AND BRUCE A. MCCLANE 1 *

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2000, p. 3234 3240 Vol. 66, No. 8 0099-2240/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved. Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes MAHFUZUR R. SARKER, 1 ROBERT P. SHIVERS, 1 SHAUNA G. SPARKS, 1 VIJAY K. JUNEJA, 2 AND BRUCE A. MCCLANE 1 * Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, 1 and Eastern Regional Research Center, Agriculture Research Service, United States Department of Agriculture, Wyndmoor, Pennsylvania 19038 2 Received 3 March 2000/Accepted 8 May 2000 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. * Corresponding author. Mailing address: E1240 BSTWR, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Phone: (412) 648-9022. Fax: (412) 624-1401. E-mail: bamcc@pop.pitt.edu. Clostridium perfringens type A food poisoning currently is the second most commonly reported food-borne disease in the United States (19, 20). Substantial experimental and epidemiologic evidence (19 21) now indicates that most, if not all, gastrointestinal symptoms of this food-borne disease are caused by C. perfringens enterotoxin (CPE), a single 35-kDa polypeptide. CPE-positive strains of C. perfringens are also recognized as the cause of several non-food-borne human gastrointestinal illnesses, including antibiotic-associated diarrhea and sporadic diarrhea (8). Several surveys (14, 18, 22) have indicated that less than 5% of the global C. perfringens population carries the enterotoxin gene (cpe). Recent studies (9 11) have shown that cpe-positive isolates can carry the cpe gene on either the chromosome or a plasmid. Interestingly, all C. perfringens type A food poisoning isolates that have been genotyped to date have been shown to carry a chromosomal copy of the cpe gene, while all human non-food-borne gastrointestinal disease isolates genotyped to date have been found to carry the cpe gene on a plasmid (10, 11). The basis for the associations between certain cpe genotypes and food-borne versus non-food-borne CPE-associated diseases remains unknown. However, at least three possible explanations for this phenomenon can be envisioned. First, since cooked meat products are the predominant food vehicles for C. perfringens type A food poisoning (2, 3), vegetative cells or spores of C. perfringens isolates carrying a chromosomal cpe gene might be strongly associated with food poisoning because these isolates are more heat resistant and thus can survive better in cooked or warmed foods than cells or spores of C. perfringens isolates carrying a plasmid cpe gene. Alternatively, since heat has been shown to cure some plasmids (17), it is possible that plasmid cpe isolates do not commonly cause food poisoning because exposure to heat induces loss of the cpe plasmid, rendering the isolates avirulent. A final possibility is that chromosomal cpe isolates are strongly associated with food poisoning because heating induces migration of a plasmid cpe gene to the bacterial chromosome, thereby converting plasmid cpe isolates to chromosomal cpe isolates. The possibility of cpe gene mobility is supported by the results of recent studies (5, 6) suggesting that the cpe gene can be associated with a transposon, which could conceivably mobilize the cpe gene under environmental pressure. To evaluate the three hypotheses described above, in this study we compared the heat resistance properties of both vegetative cells and spores of C. perfringens food poisoning isolates carrying a chromosomal cpe gene with the heat resistance properties of cells and spores of C. perfringens isolates carrying a plasmid copy of the cpe gene. Western blot analyses and Southern blot studies were also performed to detect whether 3234

VOL. 66, 2000 HEAT SENSITIVITY OF ENTEROTOXIGENIC C. PERFRINGENS 3235 TABLE 1. Vegetative cell heat resistance of cpe-positive C. perfringens isolates a Isolate Origin D value at 55 C (min) Food poisoning isolates carrying a chromosomal cpe gene NCTC 8239 Europe, 1950s b 16.5 191-10 United States, 1990s c 11.2 C1841 United States, 1980s b 13.6 FD1041 United States, 1980s b 12.5 NCTC 10239 Europe, 1950s b 12.3 E13 United States, 1960s d 12.1 Avg e 13.1 0.9 Isolates carrying a plasmid cpe gene F5603 Humans, Europe, 1980s b 5.6 F4969 Humans, Europe, 1980s b 9.1 NB16 Humans, Europe, 1980s b 8.6 B40 Humans, Europe, 1980s b 5.0 222 Veterinary, United States, 1990s b 6.7 153 Veterinary, United States, 1990s b 6.8 458 Veterinary, United States, 1990s b 5.0 Avg e 6.7 1.5 a The results are averages based on the results of two independent experiments. The results were very reproducible for each isolate (data not shown). b See references 9 and 10 for further information on the origin of this