, recently renamed , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for Initially, we determined the breadth of the population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange.

Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile.

BackgroundCharacterising the overlap of C. difficile genotypes in different reservoirs can improve our understanding of possible transmission routes of this pathogen. Most of the studies have focused on a comparison of the PCR ribotype 078 isolated from humans and animals. Here we describe for the first time a comparison of C. difficile genotypes isolated during longer time intervals from different sources including humans, animals and the non-hospital environment.ResultsAltogether 786 isolates from time interval 2008-2010 were grouped into 90 PCR ribotypes and eleven of them were shared among all host types and the environment. Ribotypes that were most common in humans were also present in water and different animals (014/020, 002, 029). Interestingly, non-toxigenic isolates were very common in the environment (30.8%) in comparison to humans (6.5%) and animals (7.7%). A high degree of similarity was observed for human and animal isolates with PFGE. In human isolates resistance to erithromycin, clindamycin and moxifloxacin was detected, while all animal isolates were susceptible to all antibiotics tested.ConclusionOur results show that many other types in addition to PCR Ribotype 078 are shared between humans and animals and that the most prevalent genotypes in humans have the ability to survive also in the environment and several animal hosts. The genetic relatedness observed with PFGE suggests that transmission of given genotype from one reservoir to the other is likely to occur.

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cToxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature.

BackgroundClostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory.ResultsAltogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries).ConclusionsThis results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

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Molecular typing methods for Clostridium difficile are based on gel electrophoresis of restriction fragments (endonuclease restriction analysis, REA; pulsed field gel electrophoresis PFGE; toxinotyping), PCR amplification (PCR ribotyping, arbitrarily primed PCR, multilocus variable-number tandem-repeat analysis MLVA), and sequence analysis (multilocus sequence typing MLST; slpA typing, tandem repeat sequence typing). We will describe two standard methods (PCR ribotyping predominantly used throughout Europe and PFGE which is predominantly used in North America) and will discuss the difficulties of inter-laboratory comparability and unification of typing nomenclature.