Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.Clostridium perfringens isolates are classified into five toxinotypes (A to E) based upon the production of four (α, β, ɛ, and ι) typing toxins (29). Each toxinotype is associated with different diseases affecting humans or animals (25). In livestock species, C. perfringens type C isolates cause fatal necrotizing enteritis and enterotoxemia, where toxins produced in the intestines absorb into the circulation to damage internal organs. Type C-mediated animal diseases result in serious economic losses for agriculture (25). In humans, type C isolates cause enteritis necroticans, which is also known as pigbel or Darmbrand (15, 17), an often fatal disease that involves vomiting, diarrhea, severe abdominal pain, intestinal necrosis, and bloody stools. Acute cases of pigbel, resulting in rapid death, may also involve enterotoxemia (15).By definition, type C isolates must produce alpha and beta toxins (24, 29). Alpha toxin, a 43-kDa protein encoded by the chromosomal plc gene, has phospholipase C, sphingomyelinase, and lethal properties (36). Beta toxin, a 35-kDa polypeptide, forms pores that lyse susceptible cells (28, 35). Recent studies demonstrated that beta toxin is necessary for both the necrotizing enteritis and lethal enterotoxemia caused by type C isolates (33, 37). Besides alpha and beta toxins, type C isolates also commonly express beta2 toxin, perfringolysin O, or enterotoxin (11).There is growing appreciation that naturally occurring plasmids contribute to both C. perfringens virulence and antibiotic resistance. For example, all typing toxins, except alpha toxin, can be encoded by genes carried on large plasmids (9, 19, 26, 30-32). Other C. perfringens toxins, such as the enterotoxin or beta2 toxin, can also be plasmid encoded (6, 8, 12, 34). Furthermore, conjugative transfer of several C. perfringens antibiotic resistance plasmids or toxin plasmids has been demonstrated, supporting a key role for plasmids in the dissemination of virulence or antibiotic resistance traits in this bacterium (2).Despite their pathogenic importance, the toxin-encoding plasmids of C. perfringens only recently came under intensive study (19, 26, 27, 31, 32). The first carefully analyzed C. perfringens toxin plasmids were two plasmid families carrying the enterotoxin gene (cpe) in type A isolates (6, 8, 12, 26). One of those cpe plasmid families, represented by the ∼75-kb prototype pCPF5603, has an IS1151 sequence present downstream of the cpe gene and also carries the cpb2 gene, encoding beta2 toxin. A second cpe plasmid family of type A isolates, represented by the ∼70-kb prototype pCPF4969, lacks the cpb2 gene and carries an IS1470-like sequence, rather than an IS1151 sequence, downstream of the cpe gene. The pCPF5603 and pCPF4969 plasmid families share an ∼35-kb region that includes transfer of a clostridial plasmid (tcp) locus (26). The presence of this tcp locus likely explains the demonstrated conjugative transfer of some cpe plasmids (5) since a similar tcp locus was shown to mediate conjugative transfer of the C. perfringens tetracycline resistance plasmid pCW3 (2).The iota toxin-encoding plasmids of type E isolates are typically larger (up to ∼135 kb) than cpe plasmids of type A isolates (19). Plasmids carrying iota toxin genes often encode other potential virulence factors, such as lambda toxin and urease, as well as a tcp locus (19). Many iota toxin plasmids of type E isolates share, sometimes extensively, sequences with cpe plasmids of type A isolates (19). It has been suggested that many iota toxin plasmids evolved from the insertion of a mobile genetic element carrying the iota toxin genes near the plasmid-borne cpe gene in a type A isolate, an effect that silenced the cpe gene in many type E isolates (3, 19).Plasmids carrying the epsilon toxin gene (etx) vary from ∼48 kb to ∼110 kb among type D isolates (32). In part, these etx plasmid size variations in type D isolates reflect differences in toxin gene carriage. For example, the small ∼48-kb etx plasmids present in some type D isolates lack both the cpe gene and the cpb2 gene. In contrast, larger etx plasmids present in other type D isolates often carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Thus, the virulence plasmid diversity of type D isolates spans from carriage of a single toxin plasmid, possessing from one to three distinct toxin genes, to carriage of three different toxin plasmids.In contrast to the variety of etx plasmids found among type D isolates, type B isolates often or always share the same ∼65-kb etx plasmid, which is related to pCPF5603 but lacks the cpe gene (27). This common etx plasmid of type B isolates, which carries a cpb2 gene and the tcp locus, is also present in a few type D isolates. Most type B isolates surveyed to date carry their cpb gene, encoding beta toxin, on an ∼90-kb plasmid, although a few of those type B isolates possess an ∼65-kb cpb plasmid distinct from their ∼65-kb etx plasmid (31).To our knowledge, the cpb gene has been mapped to a plasmid (uncharacterized) in only a single type C strain (16). Furthermore, except for the recent localization of the cpe gene to plasmids in type C strains (20), plasmid carriage of other potential toxin genes in type C isolates has not been investigated. Considering the limited information available regarding the toxin plasmids of type C isolates, our study sought to systematically characterize the size, diversity, and toxin gene carriage of toxin plasmids in a collection of type C isolates. Also, to gain insight into possible mobilization of the cpb gene by insertion sequences or conjugative transfer, the presence of IS1151 sequences or the tcp locus on type C toxin plasmids was investigated.     

Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates

Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.     

Clostridium perfringens Type A Enterotoxin (CPE): More Than Just Explosive Diarrhea

Abstract

The bacterial pathogen Clostridium perfringens is the most prolific toxin-producing species within the clostridial group. The toxins are responsible for a wide variety of human and veterinary diseases, many of which are lethal. C. perfringens type A strains are also associated with one of the most common forms of food-borne illness (FBI). The toxicosis results from the production and gastrointestinal absorption of a protein-enterotoxin known as CPE. The regulation, expression, and mechanism of action of CPE has been of considerable interest as the protein is unique. CPE expression is sporulation associated, although the mechanism of cpe-gsne regulation is not fully elucidated. Cloning studies suggest the involvement of global regulators, but these have not been identified. Although very few type A strains are naturally enterotoxigenic, the cpe gene appears highly conserved. In FBI strains, cpe is chromosomally encoded; whereas in veterinary strains, cpe may be plasmid-encoded. Variation in cpe location suggests the involvement of transposable genetic element(s). CPE-like proteins are produced by some C. perfringens types C and D; and silent remnants of the cpe gene can be found in C. perfringens type E strains associated with the iota toxin gene. CPE has received attention for its biomedical importance. The toxin has been implicated in sudden infant death syndrome (SIDS) because of its superantigenic nature. CPE can destroy a wide variety of cell types both in vitro and in vivo, suggesting that it could have potential in the construction of immunotoxins to neoplastic cells. It is obvious that CPE is an interesting protein that deserves continued attention.     

Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.     

Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

Clostridium perfringens type C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates are cpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores with D₁₀₀ values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomal cpe gene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both the cpe and cpb genes can be mobilized in Darmbrand isolates. These results suggest that C. perfringens type A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomal cpe gene.     

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.     

Evidence That the Enterotoxin Gene Can Be Episomal in Clostridium perfringens Isolates Associated with Non-Food-Borne Human Gastrointestinal Diseases

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C. perfringens isolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome. Collectively, these results provide the first evidence that distinct subpopulations of cpe-positive C. perfringens isolates may be responsible for C. perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations of cpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.     

Clostridium perfringens type A strains carrying a plasmid-borne enterotoxin gene (genotype IS1151-cpe or IS1470-like-cpe) as a common cause of food poisoning

The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). These results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning.     

