Further Characterization of Clostridium perfringens Small Acid Soluble Protein-4 (Ssp4) Properties and Expression

Background

Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36.

Methodology/Principal Findings

The current study has further characterized Ssp4 properties and expression. Spores made by cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore''s DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator.

Conclusions/Significance

Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to C. perfringens type A food poisoning transmission.     

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge

Background  

Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated.     

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

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.     

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

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. perfringens isolates associated with food poisoning carry a chromosomal cpe 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 chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (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 (D values) at 55°C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100°C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe 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.     

Further Comparison of Temperature Effects on Growth and Survival of Clostridium perfringens Type A Isolates Carrying a Chromosomal or Plasmid-Borne Enterotoxin Gene

Clostridium perfringens type A isolates can carry the enterotoxin gene (cpe) on either their chromosome or a plasmid, but food poisoning isolates usually have a chromosomal cpe gene. This linkage between chromosomal cpe isolates and food poisoning has previously been attributed, at least in part, to better high-temperature survival of chromosomal cpe isolates than of plasmid cpe isolates. In the current study we assessed whether vegetative cells and spores of chromosomal cpe isolates also survive better than vegetative cells and spores of plasmid cpe isolates survive when the vegetative cells and spores are subjected to low temperatures. Vegetative cells of chromosomal cpe isolates exhibited about eightfold-higher decimal reduction values (D values) at 4°C and threefold-higher D values at −20°C than vegetative cells of plasmid cpe isolates exhibited. After 6 months of incubation at 4°C and −20°C, the average log reductions in viability for spores of plasmid cpe isolates were about fourfold and about threefold greater, respectively, than the average log reductions in viability for spores from chromosomal cpe isolates. C. perfringens type A isolates carrying a chromosomal cpe gene also grew significantly faster than plasmid cpe isolates grew at 25°C, 37°C, or 43°C. In addition, chromosomal cpe isolates grew at higher maximum and lower minimum temperatures than plasmid cpe isolates grew. Collectively, these results suggest that chromosomal cpe isolates are commonly involved in food poisoning because of their greater resistance to low (as well as high) temperatures for both survival and growth. They also indicate the importance of proper low-temperature storage conditions, as well as heating, for prevention of C. perfringens type A food poisoning.     

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

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.     

The enteric toxins of Clostridium perfringens

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (~25–35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, -toxin (a bioterrorism select agent) and -toxin, cause veterinary enterotoxemias when absorbed from the intestines; - and -toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, 2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

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Occurrence of Clostridium perfringens from different cultivated soils

The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole.When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality.     

Detection and typing of Clostridium perfringens from retail chicken meat parts

The objectives of the present work were to investigate the presence of Clostridium perfringens in chicken meat parts (breast, wing, drumstick and leg quarter) by culture methods and to detect the cpa, cpb, etx, iA, cpe and cpb2 toxin genes by multiplex PCR. A total of 200 samples, the raw chicken breasts (n: 50), wings (n: 50), drumsticks (n: 50) and leg quarters (n: 50), were collected from various retail stores. Our results demonstrated that 47 of 50 wing samples (94%), 40 of 50 leg quarter samples (80%), 34 of 50 drumstick samples (66%) and 33 of 50 breast samples (66%) were found to be contaminated with Cl. perfringens. 558 positive isolates obtained from these samples were identified as Cl. perfringens based on the microscopic examination and biochemical tests. It was detected that 545 (97·6%) of 558 Cl. perfringens isolates carried only cpa toxin gene (type A), 12 (2·1%) of them carried both cpa and cpb2 toxin gene (type A‐cpb2), one (0·1%) of them carried both cpa and cpe toxin genes (type A‐cpe), according to the multiplex PCR results, targeted cpa, cpb, cpb2, cpe, etx and iA genes.

Significance and Impact of the Study

This study is the first report of detection of cpe and cpb2 toxin genes in Clostridium perfringens isolated from chicken meats in Turkey. The multiplex PCR protocol described in this study is useful for rapid detection of Clostridium perfringens toxin genes simultaneously in one‐step PCR.     

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments

Aims: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. Methods: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species‐specific PCR, along with full‐length sequencing of the 16S rRNA gene for a subset of isolates. Results: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species‐specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. Conclusions: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99·5% similarity). Significance and Impact of the Study: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.     

Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Background

Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.

Methodology/Principal Findings

An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.

Conclusions/Significance

The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.     

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

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 (NaP_i) 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 NaP_i. Although gerKB spores germinated to a similar extent as wild-type spores with NaP_i, 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 NaP_i. In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaP_i germination. In sum, this study identified NaP_i 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 NaP_i-induced spore germination.Clostridium perfringens is a gram-positive, anaerobic, spore-forming, pathogenic bacterium that causes a wide array of gastrointestinal (GI) diseases in both animals and humans (14, 15). However, Clostridium perfringens type A food poisoning (FP) is the most common C. perfringens-associated illness among humans and is currently ranked as the third most commonly reported food-borne disease (14). Mostly type A isolates that produce the C. perfringens enterotoxin have been associated with C. perfringens-related GI illnesses (14). C. perfringens cpe-positive isolates can carry the cpe gene on either the chromosome or a plasmid (3, 4). Interestingly, the majority of C. perfringens type A FP isolates carry a chromosomal copy of the cpe gene, while all non-food-borne (NFB) GI disease isolates carry a plasmid copy of cpe (3, 4, 11, 29). The genetic differences involved in the pathogenesis differences between C. perfringens FP and NFB isolates seem to involve more factors than the simple location of the cpe gene. For example, spores of FP isolates are strikingly more resistant than spores of NFB isolates to heat (100°C) (27), cold (4°C), and freezing (−20°C) temperatures (12) and to chemicals used in food industry settings (13), making FP spores more suited for FP environments. Under favorable environmental conditions, these dormant spores germinate to return to active growth, proliferate to high numbers, and then produce toxins to cause disease (14).Bacterial spores germinate when they sense the presence of nutrients (termed germinants) in the environment through their cognate receptors located in the spore inner membrane (18). For C. perfringens, some nutrients that initiate germination include l-asparagine, KCl, a mixture of l-asparagine and KCl, and a 1:1 chelate of Ca²⁺ and dipicolinic acid (DPA) (Ca-DPA) (20). The main receptor(s) involved in sensing these compounds is the GerKA and/or GerKC receptor(s), which is required for l-asparagine and Ca-DPA and only partially required for KCl and an l-asparagine-KCl mixture (20, 21). Upon binding of the germinant to its cognate receptor, a variety of biophysical events take place, including the release of monovalent ions (i.e., Na⁺, K⁺, and Li⁺) followed by the release of the spore''s large depot of Ca-DPA (28). In Bacillus subtilis, release of Ca-DPA acts as a signal for activation of the cortex-lytic enzyme CwlJ (17). In contrast, Ca-DPA release from the spore core has no role in triggering cortex hydrolysis during C. perfringens spore germination (19, 22, 23); instead, Ca-DPA induces germination via the GerKA and/or GerKC receptor(s) (20, 21). Degradation of the cortex in both species leads to hydration of the spore core up to levels found in growing bacteria, allowing resumption of enzymatic activity and metabolism, and consequently outgrowth (22, 28).The ability of bacterial spores to sense different nutrients appears to be tightly regulated by their adaptation to different environmental niches. For example, spores of FP isolates, but not NFB isolates, are capable of germinating with KCl (20), an intrinsic mineral of meats that are most commonly associated with FP, suggesting an adaptation of FP isolates to FP environments. In addition, the level of inorganic phosphate (P_i) is also significant in meat products (42 to 60 mM) (USDA [http://fnic.nal.usda.gov/nal_display/index.php?info_center=4&tax_level=1&tax_subject=242]). Similarly, sodium ions are also present in meats (∼30 mM), especially in processed meat products (∼300 to 400 mM) (USDA). Consequently, in this study we found that Na⁺ and P_i at ∼100 mM and pH 6.0 are unique cogerminants for spores of C. perfringens type A FP isolates, act through the GerKA and/or GerKC and GerAA receptors, and also require the presence of the putative Na⁺/K⁺-H⁺ antiporter, GerO, for normal germination.     

qPCR quantification and genetic characterization of Clostridium perfringens populations in biosolids composted for 2 years

