as a source of hard fibers, and Brazil is the largest producer of sisal. Nonetheless, the process of removing the fibers of the sisal leaf generates 95% waste. In this study, we applied chemical sequential steps (hydrothermal extraction, precipitation, liquid-liquid extraction, crystallization, SiO2 and Sephadex LH 20 column chromatography to obtain pectin, mannitol, succinic acid, kaempferol and a mixture of saponins as raw chemicals from sisal biomass. The structural identification of these compounds was performed though spectrometric methods, such as Infrared (IR, Ultraviolet (UV, Mass spectrometry (MS and Nuclear magnetic resonance (NMR. All the sisal chemicals found in this work are used by both the chemical and pharmaceutical industries as excipients or active principles in products.

  14. Chemicals from Agave sisalana biomass: isolation and identification.

    Science.gov (United States)

    Santos, Jener David Gonçalves; Vieira, Ivo Jose Curcino; Braz-Filho, Raimundo; Branco, Alexsandro

    2015-04-20

    Agave sisalana (sisal) is known worldwide as a source of hard fibers, and Brazil is the largest producer of sisal. Nonetheless, the process of removing the fibers of the sisal leaf generates 95% waste. In this study, we applied chemical sequential steps (hydrothermal extraction, precipitation, liquid-liquid extraction, crystallization, SiO2 and Sephadex LH 20 column chromatography) to obtain pectin, mannitol, succinic acid, kaempferol and a mixture of saponins as raw chemicals from sisal biomass. The structural identification of these compounds was performed though spectrometric methods, such as Infrared (IR), Ultraviolet (UV), Mass spectrometry (MS) and Nuclear magnetic resonance (NMR). All the sisal chemicals found in this work are used by both the chemical and pharmaceutical industries as excipients or active principles in products.

  15. [Isolation and identification of alkaloids form Menispermum dauricum growing in Xianning].

    Science.gov (United States)

    Pan, X; Hu, C; Zeng, F; Zhang, S; Xu, J

    1998-09-01

    The alkaloids of rhizoma of Menispermum dauricum DC growing in Xianning have been subjected to isolation and identification. The results showed that its two major constituents, which are only next of dauricine in content, are dauricinoline and daurinoline, instead of the commonly found daurisoline in the same plant materials from North China.

  16. Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum

    DEFF Research Database (Denmark)

    Atabay, H.I.; Corry, J.E.L.; On, Stephen L.W.

    1998-01-01

    %) suggests that routine isolation and identification methods should be amended to enable a thorough evaluation of its role in human gastroenteritis and avian hepatitis. Some phenotypic characters useful in identifying poultry campylobacteria are presented which could be utilized, along with other technique...

  17. Isolation and identification of euphol and ß-sitosterol from the dichloromethane extracts of Synadenium glaucescens

    CSIR Research Space (South Africa)

    Nyigo, VA

    2016-06-01

    Full Text Available 2016; 5(3): 100-104May- June© 2016, All rights reserved Isolation and identification of euphol and β-sitosterol from the dichloromethane extracts of Synadenium glaucescens Vitus A. Nyigo, Xolani Peter, Faith Mabiki, Hamisi M. Malebo, Robinson H...

  18. Fusarium proliferatum isolated from garlic in Spain: identification, toxigenic potential and pathogenicity on related Allium species

    Directory of Open Access Journals (Sweden)

    Daniel PALMERO

    2012-05-01

    Full Text Available Fusarium proliferatum has been reported on garlic in the Northwest USA, Spain and Serbia, causing water-soaked tan-colored lesions on cloves. In this work, Fusarium proliferatum was isolated from 300 symptomatic garlic bulbs. Morphological identification of Fusarium was confirmed using species-specific PCR assays and EF-1α sequencing. Confirmation of pathogenicity was conducted with eighteen isolates. Six randomly selected F. proliferatum isolates from garlic were tested for specific pathogenicity and screened for fusaric acid production. Additionally, pathogenicity of each F. proliferatum isolate was tested on healthy seedlings of onion (Allium cepa, leek (A. porrum, scallions (A. fistulosum, chives (A. schoenoprasum and garlic (A. sativum. A disease severity index (DSI was calculated as the mean severity on three plants of each species with four test replicates. Symptoms on onion and garlic plants were observed three weeks after inoculation. All isolates tested produced symptoms on all varieties inoculated. Inoculation of F. proliferatum isolates from diseased garlic onto other Allium species provided new information on host range and pathogenicity. The results demonstrated differences in susceptibility with respect to host species and cultivar. The F. proliferatum isolates tested all produced fusaric acid (FA; correlations between FA production and isolate pathogenicity are discussed. Additionally, all isolates showed the presence of the FUM1 gene suggesting the ability of Spanish isolates to produce fumonisins.

  19. Molecular characterization of Histoplasma capsulatum isolated from an outbreak in treasure hunters Histoplasma capsulatum in treasure hunters

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    Muñoz Bertha

    2010-09-01

    Full Text Available Abstract Background In Mexico, primary pulmonary histoplasmosis is the most relevant clinical form of the disease. The geographical distribution of specific strains of Histoplasma capsulatum circulating in Mexico has not been fully established. Outbreaks must be reported in order to have current, updated information on this disease, identifying new endemic areas, manner of exposure to the fungi, and molecular characterization of the causative agents. We report a recent outbreak of histoplasmosis in treasure hunters and the molecular characterization of two isolates obtained from these patients. Methods Six patients admitted to the National Institute of Respiratory Diseases (INER in Mexico City presented severe respiratory symptoms suggestive of histoplasmosis. They acquired the infection in the Veracruz (VZ endemic zone. Diagnosis was made by X-ray and Computed tomography (CT, liver function, immunological techniques, and culture. Identification of H. capsulatum isolates was confirmed by using Polymerase chain reaction (PCR was conducted with a probe from the M antigen, and the isolates were characterized by means of Random amplification of polymorphic DNA (RAPD-PCR employed the 1253 oligonucleotide and a mixture of oligonucleotides 1281 and 1283. These were compared to eight reference strain isolates from neighboring areas. Results X-ray and CT revealed disseminated micronodular images throughout lung parenchyma, as well as bilateral retrocaval, prevascular, subcarinal, and hilar adenopathies, hepatosplenomegaly, and altered liver function tests. Five of the six patients developed disseminated histoplasmosis. Two H. capsulatum strains were isolated. The same band profile was detected in both strains, indicating that both isolates corresponded to the sole H. capsulatum strain. Molecular characterization of the isolates was similar in 100% with the EH-53 Hidalgo human (HG strain (reference strain integrated into the LAm A clade described for

  20. ISOLATION AND IDENTIFICATION OF FLAVONOIDS FROM IRANIAN SENNA

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    A.R GHANADI

    2000-12-01

    Full Text Available Background. Senna italica Miller or Cassia italica (Miller Sprengel is a native medicinal plant growing in south of Iran and has laxative and cathartic effects. Anthraquinone compounds have been partly associated with the purgative effects of the plant. One of the side effects of anthraquinones is griping and presence of flavonoid compounds in the plant may decrease this effect. Methods. In this study, the plant after being collected, was extracted by a soxhlet method. The isolation of flavonoids was carried out by means of preparative Thin Layer Chromatography of extract of the plant leaves. The structure of flavonoids was determined by the UV-Vis. techniques in methanol and by addition of the shift reagents and hydrolysis. The quantitative determinations of flavonoids in plant were also performed using an UV-Vis. spectroscopy method. Results and Discussion. Rutin or 5, 7, 3', 4' tetrahydroxy-flavone 3-O-rhamnoglucoside and two flavonoids from aurone group were isolated and identified from the leaves of Senna italica. The percent of flavonoids in Iranian Senna determined 1.5 fold than Cassia angustifolia. These flavonoids may decrease the griping effects of Senna anthraquinones

  1. Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

    Science.gov (United States)

    Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

    2015-09-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.

  2. Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food.

    Science.gov (United States)

    Crispim, S M; Nascimento, A M A; Costa, P S; Moreira, J L S; Nunes, A C; Nicoli, J R; Lima, F L; Mota, V T; Nardi, R M D

    2013-01-01

    Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS); they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g). 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates), Lactobacillus delbrueckii (18 isolates), Lactobacillus casei (9 isolates), Lactobacillus reuteri (6 isolates), Lactobacillus brevis (3 isolates), Lactobacillus gasseri (2 isolates), Lactobacillus nagelii (1 isolate), and Lactobacillus plantarum group (18 isolates). recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates) and Lactobacillus paraplantarum (3 isolates). Genomic diversity was investigated by molecular typing with rep (repetitive sequence)-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus). The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

  3. Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food

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    S.M. Crispim

    2013-01-01

    Full Text Available Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS; they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g. 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates, Lactobacillus delbrueckii (18 isolates, Lactobacillus casei (9 isolates, Lactobacillus reuteri (6 isolates, Lactobacillus brevis (3 isolates, Lactobacillus gasseri (2 isolates, Lactobacillus nagelii (1 isolate, and Lactobacillus plantarum group (18 isolates. recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates and Lactobacillus paraplantarum (3 isolates. Genomic diversity was investigated by molecular typing with rep (repetitive sequence-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus. The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

  4. Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis.

    Science.gov (United States)

    Moreira, Sabrina F I; Bailão, Alexandre M; Barbosa, Mônica S; Jesuino, Rosalia S A; Felipe, M Sueli Soares; Pereira, Maristela; de Almeida Soares, Célia Maria

    2004-01-30

    Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.

  5. Morphology, Pathogenicity and Molecular Identification of Fusarium spp. Associated with Anise Seeds in Serbia

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    Snežana PAVLOVIĆ

    2016-12-01

    Full Text Available Anise (Pimpinella anisum L. is an important medicinal spice plant that belongs to the family Apiaceae. Anise seeds are rich in essential oils and this is a reason why anise production in Serbia has increased over the last decade. During a routine health inspection on anise seeds collected from three localities in the province of Vojvodina (Mošorin, Veliki Radinci and Ostojićevo during 2012 and 2013, it was found out that Fusarium spp. were a commonly observed fungi. The presence of Fusarium fungion the seed samples ranged from 3.75-13.75%. The aim of this study was to isolate and identify the strains of Fusarium species present on anise seed samples as it is necessary that commercially used anise seeds are completely free of Fusarium. Based on morphological, microscopic characteristics and a molecular identification by sequencing of TEF gene, the presence of the following species was confirmed on the anise seeds: F. tricinctum, F. proliferatum, F. equiseti, F. oxysporum, F. sporotrichoides, F. incarnatum and F. verticillioides. According to our knowledge and research, this is the first report of F. tricinctum and F. sporotrichoides as pathogens on anise seeds in the world. All seven isolates of Fusarium species are pathogenic to the anise seedlings, while the most virulent species were F. oxysporum, F. tricinctum and F. incarnatum.

