The Relative Role of Toxins A and B in the Virulence of Clotridioides difficile.

Most pathogenic strains of C. difficile possess two large molecular weight single unit toxins with four similar functional domains. The toxins disrupt the actin cytoskeleton of intestinal epithelial cells leading to loss of tight junctions, which ultimately manifests as diarrhea in the host. While initial studies of purified toxins in animal models pointed to toxin A (TcdA) as the main virulence factor, animal studies using isogenic mutants demonstrated that toxin B (TcdB) alone was sufficient to cause disease. In addition, the natural occurrence of TcdA-/TcdB+ (TcdA-/B+)mutant strains was shown to be responsible for cases of C. difficile infection (CDI) with symptoms identical to CDI caused by fully toxigenic (A+/B+) strains. Identification of these cases was delayed during the period when clinical laboratories were using immunoassays that only detected TcdA (toxA EIA). Our hospital laboratory at the time performed culture as well as toxA EIA on patient stool samples. A total of 1.6% (23/1436) of all clinical isolates recovered over a 2.5-year period were TcdA-/B+ variants, the majority of which belonged to the restriction endonuclease analysis (REA) group CF and toxinotype VIII. Despite reports of serious disease due to TcdA-/B+ CF strains, these infections were typically mild, often not requiring specific treatment. While TcdB alone may be sufficient to cause disease, clinical evidence suggests that both toxins have a role in disease.

International typing study of Clostridium difficile.

We report the results of an international Clostridium difficile typing study to cross reference strain designations for seven typing methodologies and facilitate inter-laboratory communication. Four genotypic and three phenotypic methods were used to type 100 isolates and compare the results to 39 PCR ribotypes identified among the collection.

Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production.

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.

New approach to the management of Clostridium difficile infection: colonisation with non-toxigenic C. difficile during daily ampicillin or ceftriaxone administration.

Non-toxigenic strains of Clostridium difficile are highly effective in preventing toxigenic C. difficile infection in hamsters when given following a single dose of an antimicrobial agent. The goal of this study was to determine the ability of non-toxigenic C. difficile to colonise hamsters during administration of an antibiotic to which the organisms are resistant - ceftriaxone - and an antibiotic to which they are susceptible - ampicillin - and to determine if non-toxigenic colonisation is protective against toxigenic strain challenge. Groups of four or five hamsters were administered daily ceftriaxone 60 mg/kg/d intraperitoneally or ampicillin 60 mg/kg/d orally for 5 days. Three non-toxigenic strains of C. difficile, M3, M23, and T7 (MICs 96-128 mg/L) were each given orally at a dose of 1 x 10(6) spores to groups of five animals 3h after the first dose of ceftriaxone. All animals were colonised successfully by day 3 of the study and when challenged with 1 x 10(6) spores of toxigenic strain J9 (MIC >256 mg/L) on day 3 all animals survived, whereas the control animal given ceftriaxone, but not non-toxigenic C. difficile, died within 48h of challenge. When groups of four hamsters were given ampicillin, administration of non-toxigenic strain M3 (MIC 2 mg/L) or toxigenic strain J9 (MIC 0.75 mg/L) at 1 x 10(6) spores did not result in any colonisation or infection of the animals until day 8, 3 days after the last ampicillin dose. A protection study was designed by giving M3 spores to groups of five animals daily for 5 days beginning on day 1, 3, or 5 of ampicillin. Toxigenic challenge was given with J9 spores on day 3 of each M3 regimen. M3 colonised all animals by day 8 and none became infected with J9. Colonisation by non-toxigenic C. difficile is an effective prevention strategy during antibiotic administration of ceftriaxone or ampicillin, but multiple-day administration is required for ampicillin and colonisation does not occur until several days after the drug is discontinued.

Genotypic and phenotypic analysis of Clostridium difficile correlated with previous antibiotic exposure.

To analyze Clostridium difficile susceptibility results and genotypes in relation to antibiotic exposures that precipitated C. difficile-associated diarrhea (CDAD), we examined 83 nosocomial C. difficile isolates recovered at a tertiary care center in Boston, Massachusetts. MICs were determined by E-test methodology using modified Brucella agar. Isolates were genotyped by pulsed-field gel electrophoresis and restriction enzyme analysis. Antibiotic susceptibilities were: ciprofloxacin (0%), clindamycin (59%), trovafloxacin (63%), ceftriaxone (73%), piperacillin/tazobactam (100%), metronidazole (100%), and vancomycin (100%). The two most common strain groups, isolated from a total of 33 patients, were much more likely to be resistant to clindamycin, erythromycin, and trovafloxacin than other strain groups [79% (26 of 33) versus 2% (1 of 50), respectively]. Clindamycin exposure was strongly associated with CDAD caused by isolates that exhibited multiple resistance to clindamycin, erythromycin, and trovafloxacin (prevalence odds ratio, 4.2; 95% confidence interval, 1.1-16.8), whereas other antimicrobials did not yield significant associations. Resistance of specific C. difficile strains to clindamycin and other antimicrobial agents may contribute to their hospital dissemination and explain, in part, the propensity of clindamycin to trigger nosocomial outbreaks.

