ribotyping
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A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (Svec et al., Folia Microbiol 54:53-58, 2009) was characterized by automated ribotyping performed by the RiboPrinter microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.
- MeSH
- automatizace MeSH
- genotyp MeSH
- Lactobacillus klasifikace izolace a purifikace MeSH
- lidé MeSH
- ribotypizace metody MeSH
- shluková analýza MeSH
- technika náhodné amplifikace polymorfní DNA metody MeSH
- techniky typizace bakterií metody MeSH
- zubní kaz mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The Staphylococcus strains acquired from scrapings from hospital environments were identified to the species level based on their biochemical properties. From the monitored sample the Staphylococcus epidermidis strains were selected for more accurate typing and tested on their virulence factor and ribotyped. The biotyping of S. epidermidis did not show any considerable intraspecific variation of these isolates and there were no atypical reactions, with the exception of three strains (out of 33). In contrast, the results of ribotyping showed greater heterogeneity of strains and unequivocally demonstrated the relation between the ribotype and the place of sample drawing. In addition to this fact, the found ribotypes repeat in the same environment in the long-term which suggests the occurrence and persistence of the same strains of conditionally pathogenic bacteria in hospital environment. We showed that ribotyping is a suitable method for precise and reliable detection of some coagulase-negative staphylococci.
- MeSH
- fenotyp MeSH
- finanční podpora výzkumu jako téma MeSH
- infekce spojené se zdravotní péčí etiologie genetika mikrobiologie MeSH
- koagulasa antagonisté a inhibitory izolace a purifikace MeSH
- lidé MeSH
- ribotypizace metody využití MeSH
- Staphylococcus epidermidis cytologie genetika izolace a purifikace MeSH
- Staphylococcus haemolyticus genetika izolace a purifikace MeSH
- Staphylococcus hominis cytologie genetika izolace a purifikace MeSH
- techniky typizace bakterií metody využití MeSH
- Check Tag
- lidé MeSH
The ribotyping of Clostridioides difficile is one of the basic methods of molecular epidemiology for monitoring the spread of C. difficile infections. In the Czech Republic, this procedure is mainly available in university hospitals. The introduction of ribotyping in a tertiary health care facility such as Liberec Regional Hospital not only increases safety in the facility but also supports regional professional development. In our study, 556 stool samples collected between June 2017 and June 2018 were used for C. difficile infection screening, followed by cultivation, toxinotyping, and ribotyping of positive samples. The toxinotyping of 96 samples revealed that 44.8% of typed strains could produce toxins A and B encoded by tcdA and tcdB, respectively. The ribotyping of the same samples revealed two epidemic peaks, caused by the regionally most prevalent ribotype 176 (n = 30, 31.3). C. difficile infection incidence ranged between 5.5 and 4.2 cases per 10,000 patient-bed days. Molecular diagnostics and molecular epidemiology are the two most developing parts of clinical laboratories. The correct applications of molecular methods help ensure greater safety in hospitals.
Cíl práce: Porovnání senzitivity a specificity dvou komerčních testů na stanovení Clostridium difficile GDH (glutamát dehydrogenázy) a toxinů A/B. Materiál a metodiky: Bylo testováno 86 stolic pacientů hospitalizovaných ve FN Motol. GDH a toxiny A/B byly paralelně vyšetřovány dvěma testy: C. difficile Quik Chek Complete?, Techlab,USA) a Liaison? C. difficile GDH a Toxins AαB (DiaSorin USA). Současně byly ze stolic izolovány nukleové kyseliny pomocí UltraClean? Fecal DNA (MoBio Laboratories, USA). Pro metodu PCR byla použita komerční souprava C. difficile Elite MGB? kit (Nanogen, Itálie). Anaerobní kultivace na selektivním médiu pro C. difficile (Oxoid) proběhla u všech vzorků vykazující pozitivitu alespoň jednoho testu. Čisté izoláty byly molekulárně analyzovány ribotypizací. Výsledky: GDH a toxin A/B negativních oběma metodami bylo 36 vzorků (42 %). GDH a toxin pozitivních oběma metodami bylo 20 vzorků (23 %). GDH pozitivních a toxin negativních oběma metodami, ale PCR pozitivních bylo 9 vzorků (10 %). GDH pozitivních a toxin negativních oběma metodami a PCR negativních bylo 11 vzorků (13 %). GDH pozitivní, toxin pozitivní pouze testem Liaison? bylo 6 vzorků (7 %). GDH pozitivní pouze Liaison? byly 4 vzorky (5 %). K selhání kultivace došlo u 11 vzorků (13), z toho u 7 vzorků byl pozitivní test PCR. PCR reakce byla inhibována v 5 případech (6 %). Zachycené ribotypy toxigenní: AI-3, 001, 002, 012,014, 017, 020, 049, 054, 078, 176, 203, 413 a netoxigenní: AI-34, AI-61, 010, 485, 495, 596. Závěr: Stanovení toxinu A/B testem Liaison? vykazuje u námi vyšetřeného souboru o 7 % vyšší senzitivitu. Dvoukrokové uspořádání testů také přináší ekonomickou úsporu, která může být využita např. k zavedení PCR metod do diagnostického algoritmu laboratoře.