isolate. c Food poisoning strain 191-10 was kindly provided by Jared Kerr of the Centers for Disease Control and Prevention. d See reference 15 for further information on the origin of food poisoning strain E13. e Average D value of each cpe genotype standard error. heat can cure the cpe plasmid or affect either CPE expression or the cpe genotype. MATERIALS AND METHODS Bacteria and growth conditions. The C. perfringens isolates used in this study are described in Table 1. Starter vegetative cultures (6 ml) of each C. perfringens isolate were prepared by overnight growth at 37 C in fluid thioglycolate (FTG) medium (Difco). Sporulating cultures of C. perfringens were prepared by inoculating 0.2 ml of a starter FTG medium culture into 10 ml of Duncan-Strong (DS) sporulation medium (18), which was then incubated for 24 h at 37 C. The presence of sporulating cells in each DS medium culture was confirmed by phase-contrast microscopy. Determination of D values for C. perfringens vegetative cells. To determine the heat sensitivity of vegetative cells of each C. perfringens isolate surveyed, a 0.2-ml aliquot of an overnight FTG medium starter culture, prepared as described above, was inoculated into 6 ml of FTG medium, and the resulting culture was grown for 2hat37 C. After mixing, a 0.1-ml aliquot of the 2-h FTG medium culture was aseptically withdrawn and serially diluted (dilutions range, 10 2 to 10 7 ) with sterile FTG medium. The diluted samples were then immediately plated onto brain heart infusion (BHI) agar plates to determine the total number of vegetative cells present in each FTG medium culture at the start of heating (i.e., at the zero-time point of the experiment). The remainder of each 2-h FTG medium culture was then heated at temperatures ranging from 55 to 61 C. After mixing, a 0.1-ml aliquot of each heated culture was removed. At 55 C, the aliquots were removed every 5 min for up to 30 min. At higher temperatures, aliquots were removed every 1 min for up to 10 min. The aliquots were diluted (dilutions range, 10 2 to 10 7 ) with sterile FTG medium, and the dilutions were immediately plated onto BHI agar plates. After 16 to 20 h of anaerobic incubation, colonies on the BHI agar plates were counted to determine the number of viable CFU that were present per milliliter of heated culture at each time point. The CFU values were then graphed to determine decimal reduction value (D value) (i.e., the time that a culture had to be kept at a given temperature to obtain a 90% reduction in viable cell numbers) for vegetative cultures of each C. perfringens isolate. Determination of D values for C. perfringens spores. The heat sensitivity of C. perfringens spores was determined as described previously (23), with minor modifications (in these experiments, spores were not cleaned so they were free of debris prior to heating to better simulate conditions present during cooking of contaminated foods). Briefly, DS medium cultures prepared and grown for 24 h as described above were heat shocked at 75 C for 20 min, which killed the remaining vegetative cells and facilitated spore germination (23; data not shown). A 0.1-ml aliquot of each heat-shocked DS medium culture was then serially diluted with sterile FTG medium to obtain dilutions ranging from 10 2 to 10 7. Each dilution was plated onto BHI agar plates to establish the number of viable spores per milliliter of DS medium culture at the start of heating (i.e., at the zero-time point of the experiment). The remainder of each heat-shocked DS medium culture was then heated at either 90 or 100 C for time periods ranging from 1 min to 6 h depending on the individual isolate and the temperature being used. At each time point, the heated DS medium culture was mixed, and a 0.1-ml aliquot was withdrawn and diluted (dilution range, 10 2 to 10 7 ) with sterile FTG medium. The dilutions were then plated onto BHI agar plates, which were incubated anaerobically at 37 C for 16 to 20 h. Colonies which developed from germinated spores that survived heating were counted to determine the number of viable spores present per milliliter of each heated DS medium culture. The data were then graphed to determine D values for spores of each isolate tested. Preparation of DIG-labeled cpe probes for Southern blot experiments. As described previously (12), a 639-bp digoxigenin (DIG)-labeled, double-stranded, cpe-specific DNA gene probe was prepared by a two-step PCR amplification method by using a primer set consisting of 5 -GGTACCTTTAGCCAATCA-3 (primer 2F) and 5 -TCCATCACCTAAGGACTG-3 (primer 5R). Restriction fragment length polymorphism (RFLP) Southern blot analyses. BHI agar colonies arising from control and heated FTG or DS medium cultures of C. perfringens