  6. Proteomic identification of gender molecular markers in Bothrops jararaca venom.

    Science.gov (United States)

    Zelanis, André; Menezes, Milene C; Kitano, Eduardo S; Liberato, Tarcísio; Tashima, Alexandre K; Pinto, Antonio F M; Sherman, Nicholas E; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

    2016-04-29

    Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being

  7. An optimised protocol for molecular identification of Eimeria from chickens.

    Science.gov (United States)

    Kumar, Saroj; Garg, Rajat; Moftah, Abdalgader; Clark, Emily L; Macdonald, Sarah E; Chaudhry, Abdul S; Sparagano, Olivier; Banerjee, Partha S; Kundu, Krishnendu; Tomley, Fiona M; Blake, Damer P

    2014-01-17

    Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.

  8. Isolation and identification of acetogenic bacteria obtained from deer rumen and their potential for methanogenesis inhibitor

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    Amlius Thalib

    2008-10-01

    Full Text Available Methanogenesis can be inhibited by various chemicals through different mechanism reaktion. The use of acetogenic bacteria as H2 sink is assumed to be a promising approach. Isolation and identification of acetogenic bacteria obtained from deer rumen had been conducted. Two types of media used for isolation were hydrogen-carbondioxide utilizing acetogens and carbonmonoxide utilizing acetogens. Identification of species of acetogens isolates was based on descriptions of morphology, Gram type, motility, bioreaction results, and oksygen requirement. The compositions of methane and volatile fatty acids (VFA were determined on minimal media or added with sheep rumen liquid innoculated with pure isolates. The identification results showed that the isolate cultured on media of hydrogen-carbondioxide utilizing acetogens was Acetoanaerobium noterae and the ones cultured on media of carbonmonoxide utilizing acetogens was Acetobacterium woodii. Inoculumn of A. noterae and A. woodii could decreased the composition of methane resulted from substrate fermented by fresh rumen liquid of sheep (CRDF, that is culture of A. noterae added FPM and defaunator decreased methane production by 28.8% (P CH3COOH + 2H2O by which reduction of CO2 with H2 producing CH4 can be inhibited or decreased. Their function as methanogenesis inhibitor would be more significant when they are combined with microbial growth factors and defaunator.

  9. Isolation and identification methods of Rothia species in oral cavities.

    Science.gov (United States)

    Tsuzukibashi, Osamu; Uchibori, Satoshi; Kobayashi, Taira; Umezawa, Koji; Mashimo, Chiho; Nambu, Takayuki; Saito, Masanori; Hashizume-Takizawa, Tomomi; Ochiai, Tomoko

    2017-03-01

    Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable

  10. Identification and antimicrobial susceptibility patterns of Staphylococcus spp. isolated from canine chronic otitis externa

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    Silva N.

    2001-01-01

    Full Text Available Swab samples obtained from 96 dogs with chronic otitis externa were cultured for the isolation of Staphylococcus species. Of 57 staphylococcal strains, 41 (72% were coagulase-negative (CNS. The identification of staphylococci strains was made by standard procedures for the routine identification of staphylococci in clinical practice. S. sciuri was the most frequent species isolated (22.8% from chronic otitis externa in dogs followed by S. intermedius (12.3%, S. auricularis (10.5% and S. aureus (8.8%. Three (5.2% CNS strains could not be identified. Bacterial isolates were susceptible to enrofloxacin, gentamicin, cephalothin, chloramphenicol and neomycin. Resistance was most common to penicillin G, oxacillin and ampicillin.

  11. Molecular identification of Nocardia species using the sodA gene

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    K. Sánchez-Herrera

    2017-09-01

    Full Text Available Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp, hsp65 (401 bp, secA1 (494 bp, gyrB (1195 bp and rpoB (401 bp. The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.

  12. Leukaemomycin, in antibiotic with antitumor activity. II. Isolation and identification.

    Science.gov (United States)

    Strauss, D; Fleck, W

    1975-01-01

    A Streptomyces strain belonging to Streptomyces griseus (Krainsky) Waksman et Henrici 1948 sensu Hütter (1967) was found to produce the red-pigment antibiotic leukaemomycin. The antitumor active antibiotic was isolated from the culture broth and purified by ion-exchange processes. The crude base of leukaemomycin mainly consists of 4 components with biological activity. The base-complex was separated by counter-current distribution into the biological active leukaemomycins B1, B2, C, and D. The single components of leukaemomycin were compared with the anthracycline antibiotics daunomycin, dihydrodaunomycin, and adriamycin. The analytical procedures allowed to state the identity of leukaemomycin B1 with rubomycin B, leukaemomycin C with daunomycin, leukaemomycin D with dihydrodaunomycin and allowed to suggest the identity of leukaemomycin B2 with daunosaminyldaunomycin. Formulas and physicochemical data are given. Bio-assay methods are described to determine leukaemomycin B complex beside leukaemomycin C.

  13. Isolation and identification of thiohomosildenafil and thiosildenafil in health supplements.

    Science.gov (United States)

    Zou, Peng; Hou, Peiling; Oh, Sharon Sze-Yin; Chong, Y M; Bloodworth, Bosco Chen; Low, Min-Yong; Koh, Hwee-Ling

    2008-06-09

    Two unknown compounds are detected and isolated from health supplements for the enhancement of sexual function. The structures of the unknown compounds are elucidated using high-resolution MS, ESI-MS/MS, NMR, UV and IR. One compound is identified as an analogue of sildenafil in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety, and an ethyl group instead of a methyl group is attached to the piperazinyl nitrogen. Hence, this compound is named thiohomosildenafil. Another compound is also a sildenafil analogue in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety. This compound is named thiosildenafil. Both the two compounds are first detected in health supplements. The UV, IR and completely assigned NMR data of thiohomosildenafil and thiosildenafil are first reported.

  14. Isolation and Identification of Novel Compounds from Mulgedium tatarica DC.

    Institute of Scientific and Technical Information of China (English)

    REN Yu-Lin; ZHOU Ya-Wei; YE Yun-Hua

    2003-01-01

    @@ Our study focused on the chemical constituents of Mulgedium tatarica that was collected from Inner Mongolia autonomous region of China. It is not only a folk medicinal herb but also a wild vegetable. It was reported that this plant showed several bioactivities, such as diuretic action, stimulating milk secretion, analgesia and haemorrhoidolysis. [1 ] Fatty acids and other volatile components of Mulgedium tatarica were detected by GC-MS for the first time. [2] From the ethanol extract of the whole plant of Mulgedium tatarica DC., two new compounds, named tataricoside and mulgedin A, have been isolated and characterized. Their structures were identified by UV, IR,HRFABMS, 1H NMR, 13C NMR, HMQC, HMBC and 1H-1H COSY spectral analysis.

  15. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  16. Molecular Identification of Bacteria Involved in Degradation of Crude ...

    African Journals Online (AJOL)

    the 16s rRNA gene of the isolates using DNA sequencing technique. Isolate 2, 3 ... microbial biodegradation of pollutants has intensified in recent years as humanity strives to find sustainable ..... volume of extracted oil was deducted from the.

  17. Isolation and Identification of Active Compound Cause Light Emmitting of Bacterial Photobacterium phosphoreum Isolated from the Indonesia Jepara Marine Squid

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    Idam Arif

    2005-04-01

    Full Text Available This research carried out to study the bioluminescence process of bacterial Photobacterium phosphoreum isolated from Indonesia marine squid. The method used in the present study involved isolation, purification, electrophoresis, and the absorbance and light intensity measurement. This result show that the luciferace enzyme of bacterial Photobacterium phosphoreum or called LBPP catalyzes the emission of visible light from the reaction of reduced flavin mononucleotide (FMNH2, molecular oxygen (O2, and an aldehyde (RCOH. The electrophoresis data show that LBPP comprised of two different subunits α and βwith 41kD and 38 kD molecular weights. The absorb pattern showed that the bioluminescence process centered around 516 nm and are consistent with the fluorescence data. This result concluded that the excitation state formed after LBPP bind subtracts and the ground state formed after LBPP releases product and visible light.

  18. Molecular typing of Brucella species isolates from livestock and human.

    Science.gov (United States)

    Nagalingam, Mohandoss; Shome, Rajeswari; Balamurugan, Vinayagamurthy; Shome, Bibek Ranjan; NarayanaRao, Krishnamsetty; Vivekananda; Isloor, Shrikrishna; Prabhudas, Krishnamsetty

    2012-01-01

    Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

  19. Isolation, screening and identification of novel isolates of Actinomycetes from India for antimicrobial applications

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    Vineeta Singh

    2016-12-01

    Full Text Available The search for novel bioactive compounds from the natural environment has been rapidly increased with the increase in multi-drug resistant (MDR pathogens. In the present study, the antimicrobial potential of novel actinomycetes has been evaluated by initial screening of six soil samples. Primary and secondary screening was performed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans, Candida tropicalis, Trichophyton rubrum, and other MDR bacterial and fungal test strains, and at the end thirteen active isolates were selected for further study. Microbial strains were identified on the basis of growth conditions and other biochemical characters. Five most active microbial strains were identified using 16S rRNA sequence homology and designated as Streptomyces xanthophaeus MTCC 11938, Streptomyces variabilis MTCC 12266, Streptomyces xanthochromogenes MTCC 11937, Streptomyces levis EU 124569 and Streptomyces sp. NCIM 5500. Four antibacterial and three antifungal compounds isolated from the above five isolates were purified and partially characterized using UV absorption and IR spectra. Two antibacterial metabolites, belong to chromone and peptide antibiotic, respectively. The antifungal compounds were found to be of non-polyene nature. In conclusion, we study the isolation of novel bacterial strains of actinomycetes for producing novel compounds having antibacterial and antifungal activities from the unexplored agro-ecological niches of India. Also, this study paves the way for further characterization of these isolates of Streptomyces sp. for their optimum utilization for antimicrobial purposes.

