Phenotypic and Genotypic Analysis of Clostridium difficile Isolates: a Single-Center Study

Abstract

f Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution's C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level. The most common C. difficile strain that has emerged in the past decade in North America and some areas in Europe has been classified as 027 by ribotyping, NAP1 by pulsed-field gel electrophoresis (PFGE), BI by restriction endonuclease analysis, and ST-1 by multilocus sequence typing (MLST). ST-1 strains account for half of the sporadic hospital-associated CDI in some settings (5). Some studies have reported that ST-1 strains elaborate C. difficile toxins (TCDs) at high concentrations; its purported hypervirulence is plausibly related to this trait. This strain has single and 18-bp deletions of tcdC, a negative regulator of tcdA and tcdB. These mutations cause premature stops, and this truncation is believed to cause toxin overproduction (6, 7). However, this assumption was challenged by recent studies showing no significant difference in toxin production between hypervirulent and nonhypervirulent C. difficile strains, and no association of the tcdC genotype and toxin production (8, 9).C. difficile strains containing cdtA and cdtB binary toxin genes are associated with greater mortality in their hosts than strains in which these genes are absent (10). However, it is not clear if the binary toxin genes increase the virulence of ST-1 or if they are simply epidemiologic markers of C. difficile...

Epidemiology, Diagnosis, and Management of Clostridium difficile Infection in Patients with Inflammatory Bowel Disease

“…Some strains, most notably including the epidemic strains known by their various classification schemata as ribotype 027, North American pulsed field gel electrophoresis pattern 1, or restriction endonuclease pattern BI, also produce the binary toxin, CDT (17). Although the precise pathophysiologic role of binary toxin is not known, its presence is associated with increased severity of CDI and mortality (18–20). …”

Clinical Utility of Laboratory Detection of Clostridium difficile Strain BI/NAP1/027

“…Because AF/244 is both cdt gene positive and tcdC⌬117 positive, it has been presumptively identified as BI/NAP1/027 by commercially available NAATs (28). With use of WGS, Zhou et al additionally identified 3 unique non-BI/NAP1/027 sequence types (ST-41 and 2 novel sequence types) that are positive for tcdA, tcdB, cdtA, cdtB, and tcdC⌬117 (29). Although the stool specimens from which these isolates were derived were not tested with a NAAT designed to identify BI/NAP1/027, specimens with these genotypes would presumptively be identified as BI/NAP1/027 with the commercially available NAATs.…”

Ceftolozane-Tazobactam Activity against Phylogenetically Diverse Clostridium difficile Strains

“…Currently, there are no categorical interpretative criteria for ceftolozane-tazobactam MICs for Clostridium species. It is well documented that the overall antimicrobial resistance profiles of C. difficile strains can vary widely among different strain types (10)(11)(12). Unfortunately, no C. difficile strain typing data were available as part of the Snydman et al study (2), and thus it was unknown if this profile was generalizable to different C. difficile strain types.…”

“…Of note, our test set included 26 C. difficile ribotype 027 (NAP1/BI/ST-1) strains and 3 C. difficile ri-botype 078 isolates. These strain types are currently among the most common ribotypes in North America and parts of Europe (18,19); the toxigenic status of each ribotype was determined by phenotypic (i.e., toxin enzyme immunoassays) and/or genotypic methods (3,10,13,14).…”

“…The isolates investigated were recovered from fecal specimens or rectal swab specimens from patients at Barnes-Jewish Hospital from January 2010 to July 2012. Specimens were collected as part of other ongoing studies to assess C. difficile diagnostic assays, C. difficile infection, or asymptomatic colonization (3,10,13,14). C. difficile isolates were recovered in culture (15) and ribotyped (16), as previously described.…”