were inoculated into FTG medium and the resulting cultures were grown overnight at 37 C. A 0.2-ml aliquot of each starter FTG medium culture was then inoculated into 10 ml of TGY medium (12), which was also incubated overnight at 37 C. Total C. perfringens DNA was isolated from the overnight TGY medium cultures by using a previously described protocol (12). The isolated DNA samples were then digested to completion with NruI, separated by electrophoresis on 0.8% agarose gels, transferred to positively charged nylon membranes (Boehringer Mannheim), and UV fixed to the membranes (21). DIG-labeled cpe probes were hybridized to the blots, as described in The Genius System Users Guide for Filter Hybridization (Boehringer Mannheim). Hybridized probes were then detected with a DIG-chemiluminescence detection system by using the CSPD substrate (Boehringer Mannheim). PFGE Southern blot analyses. BHI agar colonies arising from control and heated DS or FTG medium C. perfringens cultures were grown overnight in FTG medium at 37 C. A 0.2-ml aliquot of each FTG medium culture was then inoculated into 10 ml of TGY, and the resulting TGY medium culture was incubated overnight at 37 C. Bacterial cells from each overnight TGY medium culture were collected by centrifugation, and the pelleted cells were used to prepare genomic C. perfringens DNA in agarose plugs, as previously described (7, 10, 16). A 100- l aliquot of each agarose plug was then analyzed by pulsed-field gel electrophoresis (PFGE) by using 1% agarose gels prepared with PFGE grade agarose (Bio- Rad). PFGE was performed with a Bio-Rad CHEF-DR II apparatus by using pulse times ramped from 50 to 90 s over a 22-h period (4). After PFGE, the gels were subjected to cpe Southern analysis by using the procedure described above for RFLP Southern analysis. Western blot analysis. For each isolate, at least five isolated BHI agar colonies that survived the Table 1 heating experiments, along with at least five isolated BHI agar colonies that survived the Table 2 heating experiments, were grown individually overnight in FTG medium at 37 C. A 0.2-ml aliquot of each FTG medium culture was then inoculated into 10 ml of DS medium, and the resulting cultures were grown at 37 C for 8 h. The 8-h DS medium cultures were then examined by phase-contrast microscopy to ensure that spores were present, and

3236 SARKER ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. Heat resistance of spores produced by cpe-positive C. perfringens isolates a Isolate Origin D value at 100 C (min) Food poisoning isolates carrying a chromosomal cpe gene b NCTC 8239 Europe, 1950s 124 191-10 United States, 1990s 67 FD1041 United States, 1980s 32 E13 United States, 1960s 30 NCTC 10239 Europe, 1950s 45 Avg c 60.0 18 Isolates carrying a plasmid cpe gene F5603 Humans, Europe, 1980s 0.6 F4969 Humans, Europe, 1980s 0.5 NB16 Humans, Europe, 1980s 1.9 B40 Humans, Europe, 1980s 1.6 222 Veterinary, United States, 1990s 0.9 153 Veterinary, United States, 1990s 1.3 458 Veterinary, United States, 1990s 0.5 Avg c 1.0 0.3 a The results are averages based on the results of two experiments. The results were very reproducible for each isolate (data not shown). b Isolate C1841 is not listed because this isolate did not sporulate in DS medium. c Average D value of each cpe genotype standard error. they were sonicated until more than 95% of the cells were lysed (lysis was monitored by phase-contrast microscopy). After sonication, each DS medium culture lysate was analyzed for the presence of CPE by using a previously described CPE Western immunoblot procedure (9, 12, 18). Statistical analyses. Statistical analyses were performed with Student s t test. RESULTS Comparison of the heat sensitivities of vegetative cells of C. perfringens isolates carrying chromosomal versus plasmid cpe genes. To compare the heat sensitivities of vegetative cells of C. perfringens isolates carrying a chromosomal cpe gene and vegetative cells of C. perfringens isolates carrying a plasmid cpe gene, D values were determined for FTG medium cultures of 13 cpe-positive strains heated to 55, 57, 59, or 61 C. Figure 1 shows representative thermal death curve results obtained at FIG. 1. Thermal death curves for vegetative cells of strain E13, which carries a chromosomal cpe gene, and strain F5603, which carries a plasmid cpe gene. Vegetative (FTG medium) cultures of E13 and F5603 were heated at 55 C for specified times, and the number of viable bacteria per milliliter of each heated culture was then determined (see Materials and Methods). The results are the results of representative experiments; these results were highly reproducible (data not shown). 