Characterization of Virulence Plasmid Diversity among Clostridium perfringens Type B Isolates

The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to ∼90-kb plasmids in most type B isolates, although a few isolates carried a ∼65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located ∼3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.Isolates of the Gram-positive, spore-forming anaerobe Clostridium perfringens are classified (31) into five different types (A to E), depending upon their production of four (alpha, beta, epsilon, and iota) lethal typing toxins. All C. perfringens types produce alpha-toxin; in addition, type B isolates produce both beta- and epsilon-toxins, type C isolates produce beta-toxin, type D isolates produce epsilon-toxin and type E isolates produce iota-toxin. Except for the chromosomal alpha-toxin gene (plc), all C. perfringens typing toxins are encoded by genes resident on large plasmids (11, 22, 23, 32, 33). Large plasmids can also encode other C. perfringens toxins, such as the enterotoxin (CPE) or beta2-toxin (8, 9, 14, 35), as well as other potential virulence factors such as urease (12, 23).The large virulence plasmids of C. perfringens are only now being characterized (23, 28, 29, 33). The first analyzed, and still most studied, C. perfringens toxin plasmids are the CPE-encoding plasmids of type A isolates (14, 28). In type A isolates, most plasmids carrying the enterotoxin gene (cpe) belong to one of two families: (i) a 75.3-kb plasmid with a cpe locus containing an IS1151 element and the cpb2 gene encoding beta2-toxin or (ii) a 70.5-kb plasmid that lacks the cpb2 gene and carries a cpe locus with an IS1470-like sequence instead of an IS1151 element. Sequence comparisons (28) revealed that these two cpe plasmid families of type A isolates share a conserved region of ∼35 kb that includes the transfer of clostridial plasmid (tcp) locus, which is related to the conjugative transposon Tn916. Confirming that cpe plasmids can be conjugative, mixed mating studies have directly demonstrated transfer of the cpe plasmid from type A isolate F4969 to other C. perfringens isolates (5). A similar tcp locus is also shared by the tetracycline resistance plasmid pCW3 and several other toxin plasmids (2, 23, 28, 29, 33), as discussed below. Mutagenesis analyses demonstrated the importance of several genes in the tcp locus for conjugative transfer of pCW3 (2) and, by extension, presumably the tcp-carrying, conjugative toxin plasmids, such as the cpe plasmid of isolate F4969 (5) and some etx plasmids of type D isolates (19).Although the sequence of an iota-toxin-encoding plasmid has not yet been published, pulsed-field gel electrophoresis (PFGE) and PCR analyses determined that these plasmids are typically larger than the cpe plasmids of type A isolates (23). Specifically, iota-toxin plasmids are often ≥100 kb in size, reaching up to a size of ∼135 kb. These plasmids of type E isolates often encode, in addition to the iota-toxin, other potential virulence factors such as lambda-toxin and urease. These plasmids also carry a tcp locus, suggesting that they may be capable of conjugative transfer. Interestingly, many iota-toxin plasmids appear to be related, sometimes extensively, to the cpe plasmids of type A isolates. Consequently, it has been suggested (3, 23) that many iota-toxin plasmids arose from insertion of an iota-toxin gene-carrying mobile genetic element near the cpe gene on a tcp-carrying type A plasmid. This insertional event apparently inactivated the cpe gene, so most or all type E isolates now carry silent cpe genes (3, 23).The epsilon-toxin-encoding plasmids of type D isolates show considerable size variations (33), ranging from ∼48 kb to ∼110 kb. These size variations in type D etx plasmids reflect, in part, differences among their toxin gene carriage. The small 48-kb etx plasmids present in some type D isolates typically lack either the cpe gene or the cpb2 gene (encoding beta2-toxin), while the larger (>75-kb) etx plasmids found in other type D isolates can also carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Consequently, some type D isolates carry a toxin plasmid encoding only etx, other type D isolates carry a toxin plasmid with up to three different functional toxin genes (etx, cpb2, and cpe), and the remaining type D isolates carry their etx, cpe, and cpb2 genes on up to three distinct plasmids.C. perfringens type B isolates uniquely produce both beta- and epsilon-toxins, the two most lethal C. perfringens toxins (13). These bacteria are important pathogens of sheep but also cause disease in goats, calves, and foals (26). For unknown reasons, diseases caused by C. perfringens type B isolates apparently are restricted to certain geographic regions (24, 25, 26). C. perfringens type B enterotoxemias initiate when these bacteria proliferate in the gut, accompanied by toxin production. Those toxins initially affect the intestines but later are absorbed and act systemically. Studies from our group (13) showed that beta- and epsilon-toxins each contribute to lethality in a mouse model involving intravenous injection of type B culture supernatants.There has been characterization of only one type B virulence plasmid to date. Our recent study (29) showed that most, if not all, type B isolates carry a common etx plasmid of ∼65 kb that also possesses a tcp locus and a cpb2 gene, although not the cpb gene encoding beta-toxin. Interestingly, the type B etx plasmid is highly (80%) related to the ∼75-kb cpe- and cpb2-carrying plasmid found in some type A isolates (28). The ∼65-kb etx plasmid present in most, if not all, type B isolates is also carried by a minority of type D isolates (29).The absence of the cpb gene from their etx plasmids suggested that most type B isolates might carry additional virulence plasmids. Therefore, the current study was performed to better address virulence plasmid carriage and diversity among type B disease isolates.     

