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

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.

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

Journal of Bacteriology ◽

10.1128/jb.188.4.1585-1598.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1585-1598 ◽

Cited By ~ 63

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.

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

Infection and Immunity ◽

10.1128/iai.70.8.4261-4272.2002 ◽

2002 ◽

Vol 70 (8) ◽

pp. 4261-4272 ◽

Cited By ~ 51

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.

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Comparative Effects of Osmotic, Sodium Nitrite-Induced, and pH-Induced Stress on Growth and Survival of Clostridium perfringens Type A Isolates Carrying Chromosomal or Plasmid-Borne Enterotoxin Genes

Applied and Environmental Microbiology ◽

10.1128/aem.01911-06 ◽

2006 ◽

Vol 72 (12) ◽

pp. 7620-7625 ◽

Cited By ~ 48

Author(s):

Jihong Li ◽

Bruce A. McClane

Keyword(s):

Clostridium Perfringens ◽

Food Poisoning ◽

Type A ◽

Food Preservation ◽

Vegetative Cells ◽

Growth And Survival ◽

Resistance Phenotype ◽

Enhanced Resistance ◽

Enterotoxin Genes ◽

High And Low Temperatures

ABSTRACT About 1 to 2% of Clostridium perfringens isolates carry the enterotoxin gene (cpe) necessary for causing C. perfringens type A food poisoning. While the cpe gene can be either chromosomal or plasmid borne, food poisoning isolates usually carry a chromosomal cpe gene. Previous studies have linked this association between chromosomal cpe isolates (i.e., C-cpe isolates) and food poisoning, at least in part, to both the spores and vegetative cells of C-cpe isolates being particularly resistant to high and low temperatures. The current study now reveals that the resistance phenotype of C-cpe isolates extends beyond temperature resistance to also include, for both vegetative cells and spores, enhanced resistance to osmotic stress (from NaCl) and nitrites. However, by omitting one outlier isolate, no significant differences in pH sensitivity were detected between the spores or vegetative cells of C-cpe isolates versus isolates carrying a plasmid-borne cpe gene. These results indicate that both vegetative cells and spores of C-cpe isolates are unusually resistant to several food preservation approaches in addition to temperature extremes. The broad-spectrum nature of the C-cpe resistance phenotype suggests these bacteria may employ multiple mechanisms to persist and grow in foods prior to their transmission to humans.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00822-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6299-6305 ◽

Cited By ~ 24

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.

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Enterotoxin Plasmid from Clostridium perfringens Is Conjugative

Infection and Immunity ◽

10.1128/iai.69.5.3483-3487.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3483-3487 ◽

Cited By ~ 78

Author(s):

Sigrid Brynestad ◽

Mahfuzur R. Sarker ◽

Bruce A. McClane ◽

Per Einar Granum ◽

Julian I. Rood

Keyword(s):

Clostridium Perfringens ◽

Gastrointestinal Disease ◽

Intestinal Flora ◽

Food Poisoning ◽

Donor Cell ◽

Cell Contact ◽

Conjugative Transfer ◽

Chloramphenicol Resistance ◽

Food Borne

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

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

Journal of Clinical Microbiology ◽

10.1128/jcm.36.1.30-36.1998 ◽

1998 ◽

Vol 36 (1) ◽

pp. 30-36 ◽

Cited By ~ 116

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.

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Prevalence and Characterization of Enterotoxin Gene-Carrying Clostridium perfringens Isolates from Retail Meat Products in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.00783-08 ◽

2008 ◽

Vol 74 (17) ◽

pp. 5366-5372 ◽

Cited By ~ 42

Author(s):

Yasuhiro Miki ◽

Kazuaki Miyamoto ◽

Ikuko Kaneko-Hirano ◽

Kanako Fujiuchi ◽

Shigeru Akimoto

Keyword(s):

Clostridium Perfringens ◽

Food Poisoning ◽

Meat Products ◽

Disease Outbreaks ◽

Enterotoxin Gene ◽

Food Borne ◽

Causative Agents ◽

Retail Meat ◽

Meat Samples

ABSTRACT Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. It is thought that C. perfringens food poisoning isolates typically carry the enterotoxin gene (cpe) on their chromosome, while isolates from other GI diseases, such as antibiotic-associated diarrhea, carry cpe on a transferable plasmid. However, food-borne GI disease outbreaks associated with C. perfringens isolates carrying plasmid-borne cpe (plasmid cpe isolates) were recently reported in Japan and Europe. To investigate whether retail food can be a reservoir for food poisoning generally, we evaluated Japanese retail meat products for the presence of two genotypes of enterotoxigenic C. perfringens. Our results demonstrated that approximately 70% of the Japanese retail raw meat samples tested were contaminated with low numbers of C. perfringens bacteria and 4% were contaminated with cpe-positive C. perfringens. Most of the cpe-positive C. perfringens isolates obtained from Japanese retail meat carried cpe on a plasmid. The plasmid cpe isolates exhibited lower spore heat resistance than did chromosomal cpe isolates. Collectively, these plasmid cpe isolates might be causative agents of food poisoning when foods are contaminated with these isolates from equipment and/or the environment after cooking, or they may survive in food that has not been cooked at a high enough temperature.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01075-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7218-7224 ◽

Cited By ~ 41

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.

