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Published in final edited form as: Anaerobe. 2008 Feb 7;14(2):102–108. doi: 10.1016/j.anaerobe.2008.01.003

Clostridium perfringens toxin genotypes in the feces of healthy North Americans

Robert J Carman 1, Sameera Sayeed 2, Jihong Li 2, Christopher W Genheimer 1, Megan F Hiltonsmith 1, Tracy D Wilkins 1, Bruce A McClane 2
PMCID: PMC2444017  NIHMSID: NIHMS50397  PMID: 18353695

Abstract

We investigated the frequency of C. perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of ~106 cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.

Keywords: Clostridium perfringens, health, genotype, enterotoxin, beta2

1. INTRODUCTION

Clostridium perfringens is a Gram positive, spore-forming, rod-shaped anaerobe. This bacterium is commonly classified into one of five types depending on production of alpha, beta, epsilon and iota toxins. Its primary habitat is the bowel of some healthy humans and probably some individuals of most other warm-blooded animal species. It is also found in soil where animals congregate and where manure has been used to boost agricultural fertility.

Ostensibly a non-pathogenic member of the healthy human gut flora, the ecology of C. perfringens is complicated by its carriage of various combinations of toxin genes and by the location of these genes on either the chromosome or a plasmid. For example, type A strains carrying the cpe gene encoding the C. perfringens enterotoxin (CPE) cause, under suitable conditions, food poisoning [1,2,3], sporadic out-patient diarrhea [4,5] and nosocomial antibiotic associated diarrhea [6–16]. Since the cpe gene in food poisoning isolates is usually chromosomal while in other diarrheas it is predominantly plasmid-borne [3,17,18], identifying reservoirs for the type A plasmid and chromosomal cpe isolates is important for understanding disease transmission and for rational design of intervention strategies to block transmission of virulent strains. It has been clearly shown that American foods can be contaminated with chromosomal cpe isolates at the time of retail purchase [19] but it is not yet clear how or when those foods become contaminated. However, two recent studies reported the presence of a few chromosomal cpe isolates in healthy Japanese and Finnish people, possibly suggesting healthy people as a potential reservoir for chromosomal cpe food poisoning isolates.

Though no longer as widely accepted as before, the relative levels of C. perfringens type A in human feces is thought to be helpful in establishing a causal role for this bacterium in cases of diarrhea, especially food poisoning. Thus, while counts below 105 to 107/g were considered “normal”, higher counts were said to be clinically relevant, though it was sometimes unclear whether this meant only spores or all C. perfringens. Consequently, reports of C. perfringens in healthy humans’ feces ranging anywhere from none to 108/g feces are confusing and contradictory. For example, with good anaerobic technique coupled with non-selective, feces-mimicking medium, the mean carriage rate among 191 healthy adults was 109.6 cfu/g, though surprisingly only 8 subjects (~4%) were colonized (the late WEC Moore, VPI Anaerobe Lab, VA Tech, Blacksburg, VA, Personal communication). Using a variety of selective and differential media, Harmon and Kautter [20] found that even their best medium missed some positives recovered by one or more of the less effective media. Thus without paying close attention to the details, reports of counts that range anywhere from none to 108/g are difficult to interpret. Nor is it clear why an individual's carriage rate, though stable for weeks at a time, may still vary considerably over 12 months [21]. Furthermore the carriage of particular strains is sporadic and individuals may carry more than one strain at a time [22]. Finally, age affects carriage rates; C. perfringens is more abundant in healthy neonates and the elderly than in adults. Tonooka et al., [23] found C. perfringens in 28% of healthy month old babies, with rates highest among those receiving formula milk, while carriage rates went up significantly in the elderly [24].

Our studies were designed to assess the relative incidence of the different toxin genotypes of C. perfringens in healthy North Americans in order to test the hypothesis that healthy people are reservoirs for type A chromosomal cpe isolates capable of causing food poisoning or for type A plasmid cpe isolates capable of causing non-foodborne gastrointestinal diseases. We looked at the rates of carriage in individuals, the relative abundance of spores and vegetative cells in feces, the presence or not of toxins in stool, the presence of cpe and cpb2 genes, and, lastly, the type of cpe locus present in recovered type A cpe-positive isolates.

