Virulence plasmid diversity in Clostridium perfringens type D isolates

Clostridium perfringens type D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding epsilon-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, including C. perfringens enterotoxin and beta2 toxin, encoded by the cpe and cbp2 genes, respectively. In the current study we demonstrated that the etx, cpe, and cpb2 genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that the etx gene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. The etx plasmids also typically carried IS1151 and tcp open reading frames (ORFs) known to mediate conjugative transfer of C. perfringens plasmid pCW3. PCR studies revealed that other than their tcp ORFs, etx plasmids of type D isolates do not carry substantial portions of the conserved or variable regions in the cpe plasmids of type A isolates. Southern blotting also demonstrated that in type D isolates the cpe and cpb2 genes are sometimes present on the etx plasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids, (ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) many etx plasmids should be capable of conjugative transfer.     

Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence

Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans. In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene. In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence. When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C. perfringens food poisoning in Japan. The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C. perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.     

Development of a duplex PCR genotyping assay for distinguishing Clostridium perfringens type A isolates carrying chromosomal enterotoxin (cpe) genes from those carrying plasmid-borne enterotoxin (cpe) genes

About 5% of Clostridium perfringens type A isolates carry the cpe gene encoding the C. perfringens enterotoxin. Those cpe-positive type A isolates are important causes of food-poisoning and non-food-borne cases of diarrheas in humans, as well as certain veterinary cases of diarrhea. Previous studies have determined that the enterotoxigenic type A isolates causing both non-food-borne human gastrointestinal disease and veterinary disease carry their cpe genes on plasmids, while the type A isolates causing human food poisoning carry a chromosomal cpe gene. The present study reports on the successful development of a duplex PCR assay that can rapidly genotype enterotoxigenic type A isolates (i.e., determine whether those cpe-positive isolates carry a chromosomal or a plasmid-borne cpe gene). The availability of this rapid cpe genotyping assay capable of handling large numbers of samples provides a powerful new investigative tool for diagnostic, epidemiologic, and basic research purposes.     

Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR

Clostridium perfringens strains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates of C. perfringens were obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates were C. perfringens alpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtyping C. perfringens libraries from specific epidemiological or production environment settings.     

Comparison of virulence plasmids among Clostridium perfringens type E isolates

Clostridium perfringens type E isolates produce iota-toxin, which is encoded by iap and ibp genes. Using Southern blot analyses, the current study identified iap/ibp plasmids of approximately 97 or approximately 135 kb among eight type E isolates. For most of these isolates, their iap/ibp plasmid also encoded urease and lambda-toxin. However, the beta2-toxin gene, if present, was on a different plasmid from the iap/ibp plasmid. For all isolates, the iap/ibp plasmid carried a tcp locus, strongly suggesting that these plasmids are conjugative. Overlapping PCR analyses demonstrated some similarity between the iap/ibp plasmids and enterotoxin-encoding plasmids of type A isolates. Additional PCR analyses demonstrated that the iap/ibp locus is located near dcm sequences, an apparent plasmid hot spot for toxin gene insertion, and that two IS1151-related sequences are present in the iap/ibp locus. To begin testing whether those IS1151-like sequences can mobilize iap/ibp genes, a PCR assay was performed that amplifies a product only from circular DNA forms that could represent transposition intermediates. This PCR assay detected circular forms containing iap/ibp genes and silent enterotoxin gene sequences, with or without an IS1151-like sequence. Collectively, these results suggest that a mobile genetic element carrying iap/ibp has inserted onto a tcp-carrying enterotoxin plasmid in a type A isolate, creating a progenitor iap/ibp plasmid. That plasmid then spread via conjugation to other isolates, converting them to type E. Further iap/ibp plasmid diversity occurred when either the iap/ibp genes later remobilized and inserted onto other conjugative plasmids or some iap/ibp plasmids acquired additional DNA sequences.     

Enterotoxin plasmid from Clostridium perfringens is conjugative

Clostridium perfringens enterotoxin is the major virulence factor involved in the pathogenesis of C. perfringens type A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene, cpe, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the cpe plasmid encodes its own conjugative transfer, a C. perfringens strain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to the cpe gene had been replaced by the chloramphenicol resistance gene catP, was used as a donor in matings with several cpe-negative C. perfringens isolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 x 10(-2) to 4.6 x 10(-4) transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype C. perfringens tetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the cpe plasmid occurred in vivo, it would have the potential to convert cpe-negative C. perfringens strains in normal intestinal flora into strains capable of causing gastrointestinal disease.     

Clostridium perfringens urease genes are plasmid borne.

