Phenotypic evaluation of Clostridium difficile PCR ribotypes 078, 027, and 002: antimicrobial susceptibilities, cytotoxin production profiles, and biofilm formation.

Objectives: Specific Clostridium difficile ribotypes have been designated as hypervirulent, e.g. PCR ribotypes 027 (NAP1/BI) and 078 (NAP7/BK), principally based upon clinical observations of C. difficile infection (CDI) severity. Although early ribotype 027 studies highlighted potential elevated toxin production in vitro, subsequent research has not corroborated these findings when comparing with other prevalent C. difficile ribotypes. We assessed the in vitro phenotypic characteristics of three common ribotypes (002, 027, and 078), collected from diverse sources, to determine potential traits that may contribute to their high clinical prevalence. Methods: Three ribotype groups of C. difficile isolates were obtained from 13 NHS hospitals: 002 (n=11, 6 locations), 027 (n=11, 5 locations), and 078 (n=11, 5 locations), plus ATCC 700057 and internal control (E4, ribotype 010) control strains. Antimicrobial susceptibilities to: linezolid, ciprofloxacin, moxifloxacin, metronidazole, vancomycin, erythromycin, tetracycline, and clindamycin were determined using an agar incorporation method. Growth kinetics were assessed in BHIS broth, maximum specific growth rates (μmax) calculated, and cytotoxin titres (log10-relative units, RU) determined using Vero cell cytotoxicity assays. Biofilm formation was quantified in a 96-well microtitre plate assay with crystal violet staining. Results: Geometric mean MICs (GM-MICs) differed between ribotype groups. Ribotype 027 isolates were significantly more resistant to ciprofloxacin (68.2 mg/L), moxifloxacin (32 mg/L), erythromycin (128 mg/L), and less susceptible to metronidazole (1.37 mg/L) than ribotypes 078 and 002 (P<0.001). Interestingly, GM-MIC of clindamycin was significantly higher for ribotype 002 (3.31 mg/L, 64% of isolates were resistant at 8 mg/L) than for ribotypes 027 (0.44 mg/L) and 078 (0.09 mg/L) (P<0.001). Ribotype 078 isolates were generally more susceptible as assessed by GM-MICs, although intermediate resistance to tetracycline (8 mg/L) was observed in 54% of isolates (4 locations). Maximum specific growth rates in BHIS were: 0.84 h-1 (002), 0.67 h-1 (027), and 0.36 h-1 (078), with median 72 h cytotoxin titres substantially greater in 078 isolates (3 RU) compared to 027 and 002 (1 RU). Ribotype 078 isolates produced substantially greater levels of biofilm than 027 and 002 groups. Conclusion: There are marked differences between C. difficile ribotypes with regard to antimicrobial susceptibility, growth (biofilm and planktonic) and cytotoxin production. Ribotype 027 was generally more antibiotic-resistant; and ribotype 078 was less susceptible to tetracycline. Ribotype 078 produced substantially greater amounts of cytotoxin than the other ribotypes, despite little difference in culture biomass, and demonstrated increased biofilm formation, which could contribute to enhanced virulence if found in vivo. Ribotype 002 had the greatest μmax and levels of resistance to clindamycin; this is now the most prevalent UK C. difficile clinical ribotype, and further studies are warranted to explain this emergence.