2. MATERIALS AND METHODS

2.1. Fecal samples

TechLab Inc IRB #1 approved the collection of Informed Consent, self-reported information about age, diet and health, and a fecal sample from 43 healthy subjects. We recruited only those donors reporting themselves in good general health, free of underlying intestinal illnesses, having taken no antibiotics for at least 12 weeks and being free of diarrhea over the same period. 20 donors were males, 23 were females. Donors provided their first feces of the day. Immediately on arrival at the laboratory, usually within 60 min of defecation, feces were homogenized, dispensed aseptically under anaerobic gasses (90% nitrogen, 10% carbon dioxide) into airtight glass tubes. The samples were then frozen (−70 °C), usually within 120 min of defecation. At the time of use, they were thawed at room temperature while still anaerobic. All assays run on a fecal sample (as opposed to the subsequent testing of isolated bacteria) were done on the same day.

2.2. Fecal toxins

We used the C. perfringens Enterotoxin TEST (TechLab, Inc., Blacksburg, VA), a research-use-only assay, for the detection of fecal enterotoxin. This is a polyclonal antibody based assay that detects both intact, cytotoxic CPE and fragments of CPE that, though no longer biologically active, are still immunoreactive. Thus a positive would not automatically mean that sample would also be cytotoxic. Cytotoxic activity in feces was detected using Vero cells [25]. Some samples were sufficiently voluminous and liquid to be readily filterable. The others were centrifuged (8000 × g, 10 min, 4 °C) to generate a supernatant with which to work. All were filtered (0.22 µm pore size). Serial doubling dilutions were prepared and loaded into wells to give in-the-well dilutions of 1/20 to 1/2560. The reciprocal highest dilution producing a cytopathic effect within 48 h was the titer. A second 8-well row was used to neutralize any cytotoxic activity by adding 50 µL of a 1/50 dilution of goat anti-C. perfringens enterotoxin (the concentrated component that is diluted for inclusion in the C. perfringens Enterotoxin Test, TechLab, Inc.) in PBS to each well. Neutralization of a cytotoxic sample would indicate the presence of active CPE. This assay would reveal incidentally all cytotoxicities active on Vero cells. Any C. perfringens alpha toxin, which is active on Vero cells, would thus be apparent in this assay.

2.3. Clostridium perfringens counts

Aseptically and anaerobically, 10-fold dilution series (out to 10−6) were made in pre-reduced anaerobically sterilized (PRAS) diluents [26; Anaerobe Systems, Morgan Hill, CA]. The total count was obtained by spreading 100 µL from serial dilutions onto PRAS blood agar (Hardy Diagnostics, Santa Maria, CA). To select for spores by eliminating vegetative cells, we pre-incubated 1mL aliquots of each dilution of a sample in an equal volume of non-denatured 95% ethanol for 60 minutes at room temperature [6]. Inocula (200 µL, equal to 100 µL of diluted feces) were plated on to blood agar. Both sets of plates were incubated anaerobically at 37 °C for 24 h. C. perfringens was presumptively identified based on colonial and cellular morphology, patterns of hemolysis and Gram stain. Counts were adjusted for dilution and the mass of feces added to the initial tube to give count/g feces. The minimum level of detection of vegetative cells or spores or both was ~102 cells/g feces, depending on the precise mass of stool in the first dilution tube. Up to five isolates from each positive sample were subcultured in PRAS chopped meat broths (Anaerobe Systems) to provide stock cultures for subsequent studies. Each isolate was confirmed as C. perfringens using PCR and species-specific 16S rRNA primers [23]. The mean bacterial counts and their standard deviations were calculated using absolute numbers that, for presentation, were transformed into log10 count/g.

2.4. Toxin genotyping of Clostridium perfringens

Template DNA for PCR genotyping analysis were prepared as described before [19]. Each PCR mixture contained 2 µL of template DNA, 17 µl of TAQ Complete 1.1 Master Mix (Gene Choice), and 0.5 µL of each primer pair (1 µM final concentration). The multiplex PCR reactions were performed in a Techne (Burkhardtsdorf, Germany) thermocycler using previously described conditions and primer sets capable of detecting the presence of genes encoding alpha toxin, beta toxin, epsilon toxin, iota toxin, enterotoxin, and beta2 toxin [27]. PCR products were separated on 2% agarose gels and visualized with ethidium bromide staining.

The above prepared template DNA was also used for multiplex PCR cpe genotyping analysis. Primers and amplification conditions were used as described before [28] to identify the enterotoxin gene (cpe) locus present in each recovered type A cpe-positive C. perfringens fecal isolate. Specifically, this assay can distinguish between those isolates carrying a chromosomal cpe locus flanked by IS1470 sequences, a pCPF4969-like plasmid with an IS1470-like sequence downstream of cpe, or a pCPF5603-like plasmid with an IS1151 sequence downstream of cpe.

