B-156. Growth, Toxin Production and Sporulation of Hypervirulent Clostridium difficile Strains

M. Merrigan1, A. Venugopal1, D. Gerding2, G. Vedantam1,2;
1Loyola Univ., Maywood, IL, 2Hines VA Hosp., Hines, IL.

Background: Clostridium difficile is an opportunistic nosocomial gut pathogen, which produces two toxins (TcdA and TcdB) during the stationary phase of growth. Recently, “hypervirulent” (HV) C. difficile strains have caused diarrheal disease epidemics worldwide. The molecular basis of hypervirulence is poorly understood; one hypothesis is that increased disease severity is due to HV strains producing toxins at all growth phases and at amounts higher than non-HV strains. In this study, we determined the rates of growth and sporulation, and quantitated toxin production of HV and non-HV C. difficile strains to address this hypothesis. Methods/Results: Four HV C. difficile strains (BI designation) of geographically distinct origin, and four genetically distinct toxigenic non-HV strains were tested. All 8 strains showed similar growth profiles in an enriched medium. For toxin quantitation, culture supernates were collected at mid-exponential phase (6hrs), early stationary phase (12hrs), late stationary phase (15hrs), and at 18, 24, and 48 hours of growth. Toxin levels were determined using a TcdA/B ELISA kit. Bacteria were also gram-stained to semi-quantitatively determine spore accumulation over time. We found that in 8/8 strains, toxins were undetectable at exponential phase. Toxin levels began to accumulate for all strains at 15hrs and peaked at 48 hrs. Toxin producers could be divided into three distinct groups: low; <1 mg/ml total toxin (strain 630), medium; 1-50 mg/ml (strains J9, K14, BI6, BI8) and high; >50 mg/ml (strains BI17, BI23, VPI10463). HV strains fell into both medium and high toxin producing groups, which underscores the importance of comparator strains to determine alterations in toxin production. The highest toxin producer in this study was the non-HV strain VPI 10463. Sporulation began at 24 hours for 3/8 strains, with all strains sporulating by 48 hours. Two HV strains had significantly more spores than all other strains at 48 hours.Conclusions: The HV phenotype in C. difficile is not solely caused by increased or early toxin production. A synergistic combination of multiple dysregulated factors likely causes the severe disease outcomes associated with HV C. difficile.