Although many bacteria are ureolytic, and in some cases urease acts as a virulence factor, the urease phenotype has not been analyzed in the anaerobic pathogen Clostridium perfringens. In this study, approximately 2% of C. perfringens strains, representing the principal biotypes, were found to harbor the urease structural genes, ureABC, and these were localized on large plasmids that often encode, in addition, the lethal epsilon or iota toxins or the enterotoxin. This represents the first report of a plasmid-encoded urease in a gram-positive bacterium. The C. perfringens enzyme was highly similar to the ureases of other bacteria and cross-reacted with antibodies raised against the urease purified from Helicobacter pylori. Urease production was inhibited by urea and induced under growth conditions where the availability of nitrogen sources was limiting. To date, this form of regulation has been observed only for chromosomal ureABC genes.     

PCR identification of the plasmid-borne enterotoxin gene (cpe) in Clostridium perfringens strains isolated from food poisoning outbreaks

It is generally accepted that Clostridium perfringens strains associated with food poisoning carry their enterotoxin gene, cpe, on the chromosome, while C. perfringens strains isolated from non-food-borne diseases, such as antibiotic-associated diarrhea and sporadic diarrhea, carry cpe on the plasmid. However, we recently encountered a food poisoning outbreak caused by C. perfringens bearing a plasmid cpe. We therefore investigated a total of 31 clinical and non-clinical C. perfringens strains to locate the cpe gene by PCR. The cpe of nine heat-sensitive (100 degrees C for 10min) strains isolated from three outbreaks of food poisoning were located on the plasmid, while those of six heat-resistant strains from other food poisoning outbreaks were located on the chromosome. Moreover, the cpe of 5 heat-sensitive strains isolated from healthy human feces and those of 11 heat-sensitive soil strains were also located on the plasmid. These findings indicate that heat-sensitive, cpe-plasmid-borne C. perfringens strains should not be disregarded as causative agents of food poisoning.     

Proteolysis of Clostridium perfringens type A enterotoxin during purification.

The small satellite bands of enterotoxin frequently seen in polyacrylamide gels following purification of Clostridium perfringens enterotoxin were found to be due to endogenous protease activity and were not present if phenylmethylsulfonyl fluoride (PMSF; 1 mM) and EDTA (10 mM) were used in the purification protocol. The use of PMSF was avoided by passing gel filtration-purified enterotoxin material through DEAE-Sephacel. This modified protocol resulted in an 11.4-fold purification of enterotoxin and a 26.8% yield. Contrary to previous reports (B. R. Dasgupta and M. W. Pariza, Infect. Immun. 38: 592-597, 1982), if PMSF and EDTA were included during purification, we were unable to detect the novel enterotoxin ET-1 produced by strain NCTC 10240. C. perfringens proteases cleaved homogeneous enterotoxin into two additional fragments, suggesting that ET-1 was a product of endogenous protease action during purification.     

Highly conserved alpha-toxin sequences of avian isolates of Clostridium perfringens

Clostridium perfringens causes necrotic enteritis in chickens, and alpha-toxin has been suggested to be a key virulence determinant. Analysis of the alpha-toxin of 25 chicken-derived C. perfringens strains demonstrated high homology to mammal-derived strains rather than to the only avian-derived C. perfringens alpha-toxin sequence reported previously.     

Clostridium perfringens type A enterotoxin: characterization of the amino-terminal region.

The amino-terminal region of the enterotoxin of Clostridium perfringens was investigated by automated sequence analysis. The primary structure results revealed that the enterotoxin is composed of a single polypeptide amino acid sequence. Computer comparison of a 20-residue sequence with a sequence library of reported proteins revealed no significant chemical similarities, indicating that the enterotoxin represents a unique polypeptide primary structure.     

Mapping of functional regions of Clostridium perfringens type A enterotoxin.

Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE). Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding. CPE sequences required for at least four epitopes were also identified.     

Competitive erythroimmunoassay for detecting Clostridium perfringens type A enterotoxin in stool specimens

A competitive erythroimmunoassay (ERIA) is described for Clostridium perfringens enterotoxin (CPE) detection in stools. This technique uses sheep red blood cells sensitized by CPE and an anti-CPE-antibody-coated plate in which the results are read by eye. ERIA is simple, rapid, economic and more sensitive (2 ng/ml) than the enzyme-linked immunosorbent assay used for evaluation. ERIA is suitable for CPE detection in stool samples protected with phenylmethylsulphonylfluoride.     

Common polysaccharide antigens from the cell envelope of Clostridium perfringens type A.

Soluble antigens were obtained by extracting five serotype strains of Clostridium perfringens type A with water at 100 degrees C. The type-specific polysaccharides were precipitated with ethanol, and the common antigens were recovered from the ethanol supernatants by concentration, dialysis, and lyophilization. Refluxing the water-extracted cell residues with 1% acetic acid followed by concentration, dialysis, and lyophilization gave additional common antigen fractions. A comprehensive, side-by-side comparison of the antigen fractions, the ethanol precipitate, the ethanol supernatant, and the acetic acid supernatant, revealed that common antigens were recovered in all three fractions, and that three distinct entities were responsible for the formation of the observed common immunoprecipitin lines; whereas many fractions possessed all three immunoprecipitin lines, others contained only one or two. The serological homology observed between the various antigen fractions was apparently a consequence of N-acetylglucosamine- and N-acetylmannosamine-containing polymers. The common antigens were presumably associated with the cell envelope and may be the type of markers sought previously by others for the serological identification of C. perfringens.     

