Phenotypic Characterisation of Clostridium difficile PCR Ribotype 078 and Comparison with PCR Ribotypes 027 and 002
Keighley, Daniel David
Clostridium difficile is an anaerobic, Gram-positive bacterium which resides in the gut of animals and humans. There are over 600 different polymerase chain reaction (PCR) ribotypes of C. difficile, some of which are pathogenic. Despite 5% of healthy humans having C. difficile within their normal gut microflora, this organism can cause illness in the elderly and immunocompromised patients. Symptoms of C. difficile infection (CDI) range from mild diarrhoea to death, and are due to two toxins (toxin A and toxin B) that the bacterium produces. Treatment for CDI includes the use of antibiotics, such as metronidazole and vancomycin, however some antibiotics, such as fluoroquinolones, can cause CDI, as they can disrupt the normal gut flora. C. difficile also produces biofilms which protect the bacteria within from antibiotic therapy. This study evaluated three genetically distinct C. difficile groups, PCR ribotypes 078, 027 and 002. All these PCR ribotypes cause disease, however PCR ribotypes 078 and 027 have been stated to be hypervirulent strains, therefore causing more severe illness. This study compared: the growth rate and pattern; cytotoxin production; susceptibility to a range of antimicrobials; biofilm production; viable and spore counts; and antimicrobial susceptibility for the three PCR ribotype groups to determine if any of these factors might contribute to the enhanced virulence status of PCR ribotype 078. These assays were completed by conducting: batch culture growth curves; cytotoxin production assays; antimicrobial susceptibility tests (agar dilution methods); a standard 96-well microtitre plate biofilm assay for biofilm quantification; and the Calgary Biofilm device (CBD) to assess biofilm formation and susceptibilities to metronidazole and vancomycin. This study found PCR ribotype 078 had higher average absorbance readings (biomass) than the PCR ribotypes 027 and 002 at the peak of growth (8 hours of incubation in an anaerobic cabinet): average OD600 readings for the PCR ribotype 078 group were 3.70 whereas the PCR ribotype 027 group had an OD600 of 2.82 and PCR ribotypes 002 3.26. PCR ribotype 002 had significantly the highest average maximum specific growth rate (μmax) (0.73 h-1) whereas PCR ribotype 078 had the lowest average μmax (0.53 h-1) (P≤0.001). PCR ribotype 078 also had significantly higher cytotoxin production than PCR ribotypes 027 and 002, with PCR ribotype 078 median cytotoxin titres of 3 log10 relative units (RU) in 72 hour cultures, whereas PCR ribotypes 027 and 002 median titres were 1 RU (P≤0.001). All the strains in each PCR ribotype group were susceptible to metronidazole and vancomycin. PCR ribotype 078 strains were susceptible to most of the antimicrobials used in this study, for example the vancomycin and metronidazole geometric mean minimum inhibitory concentration (MIC)s for PCR ribotypes 078 were vancomycin: 0.57 mg/L and metronidazole: 0.08 mg/L. The results for vancomycin were similar to the other two PCR ribotypes (P=0.79) whereas the metronidazole result were significantly different (P≤0.001) (PCR ribotypes 027: vancomycin: 0.53 mg/L and metronidazole: 1.37 mg/L; PCR ribotype 002: vancomycin: 0.53 mg/L and metronidazole: 0.18 mg/L). PCR ribotype 078 average biofilm production significantly increased over three (0.12 OD590) to six (0.28 OD590) days whereas the average for the PCR ribotype 002 group did increase but not significantly (three days: 0.07 OD590 and six days: 0.09 OD590), however biofilm production by PCR ribotype 027 strains decreased (three days: 0.11 OD590 and six days: 0.08 OD590). PCR 078 demonstrated the lowest biofilm total viable counts (5.17 log10 colony forming units (CFU)/ml) and spore counts (4.58 log10 CFU/ml) using a 96-well microtitre plate after six days of growth compared to the other two PCR ribotypes in which total viable counts were 5.87 log10/5.70 log10 CFU/ml and spore counts were 5.32 log10/5.27 log10 CFU/ml for the PCR ribotype 027/002 groups respectively (P≤0.001). The biofilm susceptibility testing results showed PCR ribotype 078 geometric mean biofilm MIC (bMIC) and minimum biofilm eradication concentrations (MBEC) for vancomycin (0.50 mg/L and 0.57 mg/L respectively) and metronidazole (0.50 mg/L and 0.55 mg/L respectively) had similar results to those of PCR ribotypes 027 (vancomycin: bMIC 0.50 mg/L and MBEC 0.50 mg/L and metronidazole: bMIC 0.50 mg/L and MBEC 0.66 mg/L) and 002 (vancomycin: bMIC 2 mg/L and MBEC 2 mg/L and metronidazole: bMIC 4 mg/L and MBEC 2 mg/L). Total viable counts and spore counts on static CBD were <100 CFU/peg for all PCR ribotypes. This study also demonstrated that agitating the CBD in a four day growth period facilitated more extensive biofilm formation compared to static CBD assays. This study has demonstrated differences in growth (planktonic and biofilm) and cytotoxin production between the three C. difficile PCR ribotype groups assessed. These results could influence the behaviour and pathogenesis of PCR ribotype 078 in CDI if these results translated into the in vivo setting. Further studies are required in order to assess the reproducibility of these data in a larger cohort of isolates of the ribotypes studied, and in isolates obtained from varied hosts (human and animal) and environmental settings.