  20. Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state.

    Science.gov (United States)

    Reinhardt, Erica L; Ramos, Patrícia L; Manfio, Gilson P; Barbosa, Heloiza R; Pavan, Crodowaldo; Moreira-Filho, Carlos A

    2008-07-01

    Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

  1. Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state

    Science.gov (United States)

    Reinhardt, Érica. L.; Ramos, Patrícia L.; Manfio, Gilson P.; Barbosa, Heloiza R.; Pavan, Crodowaldo; Moreira-Filho, Carlos A.

    2008-01-01

    Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data. PMID:24031239

  2. Microbiological and molecular characterization of Staphylococcus hominis isolates from blood.

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    Soraya Mendoza-Olazarán

    Full Text Available BACKGROUND: Among Coagulase-Negative Staphylococci (CoNS, Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec type, and genetic relatedness of clinical S. hominis isolates. METHODOLOGY: S. hominis blood isolates (n = 21 were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus. RESULTS: Of the S. hominis isolates screened, 47.6% (10/21 were categorized as strong biofilm producers and 23.8% (5/21 as weak producers. Furthermore, 81% (17/21 of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0-95%. Among mecA-positive isolates, 14 (82% harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III. CONCLUSIONS: The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor

  3. Isolation and Identification of Putative Oral Cancer Stem Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Min; ZHAO Yan-Hua; TANG Xiao-Fei

    2011-01-01

    Objective: To isolate and characterize putative cancer stem cells in Tea8113 oral squmous cell carcinoma cell line. Methods: Putative cancer stem cells were isolated by limited dilution assay in Tea8113 cell line. Biological features of putative cancer stem cells were detected by MTT assay, flow cytometry, immunofluorescence, Colony Forming Efficiency assays, cell motility assay and in vivo tumor formation experiment. Results: Compared with untreated Tea8113 cells, the putative cancer stem cells proliferated more quickly and showed heteroploid cell cycle,higher G0/G1-arrested cells, higher CFE and higher expression levels of ABCG2 belonged to tumor stem cell phenotypes. The putative cancer stem cells had stronger capacity to generate tumors in vivo. Conclusion: The holoclone cells have higher proliferation and self-renewal abilities, which may be cancer stem cells existed in Tea8113 oral squmous cell carcinoma cell line.%目的:分离鉴定口腔鳞癌细胞系Tca8113中的肿瘤干细胞.方法:利用有限稀释的方法分离Tca8113细胞系中的肿瘤干细胞.通过MTT法、流式细胞技术、细胞免疫荧光、克隆形成率分析、细胞迁移能力检测和裸鼠皮下成瘤实验确定分离得到的肿瘤干细胞的生物学特点.结果:分离得到的紧密型克隆肿瘤细胞表现为异倍体样细胞周期,大部分细胞处于G0/G1期,增殖能力、克隆形成率和体外迁移能力都明显高于未分离的肿瘤细胞.紧密型克隆肿瘤细胞肿瘤干细胞标记物ABCG2表达也高于未分离的肿瘤细胞,并且具有更强的裸鼠皮下成瘤能力.结论:我们分离得到的紧密型克隆细胞具有较强的细胞增殖和自我更新能力,可能就是口腔鳞癌细胞系Tca8113中的肿瘤干细胞.

  4. Isolation and identification of main mastitis pathogens in Mexico

    Directory of Open Access Journals (Sweden)

    H. Castañeda Vázquez

    2013-04-01

    Full Text Available The present work is a large epidemiological study aiming to detect the prevalence of subclinical mastitis and to investigate the major udder pathogens in Jalisco State, western Mexico. For this purpose, 2205 dairy cows, representing 33 Mexican dairy herds, were involved. Of 2205 cows, 752 mastitic animals were diagnosed and only 2,979 milk samples could be obtained for further investigation. All 2979 milk samples were subjected to California Mastitis Test (CMT to differentiate clinical cases from subclinical ones where 1996 samples (67 % reacted positively. Of these, 1087 samples (54.5% came from cows suffering from clinical cases of mastitis. Bacteriological identification of the causative agents revealed the presence of a major group of pathogens including the Coagulase negative staphylococci (CNS, S.aureus, S.agalactiae, Corynebacterium spp. and Coliform bacteria which were detected in 464 (15.6%, 175 (5.9%, 200 (6.8%, 417 (14% and 123 (4.1% of the 2927 investigated quarters, 295 (15.4%, 118 (15.7%, 111 (14.8%, 227 (30.2% and 109 (14.5% of the 752 examined cows and in 33 (100%, 22 (66.7%, 19 (57.6%, 30 (90.1% and 27 (81.8% of the 33 herds involved, respectively. Other pathogens could be detected in the investigated milk samples such as S. dysgalactiae (0.4%, S.uberis (0.37%, Bacillus spp. (1%, Nocardia spp. (0.6% und Candida spp. (0.1%. Meanwhile, others were present in a negligible ratio; including the Aerococcus viridans, and Enterococcus spp., Lactococcus lactis, S. bovis.

  5. Molecular characterization of Malassezia nana isolates from cats.

    Science.gov (United States)

    Castellá, Gemma; De Bellis, Filippo; Bond, Ross; Cabañes, F Javier

    2011-03-24

    Malassezia nana (M. nana) is a lipid-dependent yeast that has been isolated from cats and cows. Some sequence variability has been observed in the large subunit (LSU) and internal transcribed spacer (ITS) regions between strains isolated from cats and cows though these regions in M. nana isolates from cats alone have proven to be relatively conserved. In the present study, microsatellite PCR fingerprinting and β-tubulin gene sequence analysis were carried out on M. nana isolates from cats to investigate the genetic diversity of this species. Although a relatively small number of isolates were available, the similarity in the sequences of the β-tubulin and the microsatellite profiles indicate that a particular M. nana genotype colonizes cats. Moreover, all isolates obtained from animals with otitis externa had the same microsatellite fingerprinting pattern. Further studies of a wider population of M. nana isolates from other hosts and status disease are needed to establish that M. nana is a genetically homogeneous species. This is the first report of the characterization of the β-tubulin gene in Malassezia spp.

  6. Isolation, identification and characterization of an electrogenic microalgae strain.

    Directory of Open Access Journals (Sweden)

    Yicheng Wu

    Full Text Available Extracellular electron transfer involving microbes is important as it closely reflects the ability of cells to communicate with the environment. However, there are few reports on electron transfer mechanisms of pure microalgae and a lack of any model alga to study the transfer processes. In the present study, nine green microalgae species were isolated from wastewater and characterized in terms of their ability to transfer electrons between cells and an electrode. One species showed direct electron transfer via membrane-associated proteins and indirect electron transfer via secreted oxygen. The microalga was identified as Desmodesmus sp. based on phylogenetic analysis and electron microscopy. Electrochemical tests demonstrated that Desmodesmus sp. was able to act as a cathodic microorganism. Stable current densities of -0.24, 35.54 and 170 mA m(-2 were achieved at potentials of +0.2, -0.2 and -0.4 V, respectively, under illumination. Dissolved oxygen concentration measurement showed gradients within the microalgae biofilm: 18.3 mg L(-1 in light decreasing to 4.29 mg L(-1 in the dark. This study diversified the exoelectrogen library and provided a potential model microalga to explore the associated mechanism of extracellular electron transfer.

  7. Growth medium for the rapid isolation and identification of anthrax

    Science.gov (United States)

    Kiel, Johnathan L.; Parker, Jill E.; Grubbs, Teri R.; Alls, John L.

    2000-07-01

    Anthrax has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to design a culture technique to rapidly isolate and identify `live' anthrax. In liquid or solid media form, 3AT medium (3-amino-L-tyrosine, the main ingredient) accelerated germination and growth of anthrax spores in 5 to 6 hours to a point expected at 18 to 24 hours with ordinary medium. During accelerated growth, standard definitive diagnostic tests such as sensitivity to lysis by penicillin or bacteriophage can be run. During this time, the bacteria synthesized a fluorescent and thermochemiluminescent polymer. Bacteria captured by specific antibody are, therefore, already labeled. Because living bacteria are required to generate the polymer, the test converts immunoassays for anthrax into viability assays. Furthermore, the polymer formation leads to the death of the vegetative form and non-viability of the spores produced in the medium. By altering the formulation of the medium, other microbes and even animal and human cells can be grown in it and labeled (including viruses grown in the animal or human cells).

  8. Isolation, identification and characterization of an electrogenic microalgae strain.

    Science.gov (United States)

    Wu, Yicheng; Guan, Kai; Wang, Zejie; Xu, Bing; Zhao, Feng

    2013-01-01

    Extracellular electron transfer involving microbes is important as it closely reflects the ability of cells to communicate with the environment. However, there are few reports on electron transfer mechanisms of pure microalgae and a lack of any model alga to study the transfer processes. In the present study, nine green microalgae species were isolated from wastewater and characterized in terms of their ability to transfer electrons between cells and an electrode. One species showed direct electron transfer via membrane-associated proteins and indirect electron transfer via secreted oxygen. The microalga was identified as Desmodesmus sp. based on phylogenetic analysis and electron microscopy. Electrochemical tests demonstrated that Desmodesmus sp. was able to act as a cathodic microorganism. Stable current densities of -0.24, 35.54 and 170 mA m(-2) were achieved at potentials of +0.2, -0.2 and -0.4 V, respectively, under illumination. Dissolved oxygen concentration measurement showed gradients within the microalgae biofilm: 18.3 mg L(-1) in light decreasing to 4.29 mg L(-1) in the dark. This study diversified the exoelectrogen library and provided a potential model microalga to explore the associated mechanism of extracellular electron transfer.

  9. Molecular typing of Streptococcus agalactiae isolates from fish

    Science.gov (United States)

    The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphisms (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 49 S. agalactiae ...

  10. [Isolation identification and characterization of halotolerant petroleum-degrading bacteria].