Clostridium perfringens type C causing necrotising enteritis.

A rapidly fatal case of enteritis necroticans in a 24 year old man with diabetes was caused by Clostridium perfringens type C. The role of beta toxin in the disease is discussed. This type has not been previously described as a causative agent in necrotising bowel disease of man outside endemic areas.     

Purification of beta-toxin from Clostridium perfringens type C.

Beta-toxin was purified about 340-fold from culture supernatant fluid of Clostridium perfringens type C with a yield of about 24% in terms of biologically active beta-toxin. The purification involved ammonium sulfate fractionation, gel filtration through Sephadex G-100, isoelectrofocusing in a pH 3 to 6 gradient, and immunoaffinity chromatography. The purified beta-toxin gave a single band on polyacrylamide gel electrophoresis.     

Interferon pretreatment enhances the sensitivity of Vero cells to Clostridium perfringens type A enterotoxin

Treatment of Vero (African green monkey kidney) cells with interferon (IFN) before the addition of Clostridium perfringens type A enterotoxin (CPE) significantly increased the sensitivity of these cells to CPE. IFN pretreatment caused a subsequent two- to four-fold increase in CPE-induced membrane permeability alterations and also decreased the time of CPE treatment required before the onset of permeability alterations and morphologic damage. Enhancement of CPE activity was dependent on the amount of IFN added during pretreatment and on the duration of IFN pretreatment incubations. Potentiation of CPE activity was observed following pretreatment of Vero cells with natural human IFN-alpha or IFN-gamma or Roferon recombinant human IFN-alpha. However, pretreatment with mouse IFN did not affect CPE activity. IFN pretreatment did not grossly enlarge the size of the functional hole produced in plasma membranes by CPE. IFN pretreatment of Vero cells slightly increased CPE specific binding, but this effect occurred kinetically after the enhancement of CPE toxicity. These results suggest that IFN pretreatment enhances the action of CPE on IFN pretreated Vero cells by increasing the sensitivity of these cells to the action of CPE rather than by increasing CPE specific binding or by directly activating the CPE molecule. Additional studies are required to further clarify the mechanism by which IFN sensitized Vero cells to CPE.     

Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens.

Clostridium ramosum, C. innocuum, and C. clostridioforme are frequently isolated from clinical specimens including blood. Because of Gram stain variability, a lack of spores, and atypical colonial morphology, identification of these species is often difficult. Three anaerobe identification kits were evaluated for their abilities to identify these species. For comparison, 11 strains of C. perfringens were evaluated in parallel. By using profile numbers and codebooks, the correct genus and species were identified, as follows: with the RapID ANA II kit, 100% (20 of 20) of C. ramosum isolates, 24% (5 of 21) of C. innocuum isolates, and 50% (10 of 20) of C. clostridioforme isolates; with the AnIDent kit, 60% (12 of 20) of C. ramosum isolates, 28% (6 of 21) of C. innocuum isolates, and 90% (18 of 20) of C. clostridioforme isolates; with the ATB32A kit, 70% (14 of 20) of C. ramosum isolates, 0% (0 of 21) of C. innocuum isolates, and 40% (8 of 20) of C. clostridioforme isolates. Profile numbers that overlapped several species were obtained as follows: with the RapID ANA II kit, 0% of C. ramosum isolates, 76% of C. innocuum isolates, and 40% of C. clostridioforme isolates; with the AnIDent kit 40% of C. ramosum isolates, 62% of C. innocuum isolates, and 5% of C. clostridioforme isolates; with the ATB32A kit, 15% of C. ramosum isolates, 52% of C. innocuum isolates, and 25% of C. clostridioforme isolates. One strain of C. innocuum was misidentified by the AnIDent kit, and the remainder yielded profile numbers that were not listed in the codebooks. The MICs of 11 antimicrobial agents including penicillin G, metronidazole, clindamycin, cefoxitin, cefotetan, imipenem, meropenem, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, and vancomycin were determined by the agar dilution method. All C. perfringens strains were susceptible to all antimicrobial agents tested. Various levels of resistance to cefoxitin, cefotetan, and penicillin G were noted with C. ramosum, C. clostridioforme, and C. innocuum. In addition, resistance to clindamycin was noted with C. ramosum (5%) and C. innocuum (10%). Most strains of C. innocuum were only moderately susceptible to vancomycin (MIC at which 90% of strains are inhibited, 4 micrograms/ml).