Human Hypervirulent Clostridium difficile Strains Exhibit Increased Sporulation as Well as Robust Toxin Production

Abstract

Toxigenic Clostridium difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth and are the leading cause of antibiotic-associated diarrhea. C. difficile isolates of the molecular type NAP1/027/BI have been associated with severe disease and hospital outbreaks worldwide. It has been suggested that these "hypervirulent" strains produce larger amounts of toxin and that a mutation in a putative negative regulator (TcdC) allows toxin production at all growth phases. To rigorously explore this possibility, we conducted a quantitative examination of the toxin production of multiple hypervirulent and nonhypervirulent C. difficile strains. Toxin gene (tcdA and tcdB) and toxin gene regulator (tcdR and tcdC) expression was also monitored. To obtain additional correlates for the hypervirulence phenotype, sporulation kinetics and efficiency were measured. In the exponential phase, low basal levels of tcdA, tcdB, and tcdR expression were evident in both hypervirulent and nonhypervirulent strains, but contrary to previous assumptions, toxin levels were below the detectable thresholds. While hypervirulent strains displayed robust toxin production during the stationary phase of growth, the amounts were not significantly different from those of the nonhypervirulent strains tested; further, total toxin amounts were directly proportional to tcdA, tcdB, and tcdR gene expression. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Comparative genomic analyses of the closely related nonhypervirulent strains VPI 10463 (the highest toxin producer) and 630 (the lowest toxin producer) revealed polymorphisms in the tcdR ribosome binding site and the tcdR-tcdB intergenic region, suggesting that a mechanistic basis for increased toxin production in VPI 10463 could be increased TcdR translation and read-through transcription of the tcdA and tcdB genes. Hypervirulent isolates produced significantly more spores, and did so earlier, than all other isolates. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.Clostridium difficile is a leading bacterial nosocomial pathogen. Antibiotic treatment alters and suppresses commensal microbiota, allowing ingested C. difficile spores to germinate and colonize the gut. If the infecting strain is of the toxinproducing lineage of C. difficile (toxigenic), the resulting infection (CDI) can range from mild diarrhea to potentially fatal pseudomembranous colitis. Since 2000, highly virulent variants of toxigenic C. difficile have caused epidemics of CDI characterized by greater incidence, severity, and fatality (12, 25, 29). These "hypervirulent" (HV) strains cluster into a distinct phylogenetic group (38), as assessed by several different molecular methods (21) [33,41]). BI/ NAP1/027 strains have spread rapidly and widely in the past 10 years and have ...

Genetic markers for Clostridium difficile lineages linked to hypervirulence

“…We define hypervirulent strains as those that are associated with more severe clinical disease. In addition to the production of toxin A (TcdA) and toxin B (TcdB), the presence of binary toxin (CdtA and CdtB), altered antimicrobial resistance patterns and increased sporulation capacity can contribute to the virulence of certain C. difficile strains (Akerlund et al, 2008;Lyras et al, 2009;Merrigan et al, 2010;Schwan et al, 2009;Voth & Ballard, 2005).…”

Clostridium difficile Infection: Pathogenesis, Diagnosis and Treatment

“…Merrigan et al observed that some hypervirulent strains underwent early sporulation and produced large amounts of toxin, with greater efficiency than other strains. These researchers concluded that sporulation could contribute to the dissemination of infectious particles in the environment, thus helping toxins to confer adaptive advantages in the pathogenesis of hypervirulent strains of C. difficile [13].…”

“…These researchers have classified the tcd genes in two groups, one comprising A, B, D and E, and the other comprising C (as tcdC has the opposite orientation). It has also been found that tcdC is expressed in the exponential growth phase of the bacterium, while the other genes are expressed in the stationary phase [12,13].…”

Sepsis, the Liver and the Gut

“…Hypervirulent strains such as the molecular type NAP1/027/BI, have been found to have more robust toxin production and show an earlier spore-formation than other strains thus causing more severe infections. [119] Excessive toxin production in this strain has been traced to a mutation in the Toxin B encoding gene sequence. [120] Another emerging strain is the PCR ribotype 078, which is associated with community-associated C.difficile infection and has been isolated in animal and food products.…”