Study objective: Comparison of two commercially available tests for the detection of Clostridium difficile Glutamate Dehydrogenase (GDH) and toxins A and B for their sensitivity and specificity. Material and methods: Eighty-six stool samples from patients hospitalised in the Motol University Hospital were analysed. GDH and toxins A and B were assayed in parallel by two tests: C. difficile Quik Chek Complete? (Techlab, USA) and Liaison? C. difficile GDH and Toxins AαB (DiaSorin, USA). From the stool samples, nucleic acids were also isolated using the UltraClean? Fecal DNA kit (MoBio Laboratories, USA). The commercially available C. difficile Elite MGB? kit (Nanogen, Italy) was used for the polymerase chain reaction (PCR). Anaerobic culture on C. difficile selective medium (Oxoid) was performed for all positive samples at least in one test. Pure isolates were characterized by PCR ribotyping. Results: Thirty-six (42%) samples were GDH negative and toxin A/B negative by both tests. Twenty (23%) samples were GDH positive and toxin A/B positive by both tests. Nine (10%) samples were GDH positive and toxin negative by both tests, but were positive by PCR. Eleven (13%) samples that were GDH positive and toxin negative by both tests remained negative by PCR. Six (7%) samples only were GDH positive and toxin positive by the Liaison? test alone. Four (5%) samples were GDH-positive by theLiaison? test alone. Culture failure was observed in 11 (13%) samples, of which seven were positive by PCR. PCR was inhibited in five (6%) samples. The following toxigenic ribotypes: AI-3, 001, 002, 012,014, 017, 020, 049, 054, 078, 176, 203, and 413 and non-toxigenic ribotypes: AI-34, AI-61, 010, 485, 495, and 596 were identified. Conclusion: The Liaison? test had seven percent higher sensitivity for the detection of toxins A/B. The two-step protocol of the tests is also cost-saving. The savings can be used e.g. for incorporating the PCR techniques into the diagnostic algorithm of the laboratory.
- MeSH
- antigeny bakteriální analýza MeSH
- Clostridioides difficile * enzymologie chemie izolace a purifikace MeSH
- feces mikrobiologie MeSH
- glutamátdehydrogenasa imunologie MeSH
- imunoenzymatické techniky metody využití MeSH
- klostridiové infekce * diagnóza genetika mikrobiologie MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Clostridium difficile is a major nosocomial pathogen of present times. The analysis of 624 C. difficile strains from 11 hospitals in the Czech Republic in 2013 revealed that 40% of isolates belonged to ribotype 176. These results suggest that the incidence of CDI (C. difficile infection) in the Czech Republic has increased probably in connection with C. difficile ribotype 176. The molecular systems Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA) diagnoses toxigenic strains and supports C. difficile ribotype 027 determination based on three specific target places in the toxigenic C. difficile genome. Twenty-nine strains cultivated from stool specimens were evaluated by the Xpert systems as presumed C. difficile PCR ribotype 027 were confirmed as a C. difficile ribotype 176 based on ribotyping. A further 120 C. difficile strains of ribotype 176 were examined for the presence of genes tcdB, cdtB and deletion in position 117 in the tcdC gene. Our experience shows that due to the correspondence of the target places, C. difficile ribotype 176 may be interpreted as ribotype 027 by Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA). Further molecular analysis as ribotyping based on capillary electrophoresis is needed to differentiate between C. difficile ribotypes 027 and 176 for appropriate epidemiological situation control on local and national levels.
- MeSH
- bakteriální proteiny genetika MeSH
- Clostridioides difficile klasifikace genetika izolace a purifikace MeSH
- delece genu MeSH
- feces mikrobiologie MeSH
- infekce spojené se zdravotní péčí mikrobiologie MeSH
- lidé MeSH
- nemocnice statistika a číselné údaje MeSH
- ribotypizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- MeSH
- DNA bakterií analýza MeSH
- druhová specificita MeSH
- Enterococcus klasifikace MeSH
- Publikační typ
- srovnávací studie MeSH
The main aim of this study was to investigate the occurrence and ribotypes of Clostridium perfringens in broiler flocks reared in 2 European countries that apply European Union Regulation 1831/2003. A total of 1,532 cecum contents were collected between June 2005 and November 2006 from birds belonging to 51 intensively reared flocks produced in the Czech Republic and 41 intensive production, organic, and free-range flocks reared in Italy. Clostridium perfringens was detected in 64.7 and 82.9% of the Czech Republic and Italian flocks, respectively, at mean loads ranging between 3.65 and 4.77 log10 cfu per gram of cecum content. More than 1 ribotype was identified among isolates belonging to the same flock in 57.1 and 76.5% of the Czech Republic and Italian flocks, respectively. Moreover, common ribotypes were identified between strains belonging to 2 up to 8 different flocks. In particular, 4 ribotypes were shared between strains isolated in the 2 European countries. The results of this study report on C. perfringens occurrence and mean populations in broilers reared on diets devoid of antibiotic growth promoters. Moreover, these findings show for the first time the presence of common ribotyping profiles among isolates collected from birds reared more than 1,000 km apart.
- MeSH
- Clostridium perfringens klasifikace izolace a purifikace MeSH
- fylogeneze MeSH
- gastrointestinální obsah mikrobiologie MeSH
- klostridiové infekce epidemiologie mikrobiologie veterinární MeSH
- kur domácí * MeSH
- nemoci drůbeže epidemiologie mikrobiologie MeSH
- ribotypizace * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Itálie MeSH
