Search

Clostridium perfringens type F is a spore-forming bacterium that causes human illnesses, including food poisoning (FP) and non-foodborne gastrointestinal diseases. In this study, we evaluated the antimicrobial activities of 15 natural products against C. perfringens spore growth. Among them, garlic, onion juice, and undiluted essential oil constituents (EOCs) of clove, rosemary, and peppermint showed the strongest activity. Therefore, we examined the inhibitory effects of these products on each stage of the life cycle of C. perfringens FP strains, including spore germination, spore outgrowth, and vegetative growth, in laboratory media and chicken meat. Both clove and peppermint oils (at 0.5%; v/v) inactivated C. perfringens spore germination in nutrient-rich trypticase–glucose–yeast extract (TGY) medium. Furthermore, EOCs at (0.1–0.5%) arrested the spore outgrowth of C. perfringens in TGY medium. Interestingly, EOCs at 0.5% completely inhibited the vegetative growth of FP isolates during a 6 h incubation in TGY medium. However, even at 4-fold higher concentrations (2%), EOCs were unable to inactivate C. perfringens spore growth in contaminated chicken meat stored under abusive conditions. Although some of the natural products inhibited C. perfringens spore germination, outgrowth, and vegetative growth in TGY medium, no such inhibitory activity was observed when these products were applied to C. perfringens spore-inoculated chicken meat. [ABSTRACT FROM AUTHOR]

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, β-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not β-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

The prevalence of Clostridium perfringens spores in the environment is crucial for the pathogenesis of this bacterium because these dormant spores, upon contact with a suitable host, can return to active growth to cause disease. We evaluated the prevalence of C. perfringens spores in the Hail and Qassim regions of Saudi Arabia. Methods: A plating method was used to identify C. perfringens. PCR analyses and DNA sequencing were performed for genotypic characterization of the newly isolated C. perfringens.25 of 300 collected samples were identified as C. perfringens by selecting black colonies on selective media and monitoring b-hemolysis on blood agar plate. PCR analyses showed that all 25 isolates carry a-toxin gene (plc), but not the enterotoxin gene (cpe), further confirming that these isolates are indeed cpe-negative. Genome sequencing found that Saudi isolates are genotypically similar to the American and European isolates; no homologues of toxinotyping genes encoding b-, i-, e- and NetB-toxin were detected in 22 Saudi isolates, with the exception of 3 isolates that carry only e-toxin gene etx. Collectively, our findings suggest that C. perfringens are highly prevalent in the Hail and Qassim environment, with 22 (88%) of 25 isolates are type A and remaining 3 (12%) are type D. [ABSTRACT FROM AUTHOR]

The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment, cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of [Ca.sup.2+] and dipicolinic acid (DPA); the viability of these spores was ~[10.sup.4]-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants. DOI 10.1099/mic.0.030965-0

The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations ([K.sup.+] and/or [Na.sup.+]) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, L-asparagine, and a 1:1 chelate of [Ca.sup.2+] and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli [Na.sup.+]/[H.sup.+] antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in [K.sup.+] uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.

Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two Clostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of C. perfringens spores. C. perfringens sleC spores did not germinate completely with nutrients, KCl or a 1:1 chelate of [Ca.sup.2+] and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleC spores was [10.sup.3]-fold lower than that of wild-type spores. However, sleC spores incubated with various germinants released most of their DPA, although slower than wild-type or sleM spores, and DPA release from sleC sleM spores was very slow. In contrast, germination and viability of sleM spores were similar to that of wild-type spores, although sleC sleM spores had [10.sup.5]-fold-lower viability. These results allow the following conclusions about C. perfringens spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.

Clostridium difficile is an emerging nosocomial pathogen and one of the major causes of antibiotic-associated diarrhoea. Cases of Clostridium difficile-associated disease (CDAD) are likely initiated by the ingestion of dormant C. difficile spores, which then germinate, outgrow and rapidly proliferate to cause gastrointestinal (GI) infections. To understand the initial stages of CDAD pathogenesis, we have characterized the germination of spores from a collection of C. difficile strains, including some clinical isolates obtained from a CDAD outbreak (CDAD isolates). Spores of one laboratory strain and five CDAD isolates did not germinate with amino acids, but did germinate on a nutrient-rich medium. However, bile salts had little effect on spore germination, either alone or in a nutrient-rich medium. These spores also germinated with KCI, as well as the non-nutrient germinants dodecylamine and a 1 : 1 chelate of [Ca.sup.2+] and dipicolinic acid. An unexpected finding was that spores of most of the C. difficile strains also germinated with inorganic phosphate ([P.sub.i]) with a pH optimum of 6. The in vitro germination of spores of CDAD strains with KCI and [P.sub.i], two molecules present at significant levels in the GI tract, suggests that C. difficile spores germinate in the human body by sensing [P.sub.i] in the early segments of the duodenum and KCI in the colon.