    Science.gov (United States)

    Wu, Tao; Xie, Wen-Jun; Yi, Yan-Li; Li, Xiao-Bin; Wang, Jun; Hu, Xiang-Ming

    2012-11-01

    To obtain efficient halotolerant petroleum-degrading bacteria, 39 bacteria strains were isolated from 30 petroleum contaminated saline soil samples in Yellow River Delta, an important base of petroleum production in China. One bacterium (strain BM38) was found to efficiently degrade crude oil in highly saline environments based on a series of liquid and soil incubation experiments. According to its morphology, physiochemical characteristics and 16S rDNA sequence analysis, this strain was identified as Pseudomonas putida. Moreover, a series of liquid incubation experiments were conducted to investigate its characteristics such as halotolerance, biosurfactants production and degrading efficiency for various hydrocarbons. The salt resistance test demonstrated that strain BM38 grew well at NaCl concentrations ranging from 0.5% to 6.0%. Petroleum degradation experiments showed that strain BM38 could degrade 73.5% crude oil after 7 days in a liquid culture medium containing 1.0% NaCl and remove more than 40% of total petroleum hydrocarbons after 40 days in the soil with 0.22% and 0.61% of salinity, these results proved that the strain was effective in removing petroleum hydrocarbons. Strain BM38 could produce a bioemulsifier in a liquid culture medium. The NaCl concentration had the significant effect on the EI24 of fermentation broth, which decreased sharply if the NaCl concentration was greater than 1.0%. However, the EI24 of BM38 was still quite high in the presence of 2.0% of NaCl, and the value was 61.0%. Furthermore, this strain was also able to grow in mineral liquid media amended with hexadecane, toluene, phenanthrene, isooctane and cyclohexane as the sole carbon sources. Among these hydracarbons, strain BM38 showed relatively high ability in degrading n-alkanes and aromatic hydracarbons. The results indicated that strain BM38 had potential for application in bioremediation of petroleum-contaminated saline soil.

  11. Isolation and identification of the thermophilic alkaline desulphuricant strain

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A desulfurization strain that belongs to the thermophilic alkaline desulphuricant is designated as strain GDJ-3 and isolated from Inner Mongolia, China. The colony of the strain shows tiny, yellow, or white-yellow, and it becomes henna with the protracting of cultivated time. The cells are bacilliform (0.3 -0.6 × 1.0-1.2 μm), motive, and Gram negative. The strain GDJ-3 is able to utilize respectively the thiosulphate, sulfate, sulfite, or sulfide as sulfur source, utilize the carbon dioxide as the carbon source, and utilize the ammonium or nitrate as the nitrogen source. According to GenBank data, 16s RNA results of GDJ-3 are in good agreement with Alpha proteobacterrium sp. (97%) and Ochrobactrum sp. (98%). For GDJ-3, the optimum growth temperature is at 45℃, the optimum pH is at 8.5-8.8, and the optimum rocking speed of sorting table is at 150 r/min. Under the optimum culture condition, the cells of the strain can live for about 18 h. In the desulfurization solution, which is prepared according to the composition of DDS solution, the objectionable constituents of sodium thiosulphate and sodium sulfide were added factitiously, and the bacterial cell concentration was set at 107/mL. After the regeneration of the above desulfurization solution by the strain cells, the concentration of sodium thiosulphate was decreased by 14.75 g/L (percentage loss of content 13.21%), the concentration of sodium sulfide was decreased by 0.76 g/L (percentage loss of content 87.36%) in the desulfurization solution in 9.5 hours, and sulfur appeared. Maybe, this kind of strain can be used as the regeneration’s bacterial source of DDS solution.

  12. Isolation, morphological identification and in vitro antibacterial activity of endophytic bacteria isolated from Azadirachta indica (neem) leaves.

    Science.gov (United States)

    Singh, Ankit Kumar; Sharma, Rajesh Kumar; Sharma, Varsha; Singh, Tanmay; Kumar, Rajesh; Kumari, Dimple

    2017-05-01

    The objective of this study was to isolate endophytic bacteria from Azadirachta indica (neem) leaves, their identification and investigate their antibacterial activity against three Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes and Bacillus cereus and Gram-negative bacteria Escherichia coli, Salmonella Typhimurium and Klebsiella pneumoniae. Fresh leaves of A. indica (neem) was procured from the Department of Botany, JNKVV, Jabalpur. Five samples were taken, and each sample was divided into five subsamples and separated for further isolation of endophytic bacteria. For sterilization leaves were treated with double distilled water, 0.1% sodium hypochlorite, 0.01% bavistin, 0.05% and 70% ethanol. Sterilized leaves of the plants were embedded in Kings B (KB) petri plates and incubated at 37°C for 24 h. Characterization of the bacteria was done according to its morphology and by Gram-staining. After that, a single colony was transferred into brain heart infusion (BHI) broth and incubated at 37°C for 24 h. The antibacterial effect was studied by the disk diffusion method with known antibiotic ciprofloxacin (Ci) as standard. A total of 25 bacterial isolates from A. indica (neem) were obtained and identified morphologically. Most of the samples on KB media depicted irregular shape, flat elevation, undulated, rough, opaque, and white in color. Most of the samples on blood agar showed irregular, raise elevation, undulated, smooth, opaque and all the isolates were nonhemolytic and nonchromogenic. The growth of endophytic bacteria in BHI broth were all isolates showed turbidity. The microscopic examination revealed that maximum isolates were Gram-positive and rod shaped. Good antibacterial activity was observed against S. aureus, Streptococcus pyogenes, E. coli, Salmonella Typhimurium, and K. pneumoniae. Endophytic bacteria are present in leaves of A. indica (neem) and it possesses antibacterial activity against few Gram-positive and Gram

  13. Isolation, morphological identification and in vitro antibacterial activity of endophytic bacteria isolated from Azadirachta indica (neem leaves

    Directory of Open Access Journals (Sweden)

    Ankit Kumar Singh

    2017-05-01

    Full Text Available Aim: The objective of this study was to isolate endophytic bacteria from Azadirachta indica (neem leaves, their identification and investigate their antibacterial activity against three Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes and Bacillus cereus and Gram-negative bacteria Escherichia coli, Salmonella Typhimurium and Klebsiella pneumoniae. Materials and Methods: Fresh leaves of A. indica (neem was procured from the Department of Botany, JNKVV, Jabalpur. Five samples were taken, and each sample was divided into five subsamples and separated for further isolation of endophytic bacteria. For sterilization leaves were treated with double distilled water, 0.1% sodium hypochlorite, 0.01% bavistin, 0.05% and 70% ethanol. Sterilized leaves of the plants were embedded in Kings B (KB petri plates and incubated at 37°C for 24 h. Characterization of the bacteria was done according to its morphology and by Gram-staining. After that, a single colony was transferred into brain heart infusion (BHI broth and incubated at 37°C for 24 h. The antibacterial effect was studied by the disk diffusion method with known antibiotic ciprofloxacin (Ci as standard. Results: A total of 25 bacterial isolates from A. indica (neem were obtained and identified morphologically. Most of the samples on KB media depicted irregular shape, flat elevation, undulated, rough, opaque, and white in color. Most of the samples on blood agar showed irregular, raise elevation, undulated, smooth, opaque and all the isolates were nonhemolytic and nonchromogenic. The growth of endophytic bacteria in BHI broth were all isolates showed turbidity. The microscopic examination revealed that maximum isolates were Gram-positive and rod shaped. Good antibacterial activity was observed against S. aureus, Streptococcus pyogenes, E. coli, Salmonella Typhimurium, and K. pneumoniae. Conclusion: Endophytic bacteria are present in leaves of A. indica (neem and it possesses

  14. Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Dalsgaard, Inger; Høi, L.

    1996-01-01

    Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B- cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish...

  15. Recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers

    DEFF Research Database (Denmark)

    Andersen, Lars Dyrskjøt; Zieger, Karsten; Ørntoft, Torben Falck

    2007-01-01

    individually contributed to the management of the disease. However, the development of high-throughput techniques for simultaneous assessment of a large number of markers has allowed classification of tumors into clinically relevant molecular subgroups beyond those possible by pathological classification. Here......, we review the recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers....

  16. Spa typing and identification of pvl genes of meticillin-resistant Staphylococcus aureus isolated from a Libyan hospital in Tripoli.

    Science.gov (United States)

    Ahmed, Mohamed O; Baptiste, Keith E; Daw, Mohamed A; Elramalli, Asma K; Abouzeed, Yousef M; Petersen, Andreas

    2017-09-01

    The purpose of the study was to investigate the molecular characteristics of meticillin-resistant Staphylococcus aureus (MRSA) isolated from clinical sources in Tripoli, Libya. A total of 95 MRSA strains collected at the Tripoli medical Centre were investigated by spa typing and identification of the Panton-Valentine Leukocidin (pvl) genes. A total of 26 spa types were characterized and distributed among nine clonal complexes; CC5 (n=32), CC80 (n=18), CC8 (n=17) and CC22 (n=12) were the most prevalent clonal complexes. In total, 34% of the isolates were positive for PVL. This study demonstrated the presence of CA-MRSA and pvl positive strains in hospital settings and underlines the importance of using molecular typing to investigate the epidemiology of MRSA. Preventative measures and surveillance systems are needed to control and minimize the spread of MRSA in the Libyan health care system. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  17. Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico.

    Science.gov (United States)

    Almazán, Consuelo; González-Álvarez, Vicente H; Fernández de Mera, Isabel G; Cabezas-Cruz, Alejandro; Rodríguez-Martínez, Rafael; de la Fuente, José

    2016-03-01

    The tick-borne pathogens Ehrlichia canis and Anaplasma platys are the causative agents of canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT). Although molecular evidence of E. canis has been shown, phylogenetic analysis of this pathogen has not been performed and A. platys has not been identified in Mexico, where the tick vector Rhipicephalus sanguineus sensu lato (s.l.) is common. The aim of this research was to screen, identify and characterize E. canis and A. platys by PCR and phylogenetic analysis in dogs from La Comarca Lagunera, a region formed by three municipalities, Torreon, Gomez-Palacio and Lerdo, in the Northern states of Coahuila and Durango, Mexico. Blood samples and five engorged R. sanguineus s.l. ticks per animal were collected from 43 females and 57 male dogs presented to veterinary clinics or lived in the dog shelter from La Comarca Lagunera. All the sampled dogs were apparently healthy and PCR for Anaplasma 16S rRNA, Ehrlichia 16S rRNA, and E. canis trp36 were performed. PCR products were sequenced and used for phylogenetic analysis. PCR products were successfully amplified in 31% of the samples using primers for Anaplasma 16S rRNA, while 10% and 4% amplified products using primers for Ehrlichia 16S rRNA and E. canis trp36 respectively. Subsequent sequencing and phylogenetic analyses of these products showed that three samples corresponded to A. platys and four to E. canis. Based on the analysis of trp36 we confirmed that the E. canis strains isolated from Mexico belong to a conservative clade of E. canis and are closely related to strains from USA. In conclusion, this is the first molecular identification of A. platys and the first molecular characterization and phylogenetic study of both A. platys and E. canis in dogs in Mexico.