2.5. Detection of CPE by Western immunobloting of Clostridium perfringens culture fluids

CPE production by C. perfringens sporulating cultures of cpe-positive isolates (as detected by PCR assays) was assessed by inoculating a 0.1 mL aliquot of an overnight thioglycollate broth culture into 10 mL of Duncan-Strong sporulation medium [29,30]. Those cultures were then incubated for ~14 h, at which time sporulation was examined by phase-contrast microscopy. Sporulating cells in the cultures were sonicated (Sonicator 3000) to release their internal CPE. After centrifugation, supernatants from the sonicated sporulating culture lysates were used for CPE Western blotting [30]. For CPE Western blotting, a 100 µL aliquot of supernatant from each sporulating (prepared as described above) culture was mixed with 100 µL of SDS sample buffer and 30 µL of that mixture was then electrophoresed on 10% acrylamide gels containing SDS (no sample boiling). After electrophoresis and transfer onto nitrocellulose, CPE Western blotting was performed using a rabbit polyclonal CPE antiserum as primary antibody, followed by a goat anti-rabbit IgG peroxidase conjugate (Sigma) as secondary antibody. Blots were developed with SuperSignal West Pico Chemiluminescent substrate (Pierce).

3. RESULTS

No donor excreted C. difficile or its toxins (data not shown). All samples were negative for evidence of biologically active alpha toxin, a probable indication of good health that we discuss elsewhere in this report. They were also negative for evidence of both cytotoxic and immunoreactive CPE. All were negative for fecal lactoferrin, a biomarker of inflammation, indicating the absence of intestinal inflammation (data not shown). Thus, these negative data support the donors’ self-reported good health.

3.1. C. perfringens in stool

A combination of spores and vegetative cells of C. perfringens were present in 22 of 43 (or 51%) of healthy donors. Spores alone were found in 5 additional subjects, so altogether 27 (or 63%) subjects carried C. perfringens (Table 1). The mean incidence for total C. perfringens (vegetative cells and spores) in the 27 carriers was 106.50/g. The standard deviation, 107.15 cfu/g, exceeded the mean, reflecting the wide range of obtained fecal counts, from as high as 107.83 down to 10 individuals carrying fewer than 103/g. There were no significant differences between rates of carriage in males and females (t-test, p <0.05).

Table 1.

Mean counts of Clostridium perfringens and its spores in the feces of healthy human subjects

Mean C. perfringens count/g feces ± SD Mean spores as % of total count ± SD
Total count Spores
All samples (n=43) 106.21 ± 7.01 105.30 ± 5.92 72 ± 133
Only samples with C. perfringens (n; % of all 43 samples) 106.50 ± 7.15 (n=22; 51%) 105.50± 6.01 (n=27; 63%) 124 ± 153
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Multiplex toxin genotyping PCR analyses showed that only type A C. perfringens (i.e. PCR positive for the alpha toxin gene but negative for genes encoding beta, epsilon and iota toxins) isolates were recovered from the feces of healthy human subjects (Table 2). We recovered 34 type A isolates (each distinct within the sample from which it was recovered) from 23 individuals (Table 2). There were nine subjects who had neither cpe nor cpb2, 11 were cpb2 positive, two had both cpe and cpb2, and one had cpe only. Multiplex cpe genotyping PCR analysis showed that two people carried type A isolates with a pCPF5603 cpe locus and a cpb2 gene, presumably both on a pCPF5603-like plasmid. Lastly, one cpb2-negative type A isolate was recovered that carried a pCPF4969 cpe locus.

Table 2.

Clostridium perfringens Type A genotypes recovered from the feces of 23 healthy human subjects

Donor #. Isolate # Genotype
1 plc
2 plc
3.1 plc
3.2 plc cpb2
4.1 plc
4.2 plc cpeF4969- like
5.1 plc
5.2 plc cpb2
6.1 plc
6.2 plc cpb2
7.1 plc
7.2 plc cpb2
8 plc cpb2
11 plc
12.1 plc
12.2 plc cpb2
14.1 plc
14.2 plc cpb2
18 plc
19 plc
21 plc
25.1 plc
25.2 plc cpb2
29.1 plc
29.2 plc cpb2
30 plc
32 plc cpb2 cpeF5603
33 plc cpb2 cpeF5603
36 plc
39.1 plc
39.2 plc cpb2
40.1 plc
40.2 plc cpb2
43 plc
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All isolates were type A, i.e. they carried plc, encoding alpha toxin with its phospholipase C activity, but not beta, epsilon or iota toxin genes. Only Isolate 33 produced immunologically detectable CPE. Isolate 4.2 did not express CPE despite sporulating. Strain 32 neither sporulated nor produced CPE. All other cpe+ isolates were efficient spore formers.