Prevalence of β2-Toxigenic Clostridium perfringens in Horses with Intestinal Disorders

The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the α-, β-, β2-, and -toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin gene cpb2. β2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenic C. perfringens. No β2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the β- and -toxins and enterotoxin. The high incidence of β2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that β2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis.     

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.     

Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease.     

Evidence that membrane rafts are not required for the action of Clostridium perfringens enterotoxin

The action of bacterial pore-forming toxins typically involves membrane rafts for binding, oligomerization, and/or cytotoxicity. Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with a unique, multistep mechanism of action that involves the formation of complexes containing tight junction proteins that include claudins and, sometimes, occludin. Using sucrose density gradient centrifugation, this study evaluated whether the CPE complexes reside in membrane rafts and what role raft microdomains play in complex formation and CPE-induced cytotoxicity. Western blot analysis revealed that the small CPE complex and the CPE hexamer 1 (CH-1) complex, which is sufficient for CPE-induced cytotoxicity, both localize outside of rafts. The CH-2 complex was also found mainly in nonraft fractions, although a small pool of raft-associated CH-2 complex that was sensitive to cholesterol depletion with methyl-β-cyclodextrin (MβCD) was detected. Pretreatment of Caco-2 cells with MβCD had no appreciable effect on CPE-induced cytotoxicity. Claudin-4 was localized to Triton X-100-soluble gradient fractions of control or CPE-treated Caco-2 cells, indicating a raft-independent association for this CPE receptor. In contrast, occludin was present in raft fractions of control Caco-2 cells. Treatment with either MβCD or CPE caused most occludin molecules to shift out of lipid rafts, possibly due (at least in part) to the association of occludin with the CH-2 complex. Collectively, these results suggest that CPE is a unique pore-forming toxin for which membrane rafts are not required for binding, oligomerization/pore formation, or cytotoxicity.     

Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias

Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.Clostridium perfringens, an anaerobic, spore-forming, gram-positive rod, is a pathogen of humans and domestic or wild animals (9). The virulence of C. perfringens is mostly due to toxin secretion, which varies from strain to strain (9). This variability allows classification of C. perfringens isolates into five types (A to E), depending upon their production of four typing toxins (9, 11, 12). All five types produce alpha toxin (CPA), type B and C isolates produce beta toxin (CPB), type B and D isolates produce epsilon toxin (ETX), and type E isolates produce iota toxin (20).C. perfringens type C isolates cause highly lethal diseases, mostly in newborn animals of many mammalian species (20). These diseases originate when type C isolates proliferate and produce toxins in the intestine. Although often involving intestinal damage, death in affected animals is thought to result primarily from a toxemia following absorption of toxins from the intestine into the circulation (17, 18). C. perfringens bacteremia is not usually observed in cases of type C infection (17). In humans, C. perfringens type C isolates cause enteritis necroticans (also known as Darmbrand or Pigbel) (13). Enteritis necroticans is a highly lethal and endemic disease throughout much of Southeast Asia, but particularly in Papua New Guinea, where this disease was the leading cause of mortality in children during the 1960s (3, 5). Less frequently, this disease also occurs in diabetic patients elsewhere (8).Type C isolates produce CPB, which is a 35-kDa protein that forms pores in the membranes of susceptible cells, leading to swelling and lysis (10, 15, 19). CPB is lethal for mice, with a calculated 50% lethal dose of 310 ng per kg when administered intravenously (i.v.) (14). In addition, CPB has been shown to produce acute intestinal necrosis when inoculated into ligated intestinal loops of rabbits, the effects of which were inhibited when the toxin was mixed with a CPB monoclonal antibody (MAb) before inoculation (21). We recently constructed a series of C. perfringens type C toxin null mutants, which demonstrated that CPB, but not perfringolysin O (PFO) or CPA, is necessary and sufficient for the type C isolate CN3685 to cause intestinal damage in a rabbit ileal loop model (16). However, it was notable that none of the rabbits challenged with this potent type C isolate died during the 6-h course of those ileal loop studies (16).Despite these recent advances, the systemic lethal effects of CPB or C. perfringens type C isolates remain poorly characterized. In part, this is due to the lack of a laboratory animal model that reproduces the lethality of natural C. perfringens type C enterotoxemia (16, 21). Mice have been used to study the lethal effects of i.v. administered C. perfringens type C vegetative culture supernatants or pure CPB (2). The mouse i.v. injection model is useful for studying the systemic lethal effects of CPB and indicates the sensitivity of this species to type C toxins. However, this model differs significantly from natural type C enterotoxemias in human and animals, where toxins are produced in the gastrointestinal tract, act locally, and are then absorbed into the circulation (20). We now present the development and application of infectious intragastric (i.g.) and intraduodenal (i.d.) challenge mouse models to investigate the lethal enterotoxemias induced by C. perfringens type C infections, including a virulence evaluation of C. perfringens type C toxin mutants.     

Toxinotyping of necrotic enteritis-producing and commensal isolates of Clostridium perfringens from chickens fed organic diets

The present study determined the effect of Clostridium perfringens isolates taken from necrotic enteritis (NE) outbreaks on organic farms in a NE virulence testing model. Thirteen strains were isolated in the course of the study. Six C. perfringens field isolates were taken from a naturally occurring NE outbreak on an organic farm. Polymerase chain reaction toxinotyping was used to establish C. perfringens strains, as well as to create a toxin profile. All field isolates were found to be type A and positive for alpha, beta-2 and netB toxin genes. During the NE virulence model, digesta samples were collected before oral inoculation to define the C. perfringens found as part of the natural flora. Three of the five natural flora isolates were found to be C. perfringens type E while the other two isolates were type A; only four of five isolates were positive for either netB or beta-2 toxin genes. Two isolates collected after inoculation were C. perfringens type A positive for cpb2 and netB. All isolates were tested positive for the quorum-sensing-related gene luxS, regardless of the strain source. The presence of luxS, alpha, netB and beta-2 toxin genes seems not to be a determinant of the disease as they were present in isolates from both outbreak birds as well as healthy and pre-inoculated birds. The C. perfringens field isolates induced mild NE lesions in one-half of the birds during the challenge study. Other mechanisms must play a role in the development of the disease beyond toxinotype, potentially including intestinal ecology and health, which would account for acute disease as seen in the field outbreak.     