  18. Isolation and molecular characterization of Acanthamoeba genotypes isolated from soil sources of public and recreational areas in Iran.

    Science.gov (United States)

    Karamati, Seyed Ahmad; Niyyati, Maryam; Lorenzo-Morales, Jacob; Lasjerdi, Zohreh

    2016-12-01

    Pathogenic strains of Acanthamoeba are causative agents of a sight threating infection of the cornea known as Acanthamoeba keratitis. AK cases have been reported in Iran recently due to inappropriate usage of contact lens maintenance and most patients report a contact with contaminated sources such as dust, water or soil. Sixty soil samples were collected from public and recreational areas in the province of East Azerbaijan, Iran and checked for the presence of Acanthamoeba spp. Samples were cultured on non-nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were performed in order to investigate the pathogenic potential of isolated Acanthamoeba strains. Acanthamoeba spp. was isolated from 41.6% of soil samples and genotyping of the strains resulted in the identification of genotypes T3, T4, T5 and T11. Most of the isolates belonging to genotypes T3 and T4 showed high pathogenic potential, indicating that they might present a potential health hazard for humans and other animals in this region. To the best of our knowledge, this is the first report on the identification of genotypes T3 and T11 from soil sources in the country.

  19. Molecular identification of Paragonimus species by DNA pyrosequencing technology.

    Science.gov (United States)

    Tantrawatpan, Chairat; Intapan, Pewpan M; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Srichantaratsamee, Chutatip; Anamnart, Witthaya; Maleewong, Wanchai

    2013-06-01

    DNA pyrosequencing for PCR amplicons is an attractive strategy for the identification of microorganisms because of its short time performance for large number of samples. In this study, the primers targeting the fragment of ITS2 region of nuclear ribosomal RNA gene were newly developed for pyrosequencing-based identification of 6 Paragonimus species, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus macrorchis, Paragonimus siamensis and Paragonimus westermani. Pyrosequencing determination of 39 nucleotides of partial ITS2 region could discriminate 6 Paragonimus species, and could also detect intra-species genetic variation of P. macrorchis. This DNA pyrosequencing-based identification can be a valuable tool to improve species-level identification of Paragonimus in the endemic areas.

  20. Molecular identification and food source inference of constructive ...

    African Journals Online (AJOL)

    user

    2011-01-10

    Jan 10, 2011 ... constructive plants native to the O. sinensis habitat was established, and then feeding food habit of .... The tender roots tissue and the vegetable food residues .... identification and classification of the target plants were.

  1. Caracterização morfofisiológica e identificação molecular de isolados de Guignardia citricarpa, agente patogênico da mancha preta dos citros = Morphophysiological characterization and molecular identification of isolates of Guignardia citricarpa, a pathogenic agent of the citrus black spot

    Directory of Open Access Journals (Sweden)

    Marilda Pereira Caixeta

    2008-12-01

    Full Text Available O presente trabalho teve como objetivo identificar 11 isolados de Guignardia citricarpa, agente causal da mancha preta dos citros (MPC, obtidos de frutas cítricas sintomáticas de diferentes regiões geográficas, por meio da PCR e caracterização morfofisiológica das estruturas propagativas, esporulação e crescimento micelial emdiferentes meios de cultura, temperaturas e regimes de luz, nas condições de laboratório. Pelo teste de PCR, todos os isolados foram identificados como o patógeno G. citricarpa. Os isolados caracterizados foram submetidos às temperaturas de 20, 25 e 30ºC, em regime de luz contínua, escuro contínuo e fotoperíodos de 12 horas, durante 24 dias. Utilizaram-se os meios de cultura aveia-ágar (AA, batata-dextrose-ágar (BDA e cenoura-dextrose-ágar (CDA. Os resultados mostraram que ocorreu interação entre os diferentes meios de cultura, temperaturas e fotoperíodos. O meio de cultura que melhor estimulou o crescimento micelial foi o CDA a 25ºC sob o fotoperíodo de 12h. A maior produção de esporos (conídios foi verificada no meio BDA a 20ºC, no fotoperíodo de 12 horas. No meio CDA, não ocorreu esporulação de nenhum isolado. Sob a temperatura de 30ºC, foiverificada apenas a produção de hifas e picnídios para a maioria dos isolados, em todos os meios de cultura e fotoperíodo testados.Thepresent work aims to identify 11 isolates of Guignardia citricarpa, the causal agent of the citrus black spot (CBS, obtained from affected fruit in different geographical regions, through PCR and morphophysiological characterization of propagative structures, sporing and mycelial growth in different means of culture, temperatures and photoperiods, under laboratory conditions. Through the PCR test, all isolates were identified as being the G.citricarpa pathogen. The characterized isolates were subjected to evaluations at temperatures of 20, 25 and 30ºC, in continuous light, continuous darkness, and alternating 12

  2. Microscopic Evaluation, Molecular Identification, Antifungal Susceptibility, and Clinical Outcomes in Fusarium, Aspergillus and, Dematiaceous Keratitis

    Directory of Open Access Journals (Sweden)

    Devarshi U. Gajjar

    2013-01-01

    Full Text Available Purpose. Fusarium, Aspergillus, and Dematiaceous are the most common fungal species causing keratitis in tropical countries. Herein we report a prospective study on fungal keratitis caused by these three fungal species. Methodology. A prospective investigation was undertaken to evaluate eyes with presumed fungal keratitis. All the fungal isolates (n=73 obtained from keratitis infections were identified using morphological and microscopic characters. Molecular identification using sequencing of the ITS region and antifungal susceptibility tests using microdilution method were done. The final clinical outcome was evaluated in terms of the time taken for resolution of keratitis and the final visual outcome. The results were analyzed after segregating the cases into three groups, namely, Fusarium, Aspergillus, and Dematiaceous keratitis. Results. Diagnosis of fungal keratitis was established in 73 (35.9% cases out of 208 cases. The spectra of fungi isolated were Fusarium spp. (26.6%, Aspergillus spp. (21.6%, and Dematiaceous fungi (11.6%. The sequence of the ITS region could identify the Fusarium and Aspergillus species at the species complex level, and the Dematiaceous isolates were accurately identified. Using antifungal agents such as fluconazole, natamycin, amphotericin B, and itraconazole, the minimum inhibitory concentrations (MICs for Fusarium spp. were >32 μg/mL, 4–8 μg/mL, 0.5–1 μg/mL, and >32 μg/mL, respectively. Antifungal susceptibility data showed that Curvularia spp. was highly resistant to all the antifungal agents. Overall, natamycin and amphotericin B were found to be the most effective antifungal agents. The comparative clinical outcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was significantly good when compared with the Fusarium and Aspergillus groups (P<0.05. The time required for healing in the Fusarium group was statistically significantly less when

  3. Incidence and identification of Klebsiella planticola in clinical isolates with emphasis on newborns.

    Science.gov (United States)

    Westbrook, G L; O'Hara, C M; Roman, S B; Miller, J M

    2000-04-01

    Studies conducted in France and Germany suggest that up to 19% of clinically identified Klebsiella sp. are actually Klebsiella planticola, an environmental species that has been attributed to two cases of septicemia, with a rare isolate of Klebsiella terrigena (0. 4%) being identified. A 1-year survey of newborns on a neonatal ward, also conducted in Germany, reported that 72% of Klebsiella sp. were Klebsiella oxytoca and 8.7% were K. planticola. The tests necessary to identify these species are not found in most clinical identification schemes or in the database matrices of most commercial identification products. To determine the incidence of unrecognized K. planticola among the Klebsiella sp. isolates in our collection, we used the battery of seven supplemental tests amended from the work of Monnet and Freney to test 352 stock isolates and 84 fresh clinical isolates from four local hospitals. After testing 436 strains of Klebsiella, only one strain was identified as a possible K. planticola and none was identified as K. terrigena. We tested an additional 43 stock strains of K. oxytoca isolated from newborns by using eight biochemical tests and found one additional strain of K. planticola. The occurrence of K. planticola in our collection is far less frequent than that observed in other countries.

  4. Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach.

    Science.gov (United States)

    Kazemi-Rad, Elham; Mohebali, Mehdi; Khadem-Erfan, Mohammad Bagher; Saffari, Mojtaba; Raoofian, Reza; Hajjaran, Homa; Hadighi, Ramtin; Khamesipour, Ali; Rezaie, Sassan; Abedkhojasteh, Hoda; Heidari, Mansour

    2013-10-01

    Pentavalent antimonial compounds have been the first line therapy for leishmaniasis; unfortunately the rate of treatment failure of anthroponotic cutaneous leishmaniasis (ACL) is increasing due to emerging of drug resistance. Elucidation of the molecular mechanisms operating in antimony resistance is critical for development of new strategies for treatment. Here, we used a cDNA-AFLP approach to identify gene(s) which are differentially expressed in resistant and sensitive Leishmania tropica field isolates. We identified five genes, aquaglyceroporin (AQP1) acts in drug uptake, ATP-binding cassette (ABC) transporter (MRPA) involved in sequestration of drug, phosphoglycerate kinase (PGK) implicated in glycolysis metabolism, mitogen activated protein kinase (MAPK) and protein tyrosine phosphatase (PTP) responsible for phosphorylation pathway. The results were confirmed using real time RT-PCR which revealed an upregulation of MRPA, PTP and PGK genes and downregulation of AQP1 and MAPK genes in resistant isolate. To our knowledge, this is the first report of identification of PTP and PGK genes potentially implicated in resistance to antimonials. Our findings support the idea that distinct biomolecules might be involved in antimony resistance in L. tropica field isolates.

