Susan P. Sambol

Toxin B is essential for virulence of Clostridium difficile

Clostridium difficile is the leading cause of infectious diarrhoea in hospitals worldwide, because of its virulence, spore-forming ability and persistence. C. difficile-associated diseases are induced by antibiotic treatment or disruption of the normal gastrointestinal flora. Recently, morbidity and mortality resulting from C. difficile-associated diseases have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns. Since 2002, epidemic toxinotype III NAP1/027 strains, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are pro-inflammatory, cytotoxic and enterotoxic in the human colon. Inside host cells, both toxins catalyse the transfer of glucose onto the Rho family of GTPases, leading to cell death. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB (encoding toxin A and B, respectively) mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed after infection of hamsters with C. difficile and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically. Our work provides evidence that toxin B, not toxin A, is essential for virulence. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.

Comparison of Seven Techniques for Typing International Epidemic Strains of Clostridium difficile: Restriction Endonuclease Analysis, Pulsed-Field Gel Electrophoresis, PCR-Ribotyping, Multilocus Sequence Typing, Multilocus Variable-Number Tandem-Repeat Analysis, Amplified Fragment Length Polymorphism, and Surface Layer Protein A Gene Sequence Typing

ABSTRACT Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

In vitro activities of 15 antimicrobial agents against 110 toxigenic clostridium difficile clinical isolates collected from 1983 to 2004.

ABSTRACT The incidence and severity of Clostridium difficile-associated disease (CDAD) is increasing, and standard treatment is not always effective. Therefore, more-effective antimicrobial agents and treatment strategies are needed. We used the agar dilution method to determine the in vitro susceptibility of the following antimicrobials against 110 toxigenic clinical isolates of C. difficile from 1983 to 2004, primarily from the United States: doripenem, meropenem, gatifloxacin, levofloxacin, moxifloxacin, OPT-80, ramoplanin, rifalazil, rifaximin, nitazoxanide, tizoxanide, tigecycline, vancomycin, tinidazole, and metronidazole. Included among the isolates tested were six strains of the toxinotype III, NAP1/BI/027 group implicated in recent U.S., Canadian, and European outbreaks. The most active agents in vitro were rifaximin, rifalazil, tizoxanide, nitazoxanide, and OPT-80 with MICs at which 50% of the isolates are inhibited (MIC50) and MIC90 values of 0.0075 and 0.015 μg/ml, 0.0075 and 0.03 μg/ml, 0.06 and 0.125 μg/ml, 0.06 and 0.125 μg/ml, 0.125 and 0.125 μg/ml, respectively. However, for three isolates the rifalazil and rifaximin MICs were very high (MIC of >256 μg/ml). Ramoplanin, vancomycin, doripenem, and meropenem were also very active in vitro with narrow MIC50 and MIC90 ranges. None of the isolates were resistant to metronidazole, the only agent for which there are breakpoints, with tinidazole showing nearly identical results. These in vitro susceptibility results are encouraging and support continued evaluation of selected antimicrobials in clinical trials of treatment for CDAD.

Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease.

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.

Decreased Cure and Increased Recurrence Rates for Clostridium difficile Infection Caused by the Epidemic C. difficile BI Strain

BACKGROUND An epidemic strain of Clostridium difficile designated by restriction endonuclease analysis (REA) as group BI has caused multiple outbreaks of severe C. difficile infection (CDI). The treatment response of patients infected with this strain is uncertain. METHODS Clostridium difficile isolates were collected from 2 phase 3 clinical trials comparing fidaxomicin to vancomycin and typed using REA. Clinical cure and recurrence outcomes were analyzed by strain type of the infecting organism, BI and non-BI, using both univariate and multivariate analyses. RESULTS From 999 patients, 719 isolates were available for typing (356 fidaxomicin treated and 363 vancomycin treated). BI was the most common REA group (34% of isolates). Patients infected with BI had lower cure rates (86.6%; 214 of 247) than those infected with non-BI strains (94.3%; 445 of 472) (P < .001). The cure rate difference between the BI and non-BI patients was significant for both vancomycin (P = .02) and fidaxomicin (P = .007). BI patients had a recurrence rate of 27.4% (51 of 186), compared with a recurrence rate of 16.6% (66 of 397) in non-BI patients (P = .002). By multivariate analysis, BI infection was statistically significant as a risk factor for reduced cure (odds ratio [OR], 0.48; 95% confidence interval [CI], .27-.85; P = .030) and for increased recurrence (OR, 1.57; 95% CI, 1.01-2.45; P = .046). CONCLUSIONS The clinical cure rate of patients infected with the epidemic BI C. difficile strain is lower than the cure rate of those infected with non-BI strains whether treated with fidaxomicin or vancomycin. Similarly, the CDI recurrence rate is increased in patients with the BI strain compared with patients with other C. difficile strains.