Aim: The ability of Clostridium perfringens to survive for a long time in the environment makes it a suitable indicator of faecal pollution, but its use as a routine indicator organism in biosolids and composted biosolids has not yet been adopted. This study was performed to improve our understanding of C. perfringens persistence in composted biosolids by monitoring its presence and studying its genetic diversity. Methods and Results: A culture‐independent TaqMan qPCR assay targeting the cpn60 gene was adapted to enumerate C. perfringens in composted biosolid samples varying in age from 1 to 24 months. The pathogen was detected in all compost samples under study, but no correlation between composting time and number of cpn60 copies was observed. Rep‐PCR detected 14 different C. perfringens genotypes, all belonging to toxinotype A, which is the most common biotype found in human and animal gastrointestinal tracts. Conclusions: Composting did not significantly decrease the number of C. perfringens cells. High genetic diversity of C. perfringens isolates present in composted biosolids is reported for the first time. Significance and Impact of Study: This study evaluated tools for surveillance of composting processes, source tracking and risk assessment of composted biosolids.     

Comparative genomics of VirR regulons in Clostridium perfringens strains

Background  

Clostridium perfringens is a Gram-positive anaerobic bacterium causing severe diseases such as gas gangrene and pseudomembranosus colitis, that are generally due to the secretion of powerful extracellular toxins. The expression of toxin genes is mainly regulated by VirR, the response regulator of a two-component system. Up to now few targets only are known for this regulator and mainly in one strain (Strain 13). Due to the high genomic and phenotypic variability in toxin production by different strains, the development of effective strategies to counteract C. perfringens infections requires methodologies to reconstruct the VirR regulon from genome sequences.     

Global regulation of gene expression in response to cysteine availability in <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

Background  

Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.     

A simple method for the isolation and determination of Clostridium perfringens

Summary A method for the isolation and determination of small numbers of vegetative cells or spores of Clostridium perfringen has been developed based on enrichment under anaerobic conditions in a fluid thioglycollate medium without dextrose, containing 400 g of D-cycloserine/ml at 46°C for 18 h (PEM). It allows virtually complete recovery of vegetative cells of all strains of Clostridium perfringens tested, whereas facultative anaerobes present in food are inhibited. Undamaged Clostridium perfringens spores can also be detected by this procedure. After enrichment, isolation of Clostriduum perfringens is carried out on iron sulphite agar at 46°C for 18 h. Typical black colonies are picked and confirmed by the following tests: neutralization of the -toxin by a specific diagnostic antiserum and absence of indole, motility, and ability to liquify gelatin.     

Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis,Argentina

Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals. This bacterium can produce more than 15 toxins, one of which is its enterotoxin (CPE), that causes human food poisoning. The aim of this work was (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, (ii) to characterize the C. perfringens enterotoxigenic strains by PCR, RPLA and the slide reverse passive latex agglutination test, (iii) to type the C. perfringens strains isolated and identification by PCR and (iv) to develop a slide RPLA test. A total of 515 samples of meat food (315 fresh sausages, 100 hamburgers and 100 samples of minced meat) were studied. A 126 C. perfringens strains (24.46%) were isolated and characterized. Of these C. perfringens -positive samples, 48 contained counts higher than 2 log/g. No significant differences were observed between counts performed in iron–milk medium and tryptose–sulfite–cycloserine agar (r= 0.99). Twelve samples (9.52%) exhibited counts with MPN >5log bacteria/g. Modified Tórtora medium (Tm) with thiotone replaced by proteose peptone turned out to be the most useful medium for both sporulation and enterotoxin production. Of the 126 samples tested by PCR and RPLA, nine strains (7.14%) were enterotoxigenic. Similar results were obtained by Slide RPLA, which exhibited a sensitivity of 8 ng/mL. Of the 126 C. perfringens strains , 123 were of type A (97.20%), two were of type C (1.59%) and one of type E (0.79%). All enterotoxigenic strains were classified as type A.     

Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens

To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C. perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C. perfringens isolates from those samples were grouped by slide agglutination and by pulsed-field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C. perfringens, and not by a food-borne outbreak.     

Comparative Genomic Hybridization Analysis Shows Different Epidemiology of Chromosomal and Plasmid-Borne cpe-Carrying Clostridium perfringens Type A

Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH) analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.