Individual spore counts tended to be nearly the same or greater than the total count (data not shown). In 14 of the 27 colonized individuals the spore count was 100% or more than the total count, while spores were on average 124% of the total count in colonized individuals (Table 1).

3.2. Genotypes in individuals

We isolated and genotyped the C. perfringens type A isolates from 23 healthy donors; 11 donors carried only one type A genotype, the other 13 donors carried two. None carried three (Table 2). When only one strain was present, it was always a type A without either the cpe or cpb2 genes.

3.3. In vitro CPE production by fecal isolates

7% (3) of all 43 subjects, and 3 of 27 (11%) colonized donors, carried cpe-positive isolates. Of the 14 cpe-positive type A isolates recovered from those 3 colonized people, 12 could sporulate in vitro, as required to demonstrate CPE expression [29]. Only two of the 12 sporulating strains produced CPE levels detectable by Western blotting. The cpe open reading frame (ORF) was sequenced in two of the 12 sporulation-capable, cpe-positive isolates that failed to produce CPE. No changes from the consensus cpe ORF was detected in one of those isolates but the other carried a premature termination codon in its cpe ORF.

4. DISCUSSION

4.1. Fecal carriage

About half our donors were colonized with, on average, slightly over 106 C. perfringens/g (Table 1), a finding that lies within the broad range of reported rates. Some colonized donors carried no vegetative cells. Many had spore counts that exceeded their total counts, an odd result that needs an explanation. In this study spores were selected by alcohol wash, though it could equally have been by heat shock. Stressors, such as ethanol, are believed to enhance the rate of germination of spores [31]. This effect may help explain how a spore count could exceed its corresponding total count, especially if the majority of viable C. perfringens was already present as spores, not vegetative cells. The skew towards spores may have occurred after the samples were collected, as an artifact of the processing although we saw no evidence of this in preliminary studies of fresh and frozen aliquots of the same fecal samples. A second possibility is that the alcohol wash eliminated the inhibitory effects of the normal flora thus elevating the relative spore count (established with no background flora thanks to the wash) at the expense of the total count (established against a high and therefore inhibitory background). A third alternative is that the trend points to C. perfringens vegetative cells occurring high in the bowel, perhaps in the terminal ileum, while spores of this bacterium are more common further along the bowel, perhaps where conditions are less hospitable. Five extreme instances of this trend, spores alone (101.93 to 104.93/g), were observed during our study. Assuming there were neither within-donor nor within-sample variations of the distribution of spores, 101.9 spores/g coupled with a probable daily fecal mass of over 100 g suggests donors excreted almost 10,000 spores each day. This is indicative of colonization, not the transient passage of ingested spores.

4.2. Genotypes

CPE is the primary virulence factor for enteric diseases caused by CPE-positive type A isolates [3]. In a series of studies [32–35] the cpe gene has been localized to either the chromosome, as in the overwhelming majority of food poisoning outbreaks, or on one of two plasmid families, represented by prototype cpe plasmids pCPF5603 and pCPF4969. The latter cpe plasmid family is known to be conjugative [36]. When chromosomal, the cpe gene is flanked by the insertion sequence, IS1470. In contrast, the pCPF5603 plasmid family carries an IS1151 sequence downstream of its cpe gene. pCPF5603-like plasmids usually also carry the cpb2 gene. pCPF4969-like plasmids have an IS1470-like sequence downstream from cpe but do not themselves carry cpb2. However, cpb2 can be present on an additional plasmid in strains carrying pCPF4969-like cpe plasmids. The beta2 toxin (CPB2) produced by expressing pCPF5603-borne cpb2 is about 10-fold more active on Caco-2 cells than the equivalent gene product produced by isolates carrying pCPF4969-like plasmids and a separate cpb2 plasmid [32].

