Production of small, acid-soluble spore proteins in Clostridium perfringens nonfoodborne gastrointestinal disease isolates

The molecular basis for the differences in heat resistance between spores of Clostridium perfringens food-borne versus nonfoodborne isolates remains unknown. Since a recent study demonstrated the role of small, acid-soluble spore proteins (SASPs) in heat resistance of spores of food-borne isolates, in the current study, we evaluated the expression of SASP-encoding genes (ssp) and the production of SASPs in nonfoodborne isolates. Our results demonstrated the presence of all three ssp genes in five surveyed nonfoodborne isolates. A beta-glucuronidase assay showed that these ssp genes are expressed specifically during sporulation. Furthermore, nonfoodborne isolate F4969 produced SASPs at a level similar to that of food-borne isolate SM101. Collectively, these results suggest that the difference in the levels of heat resistance between spores of food-borne and the nonfoodborne isolates is not the result of impaired expression of ssp genes and (or) decreased production of SASPs in nonfoodborne isolates.     

Ribotyping for strain characterization of Clostridium perfringens isolates from food poisoning cases and outbreaks.

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases.     

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

Background  

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

Effect of a small, acid-soluble spore protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores

Alpha/beta-type small, acid-soluble spore proteins (SASP) are essential for the resistance of DNA in spores of Bacillus species to damage. An alpha/beta-type SASP, Ssp2, from Clostridium perfringens was expressed at significant levels in B. subtilis spores lacking one or both major alpha/beta-type SASP (alpha- and alpha- beta- strains, respectively). Ssp2 restored some of the resistance of alpha- beta- spores to UV and nitrous acid and of alpha- spores to dry heat. Ssp2 also restored much of the resistance of alpha- spores to nitrous acid and restored full resistance of alpha- spores to UV and moist heat. These results further indicate the interchangeability of alpha/beta-type SASP in DNA protection in spores.     

Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens

Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type). The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.     

Use of plasmid profiling as a typing method for epidemiologically related Clostridium perfringens isolates from food poisoning cases and outbreaks

Plasmid profiling was used for the characterization of Clostridium perfringens isolates involved in disease outbreaks. The usefulness of this technique was demonstrated by the retrospective examination of food and patient isolates from 10 cases and outbreaks from 1984 to 1991. The origin of three outbreaks could be clearly confirmed due to identical plasmid profiles in all isolates. In one outbreak identical plasmid patterns were found between one food and one patient isolates, while one plasmid was missing in the second patient isolate. In an additional two cases a relationship between food and patient isolates is likely, if the possibility of the loss of one plasmid in one of the isolated strains is considered. In one outbreak two faecal isolates could be related to an isolate from one of the two foods implicated as outbreak source; isolates from the other food and a third faecal sample could not be linked to any other isolate. The results from three outbreaks were largely inconclusive because plasmids were not present either in all or in some of the isolates.     

Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat

Background  

Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates.     

Pathogenesis of Hobbs' heat-sensitive spore forming Clostridium perfringens type A strain.

Food poisoning in man due to heat-sensitive strains of Cl. perfringens type A appeared to be mediated through enterotoxin synthesized in vivo during sporulation. A minimum of 2.0 X 10(5) vegetative cells suspended in sporulation medium was sufficient to elicit gut-loop response in rabbits. The functional disturbance in the gut as well as the structural changes were progressive and proportional to the size of the inoculum up to a dose limit of 2.0 X 10(7) vegetative cells and beyond this the changes remained steady.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

A recombinant C-terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity

Clostridium perfringens enterotoxin (CPE) is believed to be involved in several important gastrointestinal illnesses. Recent studies have identified a number of distinct molecular events which occur after CPE treatment of eukaryotic cells or isolated membranes. Additional studies are underway to determine the temporal order and intrinsic importance of each CPE event for cytotoxicity. We now demonstrate that a truncated CPE fragment binds to membranes, but is unable to insert into membranes or cause any other subsequent post-insertion event. This is the first experimental evidence supporting the importance of membrane insertion for CPE cytotoxicity. Binding of the CPE fragment is also shown to be irreversible, strongly suggesting that the irreversible binding of wild-type CPE is not due solely to insertion of CPE into membranes.     

Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning

An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.     

Kinetics of spore coat protein synthesis byClostridium perfringens type A

Spore coat proteins of several strains ofClostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent^–) strain of this organism. The results indicated that spore coat proteins from many strains ofC. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulationspecific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393–410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.     

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica

Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments.     

Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods

Currently there is only limited understanding of the reservoirs for Clostridium perfringens type A food poisoning. A recent survey (Y.-T. Lin and R. Labbe, Appl. Environ. Microbiol. 69:1642-1646, 2003) of non-outbreak American retail foods did not identify the presence of a single C. perfringens isolate carrying the enterotoxin gene (cpe) necessary for causing food poisoning. The present study revisited this issue, using revised methodology and food sampling strategies. In our survey, cpe-positive C. perfringens isolates were detected in approximately 1.4% of approximately 900 surveyed non-outbreak American retail foods. Interestingly, those enterotoxigenic isolates in non-outbreak foods appear indistinguishable from C. perfringens isolates known to cause food poisoning outbreaks: i.e., the enterotoxigenic retail food isolates all carry a chromosomal cpe gene, are classified as type A, and exhibit exceptional heat resistance. Collectively, these findings indicate that some American foods are contaminated, at the time of retail purchase, with C. perfringens isolates having full potential to cause food poisoning. Furthermore, demonstrating that type A isolates carrying a chromosomal cpe gene are the enterotoxigenic isolates most commonly present in foods helps to explain why these isolates (rather than type A isolates carrying a plasmid cpe gene or cpe-positive type C or D isolates) are strongly associated with food poisoning outbreaks. Finally, since type A chromosomal cpe isolates present in the surveyed raw foods exhibited strong heat resistance, it appears that exceptional heat resistance is not a survivor trait selected for by cooking but is instead an intrinsic trait possessed by many type A chromosomal cpe isolates.     

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of Mr 18 000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 micrograms/kg with 0.10 micrograms corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60 degrees C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.     

Changes in protein synthesis and acid tolerance in Clostridium perfringens type A in response to acid shock.

The induction of acid-shock proteins and the degree of acid resistance conferred on Clostridium perfringens by acid shock, and the kinetics of this resistance were determined. A sublethal acid shock at pH 4.5 for 20 min increased the acid tolerance of cells at least fifteenfold. The acquired tolerance was maintained for 3 h after acid treatment. The response of the microorganism to acid shock was also examined by analysis of pulse-labeled proteins. Five acid-shock proteins (molecular weights 120, 84, 58, 45 and 17 kDa) were identified by polyacrylamide gel electrophoresis.     

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.