Michelle M. Merrigan

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.

Fatal Pseudomembranous Colitis Associated with a Variant Clostridium difficile Strain Not Detected by Toxin A Immunoassay

Clostridium difficile infection should be a primary diagnostic consideration in any patient with diarrhea who has been hospitalized and has recently received antibiotics. Detection of toxin in patient stool specimens is the most important laboratory evidence for confirming a diagnosis of C. difficile diarrhea, although reliance on any one test may be insufficient to exclude the diagnosis (1). Clostridium difficile produces two large single-unit toxins (toxins A and B), which share significant functional domain homology and act by glycosylation of small guanosine triphosphatebinding proteins that are involved in cell cytoskeleton organization (2). Commercial immunoassays have been developed that detect toxin A by using the monoclonal antibody PCG-4, which recognizes epitopes encoded by a region of highly repetitive DNA sequences in the 3 end of the toxin A gene (3). In general, these immunoassays have a similar specificity but a somewhat decreased sensitivity compared with cell-culture cytotoxin assays, which primarily detect the effects of toxin B (1). In addition to using toxin testing, the clinical laboratory at our hospital in Chicago, Illinois, has increased the sensitivity of diagnostic testing for C. difficile disease by adding a culture for C. difficile. Toxin assays are also performed in vitro on the culture supernatants of recovered isolates to screen for nontoxigenic (nonpathogenic) strains. In 1998, the cell-culture cytotoxin assay that had been used at our laboratory was replaced with an immunoassay for toxin A (Clearview C. DIFF A, Wampole Laboratories, Division of Carter-Wallace, Inc., Cranbury, New Jersey) after a prospective study showed that this toxin A immunoassay had a sensitivity and specificity similar to those of the cytotoxin assay (4). We report on a patient who died of pseudomembranous colitis that was caused by a strain of C. difficile undetected by repeated toxin A immunoassays. We genetically characterize this strain of C. difficile and estimate the number of clinical laboratories in our region with test strategies that will not detect this variant strain. Case Report An 86-year-old man with multiple cardiac and pulmonary problems was admitted to the hospital for treatment of presumed inflammatory bowel disease. During a previous hospitalization 2 months earlier, the patient had developed diarrhea and had been treated for pneumonia with antibiotics. At that time, findings on stool specimen testing were negative for toxin A but the culture was positive for C. difficile. The recovered C. difficile isolate tested negative for toxin A production in vitro (using the same toxin A immunoassay used on the stool specimen) and was reported as nontoxigenic. The patient was subsequently evaluated on several occasions for lower abdominal cramps and recurrent diarrhea. Findings on colonoscopy and abdominal computed tomography were suspicious for C. difficileassociated pseudomembranous colitis, but this diagnosis was dismissed because of two additional stool specimens that were negative for toxin A and were culture positive for a presumed nontoxigenic C. difficile strain. Two months after the initial onset of symptoms, the patient returned to the hospital with diarrhea, fever (37.4 C [99.3 F]), abdominal tenderness, and an elevated leukocyte count (14.2 109 cells/L). The patient was treated with steroids for presumed inflammatory bowel disease, partly because a recent extensive gastrointestinal evaluation (which had included a stool culture for routine enteric pathogens and an ova and parasite examination) was unrevealing. The patients diarrhea improved, but the lower abdominal cramping persisted. On the 3rd day of hospitalization, the patient experienced cardiopulmonary arrest, and attempts to resuscitate him were unsuccessful. Autopsy showed diffuse pseudomembranous colitis (Figure, left panel). A stool specimen obtained at the time of autopsy was negative for toxin A but was culture positive for a presumed nontoxigenic strain of C. difficile. After the autopsy report was received, the four clinical isolates were tested in vitro by using a cell-culture cytotoxin assay. All isolates tested positive for cytotoxin. No specific treatment for C. difficile diarrhea had been administered at any time during the patients illness. (See Case Summary.) Figure. Gross findings at autopsy performed 2 months after symptom onset. Left. Right. Molecular Investigation All four clinical isolates were identical by using HindIII-restriction endonuclease analysis typing (5) (data not shown) and were designated as restriction endonuclease analysis type CF4. The restriction pattern of the four isolates matched that of a group of highly related isolates in our clinical isolate collection (5); this group of related isolates, designated as CF, is known to be toxin variant. Although strains in the CF group produce toxin B and are cytotoxic, these variant strains do not produce toxin A and are characterized by a 1.8-kb deletion in the 3 end of the toxin A gene (6) (Table). Thus, we amplified the 3 end of the toxin A gene by performing polymerase chain reaction. The amplification was done on DNA that had been isolated and purified from the C. difficile isolates of patients and controls, as previously described (6), by using the primers A-u2 and A-d1-b (the primers start at positions 5301 and 8136, respectively, on the toxin A gene). The amplified product from the