Intestinal colonization of infant hamsters with Clostridium difficile

Infant hamsters of different ages were examined for their susceptibility to enteric Clostridium difficile colonization. Intragastric administration of C. difficile to infant hamsters resulted in multiplication of the organism in the intestinal tracts of animals 4 to 12 days old; hamsters younger or older were resistant to C. difficile intestinal colonization. Toxicity to the colonized animals could not be demonstrated despite cytotoxin titers in some infant hamsters comparable to titers found in the intestinal tracts of adult hamsters with C. difficile-associated intestinal disease. When introduced into 4-day-old hamsters, C. difficile colonized the intestinal tract and remained at high levels until the animals were 13 days old, at which time the presence of intestinal C. difficile could no longer be demonstrated. The number of C. difficile required to colonize the intestinal tracts of 50% of 7-day-old hamsters was 18 viable cells. On the other hand, 10(8) viable cells of C. difficile failed to colonize the intestinal tracts of healthy, non-antibiotic-treated adult hamsters.     

An in-vitro model of colonisation resistance to Clostridium difficile infection

To investigate the importance of the normal gut flora in preventing the establishment of Clostridium difficile in vivo we have developed an in-vitro test system based on growth in faecal emulsions. Growth of C. difficile and cytotoxin production are inhibited in faecal emulsions from healthy adults, but not in sterilised emulsions; the importance of viable bacteria in the inhibitory system is evident. Generally, faecal emulsions derived from infants, children and geriatric patients were less inhibitory than those from healthy adults. Those from bottle-fed infants were significantly less inhibitory than those from breast-fed infants. Decreased levels of cytotoxin in the latter group were attributed to the acidic pH of the stools. With the different patient groups studied, faecal samples not inhibitory to C. difficile in vitro were obtained from 21% of patients with antibiotic-associated diarrhoea, 33% of those taking antibiotics but who did not have diarrhoea, 18.7% of those with diarrhoea unassociated with antibiotics, and 79% of those with C. difficile-mediated diarrhoea. In some cases inhibition was due to low faecal pH, as in some infants, and in others to other filterable substances. The degree of inhibition could not be linked to specific volatile fatty acids or enzymes.     

Passive immunization of hamsters against disease caused by Clostridium difficile by use of bovine immunoglobulin G concentrate.

Gestating Holstein cows were vaccinated with Clostridium difficile toxoid prepared from the culture filtrate of a strain that produces high levels of toxins A and B and other antigens. A bovine immunoglobulin G (IgG) concentrate was prepared from colostrum collected at parturition. The results of our studies showed that hamsters treated prophylactically with the hyperimmune bovine IgG concentrate were protected against C. difficile disease. These results suggest that orally administered hyperimmune bovine IgG specific for C. difficile culture filtrate may be useful in prophylaxis against C. difficile disease.     

Serogrouping of Clostridium difficile strains by slide agglutination.

Six different agglutinating antisera were obtained by immunizing rabbits with Formol-treated strains of Clostridium difficile. After appropriate absorption, these antisera were used to define six serogroups designated by the letters A, B, C, D, F, and G. Altogether, 315 strains of C. difficile from various origins were tested for slide agglutination by these antisera; 312 (99%) of them were agglutinated by one of these antisera. A and C were the most common serogroups. An excellent correlation, ranging from 85 to 100%, was found between the serogroup and the toxigenicity of the strains. The correlation between serogroup and sorbitol fermentation was higher, ranging from 89 to 100%. The results of this typing were compared with the clinical origin of the strains. Only strains of serogroups A, C, and D were isolated in 153 cases of antibiotic-associated diarrhea. This series included strains from three outbreaks; all the strains in two of the outbreaks belonged to serogroup C, and in the third, all the strains belonged to serogroup A. Strains of serogroups B, F, and G were only found in the stools of asymptomatic neonates or young children. In the latter samples, strains of serogroups A and D were found in the same ratio as in adults with antibiotic-associated diarrhea, but strains of serogroup C were seldom isolated. In patients treated with antineoplastic drugs and suffering from diarrhea, the distribution of the strains was the same as in cases of antibiotic-associated diarrhea.     

Virulence of ten serogroups of Clostridium difficile in hamsters

A slide agglutination technique identifying 10 serogroups of Clostridium difficile (A,B,C,D,F,G,H,I,K and X) has been described previously. In this study, we have used the hamster to compare the ability of the 10 serogroup reference strains to colonise and produce disease. Groups of four hamsters were each given a single intraperitoneal injection of either clindamycin or cefoxitin, and an oral challenge dose of C. difficile. The time taken to establish faecal colonisation and the length of survival after colonisation were monitored. All hamsters treated with cefoxitin became colonised by day 3 and those challenged with the cytotoxigenic strains of serogroups A,C,H and K developed colitis and died. Among those challenged with the non-toxigenic strains of groups B,D,I and X and the toxigenic strains of groups F and G, faecal colonisation was established without signs of disease. This demonstrates that there are differences in virulence even among toxigenic strains of C. difficile. The same phenomenon was observed after treatment with clindamycin but the pattern of colonisation was quite different with some strains. In the hamsters challenged with toxigenic strains of groups C and K and non-toxigenic strains of groups D and I, which are highly resistant to clindamycin, the response was the same as with cefoxitin. The results were different with strains which were susceptible to clindamycin. Some animals became colonised much later than those treated with cefoxitin but the mortality was similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxin-Mediated Paracellular Transport of Antitoxin Antibodies Facilitates Protection against Clostridium difficile Infection

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′)₂ fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.     

Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A.

The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.     

Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection

Molecular analysis of Clostridium difficile (28 isolates) from children (n = 128) in Oxfordshire, United Kingdom, identified eight toxigenic genotypes. Six of these were isolated from 27% of concurrent adult C. difficile-associated infections studied (n = 83). No children carried hypervirulent PCR ribotype 027. Children could participate in the transmission of some adult disease-causing genotypes.     

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.     

Survey of neuraminidase production by Clostridium butyricum, Clostridium beijerinckii, and Clostridium difficile strains from clinical and nonclinical sources.

Neuraminidase production was investigated in 57 Clostridium butyricum strains, 16 Clostridium beijerinckii strains, and 25 Clostridium difficile strains. Neuraminidase activity was found only in C. butyricum strains originating from one human newborn with neonatal necrotizing enterocolitis, two newborns with hemorrhagic colitis, one infected placenta, and one adult with peritonitis, It was concluded that neuraminidase was not a major virulence factor in C. butyricum strains.     

Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A(+)B(+)). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A.     

PCR ribotyping of clinically important Clostridium difficile strains from Hungary.

Isolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 15 different ribotypes was detected among the 65 isolates tested. The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common. Two non-toxigenic C. difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type.     

Fourteen-genome comparison identifies DNA markers for severe-disease-associated strains of Clostridium difficile

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ～ 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.     

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.     

Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters.

Hamsters were vaccinated with toxoids containing toxin A, toxin B, both toxins, or a preparation containing neither toxin of Clostridium difficile, the causative agent of antibiotic-associated cecitis in hamsters and pseudomembranous colitis in humans. To determine whether these vaccines would reduce the severity of antibiotic-associated cecitis, the hamsters were injected subcutaneously with clindamycin. Nearly all of the hamsters protected against neither toxin or only one toxin died. These animals developed enlarged hemorrhagic ceca and diarrhea, although the ceca from the animals immunized against toxin B were less hemorrhagic. The hamsters immunized against both toxins survived clindamycin treatment and had ceca of normal size and appearance. Concentrations of both toxins were lower in the ceca of the latter animals than in the unprotected animals. To determine the effects of either toxin alone on the animals, nonimmunized hamsters were injected with either purified toxin A, which produced enlarged ceca with moderate hemorrhaging, or partially purified toxin B, which produced hemorrhagic ceca of normal size. All of the hamsters injected with either toxin at concentrations found in the ceca after clindamycin treatment died. These results suggest that toxin A causes the water influx, that both toxins cause hemorrhaging to different extents in the ceca of hamsters with antibiotic-associated cecitis and that either toxin alone can cause death. These studies may help explain the etiology of pseudomembranous colitis in humans.     

Extra-intestinal infections caused by Clostridium difficile

The objective of this paper was to investigate the incidence of extra-intestinal infections caused by Clostridium difficile. During a 10-year period, the microbiology laboratory of our institution isolated 2034 isolates of C. difficile . Of the 2034 isolates, 21 (1.08%) were obtained from extra-intestinal sources. This represents an incidence of extra-intestinal isolation of four cases per 100 000 admissions. We were able to review the records of 17 patients for our study. The isolates in 12 patients were obtained from structures or fluids anatomically close to the colon and included the following infections: peritonitis in five cases (three primary and two secondary), intra-abdominal abscesses in three patients and abdominal wound infections in four cases. The infections in the other five patients were not in the anatomic vicinity of the colon. They included one case with a brain abscess, two episodes of bacteremia and two cases of foot infections (one chronic osteomyelitis). In all but one case, C. difficile isolation was obtained as part of a polymicrobial flora. The isolates were frequently non-toxigenic and the extra-intestinal infections occurred without concomitant diarrhea or prior anti-microbial therapy. Out of the 17 patients, eight died and nine survived. Death could not be directly attributed to C. difficile in any of the cases. The isolation of C. difficile outside the intestinal tract is very uncommon. Its clinical significance should be interpreted with caution.     

How to detect Clostridium difficile variant strains in a routine laboratory

Toxin A-negative, toxin B-positive strains (A–/B+) are the best studied examples of Clostridium difficile variant strains. In addition, there are some other groups of variant C. difficile strains that produce both toxins (A+/B+) or are non-cytotoxic (A–/B–) but differ from the reference strain VPI 10463 in their toxin genes. Here we describe two simple methods (amplification of the tcd A gene and amplification of the binary toxin gene cdt A) which can be used in rapid screening for variant C. difficile strains in collections or in routine laboratories.