Molecular analysis of Clostridium difficile PCR ribotype 027 isolates from Eastern and Western Canada

Abstract

The prevalence and characteristics of PCR ribotype 027 strains of Clostridium difficile have come into question following recent outbreaks in Eastern Canada and elsewhere. In order to determine the distribution of this strain in other regions in Canada, we screened a bank of 1,419 isolates recovered from three different Canadian health regions between 2000 and 2004. Among isolates from a Montreal area hospital, PCR ribotype 027 strains represented 115/153 strains (75.2%) from 2003 to 2004, but ribotype 027 strains were absent in 2000 and 2001. In Calgary, by contrast, ribotype 027 rates have remained relatively stable over 4 years of surveillance, representing 51/685 (7.4%) hospital isolates and 62/373 (16.6%) strains from the community (P < 0.001). PCR ribotype 027 accounted for 8/135 (5.9%) hospital isolates in the Fraser Health Region in 2004. repetitive extragenic palindromic PCR was used to subtype a random selection of 027 isolates from each region. All 10 of the isolates from Quebec were of a single subtype, which was also dominant among isolates from Alberta (8/10 isolates) and British Columbia (6/8 isolates). Comparative sequencing of the tcdC repressor gene confirmed the documented 18-bp deletion and identified a second, single-base-pair deletion at position 117. Both deletions were conserved across all three provinces and were identified in a United Kingdom reference strain. The presence of a frameshift in the early portion of the tcdC gene implies serious functional disruption and may contribute to the hypervirulence of the 027 phenotype. PCR ribotype 027 strains appear to be widely distributed, to predate the Montreal outbreak, and to have measurable community presence in Western Canada.

FIG. 1.

Frequency of C. difficile ribotype 027 isolates in the Calgary Health Region by month (2001 to 2004). These data were compiled during several discrete sampling intervals between the spring of 2001 and the fall of 2004 and represent the total number of isolates captured and saved by our laboratory during each month. Sampling was intermittent, however, and these figures should not be interpreted as patterns of regional incidence.

FIG. 2.

Molecular strain type analysis of 28 ribotype 027 (NAP1) isolates from Alberta, British Columbia (BC), and Quebec. Lettering was assigned to Albertan isolates in chronological order, with dates that spanned 2001 to 2004. Isolates from Quebec and British Columbia were largely obtained from single sampling intervals and were numbered in accordance with existing catalog numbers.

FIG. 3.

Theoretical impact of the Δ117 deletion on the integrity of the TcdC peptide. Residues 1 to 100 of the wild-type ATCC 43255 TcdC protein are shown at the bottom. With the Δ117 deletion and the resulting frameshift, the TcdC repressor protein in PCR ribotype 027 strains is likely prematurely truncated at position 65, with nonsense residues after position 43. It is likely, given the position of this mutation early in the transcript, that the functional capacity of the protein is severely impacted, which may correlate with the unchecked hypervirulence reported previously by Warny and others (24).