10 research outputs found
Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile
PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs
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New York City House Mice (Mus musculus) as Potential Reservoirs for Pathogenic Bacteria and Antimicrobial Resistance Determinants
House mice (Mus musculus) thrive in large urban centers worldwide. Nonetheless, little is known about the role that they may play in contributing to environmental contamination with potentially pathogenic bacteria. Here, we describe the fecal microbiome of house mice with emphasis on detection of pathogenic bacteria and antimicrobial resistance genes by molecular methods. Four hundred sixteen mice were collected from predominantly residential buildings in seven sites across New York City over a period of 13 months. 16S rRNA sequencing identified Bacteroidetes as dominant and revealed high levels of Proteobacteria. A targeted PCR screen of 11 bacteria, as indicated by 16S rRNA analyses, found that mice are carriers of several gastrointestinal disease-causing agents, including Shigella, Salmonella, Clostridium difficile, and diarrheagenic Escherichia coli. Furthermore, genes mediating antimicrobial resistance to fluoroquinolones (qnrB) and β-lactam drugs (blaSHV and blaACT/MIR) were widely distributed. Culture and molecular strain typing of C. difficile revealed that mice harbor ribotypes associated with human disease, and screening of kidney samples demonstrated genetic evidence of pathogenic Leptospira species. In concert, these findings support the need for further research into the role of house mice as potential reservoirs for human pathogens and antimicrobial resistance in the built environment
Evaluation of viral co-infections among patients with community-associated Clostridioides difficile infection.
We assessed viral co-infections in 155 patients with community-associated Clostridioides difficile infection in five U.S. sites during December 2012-February 2013. Eighteen patients (12%) tested positive for norovirus (n = 10), adenovirus (n = 4), rotavirus (n = 3), or sapovirus (n = 1). Co-infected patients were more likely than non-co-infected patients to have nausea or vomiting (56% vs 31%; p = 0.04), suggesting that viral co-pathogens contributed to symptoms in some patients. There were no significant differences in prior healthcare or medication exposures or in CDI complications
Process for multi-centre consensus method validation.
<p><b>STAGE 1:</b> 70 well characterised ribotypes disseminated from Netherlands to each laboratory for ribotyping (data (i) held locally for future comparsion/ribotype assignment, (ii) sent to UK laboratory); <b>STAGE 2:</b> 50 anonymised isolates disseminated from Netherlands to each laboratory for ribotype identification (assignments sent to UK laboratory for analysis); <b>STAGE 3:</b> 60 anonymised data files disseminated from UK to each laboratory for ribotype identification (assignments sent to UK for analysis).</p
PCR-ribotypes associated with minimum and maximum differences in DNA fragment sizes reported across participating centres.
<p>PCR-ribotypes associated with minimum and maximum differences in DNA fragment sizes reported across participating centres.</p
Analysis of five discrepant results generated in validation stage two (isolate challenge set).
<p>Analysis of five discrepant results generated in validation stage two (isolate challenge set).</p
Comparison of internal protocols from the four participating laboratories and the proposed consensus protocol.
<p>Comparison of internal protocols from the four participating laboratories and the proposed consensus protocol.</p
PCR-ribotypes with very similar profiles: (a) ribotypes 027 and 081 and (b) ribotypes 015 and 046.
<p>PCR-ribotypes 027 and 081 differ from one another by only a ~20bp difference at position d. Similarly PCR-ribotypes 015 and 046 differ by only a ~20bp difference at position b. Discriminating features between these very similar profiles are indicated (arrows) and associated fragment sizes are highlighted in bold. Relative fragment size was the only parameter used to discriminate between ribotype profiles. Relative peak heights (relative fluorescent units, y-axis) within profiles lacked reproducibility for some ribotypes and therefore this parameter was not used.</p