four isolates recovered from our patient was 1.8 kb smaller than the product from amplification of the standard toxigenic strain VPI 10463; the size of the amplified product was identical in size to the amplified product from the toxin Bpositive, toxin Anegative variant strain, type CF2 (6) (data not shown). Table 1. Diagnostic and Clinical Characteristics of Clostridium difficile Strains with Different Toxigenic Potential Table 2. Case Summary To identify additional CF isolates, we screened our collection of more than 5000 clinical isolates (6). By using restriction endonuclease analysis, we identified 58 variant CF isolates (1.7% of the 3445 total isolates typed), including 3 additional CF4 isolates from patients at two Veterans Affairs hospitals (in Minneapolis, Minnesota, and in Chicago) and a county hospital (in Chicago). At that time, the clinical laboratory at each of the three hospitals had been using both cytotoxin assays and culture for C. difficile testing. In all three patients, results on stool cytotoxin assays were positive and C. difficile diarrhea was diagnosed. Telephone Survey In September 2000, we surveyed all hospital-based laboratories in the Chicago metropolitan area to determine the type of C. difficile testing being performed. Immunoassay for toxin A was the only test being used to detect C. difficile in 31 of the 67 (46%) laboratories surveyed. Two laboratories were using a latex agglutination test for nontoxin C. difficile antigen, and the remaining laboratories performed either immunoassay for both toxins A and B or a cell-culture cytotoxin assay. Discussion Reliance on a single laboratory test despite epidemiologic and clinical clues that were highly suggestive of C. difficile infection contributed to the death of this patient. The patient initially presented with abdominal pain and diarrhea in the hospital after receiving antibiotic therapy for pneumonia. Although findings on colonoscopy and abdominal computed tomography were highly suggestive of C. difficileassociated pseudo-membranous colitis, the diagnosis was not made. Laboratory testing of stool specimens for C. difficile toxin is an accepted method for the diagnosis of C. difficile diarrhea. However, these tests are not highly sensitive compared with stool culture, and a negative result on immunoassay or on cytotoxin assay should not be used to exclude the diagnosis of C. difficile diarrhea (8). Several C. difficile strains with variations in the pathogenicity locus of the toxin gene have been reported (9-11). Rupnik and colleagues classified these variations into 15 toxinotypes by using molecular techniques (12). Toxinotype VIII strains are characterized by a 1.8-kb deletion in the toxin A gene and altered restriction sites in the toxin B gene. The toxinotype VIII strains do not produce a functional toxin A and are undetectable by toxin A immunoassays (13). Until recently, reports had suggested that toxinotype VIII strains, which also correspond to serogroup F [14], were nonpathogenic (10, 14, 15). We recently characterized a toxin variant of a C. difficile strain that we designated as restriction endonuclease analysis type CF2. The CF2 strain was found in specimens recovered from seven patients at a Veterans Affairs hospital in Minneapolis; five of the seven patients had documented cases of C. difficile diarrhea (6). Despite producing no toxin A and having genotypic characteristics nearly identical to those of strain 1470, which is the prototype strain for toxinotype VIII, type CF2 is cytotoxic and can cause disease in hamsters (although with lower colonization efficiency and mortality compared with other, fully toxigenic strains of C. difficile [16]). Recently, a nosocomial outbreak of C. difficile diarrhea due to a toxinotype-VIII variant strain was reported at a hospital in Winnipeg, Manitoba, Canada (17). Toxin-variant strains of C. difficile that appear identical or similar to toxinotype VIII are more widespread than was previously recognized (6, 17-19). Moreover, these strains can cause the full spectrum of C. difficileassociated diseases. A recent survey conducted by the United Kingdom National External Quality Assessment Scheme reported that 108 of 243 (44%) laboratories surveyed had been performing only toxin A testing for diagnosis of C. difficile (United Kingdom National External Quality Assessment Scheme. Clostridium difficile Survey. Distribution no. 1305. 10 January 2000). The results of the U.K. survey and of our survey of hospital-based laboratories in the Chic

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.

Infection of Hamsters with Epidemiologically Important Strains of Clostridium difficile

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.

International Typing Study of Toxin A-Negative, Toxin B-Positive Clostridium difficile Variants

ABSTRACT 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.

Toxin Gene Analysis of a Variant Strain of Clostridium difficile That Causes Human Clinical Disease

ABSTRACT A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3′ end of the gene, and a point mutation in the 5′ end of the gene, resulting in a premature stop codon at tcdAposition 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5′ end of the gene compared totcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3′ end of tcdA demonstrated identical 1.8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

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.

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.

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.

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.