scholarly journals Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates

2002 ◽  
Vol 70 (8) ◽  
pp. 4261-4272 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Ganes Chakrabarti ◽  
Yosiharu Morino ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Horizontal Transfer ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Open Reading Frame ◽  
Enterotoxin Gene ◽  
Genetic Element ◽  
Reading Frame ◽  
Food Borne

ABSTRACT 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.

scholarly journals Inorganic Phosphate and Sodium Ions Are Cogerminants for Spores of Clostridium perfringens Type A Food Poisoning-Related Isolates

2009 ◽  
Vol 75 (19) ◽  
pp. 6299-6305 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Pathima Udompijitkul ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Inorganic Phosphate ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Wild Type ◽  
Similar Extent ◽  
Active Growth ◽  
Food Borne

ABSTRACT Clostridium perfringens type A isolates carrying a chromosomal copy of the enterotoxin (cpe) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne cpe gene are involved in C. perfringens-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases, C. perfringens spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K+ ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na+ and inorganic phosphate (NaPi) at a pH of ∼6.0. Spores of gerKA-KC and gerAA mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaPi. Although gerKB spores germinated to a similar extent as wild-type spores with NaPi, their rate of germination was lower. Similarly, gerO and gerO gerQ mutant spores germinated slower and released less DPA than did wild-type spores with NaPi. In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaPi germination. In sum, this study identified NaPi as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the gerKA-KC operon, gerAA, and gerO are required for NaPi-induced spore germination.

scholarly journals Complete Sequencing and Diversity Analysis of the Enterotoxin-Encoding Plasmids in Clostridium perfringens Type A Non-Food-Borne Human Gastrointestinal Disease Isolates

2006 ◽  
Vol 188 (4) ◽  
pp. 1585-1598 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Derek J. Fisher ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Gastrointestinal Disease ◽  
Food Poisoning ◽  
Variable Region ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Virulence Plasmids ◽  
Food Borne

ABSTRACT Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an ∼35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the ∼43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe +/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe +/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.

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

1998 ◽  
Vol 36 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Renee E. Collie ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Pulsed Field Gel Electrophoresis ◽  
Transmission Mechanisms ◽  
Food Borne ◽  
Antibiotic Associated Diarrhea

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. perfringensisolates 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 ofcpe-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 ofcpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.

scholarly journals Prevalence of Enterotoxigenic Clostridium perfringens Isolates in Pittsburgh (Pennsylvania) Area Soils and Home Kitchens

2007 ◽  
Vol 73 (22) ◽  
pp. 7218-7224 ◽  
Author(s):  
Jihong Li ◽  
Sameera Sayeed ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
The United States ◽  
Soil Samples ◽  
Promoter Regions ◽  
Food Borne ◽  
Upstream Promoter ◽  
Type C

ABSTRACT In the United States and Europe, food poisoning due to Clostridium perfringens type A is predominantly caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe). Neither the reservoir for these isolates nor the point in the food chain where these bacteria contaminate foods is currently understood. Therefore, the current study investigated whether type A isolates carrying a chromosomal cpe gene are present in two potential reservoirs, i.e., soil and home kitchen surfaces. No C. perfringens isolates were recovered from home kitchen surfaces, but most surveyed soil samples contained C. perfringens. The recovered soil isolates were predominantly type A, but some type C, D, and E soil isolates were also identified. All cpe-positive isolates recovered from soil were genotyped as type A, with their cpe genes on cpe plasmids rather than the chromosome. However, two cpe-positive soil isolates did not carry a classical cpe plasmid. Both of those atypical cpe-positive soil isolates were sporulation capable yet failed to produce C. perfringens enterotoxin, possibly because of differences in their upstream promoter regions. Collectively these results suggest that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning, although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases.

scholarly journals Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes

2000 ◽  
Vol 66 (8) ◽  
pp. 3234-3240 ◽  
Author(s):  
Mahfuzur R. Sarker ◽  
Robert P. Shivers ◽  
Shauna G. Sparks ◽  
Vijay K. Juneja ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Gastrointestinal Disease ◽  
Food Poisoning ◽  
Meat Products ◽  
Gastrointestinal Diseases ◽  
Vegetative Cells ◽  
Food Borne ◽  
Enterotoxin Genes ◽  
Plasmid Genes ◽  
High Degree

ABSTRACT Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringensisolates associated with food poisoning carry a chromosomalcpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomalcpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpeisolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (Dvalues) at 55°C than vegetative cells of plasmid cpeisolates exhibited. Furthermore, the spores of chromosomalcpe isolates had, on average, approximately 60-fold-higherD values at 100°C than the spores of plasmidcpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained theircpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomalcpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.

scholarly journals Clostridium perfringens Type E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences

1998 ◽  
Vol 66 (9) ◽  
pp. 4531-4536 ◽  
Author(s):  
Stephen J. Billington ◽  
Eva U. Wieckowski ◽  
Mahfuzur R. Sarker ◽  
Dawn Bueschel ◽  
J. Glenn Songer ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Horizontal Transfer ◽  
Type A ◽  
Enterotoxin Gene ◽  
Ribosome Binding ◽  
Toxin Genes ◽  
Neonatal Calves ◽  
Hemorrhagic Enteritis ◽  
Episomal Dna ◽  
Genotype E

ABSTRACT 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 functionalcpe gene of type A isolates, these silent type Ecpe 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.

scholarly journals A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a ChromosomalcpeGene Belong to One Multilocus Sequence Typing Cluster

2012 ◽  
Vol 78 (19) ◽  
pp. 7060-7068 ◽  
Author(s):  
Yinghua Xiao ◽  
Arjen Wagendorp ◽  
Roy Moezelaar ◽  
Tjakko Abee ◽  
Marjon H. J. Wells-Bennik
Keyword(s):  
Clostridium Perfringens ◽  
Multilocus Sequence Typing ◽  
Food Poisoning ◽  
Distinct Group ◽  
Nationwide Survey ◽  
Type A ◽  
Evolutionary Significance ◽  
Pcr Analysis ◽  
Content Type ◽  
Food Borne

ABSTRACTOf 98 suspected food-borneClostridium perfringensisolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified asC. perfringenstype A. Using PCR-based techniques, thecpegene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)5fingerprint repetitive sequence-based PCR analysis distinguishedC. perfringensfrom other sulfite-reducing clostridia but did not allow for differentiation between various types ofC. perfringensstrains. To characterize theC. perfringensstrains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpeisolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomalcpestrains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the othercpeplasmid-carrying andcpe-negative strains. These results suggest that different groups ofC. perfringenshave undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxicC. perfringensmay allow for improved control of this organism in foods.

scholarly journals Molecular Characterization of Clostridium perfringens Isolates from Humans with Sporadic Diarrhea: Evidence for Transcriptional Regulation of the Beta2-Toxin-Encoding Gene

2005 ◽  
Vol 71 (12) ◽  
pp. 8362-8370 ◽  
Author(s):  
Ben Harrison ◽  
Deepa Raju ◽  
Helen S. Garmory ◽  
Moira M. Brett ◽  
Richard W. Titball ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Toxin Gene ◽  
Food Borne ◽  
Antibiotic Associated Diarrhea ◽  
Encoding Gene ◽  
Beta2 Toxin

ABSTRACT Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe + C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe + type A, and 12 of the 14 cpe + isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe + SD isolates. However, only 10 of the 12 cpe +/cpb2 + SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD.

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