  5. Molecular characterization of Haemophilus ducreyi isolates from different geographical locations.

    Science.gov (United States)

    Mbwana, J; Bölin, I; Lyamuya, E; Mhalu, F; Lagergård, T

    2006-01-01

    The technique of random amplified polymorphic DNA (RAPD) was adapted and optimized to study Haemophilus ducreyi isolates. A panel of 43 strains isolated from chancroid patients from different countries in Africa, Europe, North America, and Asia were characterized. The strains were also studied with respect to lipooligosaccharide (LOS) migration and immunoblotting patterns and the presence of cytolethal distending toxin genes. The RAPD method with the OPJ20 primer generated nine banding patterns (1 to 9). The majority of the isolates were clustered into two major profiles, 14 and 13 strains into profiles 1 and 2, respectively, and just a few strains revealed patterns 3 and 4. The isolates from Thailand were exceptional in that they showed greater diversity and were represented by six different RAPD patterns, i.e., patterns 3 and 5 to 9. The LOS migration and immunoblotting analyses revealed two different patterns, which indicated long and short forms of LOS; the former was found in 20/23 tested strains. Two strains that expressed the short form of LOS were grouped into RAPD pattern 4. The absence of cdtABC genes was observed in only 4/23 strains, and three of these isolates were assigned to RAPD pattern 4. Our results showed limited genotypic and phenotypic variations among H. ducreyi strains, as supported by the conserved RAPD and LOS profiles shared by the majority of the studied strains. However, the RAPD method identified differences between strains, including those from different geographic areas, which indicate the potential of RAPD as an epidemiological tool for the typing of H. ducreyi isolates in countries where chancroid is endemic.

  6. Molecular identification of uncommon clinical yeast species in Iran

    Directory of Open Access Journals (Sweden)

    Ladan Karimi

    2015-03-01

    Conclusion: We identified several rare clinical isolates selected from a big collection at the species level by ITS-sequencing. As the list of yeast species as opportunistic human fungal infections is increasing dramatically, and many isolates remain unidentified using conventional methods, more sensitive and specific advanced approaches help us to clarify the aspects of microbial epidemiology of the yeast infections.

  7. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    Science.gov (United States)

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

  8. Identification of Candida spp. isolated from vaginal swab by phenotypic methods and multiplex PCR in Duhok, Iraq

    Directory of Open Access Journals (Sweden)

    Ahmed Basheer Mohammed

    2015-11-01

    Full Text Available Background: Candida species are the second most common cause of vulvovaginitis worldwide. The purpose of this study was to identify the species of vaginal Candida isolates by using phenotypic and Multiplex PCR techniques. Methods: 91 isolates from patients admitted to Azadi hospital and Maternity hospital in Duhok city were collected. The vaginal swab specimens were inoculated on Sabouraud dextrose agar. Colonies were then sub cultured on Chromogenic Candida agar. Genomic DNA extraction was performed using a Genomic DNA Extraction kit. For rapid identification of Candida spp., specific primers based on the genomic sequence of DNA topoisomerase 11 of C. albicans, C. parapsilosis I, C. parapsilosis II, C. guilliermondi, C. dubliniensis, C. krusei, C. kefyr and C. glabrata, C. tropicalis I, C. tropicalis II, C. lusitaniae were used. The multiplex PCR products were separated by electrophoresis in 1.5% agarose gel, visualized by staining with ethidium bromide, and photographed. Results: 4 Candida species, namely C. albicans, C. glabrata, C. krusei and C. tropicalis were distinguished by Chromogenic Candida agar on the basis of colony colour and morphology. PCR with the primer mixes yielded 7 different sized of PCR products corresponding to C. albicans, C. guilliermondii, C. dubliniensis, C. glabrata, C. kefyr, C. krusei and C. tropicalis II. The analysis revealed C. glabrata and C. albicans were the most common species isolated with the percentage 40% and 30% respectively. Conclusions: This study concluded that phenotypic characteristics on selective agar medium such as chromogenic candida agar are useful for presumptive identification of Candiada spp. with the support of molecular method such as multiplex PCR. [Int J Res Med Sci 2015; 3(11.000: 3211-3216

  9. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

    Science.gov (United States)

    Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

    2015-06-01

    Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

  10. Identification at biovar level of Brucella isolates causing abortion in small ruminants of iran.

    Science.gov (United States)

    Behroozikhah, Ali Mohammad; Bagheri Nejad, Ramin; Amiri, Karim; Bahonar, Ali Reza

    2012-01-01

    To determine the most prevalent biovar responsible for brucellosis in sheep and goat populations of Iran, a cross-sectional study was carried out over 2 years in six provinces selected based on geography and disease prevalence. Specimens obtained from referred aborted sheep and goat fetuses were cultured on Brucella selective media for microbiological isolation. Brucellae were isolated from 265 fetuses and examined for biovar identification using standard microbiological methods. Results showed that 246 isolates (92.8%) were B. melitensis biovar 1, 18 isolates (6.8%) were B. melitensis biovar 2, and, interestingly, one isolate (0.4%) obtained from Mazandaran province was B. abortus biovar 3. In this study, B. melitensis biovar 3 was isolated in none of the selected provinces, and all isolates from 3 provinces (i.e., Chehar-mahal Bakhtiari, Markazi, and Ilam) were identified only as B. melitensis biovar 1. In conclusion, we found that B. melitensis biovar 1 remains the most prevalent cause of small ruminant brucellosis in various provinces of Iran.

  11. Identification at Biovar Level of Brucella Isolates Causing Abortion in Small Ruminants of Iran

    Directory of Open Access Journals (Sweden)

    Ali Mohammad Behroozikhah

    2012-01-01

    Full Text Available To determine the most prevalent biovar responsible for brucellosis in sheep and goat populations of Iran, a cross-sectional study was carried out over 2 years in six provinces selected based on geography and disease prevalence. Specimens obtained from referred aborted sheep and goat fetuses were cultured on Brucella selective media for microbiological isolation. Brucellae were isolated from 265 fetuses and examined for biovar identification using standard microbiological methods. Results showed that 246 isolates (92.8% were B. melitensis biovar 1, 18 isolates (6.8% were B. melitensis biovar 2, and, interestingly, one isolate (0.4% obtained from Mazandaran province was B. abortus biovar 3. In this study, B. melitensis biovar 3 was isolated in none of the selected provinces, and all isolates from 3 provinces (i.e., Chehar-mahal Bakhtiari, Markazi, and Ilam were identified only as B. melitensis biovar 1. In conclusion, we found that B. melitensis biovar 1 remains the most prevalent cause of small ruminant brucellosis in various provinces of Iran.

  12. Developments in techniques for the isolation, enrichment, main culture conditions and identification of spermatogonial stem cells

    OpenAIRE

    He, Yanan; Chen, Xiaoli; Zhu, Huabin; Wang, Dong

    2015-01-01

    The in vitro culture system of spermatogonial stem cells (SSCs) provides a basis for studies on spermatogenesis, and also contributes to the development of new methods for the preservation of livestock and animal genetic modification. In vitro culture systems have mainly been established for mouse SSCs, but are lacking for farm animals. We reviewed and analyzed the current progress in SSC techniques such as isolation, purification, cultivation and identification. Based on the published studie...

  13. On the Stability and Evolution of Isolated Molecular Clouds

    Science.gov (United States)

    Langer, W.; Nelson, R.

    1998-01-01

    We present the results of three dimensional hydrodynamic models of evolving, isolated, low mass, quiescent clouds and Bok gobules, where the interstellar radiation field plays an important role in the thermal and chemical evolution, and thermal pressure provides dominant support against gravitational collapse.

  14. Molecular characterization of Pseudomonas syringae pv. tomato isolates from Tanzania

    DEFF Research Database (Denmark)

    Shenge, K.C.; Stephan, D.; Mabagala, R. B.

    2008-01-01

    pathogenicity assays on tomato, carbon source utilization by the Biolog Microplate system, polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. All the P. syringae pv. tomato isolates produced bacterial speck symptoms on susceptible tomato (cv. ‘Tanya') seedlings. Metabolic...

  15. Molecular Screening of Staphylococcal Enterotoxin B Gene in Clinical Isolates

    Directory of Open Access Journals (Sweden)

    Ali Karami

    2011-01-01

    Full Text Available Objective: The role of staphylococcal enterotoxin B (SEB in food poisoning is well known,however its role in other diseases remains to be explored. The aim of this study is the molecularscreening and characterization of the SEB gene in clinically isolated strains.Materials and Methods: In this experimentally study, 300 Staphylococcus aureus (S.aureus strains isolated from clinical samples were assayed. The isolated strains wereconfirmed by conventional bacteriological methods. Polymerase chain reaction (PCRwas used to determine the enterotoxin B (ent B gene. Assessment of toxin productionin all strains that contained the ent B gene was then performed. Finally, using specificantibody against SEB, a Western-blot was applied to confirm detection of enterotoxin Bproduction.Results: Results indicated that only 5% of the 300 clinically isolated S. aureus containedthe ent B gene. All strains which contained the ent B gene produced a proteinous enterotoxinB. The results of sequence determination of the PCR product were compared withthe gene bank database and 98% similarity was achieved. The results of the Western-blotconfirmed that enterotoxin B was produced in strains that contained the ent B gene.Conclusion: The results of this study indicate that 5% of clinically isolated S. aureusstrains produce enterotoxin B. Considering that the enterotoxin B is an important superantigen,it is possible that a delay in diagnosis and lack of early proper treatment can causean incidence of late complications, particularly in staphylococcal chronic infections. Forthis reason, it is suggested that in addition to detecting bacteria, an enterotoxin B detectiontest should be performed to control its toxigenicity.