55 C for the following two cpe-positive isolates: E13, a food poisoning isolate carrying a chromosomal cpe gene, and F5603, a non-food-borne human gastrointestinal disease isolate carrying a plasmid cpe gene. From the Fig. 1 results, we calculated that the D value at 55 C for E13 is 12 min but the D value for F5603 is less than 6 min. These heat sensitivity differences were not attributable to the presence of substantial numbers of heat-resistant spores in the FTG medium culture of E13, since microscopic inspection did not reveal the presence of any spores in FTG medium cultures of either E13 or F5603. Furthermore, the differences in heat sensitivity between E13 and F5603 shown in Fig. 1 were not confined to 55 C. The D value of E13 remained approximately twice that of F5603 at 57 C (data not shown). At even higher temperatures (59 or 61 C), both strains died too quickly to reliably measure D values. Additional heating experiments (Table 1) revealed that the higher D values exhibited by E13 than by F5603 (Fig. 1) reflect a general pattern whereby the vegetative cells of plasmid cpe isolates are consistently more heat sensitive than the vegetative cells of chromosomal cpe isolates. Although the 13 isolates characterized in Table 1 (including E13 and F5603) vary considerably with respect to their geographic origins and dates of isolation, the D values measured at 55 C for the six C. perfringens chromosomal cpe isolates surveyed were, on average, twice the D values of the seven C. perfringens isolates carrying a plasmid cpe gene surveyed (the differences are statistically significant at P 0.05). It is particularly notable that the D value determined for vegetative cells of even the most heatsensitive chromosomal cpe isolate was higher than the vegetative cell D value of the most heat-resistant plasmid cpe isolate; i.e., there was no overlap between the heat sensitivities of vegetative cells of C. perfringens isolates carrying a chromosomal cpe gene and the heat sensitivities of vegetative cells of C. perfringens isolates carrying a plasmid cpe gene. The differences in D values between chromosomal and plasmid cpe isolates shown in Table 1 were not confined to 55 C, since similar differences were also detected at 57 C (data not shown). At even higher temperatures, death occurred too rapidly to reliably measure D values. Microscopic examination confirmed that only vegetative cells were present in FTG medium cultures of each isolate shown in Table 1. Consistent with this obser-

VOL. 66, 2000 HEAT SENSITIVITY OF ENTEROTOXIGENIC C. PERFRINGENS 3237 FIG. 2. Thermal death curves for sporulating cultures of strain E13, which carries a chromosomal cpe gene, and strain F5603, which carries a plasmid cpe gene. Heat-shocked DS medium cultures of E13 and F5603 were heated at 100 C for specified times, and the number of viable spores per milliliter of each culture was determined (see Materials and Methods). The results are the results of representative experiments; these results were highly reproducible (data not shown). vation, no colonies were obtained when FTG medium cultures of each isolate were heat shocked at 75 C for 20 min before plating on BHI agar plates and overnight anaerobic incubation at 37 C. Comparison of the heat sensitivities of spores produced by C. perfringens isolates carrying chromosomal versus plasmid cpe genes. Having established that the vegetative cells of the chromosomal cpe isolates surveyed consistently exhibited greater heat resistance than the vegetative cells of the plasmid cpe isolates surveyed exhibited (Fig. 1 and Table 1), we then performed experiments to evaluate the heat sensitivities of spores produced by C. perfringens isolates carrying a chromosomal cpe gene or a plasmid cpe gene. Representative thermal death curves obtained at 100 C for heat-shocked DS medium cultures of isolates E13 and F5603 are shown in Fig. 2. The results shown in Fig. 2 clearly indicate that spores of E13, a chromosomal cpe isolate, had a D value of 30 min at 100 C, while spores of F5603, a plasmid cpe isolate, had a D value of only 0.6 min. The D value for E13 spores was also significantly higher than the D value for F5603 spores at 90 C (data not shown). Phase-contrast microscopy analysis confirmed that high levels of spores were present in heat-shocked DS medium cultures of both E13 and F5603. To evaluate whether the observed differences in D values between spores of E13 and spores of F5603 reflect a pattern whereby spores of chromosomal cpe isolates are typically more heat resistant than spores of plasmid cpe isolates, D values were determined at 100 C for heat-shocked DS medium cultures of four other food poisoning isolates carrying a chromosomal cpe gene and six other isolates carrying a plasmid cpe gene (Table 2). As Table 