Finnish workers [37] recently studied C. perfringens fecal carriage in 136 healthy food workers. By amplifying cpe from enrichment broths they identified 25 (18% of the total study population) cpe+ fecal samples, whereas after enrichment only 11 (8%) of 136 samples were also culture positive for cpe+ isolates. Given the reputation of C. perfringens for ease of isolation, it is somewhat surprising that 14/25 samples were PCR positive but culture negative for cpe. In contrast, without enrichment we detected 7% of donors as carrying cpe+ isolates. Of the 11 cpe+/culture+ donors identified in the Finnish study, 10 carried both cpe− and cpe+ isolates, while one carried only cpe+ isolates. From among those 11 donors, the Finns found one with a strain carrying a chromosomally-located cpe (IS1470-cpe), several that by implication had cpe on the plasmids pCPF5603 or pCPF4969 and, lastly, two with untypeable cpe+ strains. We found no IS1470-cpe or untypeable strains in our study, only isolates with a pCPF5603 or pCPF4969 cpe locus. The Finns also saw one instance of both cpe plasmid families in the same sample; that individual also carried an additional cpe− strain, making three in total. Though we saw no instances of three genotypes in the same sample (Table 2), we often found two. Further analysis of their chromosomal DNA might resolve the strains into, perhaps, one with and one without a cpe or cpb2 plasmid or show a single strain occurring with and without a toxin-encoding plasmid.

Japanese workers have also studied the distribution of cpe+ isolates in health and disease [38]. They recovered 16 cpe+ fecal isolates from an unspecified number of healthy donors. 11 of those isolates apparently carried a pCPF5603-like cpe plasmid, three had a pCPF4969-like cpe plasmid, and two isolates had a chromosomal cpe gene. Thus all three groups of workers, us, the Finns [37] and the Japanese [38], have found both cpe plasmid families in healthy donors, at low and comparable frequencies. In contrast, Miyamoto et al. [28] found nearly equal numbers of pCPF4969-like and pCPF5603-like plasmids among European nonfoodborne gastrointestinal disease isolates but suggested a skew in favor of pCPF4969-like plasmids among North American nonfoodborne gastrointestinal disease isolates.

With our data, the presence of chromosomal cpe isolates has now been surveyed in the feces of healthy Finns, Japanese and Americans. While a few fecal samples have tested positive (at apparently low levels) for these bacteria, these survey results collectively indicate that most healthy people are not routinely colonized by chromosomal cpe type A isolates. Whether the few carriers of these bacteria represent an important reservoir for food poisoning transmission remains to be determined.

The finding of cpe+ spores in both our and the Finnish studies is important. If, as in vitro, the in vivo regulation of sporulation and enterotoxin production are co-regulated, the presence of cpe+ spores suggests possible in vivo expression of CPE. Despite all 11 colonized donors having at least one readily sporulating cpe+ isolate recovered originally as a spore, we saw no cytotoxicity from CPE. In fact no sample at all elicited any type of cytopathic effect, implying the absence of any and all cytotoxic activities. This would include the alpha toxin, the cytotoxic phospholipase C, of C. perfringens whose production is constitutive in vitro, and that by implication should have been detectable in all 27 colonized healthy donors but was not. This would be explained if the in vitro degradation of alpha toxin by proteases [39,40] also occurred in situ as a result of both host and bacterial enzymes. In fact, the capacity of stool to inhibit alpha toxin activity has been long known [41]. Reports of active alpha toxin in diarrheic stool [42] coupled with its absence from healthy feces [43] suggest an innate mechanism of resistance during health, possibly mediated by host or bacterial proteases or both. Degradation by proteases might also explain the lack of CPE cytotoxic activity in any healthy donors’ feces and arguably in the majority of symptomatic patients with CPE food poisoning [44]. Perhaps in some healthy donors, cpe expression and sporulation both occur in the ileo-cecal region but normal proteolytic activity eliminates, in a stepwise manner, firstly the biological activity and finally, even the immunoreactivity.

We saw three donors colonized with cpe+ isolates. From all three at least one readily sporulating cpe+ strain was recovered after alcohol shock. Those stools might have contained expressed CPE but did not. One possible explanation for the lack of CPE in the three donors colonized with cpe+ isolates is the inability of all isolates but one to produce CPE in vitro, as detectable by Western blot with specific monoclonal antibodies [45]. One isolate had a stop codon located within its cpe sequence (McClane and Li, unpublished data) that could explain its deficiencies in cpe expression. This isolate is interesting since it represents, to our knowledge, the first identified type A isolate carrying a defective cpe ORF. A lack of expression by the remaining cpe+ isolates may be nothing more than a measure of our lack of success in establishing the appropriate in vitro conditions for expression.