K-1 Antigen Content and the Presence of an Additional Sialic Acid-Containing Antigen Among Bacteremic K-1 Escherichia coli: Correlation with Susceptibility to Opsonophagocytosis

Eighty percent of blood culture isolates of Escherichia coli K-1 are resistant to in vitro opsonophagocytosis by normal human granulocytes and fresh serum. To determine the basis for susceptibility to phagocytosis in 20% of bacteremic K-1 E. coli, we investigated possible quantitative and qualitative immunochemical differences in the K-1 antigen content among resistant and sensitive isolates. We prepared extracts of blood culture K-1 E. coli by sonication and determined the K-1 polysaccharide content per dry weight of bacteria by rocket immunoelectrophoresis using cross-reactive equine anti-group B meningococcal sera. We assessed qualitative differences in the antigen content by crossed immunoelectrophoresis, using an immune globulin fraction and isolated immunoglobulin G (IgG) and IgM from the group B antisera. Three different resistant K-1 isolates contained a mean K-1 content of 48.5 ± 7.6 μg/mg ± standard deviation of dry bacteria, and three sensitive isolates contained 23.2 ± 5.6 μg/mg (P < 0.005). Crossed immunoelectrophoresis of extracts from both sensitive and resistant strains revealed a secondary sialic acid-containing antigen that was electrophoretically different from both the major K-1 antigen and a reference group B meningococcal antigen. This negatively charged secondary antigen was susceptible to Clostridium perfringens neuraminidase degradation and reacted only with IgG whereas the major K-1 antigen reacted only with IgM. This antigen was detected in the extracts of resistant isolates only at 10¹⁰ but not at 10⁹ colony-forming units per milliliter. This study demonstrates that (i) the degree of phagocytosis of bacteremic E. coli K-1 isolates is inversely associated with K-1 content, and (ii) more easily phagocytosed (sensitive) K-1 isolates have greater amounts of an additional sialic acid-containing antigen that appears to be unrelated to the previously described O acetyl K-1 antigen.     

Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) gene interactions with the apolipoprotein E epsilon4 allele as risk factors in Alzheimer's disease and in Parkinson's disease with coexisting Alzheimer pathology

Alzheimer's disease (AD) and Parkinson's disease (PD) are genetically heterogeneous. Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) genes may modify the risk of these disorders. We investigated whether common polymorphisms present in these genes operate as risk factors for AD and PD in Finnish subjects, independently or in concert with the apolipoprotein E ε4 allele (APOE ε4). Eighty late onset sporadic AD patients, 53 PD patients (34 of whom had concomitant AD pathology), and 67 control subjects were genotyped for the insertion (I)/deletion (D) polymorphism of DCP1 and the K variant of BCHE. In logistic regression analysis, the DCP1 *I allele in combination with APOE ε4 significantly increased the risk of AD (OR 30.0, 95% CI 7.3-123.7), compared to subjects carrying neither of the alleles. Similar analysis showed that the risk of AD was significantly increased in subjects carrying both the BCHE wild type (*WT/*WT) genotype and ε4 (OR 9.9, 95% CI 2.9-33.8), compared to those without this BCHE genotype and ε4. Further, the risk of PD with AD pathology was significantly increased for carriers of DCP1 *I and ε4 (OR 8.0, 95% CI 2.1-31.1). We thus conclude that, in Finns, interaction between DCP1 *I and ε4 increases the risk of AD as well as of PD with coexisting Alzheimer pathology, which underlines the importance of the DCP1 I/D polymorphism in the development of Alzheimer neuropathology, whereas the wild type BCHE genotype in combination with ε4 had a combined effect with regard to the risk of AD.


Keywords: Alzheimer's disease; Parkinson's disease; dipeptidyl carboxypeptidase 1; butyrylcholinesterase     

Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon

Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.     

Can Results Obtained with Commercially Available MicroScan Microdilution Panels Serve as an Indicator of β-Lactamase Production among Escherichia coli and Klebsiella Isolates with Hidden Resistance to ExpandedSpectrum Cephalosporins and Aztreonam?

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of β-lactamases that may confer resistance to newer β-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized β-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of β-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum β-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of ≥2 μg/ml were obtained only for isolates producing ESBLs or AmpC β-lactamases. Cefoxitin MICs of >16 μg/ml were obtained for all strains producing AmpC β-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of ≥4 μg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of ≥ 8 μg/ml were obtained for seven of eight producers of AmpC β-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain β-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.