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.     

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.     

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 E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type A C. perfringens isolates.     

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.     

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.     

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.     

Characterization of IS1245 for Strain Typing of Mycobacterium avium

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.     

Association of IS1016 with the hia adhesin gene and biotypes V and I in invasive nontypeable Haemophilus influenzae

A subset of invasive nontypeable Haemophilus influenzae (NTHI) strains has evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. We examined IS1016-positive invasive NTHI isolates collected as part of Active Bacterial Core Surveillance within the Georgia Emerging Infections Program for the presence or absence of hmw1 and hmw2 (two related adhesin genes that are common in NTHI but absent in encapsulated H. influenzae) and hia (homologue of hsf, an encapsulated H. influenzae adhesin gene). Isolates were serotyped using slide agglutination, confirmed as NTHI strains using PCR capsule typing, and biotyped. Two hundred twenty-nine invasive NTHI isolates collected between August 1998 and December 2006 were screened for IS1016; 22/229 (9.6%) were positive. Nineteen of 201 previously identified IS1016-positive invasive NTHI isolates collected between January 1989 and July 1998 were also examined. Forty-one IS1016-positive and 56 randomly selected IS1016-negative invasive NTHI strains were examined. The hia adhesin was present in 39 of 41 (95%) IS1016-positive NTHI strains and 1 of 56 (1.8%) IS1016-negative NTHI strains tested; hmw (hmw1, hmw2, or both) was present in 50 of 56 (89%) IS1016-negative NTHI isolates but in only 5 of 41 (12%; all hmw2) IS1016-positive NTHI isolates. IS1016-positive NTHI strains were more often biotype V (P < 0.001) or biotype I (P = 0.04) than IS1016-negative NTHI strains, which were most often biotype II. Pulsed-field gel electrophoresis revealed the expected genetic diversity of NTHI with some clustering based on IS1016, hmw or hia, and biotypes. A significant association of IS1016 with biotypes V and I and the presence of hia adhesins was found among invasive NTHI. IS1016-positive NTHI strains may represent a unique subset of NTHI strains, with characteristics more closely resembling those of encapsulated H. influenzae.     

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.     

Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.     

Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.     

Development and preliminary evaluation of a slide latex agglutination assay for detection of Clostridium perfringens type A enterotoxin

A slide latex agglutination (SLA) assay was developed for rapid screening for Clostridium perfringens type A enterotoxin (CPE). SLA specifically detected CPE added to buffer or normal feces (sensitivity limit of 1 μg CPE/g feces). Using clinical fecal samples from C. perfringens food poisoning cases, a strong correlation was shown between (1) SLA results and results from other CPE assays and (2) between SLA results and illness status.     

Stability of Mycobacterium tuberculosis IS6110 Restriction Fragment Length Polymorphism Patterns and Spoligotypes Determined by Analyzing Serial Isolates from Patients with Drug-Resistant Tuberculosis

The stability of Mycobacterium tuberculosis IS6110 fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165 M. tuberculosis isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS6110 fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS6110 restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS6110 patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS6110 fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS6110 fingerprint patterns of M. tuberculosis isolates have high degrees of stability. Compared to IS6110, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.     

Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response.Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals; the former include gas gangrene, food poisoning, necrotizing enterocolitis of infants, and enteritis necroticans (28, 37, 45). C. perfringens is an obligately anaerobic rod-shaped bacterium commonly found in the gastrointestinal tracts of both animals and humans and widely distributed in soil and sewage. The ability of C. perfringens to cause disease is associated with the production of a variety of extracellular toxins (13 different toxins have been reported so far). On the basis of differential production of toxins, the strains of C. perfringens can be divided into five types, A through E (35), of which type A and type C strains are implicated in human diseases while other types are of veterinary importance. Type A strains cause gas gangrene, the most destructive of all clostridial diseases, which is characterized by rapid destruction of tissue with production of gas (4, 42). The incidence of disease ranged from 1% or less of wounded personnel during World War II to 10% of wounded personnel during World War I (27). Hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and C. perfringens was widely recognized as the most important causal organism of the disease. Besides gas gangrene, type A strains also cause gastrointestinal diseases in humans (food poisoning, antibiotic-associated diarrhea, sporadic diarrhea, sudden infant death syndrome) and animals (diarrhea in foals and pigs, etc.). C. perfringens type C strains cause necrotic enteritis in humans and animals, in addition to enterotoxemia in sheep. Moreover, C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; therefore, efforts to develop strategies for detection and protection are warranted.Interest in a vaccine against gas gangrene has been intermittent; most efforts were made during World Wars I and II and were devoted to the therapeutic use of antisera. Such antisera, raised against toxoids of all five species of clostridia associated with gas gangrene, were shown to have benefits if they were given soon after trauma (20). Active immunization against the disease has received little attention until a few years ago (32, 43, 44). A number of clinical studies of other pathogenic bacteria, including Clostridium difficile, have highlighted the importance of nontoxin protein antigens in disease expression, especially in colonization by the bacterium (9, 12, 16, 26).Despite a sudden spurt of activity in the proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have been largely ignored. Clostridium difficile is the only clostridial species whose proteome has been analyzed to some extent (34). Proteomic methodology has been used to elucidate proteins regulated by the VirR/VirS system in Clostridium perfringens (40).To invade, multiply in, and colonize host tissues, a pathogen must be able to evade the host immune system and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance for the understanding of microbial pathogenesis. The exoproteins of Gram-positive bacteria are likely to contain some of these key factors. The term “secretome” refers to and takes into account both the protein secretion systems and the secreted proteins; in monoderm bacteria (Gram-positive cell envelope architecture), these proteins can also be found in the membrane and/or cell wall. The proteins found in the extracellular milieu of Gram-positive bacteria are hence extracellular proteins, or exoproteins, which form the exoproteome; these exoproteins are not necessarily secreted by known secretion systems (15). This terminology is used for the subproteome described in this study.Further, the correct identification of C. perfringens pathovars is critical for epidemiological studies and for the development of effective preventive measures, including vaccination. It is not well understood why C. perfringens produces illnesses that differ so greatly in severity. Specific illnesses may reflect the particular type of tissue that high numbers of the organism are able to invade. Clearly, the presence of certain toxins is one explanation, but it falls short in many instances. For example, strains that cause food poisoning may differ from those that cause gas gangrene only by the presence of an enterotoxin gene in the former, yet food-poisoning strains have never been found to cause gas gangrene. Elucidation of the exoproteins of this bacterium is likely to reveal factors responsible for the host specificity of the different C. perfringens pathovars.The present investigation was carried out with the following objectives: (i) identification of dominant exoproteins from the C. perfringens type A strain ATCC 13124 (a gas gangrene isolate and the species type strain) and comparison with those of type C strains, (ii) elucidation of immunogenic components of the exoproteome, (iii) in silico analysis of the identified proteins with respect to localization, predicted function, and likely potential as surface markers for detection and as vaccine candidates, and (iv) validation of selected candidates with respect to vaccine potential in an experimental mouse model of gas gangrene. To the best of our knowledge, ours is the first proteomic elucidation of the C. perfringens exoproteome. The identification of extracellular proteins of C. perfringens type A and type C strains in the present investigation advances our understanding of the pathogenesis of the disease and opens significant opportunities for the identification of diagnostic markers and the development of vaccines against gas gangrene in humans.     

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C_T of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C_T values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C_T values of 30 ≤ C_T <40.     

High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.     

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.     

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.