Typing Clostridium difficile strains based on tandem repeat sequences

Background: Genotyping of epidemic Clostridium difficile strains is necessary to track their emergence and spread. Portability of genotyping data is desirable to facilitate inter-laboratory comparisons and epidemiological studies. Results: This report presents results from a systematic screen for variation in repetitive DNA in the genome of C. difficile. We describe two tandem repeat loci, designated \u27TR6\u27 and \u27TR10\u27, which display extensive sequence variation that may be useful for sequence-based strain typing. Based on an investigation of 154 C. difficile isolates comprising 75 ribotypes, tandem repeat sequencing demonstrated excellent concordance with widely used PCR ribotyping and equal discriminatory power. Moreover, tandem repeat sequences enabled the reconstruction of the isolates\u27 largely clonal population structure and evolutionary history. Conclusion: We conclude that sequence analysis of the two repetitive loci introduced here may be highly useful for routine typing of C. difficile. Tandem repeat sequence typing resolves phylogenetic diversity to a level equivalent to PCR ribotypes. DNA sequences may be stored in databases accessible over the internet, obviating the need for the exchange of reference strains

PCR-ribotype distribution of Clostridium difficile in Irish pigs

Clostridium difficile is an important enteric pathogen in humans causing infections in the healthcare environment and the community. Carriage of C. difficile and C. difficile-related enterocolitis has been reported in piglets worldwide. The aim of this study was to investigate the rates of C. difficile isolation from pigs in Ireland. Faecal samples from piglet litters and sows were collected from six farms in 2015. The sows were non-diarrhoeal at the time of sampling. The diarrhoeal status of the piglets was unknown. C. difficile was isolated from 34/44 (77%) of piglet litter samples and from 33/156 (21%) of sow samples. The isolation rate in sows varied from 3 to 39% and in piglet litters from 72 to 86% depending on farm location. Toxin A and toxin B were present in 99% (66/67) of isolates; and binary toxin in 85% (57/67). Only PCR-ribotypes 078 (88%) and 193 (12%) were identified in piglets. Seven PCR-ribotypes were detected in sow C. difficile isolates: PCR-ribotypes 078 (67%), 050 (12%), 014/020 (6%), 015 (6%), 029 (3%), 035 (3%) and 193 (3%). This study shows that toxigenic C. difficile strains such as PCR-ribotype 078 can be commonly isolated from pigs at different geographical locations in Ireland. Since PCR-ribotype 078 is frequently found in humans in Ireland, this highlights the potential for interspecies transmission

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

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile

Clostridium difficile in wild rodents and insectivores in The Netherlands

With wild rodents and insectivores being present around humans and their living, working, and food production environments, it is important to gain knowledge of the zoonotic pathogens present in these animals. The enteropathogen Clostridium difficile, an opportunistic anaerobic bacteria, can be carried by both animals and humans, and is distributed globally. It is known that there is genetic overlap between human and animal sources of C. difficile. In this study, the aim was to assess the presence of C. difficile in rodents and insectivores trapped on and around pig and cattle farms in The Netherlands. In total 347 rodents and insectivores (10 different species) were trapped and 39·2% tested positive for presence of C. difficile. For all positive samples the ribotype (RT) was determined, and in total there were 13 different RTs found (in descending order of frequency: 057, 010, 029, 005, 073, 078, 015, 035, 454, 014, 058, 062, 087). Six of the ribotypes isolated from rodents and insectivores are known to be associated with human CDI; RT005, RT010, RT014, RT015, RT078, and RT087. The presence of rodents and insectivores in and around food production buildings (e.g. farms) could contribute to the spread of C. difficile in the human environment. In order to enable on-farm management for pathogen control, it is essential to comprehend the role of wild rodents and insectivores that could potentially affect the ecology of disease agents on farms. This article is protected by copyright. All rights reserved

First molecular characterisation and PCR ribotyping of Clostridium difficile strains isolated in two Algerian Hospitals

Introduction: Clostridium difficile is the major etiological agent of nosocomial antibiotics associated diarrhoea. C. difficile infection is associated with considerable morbidity and mortality among hospitalized patients worldwide. Despite its known importance, there is no study on this important pathogen in Algeria.Methodology: In this prospective study, undertaken between 2013 and 2015, faecal specimens were collected from 159 hospitalized patients with antibiotic-associated diarrhoea in two tertiary health care hospitals in Chlef, Algeria. Faecal samples were cultured on CLO plates Agar with cefoxitin, cycloserine antibiotics and sodium taurocholate. C. difficile suspected colonies were analysed by multiplex PCR for the detection of the toxin genes. C. difficile isolates were analysed by PCR ribotyping and multi-locus tandem repeat analysis. Antimicrobial susceptibility was determined by the E-test method, according to the Clinical and Laboratory Standards Institute protocol.Results: C. difficile was cultured from 11 of 159 stool specimen (6.9%). Seven strains were toxigenic, mainly represented by the 020 and 014 PCR ribotypes and four non toxigenic belong to PCR ribotype 084. All 11 isolates were susceptible to both vancomycin and metronidazole and resistant to ciprofloxacin.Conclusions: This study, which reported for the first time C. difficile ribotypes circulating in Algerian health care facilities, could paves the way for further more comprehensive studies on this important pathogen, and could be useful to the local health authorities to implement a surveillance program of C. difficile in Algeria.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Data from a survey of Clostridium perfringens and Clostridium difficile shedding by dogs and cats in the Madrid region (Spain), including phenotypic and genetic characteristics of recovered isolates

This article contains information related to a recent survey of the prevalence of fecal shedding of Clostridium perfringens and C. difficile by dogs and cats attended in veterinary clinics located in the Madrid region (Spain). Specifically, we provide detailed information about the clinics that participated in the survey, the demographic and clinic characteristics of recruited animals and the genetic and phenotypic characteristics (including antimicrobial susceptibility data), of recovered bacterial isolates

Predominance of Clostridioides difficile PCR ribotype 181 in northern Greece, 2016-2019.

Thirty-five different PCR ribotypes were identified. The most common RTs identified were: 181 (36%, 80/221), 017 (10%, 21/221), 126 (9%, 19/221), 078 (4%, 9/221) and 012 (4%, 8/221). Notably, the predominant RT181, with toxin profile tcdA + tcdB + cdtA + cdtB+, was identified in seven out of ten participating hospitals

Toxigenic Clostridium difficile PCR ribotypes in edible marine bivalve molluscs in Italy

Even though food of animal sources and different foodstuffs are well known to be potentially carrier of Clostridium difficile, fewdata are available on the occurrence of C. difficile in seafood. This work investigated the occurrence of C. difficile in edible bivalve molluscs in southern Italy. Out of the 925 investigated samples, 3.9% contained C. difficile. Eighteen strains harboured both genes for toxins A and B whereas 1 only had toxin B gene. Binary toxin genes were found in 22.2% of the isolates. The most frequently ribotypes found were 078/126 (22.2%), 010 (19.4%), and 001 (8.3%). All isolates were susceptible to metronidazole, vancomycin, fidaxomicin, and to the new semisynthetic thiopeptide antibiotic LFF571, whereas 19.4% of them were resistant to moxifloxacin, 30.5% to clindamycin, 38.8% to erythromycin, and 100% to ciprofloxacin. This study points out that edible molluscs could be a potential source of toxigenic C. difficile ribotypes and a potential risk for human health