Cfr is a radical S -adenosyl- l -methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three cfr -like genes implicated in antibiotic resistance have been described, two of which, cfr (B) and cfr (C), have been sporadically detected in Clostridium difficile . However, the methylase activity of Cfr(C) has not been confirmed.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAP _CR1 . Whole-genome sequencing and ribotyping indicated that the NAP _CR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAP _CR1 strains are resistant to fluoroquinolones due to a mutation in gyrA , and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAP _CR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAP _CR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAP _CR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

The population structure of Clostridium difficile currently comprises eight major genomic clades. For the highly divergent C-I clade, only two toxigenic strains have been reported, which lack the tcdA and tcdC genes and carry a complete locus for the binary toxin (CDT) next to an atypical TcdB monotoxin pathogenicity locus (PaLoc). As part of a routine surveillance of C. difficile in stool samples from diarrheic human patients, we discovered three isolates that consistently gave negative results in a PCR-based screening for tcdC. Through phenotypic assays, whole-genome sequencing, experiments in cell cultures, and infection biomodels we show that these three isolates (i) escape common laboratory diagnostic procedures, (ii) represent new ribotypes, PFGE-types, and sequence types within the Clade C-I, (iii) carry chromosomal or plasmidal TcdBs that induce classical or variant cytopathic effects (CPE), and (iv) cause different levels of cytotoxicity and hamster mortality rates. These results show that new strains of C. difficile can be detected by more refined techniques and raise questions on the origin, evolution, and distribution of the toxin loci of C. difficile and the mechanisms by which this emerging pathogen causes disease.

We isolated a regional toxigenic genotype of Clostridium difficile, previously found in human infection in 4 of 200 (2%) samples of retail meats for human consumption: 1 of 67 samples of beef, 2 of 66 of pork, and 1 of 67 of poultry meat. These four isolates were positive for the tcdA and tcdB genes but negative for deletion of the tcdC and cdtB genes. All strains induced cytopathic effects in HeLa cells. However, they were susceptible to some antibiotics to which clinical isolates are often resistant. All strains were susceptible to vancomycin, metronidazole, moxifloxacin, and rifampicin but resistant to clindamycin and ciprofloxacin. This first report of isolation of C. difficile in foodstuff from Latin America lends support to the notion that animal products serve as a reservoir for clinical strains of this pathogen in the community.

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1 _V ) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1 _V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC . NAP1 _V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1 _V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdB _NAP1 and TcdB _NAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdB _NAP1 induced a stronger proinflammatory response than TcdB _NAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile -induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.