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Prevalence and Characterization of Antibiotic-Resistant Staphylococcus aureus Recovered from Pasteurized Cheese Commercialized in Panama City Markets

Journal of Food Quality ◽

10.1155/2021/9923855 ◽

2021 ◽

Vol 2021 ◽

pp. 1-7

Author(s):

Fermín Mejía ◽

Nohelia Castro-del Campo ◽

Arleny García ◽

Katerine Rodríguez ◽

Humberto Cornejo ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Food Poisoning ◽

Microbiological Quality ◽

Gastrointestinal Diseases ◽

Antibiotic Resistant ◽

Panama City ◽

Antibiotic Resistance Profile ◽

White Cheese ◽

High Degree

Foodborne bacteria, with a high degree of antibiotic resistance, play an important role in the morbidity and mortality of gastrointestinal diseases worldwide. Among 250 disease-causing bacteria, Staphylococcus aureus is one of the major causes of food poisoning, and its resistance to multiple antimicrobials remains of crucial concern. Cheese is often contaminated when proper sanitary procedures are not followed during its production and marketing. This work aimed to evaluate the microbiological quality of pasteurized white cheese commercialized in Panama City. Cheese from five different brands sold in local supermarkets were selected to determine the presence of S. aureus as well as its antibiotic resistance profile. The results showed significant contamination of S. aureus with a geometric median sample of 104–107 CFU/g. Four out of five (4/5) cheese brands analyzed presented risk of food poisoning by exceeding the allowed range of consumption with a geometric median sample of 1,8 × 106–1,4 × 107 CFU/g. Fourteen different resistance phenotypes were found. Fifty-five percent (55%) of the analyzed strains were resistant to erythromycin. The data confirm a relatively high prevalence and high levels of S. aureus, most likely originated during handling in Panama City retail markets. Further studies are needed to reduce bacterial contamination and to decrease the risk of food poisoning in the consumption of pasteurized cheese.

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Growth and Sporulation Potential of Clostridium perfringens in Aerobic and Vacuum-Packaged Cooked Beef

Journal of Food Protection ◽

10.4315/0362-028x-57.5.393 ◽

1994 ◽

Vol 57 (5) ◽

pp. 393-398 ◽

Cited By ~ 32

Author(s):

V. K. JUNEJA ◽

B. S. MARMER ◽

A. J. MILLER

Keyword(s):

Clostridium Perfringens ◽

Food Poisoning ◽

Ground Beef ◽

Viable Count ◽

Anaerobic Conditions ◽

Vegetative Cells ◽

Aerobic Conditions ◽

C Storage ◽

Meat Samples ◽

Aerobic And Anaerobic

Growth of Clostridium perfringens in aerobic-and anaerobic-(vacuum) packaged cooked ground beef was investigated. Autoclaved ground beef was inoculated with ~3.0-log10 CFU/g of C. perfringens, packaged and stored at various temperatures. Vegetative cells and heat-resistant spores were enumerated by plating unheated and heated (75°C for 20 min) meat samples on tryptose-sulfite-cycloserine agar. Clostridium perfringens grew to >7 logs within 12 h at 28, 37 and 42°C under anaerobic atmosphere and at 37 and 42°C under aerobic conditions. At 28°C under aerobic conditions, growth was relatively slow and total viable count increased to >6 logs within 36 h. Similarly, growth at 15°C in air was both slower and less than under vacuum. Regardless of packaging, the organism either declined or did not grow at 4, 8 and 12°C. Spores were not found at <12°C. Spores were detected as early as 8 h at 42°C under anaerobic conditions, but in general, the type of atmosphere had little influence on sporulation at ≥28°C. Temperature abuse (28°C storage) of refrigerated products for 6 h will not permit C. perfringens growth. However, cyclic and static temperature abuse of such products for relatively long periods may lead to high and dangerous numbers of organisms. Reheating such products to an internal temperature of 65°C before consumption would prevent food poisoning since the vegetative cells were killed.

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