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of [Ca.sup.2+] and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, [Ca.sup.2+] and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of L-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of [Ca.sup.2+] and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective [alpha]/[beta]-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCI was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of [Ca.sup.2+] and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human-and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA (catabolite control protein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens.

[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].sup.-] and [[alpha].sup.-][[beta].bar].sup.-] strains, respectively). Ssp2 restored some of the resistance of [[alpha].sup.-][[beta].bar].sup.-] spores to UV and nitrous acid and of [[alpha].sup.-] spores to dry heat. Ssp2 also restored much of the resistance of [[alpha].sup.-] spores to nitrous acid and restored full resistance of [[alpha].sup.-] spores to UV and moist heat. These results further indicate the interchangeability of [alpha]/[beta]-type SASP in DNA protection in spores.

Antisense RNA is used to decrease the levels of [alpha]/[beta]-type small, acid-soluble spore proteins (SASP) in Clostridium perfringens spores, which in turn have reduced the resistance to both moist heat and ultraviolet (UV) radiation but not to dry heat. The results have highlighted the [alpha]/[beta]-type SASP's role in the resistance of Clostridium perfringens spores.

The 14 cpe(super +) Clostridium perfringens fecal isolates associated with human sporadic diarrhea (SD) in England is characterized to test the association of beta2 toxin (CPB2) with human gastrointestinal GI diseases. The results have shown that all C. perfringens plasmid cpe isolates carry cpb2 and that there is a positive correlation between cpb2 transcription levels and the amount of CPB2 produced by cpe(super +)/cpb2(super +) C. perfringens human SD isolates.

Isolates of Clostridium perfringens that have a chromosomal gene for enterotoxin are more resistant to heat than those that have a plasmid gene for enterotoxin. This may explain why isolates with the chromosomal gene are often linked to food poisoning whereas those with the plasmid gene are not.

A complete deletion of lcrV in the pYV plasmid of Yersinia enterocolitica E40 was genetically engineered and the phenotype of the mutant was characterized to elucidate the participation of LcrV in the Yop virulon. Results of the complementation studies provided evidence that the mutation was not polar with respect to yopB and yopD. However, the mutation eliminated the secretion of YopB and YopD, whereas the secretion of the other Yops was not influenced and even showed an increase.

To the Editor: The increased severity of Clostridium difficile infection is primarily attributed to the appearance of an epidemic strain characterized as PCR ribotype 027 (1). The only report that [...]

Abstract 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. Results Our current study demonstrated the presence of all three SASP-encoding genes (ssp1, 2 and 3) in five surveyed C. perfringens clinical food poisoning isolates. β-Glucuronidase assay showed that these ssp genes are expressed specifically during sporulation. Consistent with these expression results, our study also demonstrated the production of SASPs by C. perfringens food poisoning isolates. When the heat sensitivities of spores produced by a ssp3 knock-out mutant of a C. perfringens food poisoning isolate was compared with that of spores of the wild-type strain, spores of the ssp3 mutant were found to exhibit a lower decimal reduction value (D value) at 100°C than exhibited by the spores of wild-type strain. This effect was restored by complementing the ssp3 mutant with a recombinant plasmid carrying wild-type ssp3, suggesting that the observed differences in D values between spores of wild-type versus ssp3 mutant was due to the specific inactivation of ssp3. Furthermore, our DNA protection assay demonstrated that C. perfringens SASPs can protect DNA from DNase I digestion. Conclusion The results from our current study provide evidences that SASPs produced by C. perfringens food poisoning isolates play a role in protecting their spores from heat-damage, which is highly significant and relevant from a food safety perspective. Further detailed studies on mechanism of action of SASPs from C. perfringens should help in understanding the mechanism of protection of C. perfringens spores from heat-damage.