2 shows, spores produced by the chromosomal cpe isolates surveyed had, on average, approximately 60-fold-higher D values at 100 C than spores produced by isolates carrying a plasmid cpe gene had (the differences were statistically significant at P 0.01). Furthermore, the D value for spores produced by even the most heat-sensitive chromosomal cpe isolate was still at least 15-fold higher than the D value for spores produced by the most heat-resistant plasmid cpe isolate; i.e., no overlap was detected between the FIG. 3. RFLP analysis of NruI-digested total DNA isolated from strain E13, which carries a chromosomal cpe gene, and strain F5603, which carries a plasmid cpe gene. Southern blots were probed with a 639-bp DIG-labeled cpe-specific fragment. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) or DS medium (Spores) cultures of E13 are results obtained for survivors of heating for 20 min at 55 C and heating for 60 min at 100 C, respectively. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) or DS medium (Spores) cultures of F5603 are results obtained for survivors of heating for 10 min at 55 C and heating for 1 min at 100 C, respectively. The molecular sizes (in kilobase pairs [Kb]) of the DNA markers are indicated in the center. heat sensitivities of spores produced by chromosomal and plasmid cpe isolates. The differences in heat sensitivity between chromosomal and plasmid cpe isolates shown in Table 2 were not confined to 100 C; at 90 C, spores of chromosomal cpe isolates also exhibited significantly higher D values than spores of plasmid cpe isolates (data not shown). Phase-contrast microscopy analysis confirmed that moderate to high levels of spores were present in DS medium cultures of all of the isolates tested. RFLP genotyping of heat experiment survivors. To assess if exposure to high temperatures induced loss of the cpe plasmid or changed cpe genotypes (i.e., converted plasmid cpe isolates to chromosomal cpe isolates by driving the plasmid cpe gene onto the chromosome), survivors of our Table 1 and 2 heating experiments were subjected to NruI RFLP analysis, a reliable presumptive test for distinguishing between chromosomal and plasmid cpe genes (10, 11). Briefly, the basis for this assay is that the cpe sequences of isolates carrying a chromosomal cpe gene localize to 5-kb NruI DNA fragments but are present on NruI-digested DNA fragments that are more than 20 kb long in isolates carrying a plasmid cpe gene. Consistent with previous reports (10, 11), the cpe gene was present on NruI fragments that were more than 20 kb long in all control (unheated) plasmid cpe isolates used in this study (Fig. 3 shows representative RFLP blot results for isolate F5603 prior to heating). When similar RFLP analyses were performed with colonies of cpe plasmid isolates that had survived heating as either spores or vegetative cells, the NruIdigested DNA from all survivors were still able to hybridize to a cpe-specific gene probe (Fig. 3 shows representative results for heat-exposed F5603 survivors). In contrast, that same cpe probe did not hybridize (data not shown) to NruI-digested DNA isolated from ATCC 3624, a cpe-negative strain (12). These results indicate that the heating conditions used in the

3238 SARKER ET AL. APPL. ENVIRON. MICROBIOL. FIG. 4. PFGE-Southern hybridization analysis of undigested genomic DNA from strain E13, which carries a chromosomal cpe gene, and strain F5603, which carries a plasmid cpe gene. The blot was probed with a 639-bp DIG-labeled cpe-specific fragment. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) or DS medium (Spores) cultures of E13 are results obtained for survivors of heating for 20 min at 55 C and heating for 60 min at 100 C, respectively. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) or DS medium (Spores) cultures of F5603 are results obtained for survivors of heating for 10 min at 55 C and heating for 1 min at 100 C, respectively. The molecular sizes (in kilobase pairs [kb]) of the DNA markers are indicated between the two blots. The cpe probe reactive material appearing at the top of all gel lanes was found previously with both chromosomal and plasmid cpe isolates subjected to PFGE-Southern hybridization analyses (9 11); this material apparently was cpe-containing DNA that was trapped in gel wells. Table 1 and 2 experiments did not induce loss of the cpe gene from plasmid cpe isolates that survived heating. Furthermore, the cpe RFLP results shown in Fig. 3 also indicate that DNA from all of the plasmid cpe isolates that survived the Table 1 and 2 heating experiments, whether they were heated as vegetative cells or as spores, still carried the cpe gene on an NruI fragment that was more than 20 kb long (Fig. 3 