Rifampin and Rifaximin Resistance in Clinical Isolates of Clostridium difficile

ABSTRACT Rifaximin, a poorly absorbed rifamycin derivative, is a promising alternative for the treatment of Clostridium difficile infections. Resistance to this agent has been reported, but no commercial test for rifaximin resistance exists and the molecular basis of this resistance has not been previously studied in C. difficile. To evaluate whether the rifampin Etest would be a suitable substitute for rifaximin susceptibility testing in the clinical setting, we analyzed the in vitro rifaximin susceptibilities of 80 clinical isolates from our collection by agar dilution and compared these results to rifampin susceptibility results obtained by agar dilution and Etest. We found rifaximin susceptibility data to agree with rifampin susceptibility; the MICs of both antimicrobials for all isolates were either very low or very high. Fourteen rifaximin-resistant (MIC, ≥32 μg/ml) unique isolates from patients at diverse locations in three countries were identified. Molecular typing analysis showed that nine (64%) of these isolates belonged to the epidemic BI/NAP1/027 group that is responsible for multiple outbreaks and increased disease severity in the United Kingdom, Europe, and North America. The molecular basis of rifaximin and rifampin resistance in these isolates was investigated by sequence analysis of rpoB, which encodes the β subunit of RNA polymerase, the target of rifamycins. Resistance-associated rpoB sequence differences that resulted in specific amino acid substitutions in an otherwise conserved region of RpoB were found in all resistant isolates. Seven different RpoB amino acid substitutions were identified in the resistant isolates, which were divided into five distinct groups by restriction endonuclease analysis typing. These results suggest that the amino acid substitutions associated with rifamycin resistance were independently derived rather than disseminated from specific rifamycin-resistant clones. We propose that rifaximin resistance in C. difficile results from mutations in RpoB and that rifampin resistance predicts rifaximin resistance for this organism.

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.

Effectiveness of alcohol-based hand rubs for removal of Clostridium difficile spores from hands

BACKGROUND Alcohol-based hand rubs (ABHRs) are an effective means of decreasing the transmission of bacterial pathogens. Alcohol is not effective against Clostridium difficile spores. We examined the retention of C. difficile spores on the hands of volunteers after ABHR use and the subsequent transfer of these spores through physical contact. METHODS Nontoxigenic C. difficile spores were spread on the bare palms of 10 volunteers. Use of 3 ABHRs and chlorhexidine soap-and-water washing were compared with plain water rubbing alone for removal of C. difficile spores. Palmar cultures were performed before and after hand decontamination by means of a plate stamping method. Transferability of C. difficile after application of ABHR was tested by having each volunteer shake hands with an uninoculated volunteer. RESULTS Plain water rubbing reduced palmar culture counts by a mean (+/- standard deviation [SD]) of 1.57 +/- 0.11 log10 colony-forming units (CFU) per cm2, and this value was set as the zero point for the other products. Compared with water washing, chlorhexidine soap washing reduced spore counts by a mean (+/- SD) of 0.89 +/- 0.34 log10 CFU per cm2; among the ABHRs, Isagel accounted for a reduction of 0.11 +/- 0.20 log10 CFU per cm2 (P = .005), Endure for a reduction of 0.37 +/- 0.42 log10 CFU per cm2 (P = .010), and Purell for a reduction of 0.14 +/- 0.33 log10 CFU per cm2 (P = .005). There were no statistically significant differences between the reductions achieved by the ABHRs; only Endure had a reduction statistically different from that for water control rubbing (P = .040). After ABHR use, handshaking transferred a mean of 30% of the residual C. difficile spores to the hands of recipients. CONCLUSIONS Hand washing with soap and water is significantly more effective at removing C. difficile spores from the hands of volunteers than are ABHRs. Residual spores are readily transferred by a handshake after use of ABHR.