  16. Molecular studies of the Brazilian infectious bronchitis virus isolates

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    JT Abreu

    2010-06-01

    Full Text Available Avian infectious bronchitis virus (IBV isolates have been widely characterized by reverse transcription followed by polymerase chain reaction and DNA sequencing. In present study, these techniques were applied to three viral genomic regions comprising the complete and/or a partial S1 segment, S2 and nucleocapsid genes. DNA sequences from viral isolates obtained from 1972 to 1989 and from 2006 to 2008 were compared. High similarity (>90% was observed among some of the genomic segments, including S1 hypervariable region, which could suggest a common origin or ancestry. DNA sequences from S2 and N protein genes obtained from different infected tissues of the same flock were analyzed, and a clear segregation between respiratory and intestinal tract was observed. Therefore, these data suggest co-circulation of more than one viral strain in the same flock. 57.1% of DNA sequences from the S1 complete segment samples, 53.3% from the S2 fragment and 62.5% from the partial N gene were found to be different from analyzed sequences from reference strains leading to the conclusion that parte of viral isolates included in this study may be considered region specific. Considering the simultaneous analysis of the three genes, a large IBV genetic profile was observed in both old and recent isolates groups. However, most prominent diversity between viral isolates was obtained in the period from 1972 and 1989, showing the presence of a large number of variants in the state of Minas Gerais before the official approval of vaccination (1980.

  17. Isolation, identification and characterization of Bacillus cereus from the dairy environment.

    NARCIS (Netherlands)

    te Giffel, M.C.

    1997-01-01

    In this thesis the occurrence of Bacillus cereus in the milk production and processing environment was investigated. Isolates were identified biochemically and by DNA probes based on the variable regions of 16S rRNA. Further characterization was carried out using biochemical and molecular typing, in

  18. First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America.

    Science.gov (United States)

    Romero, L E; Meneses, A I; Salazar, L; Jiménez, M; Romero, J J; Aguiar, D M; Labruna, M B; Dolz, G

    2011-08-01

    The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica.

  19. Molecular Analysis of Fomitiporia mediterranea Isolates from Esca-Affected Grapevines in Southern Italy

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    C. Ciccarone

    2004-08-01

    Full Text Available Four Fomitiporia isolates representative of a collection of about 300 isolates from esca-affected grapevines in southern Italy (Apulia, Campania and Abruzzo were examined by molecular methods. The DNA of each isolate was analysed by means of polymerase chain reaction (PCR amplification using ITS5 and ITS4 primers. An amplification product of 740 bp was obtained from all isolates. The denaturated products had the same migration pattern when analysed by single-strand conformation polymorphism. The PCR fragments that included the ribosomal ITS1-5.8S-ITS2 region were sequenced. In order to ascertain the taxonomic identity of the isolates, the ITS sequences were compared with those of Fomitiporia mediterranea, F. punctata and F. robusta deposited in the GenBank. The ITS sequences of the isolates were uniform and homologous with those of the type culture of Fomitiporia mediterranea M. Fischer, to which the southern Italian isolates were compared.

  20. Isolation and Identification of Lactic Acid Bacteria Isolated from a Traditional Jeotgal Product in Korea

    Science.gov (United States)

    Cho, Gyu Sung; Do, Hyung Ki

    2006-06-01

    Seventeen lactic acid bacterial strains (LAB) were isolated using MRS agar medium from Jeotgal, a Korean fermented food, purchased at the Jukdo market of Pohang. To identify the strains isolated, they were tested by examining their cell morphologies, gram-staining, catalase activity, arginine hydrolase activity, D-L lactate form and carbohydrate fermentation. According to the phenotypic characteristics, three strains were tent atively identified as Lactobacillus spp., ten were Enterococcus spp. (or Streptococcus spp., or Pediococcus spp.) and the rest were Leuconostoc spp. (or Weissella spp.). Five strains among 17 were chosen by preliminary bacteriocin activity test. Four bacterial strains which inhibited both indicator microorganisms were identified by 16S rRNA sequencing. The results are as follows; Leuconostoc mesenteroides (HK 4), Leuconostoc mesenteroides (HK 5), Leuconostoc mesenteroides(HK 11), Streptococcus salivarius(HK 8). In order to check LAB which are showing a high survival rate in gut, we investigated three strains inhibiting both indicator microorganisms in artificial gastric acid and bile juice -all except HK8. The three strains mentioned above grew in extreme low acid conditions.

  1. Molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates.

    Science.gov (United States)

    Hou, Xiang-hua; Song, Xiu-yu; Ma, Xiao-bo; Zhang, Shi-yang; Zhang, Jia-qin

    2015-01-01

    Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR) K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs) at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL) producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38) and class II integrons (10/38). All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX' and aadA1 genes. β-lactam resistance was conferred through bla SHV (22/38), bla TEM (10/38), and bla CTX-M (7/38). The highly conserved bla KPC-2 (37/38) and bla OXA-23(1/38) alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38) and the plasmid-mediated qnrB gene (13/38) were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR) fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

  2. Molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates

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    Xiang-hua Hou

    2015-09-01

    Full Text Available Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38 and class II integrons (10/38. All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX’ and aadA1 genes. β-lactam resistance was conferred through blaSHV (22/38, blaTEM (10/38, and blaCTX-M (7/38. The highly conserved blaKPC-2 (37/38 and blaOXA-23(1/38 alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38 and the plasmid-mediated qnrB gene (13/38 were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

  3. Molecular detection and identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in spoiled wines.

    Science.gov (United States)

    Cocolin, Luca; Rantsiou, Kalliopi; Iacumin, Lucilla; Zironi, Roberto; Comi, Giuseppe

    2004-03-01

    In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

  4. Molecular typing and antifungal susceptibility of clinical and environmental Cryptococcus neoformans species complex isolates in Goiania, Brazil.

    Science.gov (United States)

    Souza, L K H; Souza Junior, A H; Costa, C R; Faganello, J; Vainstein, M H; Chagas, A L B; Souza, A C M; Silva, M R R

    2010-01-01

    A total of 124 Cryptococcus isolates, including 84 clinical strains obtained from cerebrospinal fluid from AIDS patients and 40 environmental isolates from pigeon excreta and from Eucalyptus trees, were studied. The varieties, serotypes, phospholipase activity and molecular profile of these isolates were determined. Cryptococcus neoformans var. grubii serotype A was identified in 120 isolates and Cryptococcus gattii serotype B in four isolates. The clinical isolates showed higher phospholipase activity than environmental isolates. Similar patterns of in vitro susceptibility to amphotericin B, fluconazole, itraconazole and voriconazole and no resistance were found for all isolates. Molecular type VNI (C. neoformans var. grubii) was recovered in 80 clinical and 40 environmental isolates while the type VGIII (C. gattii) was found in four clinical isolates. This study demonstrated for the first time the molecular types of clinical and environmental Cryptococcus isolates in the midwest Brazil region.

  5. Isolation and Molecular Structure of Hexacyanoruthenate(III)

    DEFF Research Database (Denmark)

    Bendix, J; Steenberg, P; Søtofte, Inger

    2003-01-01

    The [Ru(CN)(6)](3-) ion is synthesized in aqueous solution and isolated as [Ph(4)AS](3)[Ru(CN)(6)].2H(2)O (1). Compound 1 crystallizes as orange needles in the monoclinic space group P2(1)/n with cell parameters a = 11.346(2) Angstrom, b = 23.107(5) Angstrom, c = 25.015(5) Angstrom, beta = 99.55(...

  6. Molecular Identification of Streptomyces producing antibiotics and their antimicrobial activities

    Directory of Open Access Journals (Sweden)

    Latifa A. Al_husnan

    2016-12-01

    Full Text Available Five strains of Streptomyces, namely S, N, W, E and C (designations should be mentioned in detail here isolated from the rhizosphere soil cultivated with palm Alajua (date, pressed dates, AlMedina city, Saudi Arabia, were induced to produce antibiotics. Antimicrobial activities were determined on solid medium supplemented with starch. The detection was based on the formation of transparent zones around colonies. The results indicated that isolates had antibacterial activities against Staphylococcus aureus, Bacillus cereus, B. subtilis, Pseudomonas aeruginosa and also showed antifungal activity against Candida albicans and Aspergillus niger. DNA extracted from five isolates was used as template for 16s rDNA gene amplification. The expected PCR size was 1.5 kbp;1.6 kbp; 1.25 kbp; 1.25kbp and 1.0 k bp for S, N, W, E and C isolates respectively using universal 16s rDNA gene primers using direct PCR. The isolates varied morphologically on the basis of spore color, aerial and substrate mycelium formation, and production of diffusible pigment. Isolates were tested under a microscope by using slide culture technique. The results indicate that the soil of this region is source of Streptomyces having antibacterial and antifungal activity and thus better utilization of these microorganisms as biological control agents.

  7. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    OpenAIRE

    Chan, Km; Yuen, KY; Que, TI; Woo, GKS; Fung, Amy; Yip, KT; Woo, PCY; Ng, KHL; Lau, SKP

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available ...

  8. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    OpenAIRE

    Lau, S.K.P.; Ng, K H L; Woo, P C Y; Yip, K‐t; Fung, A M Y; Woo, G K S; Chan, K‐m; Que, T‐L

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available ...

  9. Isolation, molecular characterisation and mapping of four rose MLO orthologues

    Directory of Open Access Journals (Sweden)

    Helgard eKaufmann

    2012-11-01

    Full Text Available Powdery mildew is a major disease of economic importance in cut and pot roses. As an alternative to conventional resistance breeding strategies utilising single-dominant genes or QTLs, mildew resistance locus o (MLO-based resistance might offer some advantages. In dicots such as Arabidopsis, pea and tomato, loss-of-function mutations in MLO genes confer high levels of broad-spectrum resistance. Here, we report the isolation and characterisation of four MLO homologues from a large rose EST collection isolated from leaves. These genes are phylogenetically closely related to other dicot MLO genes that are involved in plant powdery mildew interactions. Therefore, they are candidates for MLO genes involved in rose powdery mildew interactions. Two of the four isolated genes contain all of the sequence signatures considered to be diagnostic for MLO genes. We mapped all four genes to three linkage groups and conducted the first analysis of alternative alleles. This information is discussed in regards to a reverse genetics approach aimed at the selection of rose plants that are homozygous for loss of function in one or more MLO genes.

