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Applied and Environmental Microbiology, August 2000, p. 3234-3240, Vol. 66, No. 8
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
190382
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.
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 heat can
cure the cpe plasmid or affect either CPE expression or the
cpe genotype.
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.
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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
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Vegetative cell heat resistance of
cpe-positive C. perfringens isolatesa
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 2 h at 37°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 107) 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).
2 to 107) 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 102 to
107. 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).
2 to
107) 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-µl 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 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).
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Statistical analyses. Statistical analyses were performed with Student's t test.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 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.
|
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.
|
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 NruI-digested 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 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.
|
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.
|
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 CPE-specific 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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 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 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?
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* 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{at}pop.pitt.edu.
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Abstract
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Materials and Methods
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Discussion
References
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Li, J., Sayeed, S., McClane, B. A.
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Raju, D., Setlow, P., Sarker, M. R.
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Harrison, B., Raju, D., Garmory, H. S., Brett, M. M., Titball, R. W., Sarker, M. R.
(2005). Molecular Characterization of Clostridium perfringens Isolates from Humans with Sporadic Diarrhea: Evidence for Transcriptional Regulation of the Beta2-Toxin-Encoding Gene. Appl. Environ. Microbiol.
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Waters, M., Raju, D., Garmory, H. S., Popoff, M. R., Sarker, M. R.
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Margosch, D., Ganzle, M. G., Ehrmann, M. A., Vogel, R. F.
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