Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164.

Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases

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

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

An outbreak of Clostridium difficile infections due to new PCR ribotype 826

__Objectives:__ To investigate an unusual outbreak of five patients with a total of eight episodes of a Clostridium difficile infection on a gastrointestinal surgical ward of a Dutch tertiary-care, university-affiliated hospital. __Methods:__ Clinical case investigations and laboratory analyses were performed. Laboratory analyses included PCR ribotyping, multiple-locus variable-number tandem repeat analysis typing, toxin typing, antimicrobial susceptibility testing and whole genome sequencing. __Results:__ The outbreak was associated with recurrent and severe disease in two of five patients. All episodes were due to a unique ribotype that was not recognized in the collection of an international network of reference laboratories and was assigned PCR ribotype 826. PCR ribotype 826 is a toxin A-, toxin B- and binary toxin-positive ribotype which according to molecular typing belongs to clade 5 and resembles the so-called hypervirulent ribotype 078. The presence of a clonal outbreak was confirmed by whole genome sequencing, yet the source of this newly identified ribotype remained unclear. __Conclusions:__ This newly identified C. difficile PCR ribotype 826 is part of clade 5 and might also have increased virulence. The recognition of this outbreak highlights the need for ongoing C. difficile infection surveillance to monitor new circulating ribotypes with assumed increased virulence

Recreational sandboxes for children and dogs can be a source of epidemic ribotypes of Clostridium difficile

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

The bacterial gut microbiota of adult patients infected, colonized or noncolonized by clostridioides difficile

Gut microbiota composition in patients with Clostridioides difficile colonization is not well investigated. We aimed to identify bacterial signatures associated with resistance and susceptibility to C. difficile colonization (CDC) and infection (CDI). Therefore, gut microbiota composition from patients with CDC (n = 41), with CDI (n = 41), and without CDC (controls, n = 43) was determined through 16S rRNA gene amplicon sequencing. Bacterial diversity was decreased in CDC and CDI patients (p < 0.01). Overall microbiota composition was significantly different between control, CDC, and CDI patients (p = 0.001). Relative abundance of Clostridioides (most likely C. difficile) increased stepwise from controls to CDC and CDI patients. In addition, differential abundance analysis revealed that CDI patients' gut microbiota was characterized by significantly higher relative abundance of Bacteroides and Veillonella than CDC patients and controls. Control patients had significantly higher Eubacterium hallii and Fusicatenibacter abundance than colonized patients. Network analysis indicated that Fusicatenibacter was negatively associated with Clostridioides in CDI patients, while Veillonella was positively associated with Clostridioides in CDC patients. Bacterial microbiota diversity decreased in both CDC and CDI patients, but harbored a distinct microbiota. Eubacterium hallii and Fusicatenibacter may indicate resistance against C. difficile colonization and subsequent infection, while Veillonella may indicate susceptibility to colonization and infection by C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes

Clostridium difficile infection in an endemic setting in the Netherlands

The purpose of this investigation was to study risk factors for Clostridium difficile infection (CDI) in an endemic setting. In a 34-month prospective case–control study, we compared the risk factors and clinical characteristics of all consecutively diagnosed hospitalised CDI patients (n = 93) with those of patients without diarrhoea (n = 76) and patients with non-CDI diarrhoea (n = 64). The incidence of CDI was 17.5 per 10,000 hospital admissions. C. difficile polymerase chain reaction (PCR) ribotype 014 was the most frequently found type (15.9%), followed by types 078 (12.7%) and 015 (7.9%). Independent risk factors for endemic CDI were the use of second-generation cephalosporins, previous hospital admission and previous stay at the intensive care unit (ICU). The use of third-generation cephalosporins was a risk factor for diarrhoea in general. We found no association of CDI with the use of fluoroquinolones or proton pump inhibitors (PPIs). The overall 30-day mortality among CDI patients, patients without diarrhoea and patients with non-CDI diarrhoea was 7.5%, 0% and 1.6%, respectively. In this endemic setting, risk factors for CDI differed from those in outbreak situations. Some risk factors that have been ascribed to CDI earlier were, in this study, not specific for CDI, but for diarrhoea in general. The 30-day mortality among CDI patients was relatively high

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

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 RTs isolated from rodents and insectivores are known to be associated with human C. difficile infection; 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. Significance and Impact of the Study: This study shows that rodents and insectivores in and around food production buildings (e.g. farms) can carry Clostridium difficile ribotypes associated with human C. difficile infection (CDI). C. difficile spores in rodent and insectivore droppings are able to survive in the environment for prolonged periods, leading to host-to-host exposure and transmission. Therefore we can state that rodent and insectivore presence on farms is a risk for zoonotic pathogen transmission of C. difficile.</p