  10. Isolation, Molecular Characterization, and Mapping of Four Rose MLO Orthologs.

    Science.gov (United States)

    Kaufmann, Helgard; Qiu, Xianqin; Wehmeyer, Juliane; Debener, Thomas

    2012-01-01

    Powdery mildew is a major disease of economic importance in cut and pot roses. As an alternative to conventional resistance breeding strategies utilizing single-dominant genes or QTLs, mildew resistance locus o (MLO)-based resistance might offer some advantages. In dicots such as Arabidopsis, pea, and tomato, loss-of-function mutations in MLO genes confer high levels of broad-spectrum resistance. Here, we report the isolation and characterization of four MLO homologs from a large rose EST collection isolated from leaves. These genes are phylogenetically closely related to other dicot MLO genes that are involved in plant powdery mildew interactions. Therefore, they are candidates for MLO genes involved in rose powdery mildew interactions. Two of the four isolated genes contain all of the sequence signatures considered to be diagnostic for MLO genes. We mapped all four genes to three linkage groups and conducted the first analysis of alternative alleles. This information is discussed in regards to a reverse genetics approach aimed at the selection of rose plants that are homozygous for loss-of-function in one or more MLO genes.

  11. Molecular identification of marine symbiont bacteria of gastropods from the waters of the Krakal coast Yogyakarta and its potential as a Multi-Drug Resistant (MDR) antibacterial agent

    Science.gov (United States)

    Bahry, Muhammad Syaifudien; Pringgenies, Delianis; Trianto, Agus

    2017-01-01

    The resistance of pathogenic bacteria may occur to many types of antibiotics, especially in cases of non-compliance use of antibiotics, which likely to allow the evolution of Multi-Drug Resistant (MDR) bacteria. Gastropods seas are marine invertebrates informed capable of production of secondary metabolites as antibacterial MDR. The purpose of the study was the isolation and identification of gastropod symbiont bacteria found in the waters of Krakal, Gunung Kidul, Yogyakarta, which has the ability to produce antibacterial compounds against MDR(Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, MRSA (methicillin-Resistant Staphylococcus aureus), Staphylococcus aureus, and Staphylococcus homunis) molecular. Stages of this research began with the isolation of bacteria, bacteria screening for anti-MDR compound, mass culture, and extraction, antibacterial activity test, DNA extraction, amplification by PCR 16S rDNA and sequencing. The results of the study showed that 19 isolates of bacteria were isolated from three species of gastropods namely Littorina scabra, Cypraea moneta and Conus ebraeus. Among them, 4 isolates showed activity against MDR test bacteria (E. coli, E. cloacae, K. pneumoniae, S. aureus and S. homunis). The highest activity was displayed by code LS.G1.8 isolate with the largest inhibition zone 15.47±0.45mm on S. humonis at 250 µg/disk concentration. Isolate CM.G2.1 showed largest inhibition zone, with 21.5±0.07mm on MRSA at 1000 µg/disk concentration and isolate the largest inhibition zone CM.G2.5 14.37±0.81mm on MRSA 14.37±0.81mm at concentrations 1000 µg/disk. The molecular identification of isolates LS.G1.8 has 99% homology with Bacillus subtilis and isolates CM.G2.1 has 99% homology with Bacillus pumillus.

  12. Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing.

    Science.gov (United States)

    Kuhnert, P; Boerlin, P; Emler, S; Krawinkler, M; Frey, J

    2000-12-01

    Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

  13. Isolation and Identification of Virus dsRNA from Strawberry Plants

    Institute of Scientific and Technical Information of China (English)

    LI He; DAI Hong-yan; ZHANG Zhi-hong; GAO Xiu-yan; DU Guo-dong; ZHANG Xin-yu

    2007-01-01

    The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid (dsRNA) and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction (RT-PCR). The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ (DNase Ⅰ ), but evidently, it became resistant to ribonuclease A (RNase A) only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

  14. Molecular characterization of Borrelia burgdorferi sensu lato strains isolated in the area of Belgrade, Serbia Caracterização molecular de cepas de Borrelia burgdorferi sensu lato isoladas na região de Belgrado, Sérvia

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    Elizabeta S. Ristanovic

    2007-03-01

    Full Text Available This is the first report of the molecular characterization and identification of Borrelia burgdorferi sensu lato strains isolated in Serbia. Isolates A1, A2 and M1, from Ixodes ricinus, belong to Borrelia burgdorferi sensu stricto, while isolate K1 from Apodemus flavicollis is a mixture of Borrelia afzelii and B. burgdorferi s.s.Trata-se do primeiro relato de identificação e caracterização molecular de cepas de Borrelia burgdorferi sensu lato isoladas na região de Belgrado, Sérvia. As cepas A1, A2 e M1, isoladas de Ixodes ricinus, pertencem à Borrelia burgdorferi sensu stricto, enquanto a cepa K1, isolada de Apodemus flavocollis é uma mistura de Borrelia afzelii e B. burgdorferi s.s.

  15. Isolation, identification and screening of antimicrobial thermophilic Streptomyces sp. Al-Dhabi-1 isolated from Tharban hot spring, Saudi Arabia.

    Science.gov (United States)

    Al-Dhabi, Naif Abdullah; Esmail, Galal Ali; Duraipandiyan, Veeramuthu; Valan Arasu, Mariadhas; Salem-Bekhit, Mounir M

    2016-01-01

    The strain Streptomyces sp. Al-Dhabi-1 was isolated from soil sediments collected from Tharban hot spring in the southern west of Saudi Arabia using actinomycetes isolation agar and starch casein agar at 55 °C. Identification of the isolate was done according to morphological, physiological and biochemical characteristics and 16S rRNA sequence similarity as well. 16S rRNA sequence and blast analyses confirmed that the isolate belonging to the genus Streptomyces. The sequence was submitted to GenBank with accession number (KF815080). Ethyl acetate extract of Streptomyces sp. Al-Dhabi-1 showed good antimicrobial activities against tested pathogenic microbes. Minimum inhibitory concentration results showed that the best values were observed against S. agalactiae (<0.039 mg/ml) and Klebsiella pneumonia (0.125 mg/ml). Minimum inhibitory concentration of Al-Dhabi-1 against fungi; Cryptococcus neoformans (0.078 mg/ml), C. albicans (0.156 mg/ml), A. niger (0.625 mg/ml), and T. mentagrophytes (0.156 mg/ml). GC-MS analysis was used for the chemical profile of ethyl acetate extract. Benzeneacetic acid (16.02 %) and acetic acid 2-phenylethyl ester (10.35 %) were the major compounds among 31 substances found the ethyl acetate extract. According to the results of antimicrobial activity against pathogenic microbes, it is clear that the actinomycetes from hot springs with extreme environments are promising source for antimicrobial compounds.

  16. Pseudomonas isolation and identification: an introduction to the challenges of polyphasic taxonomy.

    Science.gov (United States)

    Sandman, Kathleen; Ecker, Christopher

    2014-12-01

    The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges inherent in contemporary bacterial taxonomy. In this ten-laboratory period series of exercises, students isolate a strain of Pseudomonas from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes. Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species. Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of Pseudomonas and bacterial taxonomy increased following the exercises.

  17. Pseudomonas Isolation and Identification: An Introduction to the Challenges of Polyphasic Taxonomy †

    Science.gov (United States)

    Sandman, Kathleen; Ecker, Christopher

    2014-01-01

    The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges inherent in contemporary bacterial taxonomy. In this ten-laboratory period series of exercises, students isolate a strain of Pseudomonas from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes. Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species. Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of Pseudomonas and bacterial taxonomy increased following the exercises. PMID:25574295

  18. Pseudomonas Isolation and Identification: An Introduction to the Challenges of Polyphasic Taxonomy

    Directory of Open Access Journals (Sweden)

    Kathleen Sandman

    2014-09-01

    Full Text Available The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges in contemporary bacterial taxonomy.  In this ten-laboratory period series of exercises, students isolate a strain of Pseudomonas from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes.  Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species.  Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of Pseudomonas and bacterial taxonomy increased following the exercises.

  19. Isolation and identification of curcumin and bisacurone from rhizome extract of temu glenyeh (Curcuma soloensis. Val)

    Science.gov (United States)

    Vitasari, Rista A.; Wibowo, Fajar R.; Marliyana, Soerya D.; Widyo Wartono, M.

    2016-02-01

    Temu glenyeh (Curcuma soloensis. Val) is one of the medicinal plants that grow in Surakarta. This plant is similar with C. longa and C. Xanthoriza. Chemical constituents from an extract of the plant have never been studied. In this paper, we report the isolation of a terpenoid and curcumin from the rhizome of C. soloensis. The isolation was employed by soxhlet apparatus using acetone as solvent. The fractionation and purification of the compound from the acetone extracts were undertaken by vacuum liquid chromatography and flash chromatography. Identification of compounds used spectroscopy methods, such as FTIR, NMR (1H NMR, 13C NMR, COSY, HSQC and HMBC) and GC-MS. Isolated compounds were identified as curcumin (1) and bisacurone (2).

  20. Isolation and Identification of Vancomycin Resistant Staphylococcus aureus from Post Operative Pus Sample

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    Subhankari Prasad Chakraborty1, Santanu KarMahapatra1, Manjusri Bal2 and Somenath Roy1*

    2011-04-01

    Full Text Available Staphylococcus aureus is most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections and pneumonia. Recently, S. aureus have evolved resistance to both synthetic and traditional antibiotics. This study was carried out to isolate pathogenic S. aureus from post-operative pus sample, and VRSA was identified by evaluation of resistance patterns using conventional antibiotics. Thirty post operative pus samples were collected from nearby Hospital and species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene. Antibiotic susceptibility tests were carried out by MIC, MBC, DAD test and BHI vancomycin screening agar. VRSA were confirmed by PCR amplification of the vanA and vanB genes. From this study, it was observed that isolated S. aureus strains are pathogenic; 30% of strains were resistant to penicillin G, ampicillin and erythromycin; 26.67% strains were resistant to cephotaxime, gentamycin, streptomycin, tetracycline, chloramphenicol, norfloxacin, methicillin and vancomycin.

  1. Isolation, Identification of Heat Resistant Moulds in Margarine and Determination of Their Heat Resistance

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    A. S. Demirci

    2006-09-01

    Full Text Available In this study, moulds that cause problems in a margarine production plant which is located in Trakyaregion have been isolated and identified. In addition to, their heat resistance and lipolytic activity werestudied. For this purpose, margarine samples from various production lots and process water samples fromproduction plant were taken aseptically, transported immediately to the laboratory and analyzed. In thisresearch, two different heat resistant mould strains have been isolated from margarines and process water.After identification of this moulds, their heat resistances at different temperatures have been investigated.Mould isolates were identified as heat resistant Aspergillus fumigatus, Paecilomyces variotii based onmacroscopic and micro