Of our 43 subjects, 8 (23%) were colonized with isolates carrying the cpb2 gene (Table 2). Conversely, Fisher et al., [32] have shown that over 75% of C. perfringens recovered from cases of sporadic diarrhea or nosocomial antibiotic associated diarrhea (AAD) caused by C. perfringens were cpb2+ and almost all the cpb2+ isolates they studied expressed CPB2 in vitro. However, some care should be taken with these data since the North American AAD isolates included as part of that survey came mainly from two nosocomial outbreaks of C. perfringens AAD that, if typical of earlier outbreaks (6), may be associated with a single strain at each site. Apparently cpb2 may be found in health or in disease and the possible involvement of beta2 toxin in human GI disease remains intriguing but unproven. The toxin can clearly damage Caco-2 human enterocyte-like cells [32]. However, there are no published reports, successful or not, of attempts to fulfill Koch’s Postulates using strains that produce CPB2, nor have we been able, thus far, to show any enterotoxic effect of partially-purified or purified CPB2 in rabbit ileal loops (M.R. Sarker and R.J. Carman, unpublished data; D.J. Fisher, F.A. Uzal and B.A. McClane, unpublished data). Despite this, a body of literature is developing that may support a clinical role for CPB2.

5. CONCLUSIONS

We saw no evidence that healthy humans are a reservoir for the chromosomal-cpe typical of isolates from instances of food poisoning. About half of all subjects were colonized with C. perfringens type A, with on average about 106/g feces, though with a broad variance. Often spores outnumbered vegetative cells. A variety of type A genotypes were found, often more than one per person. There was no evidence for the accumulation of detectable alpha, CPE or CPB2 toxins during health. Though recovered from healthy individuals, some type A genotypes (cpe+, cpb2+, conjugative plasmid) were suggestive of potential virulence.

ACKNOWLEDGMENTS

This research was generously supported, in part, by National Research Initiative Competitive Grant 2005-53201-15387 from the USDA Cooperative State Research, Education and Extension Service and by T32 AI060525-01A1 (a Ruth L. Kirschstein National Service Award) from the National Institute of Allergy and Infectious Diseases.