shows representative results for isolate F5603). These results strongly suggest that the cpe gene remained on a plasmid in plasmid cpe isolates that survived heating; i.e., heating did not induce migration of the plasmid cpe gene to the chromosome. Similar cpe RFLP experiments were also performed to evaluate whether heat induced loss of the cpe gene from chromosomal cpe isolates. These experiments (Fig. 3 shows representative RFLP results for isolate E13) indicated that the cpe gene localizes to a 5-kb NruI fragment in chromosomal cpe isolates both before and after heating; i.e., heating did not result in loss of the chromosomal cpe gene or induce migration of this gene to a plasmid. PFGE genotyping analysis of selected heating experiment survivors. The cpe RFLP genotyping results presented above strongly suggested that heating does not induce migration of the plasmid cpe gene to the chromosome. To confirm these findings, selected survivors of Table 1 and 2 heating experiments were also genotyped by performing a PFGE Southern blot analysis (Fig. 4). The basis of this assay is that because of the relatively smaller size of plasmid DNA than of chromosomal DNA, some cpe-containing DNA can enter pulsed-field gels when it is located on a plasmid but not when it is located on the chromosome. PFGE genotyping experiments showed that prior to heat exposure, some cpe-containing DNA from isolate F5603 entered the gel, which is consistent with results of previous genotyping studies (10) which indicated that this isolate carries a plasmid cpe gene. After heating, whether as vegetative cells or as spores, some cpe-containing DNA of F5603 was still able to enter the pulsed-field gels, and this cpe DNA comigrated with the cpe-containing plasmid DNA from unheated cultures of F5603. These results supported the RFLP results shown in Fig. 3 by confirming that the cpe gene was retained on a plasmid in F5603 survivors of both Table 1 and Table 2 heating experiments. Similar PFGE Southern blot results were also obtained with heated and control cultures of F4969, which also carries a plasmid cpe gene (data not shown). When unheated cultures of isolate E13 were similarly genotyped by PFGE (Fig. 4), no cpe-containing DNA entered the pulsed-field gels, indicating that this isolate carries a chromosomal cpe gene. Heating did not induce migration of the cpe gene of E13 to a plasmid, since no cpe-containing DNA from survivors of this strain, whether heated as spores or as vegetative cells, entered pulsed-field gels in the Fig. 4 experiments. Similar PFGE Southern blot results were also obtained with heated and control cultures of NCTC 10239, which carries a chromosomal cpe gene (data not shown). Western immunoblot analysis of CPE expression by C. perfringens colonies that survived heating. While our RFLP- PFGE Southern blot results confirmed that the cpe gene was present at its original chromosomal or plasmid location in all survivors of Table 1 and 2 heating experiments, it was still possible that heating might affect an isolate s ability to express CPE. To evaluate this possibility, survivors of Table 1 and 2 heating experiments were characterized by performing a CPEspecific Western immunoblot analysis. Consistent with previous reports demonstrating that CPE expression is strongly associated with sporulation (12, 13), CPE-specific Western immunoblotting did not reveal any CPE expression by vegetative cultures of control (unheated) cpe-positive isolates (data not shown). However, all control cpe-positive strains did produce CPE (Fig. 5 shows representative results for isolates F5603 and E13) when they were grown in DS sporulation medium. In similar CPE-Western immunoblot analyses we detected CPE expression by all survivors of Table 1 and 2 heating experiments when these survivors were grown in DS medium (Fig. 5 shows representative results) but not when they were grown in FTG medium (data not shown); i.e., heating did not induce loss of CPE expression. DISCUSSION The major contribution of this study was to provide evidence which suggests that vegetative cells and spores of C. perfringens food poisoning isolates carrying a chromosomal cpe gene are significantly more heat resistant than vegetative cells and spores of C. perfringens isolates carrying a plasmid cpe gene are. Several previous surveys (1, 23) have revealed differences in heat sensitivity between spores produced by different C. perfringens isolates. Furthermore, in at least one previous study (1) the workers reported that C. perfringens food poisoning isolates are typically more heat resistant than CPE-negative C. perfringens isolates are. However, to our knowledge, our study is the first study to directly compare and note differences between the heat sensitivities of C. perfringens isolates carrying a chromosomal cpe gene and C. perfringens isolates carrying a plasmid cpe gene. The importance of the differences in heat resistance detected in our study is supported by the fact that no heat-induced effects on possession of the cpe plasmid, cpe genotype, or CPE expression were observed in this study. How could possession of heat resistance explain, at least in

