Emergence of an outbreak-associated Clostridium difficile variant with increased virulence

Abstract

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 NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 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. NAPCR1 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 NAPCR1 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, NAPCR1 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.

FIG 1

Epidemic curve for a CDI outbreak at a tertiary care hospital in Costa Rica, showing the numbers of CDI cases diagnosed (through clinical evidence and toxin detection) at San Juan de Dios Hospital during a 28-month period in 2008 to 2010.

FIG 2

Molecular characterization of C. difficile isolates. (A) C. difficile strains (n = 57) isolated during the outbreak were typed by PFGE. Sixteen different SmaI macrorestriction patterns were detected and classified into the indicated NAP types. A previously undescribed NAP type was highly represented and was designated NAP_CR1. (B) A phylogenomic tree based on core SNPs depicts the high level of genomic similarity of NAP_CR1 strains (in bold) and their phylogenetic relationships to C. difficile 630, two NAP1 strains (R20291 and CD196), a NAP4 strain, and a NAP9 strain (M68). The scale distances correspond to the average number of substitutions per site.

FIG 3

Kaplan-Meier survival curves for hamsters infected with different clinical genotypes of C. difficile. Groups of 5 Syrian Golden hamsters previously treated with clindamycin were orally inoculated with spores from the indicated genotypes. Hamsters were monitored at 12-h intervals for signs of C. difficile infection, and the numbers of dead animals were recorded. C. difficile isolates obtained from fecal pellets were typed by PFGE to confirm the inoculated strain. *, P < 0.05 (Mantel-Cox test).

FIG 4

Quantification of histopathological effects of bacteria-free supernatants of the C. difficile genotypes in the murine ligated ileal loop model. Bacteria-free supernatants (48-h growth) of representative strains from the indicated genotypes were prepared in TYT broth. Six to 8 mice per group were inoculated with 0.3 ml of the indicated supernatant in ligated ileal loops. Four hours after inoculation, the mice were sacrificed and the severity of the histopathological alterations was scored on coded slides, using a histopathological score (HS) scale of 1 (mild) to 3 (severe) for neutrophil infiltration (A), edema (B), and epithelial damage (C); the general damage induced in the indicated groups was determined as the median of all scores (D). Non-Tox, nontoxigenic. *, P < 0.05, compared to the groups without asterisks (Kruskal-Wallis test and Dunn's multiple-comparison test).

FIG 5

Quantification of toxin production by the different genotype groups. (A) Twenty-four-hour bacteria-free supernatants were titrated in 10-fold dilutions on HeLa cell monolayers. Twenty-four hours after inoculation with the indicated supernatant, the dilution inducing a cytopathic effect (CPE) for 50% of the cells was calculated by visual examination under a microscope. Each bar represents the CPE₅₀ of one strain. (B) Proteins from bacteria-free supernatants obtained at the indicated times were precipitated and separated by 7.5% SDS-PAGE. Proteins were electrotransferred to PVDF membranes and probed with monoclonal antibodies against TcdA and TcdB. (C) Total RNA was prepared from the indicated strains at 5 and 8 h during the growth cycle. RNA was retrotranscribed, and cDNA was quantified by real-time PCR using primers specific for tcdA and tcdB. *, P < 0.05 (one-way analysis of variance [ANOVA] with Bonferroni's correction).