Footnotes

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REFERENCES

  • 1.Klein E. Uber einen pathogenen anaeroben Darmbacillus, Bacillus enteritidis sporogenes. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Orig. 1895;18:737–743. [Google Scholar]
  • 2.McClung LS. Human food poisoning due to growth of Clostridium perfringens (C. welchii) in freshly cooked chicken: Preliminary note. J Bacteriol. 1945;50:229–231. [PMC free article] [PubMed] [Google Scholar]
  • 3.Sarker MR, Carman RJ, McClane BA. Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens Type A human intestinal disease isolates to affect rabbit ileal loops. Molec Microbiol. 1999;33:946–958. doi: 10.1046/j.1365-2958.1999.01534.x. [DOI] [PubMed] [Google Scholar]
  • 4.Brett MM, Rodhouse JC, Donovan TJ, Tebbutt GM, Hutchinson DN. Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhoea. J Clin Pathol. 1992;145:609–611. doi: 10.1136/jcp.45.7.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mpamugo O, Donovan T, Brett MM. Enterotoxigenic Clostridium perfringens as a cause of sporadic cases of diarrhoea. J Med Microbiol. 1995;43:442–445. doi: 10.1099/00222615-43-6-442. [DOI] [PubMed] [Google Scholar]
  • 6.Abrahao C, Carman RJ, Hahn H, Liesenfeld O. Similar frequency of detection of Clostridium perfringens enterotoxin and Clostridium difficile toxins in patients with antibiotic associated diarrhea. Eur J Clin Microbiol. 2001;20:676–677. doi: 10.1007/s100960100571. [DOI] [PubMed] [Google Scholar]
  • 7.Asha NJ, Tompkins D, Wilcox MH. Comparative analysis of prevalence, risk factors, and molecular epidemiology of antibiotic-associated diarrhea due to Clostridium difficile, Clostridium perfringens, and Staphylococcus aureus. J Clin Microbiol. 2006;44:2785–2791. doi: 10.1128/JCM.00165-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Asha NJ, Wilcox MH. Laboratory diagnosis of Clostridium perfringens antibiotic- associated diarrhoea. J Med Microbiol. 2002;51:891–894. doi: 10.1099/0022-1317-51-10-891. [DOI] [PubMed] [Google Scholar]
  • 9.Borriello SP, Barclay FE, Welch AR, Stringer MF, Watson GN, Williams RK, Seal DV, Sullens K. Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens. J Med Microbiol. 1985;20:363–372. doi: 10.1099/00222615-20-3-363. [DOI] [PubMed] [Google Scholar]
  • 10.Borriello SP, Carman RJ. Association of toxigenic Clostridium spiroforme with iota toxin positive enterotoxemia in rabbits. J Clin Microbiol. 1983;17:414–418. doi: 10.1128/jcm.17.3.414-418.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Boriello SP, Larson HE, Welch AR, Barclay F, Stringer MF, Bartholomew BA. Enterotoxigenic Clostridium perfringens: a possible cause of antibiotic- associated diarrhoea. Lancet. 1984;i:305–307. doi: 10.1016/s0140-6736(84)90359-3. [DOI] [PubMed] [Google Scholar]
  • 12.Boriello SP, Williams RKT. Treatment of Clostridium perfringens enterotoxin-associated diarrhoea with metronidazole. J Infect. 1985;10:65–67. doi: 10.1016/s0163-4453(85)80013-x. [DOI] [PubMed] [Google Scholar]
  • 13.Hancock P. Antibiotic-associated diarrhoea C. difficile or C. perfringens. Rev Med Microbiol. 1997;8 Suppl 1:566–567. [Google Scholar]
  • 14.Larson HE, Borriello SP. Infectious diarrhea due to Clostridium perfringens. J Infect Dis. 1988;157:390–391. doi: 10.1093/infdis/157.2.390. [DOI] [PubMed] [Google Scholar]
  • 15.Modi N, Wilcox MH. Evidence for antibiotic induced Clostridium perfringens diarrhoea. J Clin Pathol. 2001;54:748–751. doi: 10.1136/jcp.54.10.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Samuel SC, Hancock P, Leigh DA. An investigation into Clostridium perfringens enterotoxin associated diarrhoea. J Hosp Infect. 1981;18:219–230. doi: 10.1016/0195-6701(91)90146-y. [DOI] [PubMed] [Google Scholar]
  • 17.Collie RE, McClane BA. Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non-foodborne human gastrointestinal diseases. J Clin Microbiol. 1998;36:30–36. doi: 10.1128/jcm.36.1.30-36.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Cornillot E, Saint-Joanis B, Daube G, Katayama S, Granum PE, Canard B, Cole ST. The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne. Mol Microbiol. 1995;15:639–647. doi: 10.1111/j.1365-2958.1995.tb02373.x. [DOI] [PubMed] [Google Scholar]
  • 19.Wen Q, McClane BA. Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods. Appl Environ Microbiol. 2004;70:2685–2691. doi: 10.1128/AEM.70.5.2685-2691.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Harmon SM, Kautter DA. Enumeration of Clostridium perfringens spores in human feces: comparison of four culture media. J Assoc Anal Chem. 987;70:994–996. [PubMed] [Google Scholar]
  • 21.Torres-Anjel MJ, Riemann HP, Brant P. Enterotoxigenic Clostridium perfringens type A in selected humans. II. A cohort study. Rev Latinoam Microbiol. 1975;17:199–205. [PubMed] [Google Scholar]
  • 22.Stringer MF, Watson GN, Gilbert RJ, Wallace JG, Hassall JE, Tanner EL, Webber PP. Faecal carriage of Clostridium perfringens. J Hyg (Lond) 1985;95:277–288. doi: 10.1017/s0022172400062707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tonooka T, Sakata S, Kitahara M, Hanai M, Ishizeki S, Takada M, Sakamoto M, Benno Y. Detection and quantification of four species of the genus Clostridium in infant feces. Microbiol Immunol. 2005;49:987–992. doi: 10.1111/j.1348-0421.2005.tb03694.x. [DOI] [PubMed] [Google Scholar]
