Hypervirulent Clostridium difficile PCR-ribotypes exhibit resistance to widely used disinfectants

Abstract

The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings.

Figure 1. Vegetative cells and Spore counts of C. difficile PCR ribotypes 012, 017 and 027.

A) Total cell counts and spore counts were obtained by plating cultures and heat resistant samples of C. difficile on blood plates containing 0.1% taurocholate. B) Percentage spore counts were obtained by calculating the number of heat resistant spores as a proportion of the total cell counts. Data consists of three biological and two technical replicates from separate cultures. Student T-tests were performed between total counts and spores for each strain and significant differences are marked with a bracket * (p<0.05). A comparison for percentage survival of spores was performed using linear regression and a partial F-test, where M68 was the reference strain, a significant difference (p<0.01) in spore production between the three strains is marked with a bracket **.

Figure 2. Exposure of C. difficile PCR-ribotype 012, 017 and 027 strains to disinfectants.

Percentage survival after 30 minute exposure to A) Actichlor® at 5000 ppm, 1500 ppm, 1000 ppm and 500 ppm. B) Haztab® at 5000 ppm, 1500 ppm, 1000 ppm and 500 ppm. C) Perasafe® at 2%, 1.62%, 1% and 0.81%. D) Virkon® at 2%, 1.5%, 1% and 0.5%. E) Biocleanse® at 20%, 10%, 5% and 2.5%. F) Newgenn® at 0.8%, 0.4%, 0.2% and 0.1%. G) Proceine 40® at 6%, 0.6% and 0.06%. H) Steri 7® at 100%, 80%, 40% and 20%. I) Hibiscrub® at 50%. The survival was calculated as a percentage of the heat resistant spore counts from unchallenged cultures. Data consists of three biological and two technical replicates from separate cultures. 2) and an interaction test (Chi²) was performed using the statistical program Stata 12. Bracket ∧ indicate a significant difference in concentration independent of PCR-ribotype (p<0.01), Bracket * indicates a significant difference between PCR-ribotypes independent of concentration (p<0.01) and Bracket ∧* indicates a significant difference between PCR-ribotypes in a concentration dependent manor (p<0.01).