Prevention of fatal Clostridium difficile-associated disease during continuous administration of clindamycin in hamsters.

Clostridium difficile-associated disease (CDAD) due to toxigenic strains is prevented in hamsters by colonization by nontoxigenic C. difficile after administration of clindamycin (Cm). To prevent CDAD during treatment with antibiotics, we gave a Cm-resistant nontoxigenic C. difficile strain, M13 (minimal inhibitory concentration [MIC], >256 microg/mL), and a Cm-susceptible strain, M3 (MIC, 0.5 microg/mL), to hamsters receiving Cm daily for days 1-5. Either M13 or M3 was given orogastrically (1 x 10(6) spores/day) to each hamster in 3 groups of 5 each, on either day 3, days 3-5, or days 3-7. M13 colonized at a higher rate and faster than did M3 (P<.001). When hamsters were challenged by toxigenic strain B1 on days 5, 7, or 9, M13 prevented CDAD in 100% of the hamsters. M3 protected no hamsters challenged by B1 on day 5, 20% on day 7, and 100% on day 9. M13 contains the erm(B) resistance gene but not the mobilizable element Tn5398. The benefits of use of Cm-resistant nontoxigenic C. difficile to prevent CDAD must be balanced against the risk that resistance might be transferred to other enteric bacteria.

International typing study of toxin A-negative, toxin B-positive Clostridium difficile variants.

Clinically important strains of Clostridium difficile that do not produce toxin A but produce toxin B and are cytotoxic (A(-)/B(+)) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A(-)/B(+) C. difficile isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A(-)/B(+) variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with C. difficile disease in the United Kingdom and the United States. C. difficile A(-)/B(+) variants of apparent clonal origin are widely distributed in Europe and North America.

Susceptibility of hamsters to human pathogenic Clostridium difficile strain B1 following clindamycin, ampicillin or ceftriaxone administration.

Clindamycin-treated hamsters are predictably susceptible to infection with pathogenic strains of Clostridium difficile. This animal model parallels most of the important aspects of human C. difficile associated disease (CDAD). In humans, almost any antibiotic may precipitate CDAD, but clindamycin, ampicillin and second-and third-generation cephalosporins are implicated most often. We studied the effect of ampicillin and ceftriaxone compared to clindamycin on the susceptibility of hamsters to challenge with C. difficile strain designated B1 by restriction endonuclease typing, an epidemic strain from one hospital. Hamsters were highly susceptible to CDAD following a single dose of clindamycin (30 mg/kg orogastrically) from 1 to 4 days when challenged with 100 colony-forming units (CFU) of spores of epidemic CD strain B1. Ampicillin was given orogastrically at 60 mg/kg to groups of three hamsters that were challenged with 10000 CFU of CD strain B1 spores on days 1-4 following ampicillin. Hundred percent CDAD mortality occurred in all groups on each challenge day. Ceftriaxone, given intraperitoneally at 60 mg/kg, induced susceptibility to CDAD for a more limited time course and at a higher CD inoculum, producing 100% mortality when hamsters were challenged with 10000 CFU of CD strain B1 on day 1 following ceftriaxone, 33% mortality at day 2, and no CDAD when challenged on days 3 and 4 following ceftriaxone. Hamsters are susceptible to CD infection for at least 4 days following ampicillin and clindamycin, but ceftriaxone has a shorter duration of susceptibility.

Colonization for the prevention of Clostridium difficile disease in hamsters.

Studies suggest that asymptomatic colonization with Clostridium difficile (CD) decreases the risk of CD-associated disease (CDAD) in humans. A hamster model was used to test the efficacy of colonization with 3 nontoxigenic CD strains for preventing CDAD after exposure to toxigenic CD. Groups of 10 hamsters were given 10(6) nontoxigenic CD spores 2 days after receiving a single dose of clindamycin. Five days later, the hamsters were given 100 spores of 1 of 3 toxigenic CD strains previously shown to cause mortality within 48 h. Each nontoxigenic strain prevented disease in 87%-97% of hamsters that were challenged with toxigenic strains. Failure to prevent CDAD was associated with failure of colonization with nontoxigenic CD. Colonization with nontoxigenic CD strains is highly effective in preventing CDAD in hamsters challenged with toxigenic CD strains, which suggests that use of a probiotic strategy for CDAD prevention in humans receiving antibiotics might be beneficial.