  • 24.Benno Y, Endo K, Mizutani T, Namba Y, Komori T, Mitsuoka T. Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl Environ Microbiol. 1989;55:1100–1105. doi: 10.1128/aem.55.5.1100-1105.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.McClane BA, McDonel JL. The effects of Clostridium perfringens enterotoxin on morphology, viability, and macromolecular synthesis in Vero cells. J Cell Physiol. 1979;99:191–200. doi: 10.1002/jcp.1040990205. [DOI] [PubMed] [Google Scholar]
  • 26.Holdeman LV, Cato EP, Moore WEC, editors. Anaerobe Lab Manual. 4th edition. Blacksburg, VA: Anaerobe Laboratory, Virginia Polytechnic Institute and State University; 1977. [Google Scholar]
  • 27.Garmory HS, Chanter N, French NP, Bueschel D, Songer JG, Titball RW. Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined using genotyping and subtyping PCR assays. Epidemiol Infect. 2000;124:61–67. doi: 10.1017/s0950268899003295. Erratum in: Epidemiol Infect 2000;125:227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Miyamoto K, Wen Q, McClane BA. 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. J Clin Microbiol. 2004;42:1552–1558. doi: 10.1128/JCM.42.4.1552-1558.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Duncan CL, Strong DH, Sebald M. Sporulation and enterotoxin production by mutants of Clostridium perfringens. J Bacteriol. 1972;110:378–391. doi: 10.1128/jb.110.1.378-391.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kokai-Kun JF, Songer JG, Czeczulin JR, Chen F, McClane BA. Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol. 1994;32:2533–2539. doi: 10.1128/jcm.32.10.2533-2539.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Craven SE, Blankenship LC. Activation and injury of Clostridium perfringens spores by alcohols. Appl Environ Microbiol. 1985;50:249–256. doi: 10.1128/aem.50.2.249-256.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Fisher DJ, Miyamoto K, Harrison B, Akimoto S, Sarker MR, McClane BA. Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene. Mol Microbiol. 2005;56:747–762. doi: 10.1111/j.1365-2958.2005.04573.x. [DOI] [PubMed] [Google Scholar]
  • 33.Miyamoto K, Chakrabarti G, Morino Y, McClane BA. Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates. Infect Immun. 2002;70:4261–4272. doi: 10.1128/IAI.70.8.4261-4272.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Miyamoto K, Fisher DJ, Li J, Sayeed S, Akimoto S, McClane BA. Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates. J Bacteriol. 2006;188:1585–1598. doi: 10.1128/JB.188.4.1585-1598.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sparks S, Carman RJ, Sarker MR, McClane BA. Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America. J Clin Microbiol. 2001;39:883–888. doi: 10.1128/JCM.39.3.883-888.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Brynestad S, Sarker MR, McClane BA, Granum PE, Rood JI. Enterotoxin plasmid from Clostridium perfringens is conjugative. Infect Immun. 2001;69:3483–3487. doi: 10.1128/IAI.69.5.3483-3487.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Heikinheimo A, Lindstrom M, Granum PE, Korkeala H. Humans as reservoir for enterotoxin gene-carrying Clostridium perfringens type A. Emerg Infect Dis. 2006;12:1724–1729. doi: 10.3201/eid1211.060478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tanaka D, Kimata K, Shimizu M, Isobe J, Watahiki M, Karasawa T, et al. Genotyping of Clostridium perfringens Isolates collected from food poisoning outbreaks and healthy individuals in Japan based on the cpe locus. Jpn J Infect Dis. 2007;60:68–69. [PubMed] [Google Scholar]
  • 39.Park KB, Labbe RG. Proteolysis of Clostridium perfringens type A enterotoxin during purification. Infect Immun. 1990;58:1999–2001. doi: 10.1128/iai.58.6.1999-2001.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Sato H, Yamakawa Y, Ito A, Murata R. Effect of protease on the production of Clostridium perfringens alpha toxin. Jpn J Med Sci Biol. 1977;30:44–46. [PubMed] [Google Scholar]
  • 41.Goudie JG. The nature of a neutralising substance for Clostridium welchii alpha-toxin in faeces. J Pathol Bacteriol. 1959;78:17–28. doi: 10.1002/path.1700780104. [DOI] [PubMed] [Google Scholar]
  • 42.Carman RJ, Lewis JCM. Recurrent diarrhoea in a dog associated with Clostridium perfringens Type A. Vet Rec. 1983;112:342–343. doi: 10.1136/vr.112.15.342. [DOI] [PubMed] [Google Scholar]
  • 43.Yamagishi T, Serikawa T, Morita R, Nakamura S, Nishida S. Persistent high numbers of Clostridium perfringens in the intestines of Japanese aged adults. Jpn J Microbiol. 1976;20:397–403. doi: 10.1111/j.1348-0421.1976.tb01005.x. [DOI] [PubMed] [Google Scholar]
  • 44.Berry PR, Rodhouse JC, Hughes S, Bartholomew BA, Gilbert RJ. Evaluation of ELISA, RPLA, and Vero cell assays for detecting Clostridium perfringens enterotoxin in faecal specimens. J Clin Pathol. 1988;41:458–461. doi: 10.1136/jcp.41.4.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wnek AP, Strouse RJ, McClane BA. Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin. Infect Immun. 1985;50:442–448. doi: 10.1128/iai.50.2.442-448.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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