VOL. 66, 2000 HEAT SENSITIVITY OF ENTEROTOXIGENIC C. PERFRINGENS 3239 heat resistance are interesting and clearly deserve further study. If additional studies confirm that most food poisoning isolates carry a chromosomal cpe gene and possess heat resistance, then it would be important to determine whether the heat resistance properties of food poisoning isolates and a chromosomal cpe gene are acquired separately or if acquisition of these two virulence traits is somehow linked. The physiologic mechanism(s) responsible for the differences in heat resistance between chromosomal and plasmid cpe isolates also deserves study. For example, might some gene(s) on the cpe plasmid confer heat sensitivity? ACKNOWLEDGMENTS FIG. 5. CPE Western immunoblot analysis of sporulating cultures of unheated and heated C. perfringens isolates. The representative results shown are the results obtained for DS medium lysates of strain E13 (which carries a chromosomal cpe gene) and strain F5603 (which carries a plasmid cpe gene), which were analyzed for CPE expression by an immunoblot analysis performed with CPE antiserum. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) and DS medium (Spores) cultures of E13 are results obtained for survivors of heating for 20 min at 55 C and heating for 60 min at 100 C, respectively. The representative results shown for heated (lanes H) and unheated (lanes U) FTG medium (Veg) or DS medium (Spores) cultures of F5603 are results obtained for survivors of heating for 10 min at 55 C and heating for 1 min at 100 C, respectively. The molecular sizes of the protein markers (in kilodaltons) are indicated on the left. part, the strong association between chromosomal cpe isolates and C. perfringens type A food poisoning? If vegetative cells and spores of chromosomal cpe isolates typically possess greater heat resistance than vegetative cells and spores of plasmid cpe isolates possess, this fact should favor survival of the chromosomal cpe isolates in improperly warmed or incompletely cooked foods. Enhanced survival under inadequate warming or incomplete cooking conditions would be a highly desirable trait for a C. perfringens food poisoning isolate given that (i) cooked meat and poultry products, as well as cooked meat stews, are the major food vehicles for C. perfringens type A food poisoning (2, 3) and (ii) improper holding temperatures and incomplete cooking of foods are recognized as major contributing factors for the development of 75 to 100 and 20 to 50% of C. perfringens type A food poisoning outbreaks, respectively (2, 3). The differences in heat sensitivity between chromosomal and plasmid cpe isolates detected in this study could also contribute to our understanding of the pathogenesis of C. perfringens type A food poisoning. While CPE expression appears to be necessary for a C. perfringens isolate to cause food poisoning (19 21), previous observations indicating that plasmid cpe isolates rarely, if ever, cause food poisoning imply that the presence of a cpe gene is not enough to cause food poisoning. If this finding is coupled with our results suggesting that chromosomal cpe isolates linked to food poisoning possess greater heat resistance than plasmid cpe isolates possess, heat resistance also appears to be a potentially important virulence trait for C. perfringens food poisoning isolates. Of course, C. perfringens isolates with a plasmid cpe gene and low heat resistance might occasionally cause food poisoning outbreaks, particularly when uncooked foods or cooked foods kept under grossly inadequate conditions are involved. The observations suggesting that C. perfringens food poisoning isolates typically possess both a chromosomal cpe gene and This research was supported by USDA grant 9802822 from the Ensuring Food Safety Research Program and by Public Health Service grant AI 19844-17 from the National Institute of Allergy and Infectious Diseases (both to B.A.M.). We thank Jared Kerr at the Centers for Disease Control and Prevention for providing isolate 191-10. REFERENCES 1. Ando, Y., T. Tsuzuki, H. Sunagawa, and S. Oka. 1985. Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens. Microbiol. Immunol. 29:317 326. 2. Bean, N. H., J. S. Goulding, C. Lao, and F. J. Angulo. 1996. 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