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Abstract C. perfringens type F isolates carrying enterotoxin gene (cpe) on the chromosome (C- cpe isolates) are mostly associated with food poisoning, while isolates carrying plasmid-borne cpe (P- cpe isolates) with non-food-borne gastrointestinal diseases. Spore germination is considered the most essential step for initiation of these diseases. Identifying the most effective germinants for spores of C- cpe and P- cpe isolates should help developing novel strategies involving induction of spore germination followed by inactivation of germinated spores with mild treatments. In this study, we showed that (i) l -lysine (pH 6.0) triggered germination of spores of all tested C- cpe and P- cpe isolates; although extremely low concentration of l -lysine (5–10 mM) induced germination of C- cpe spores, 10-fold higher concentration (50 mM) was required for P- cpe spore germination; (ii) P- cpe strain F4969 gerKC spores did not germinate, C- cpe strain SM101 gerKC spores germinated extremely poorly and these gerKC spores released significantly less DPA as compared to wild type spores; and these defects were restored to a nearly wild-type level by complementing gerKC spores with wild-type gerKC ; and (iii) F4969 gerAA spores also did not germinate, and released less DPA than wild-type spores in presence of l -lysine (pH 6.0); and these defects were restored partially (germination) and fully (DPA release) by complimenting gerAA spores with wild-type gerAA. Collectively, our current study identified l -lysine as a universal germinant for spores of both C- cpe and P- cpe isolates and provided evidence that GerKC (from SM101 or F4969) and F4969 GerAA play major roles in l -lysine-induced germination. Highlights • l -lysine (pH 6.0) is a universal germinant for spores of C -cpe and P -cpe isolates. • GerKC is the main GR protein involved in l -lysine germination of spores of C- cpe and P- cpe isolates. • GerAA plays a role in l -lysine germination of spores of P- cpe isolates. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a spore-forming bacterium and a major cause of bacterial food-borne illness. In this study, we evaluated the inhibitory effects of chitosan against spore germination, spore outgrowth and vegetative growth of C . perfringens food poisoning (FP) isolates. Chitosan of differing molecular weights inhibited germination of spores of all tested FP isolates in a KCl germinant solution containing 0.1 mg/ml chitosan at pH 4.5. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C . perfringens spores in Tripticase-yeast extract-glucose (TGY) medium. Furthermore, chitosan (1.0 mg/ml) was bacteriostatic against vegetative cells of C . perfringens in TGY medium. Although chitosan showed strong inhibitory activities against C . perfringens in laboratory medium, higher levels (2.0 mg/g) were required to achieve similar inhibition of spores inoculated into chicken meat. In summary, the inhibitory effects of chitosan against C . perfringens FP isolates was concentration dependent, and no major difference was observed when using different molecule weight chitosan as an inhibitor. Our results contribute to a better understanding on the potential application of chitosan in cooked meat products to control C . perfringens -associated disease. [ABSTRACT FROM AUTHOR]

Abstract: Clostridium perfringens is the causative agent of a variety of histotoxic infections in humans and animals. Studies on the early events of C. perfringens infections have been largely focused on the interactions between their vegetative cells and macrophages. Consequently, in the current study we have examined the interactions between C. perfringens spores and Raw 264.7 macrophages. Raw 264.7 cells were able to interact and phagocytose Clostridium perfringens spores of a food poisoning isolate, strain SM101, and a non-food borne isolate, strain F4969, albeit to different extents. Phagocytosis and to a lesser extent, association, of C. perfringens spores by Raw 2647 macrophages was completely inhibited in presence of cytochalasin D. Complement increased association and phagocytosis of C. perfringens spores by Raw 264.7 macrophages. Survival of C. perfringens spores during macrophage infection seems to depend on the ability of spore germination during infection as: (i) F4969 spores germinated during infection with Raw 264.7 macrophages and subsequently killed by macrophages; and (ii) SM101 spores remained dormant inside Raw 264.7 macrophages and thus survived up to 24 h of infection. The in vitro spore-resistance factors, α/β-type SASP, SpmA/B proteins and spore’s core water content, seems to play no role in mediating SM101 spore-resistance to macrophages. Collectively, these results might well have implications in understanding the initial stages of infections by C. perfringens spores. [Copyright &y& Elsevier]

Abstract: Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host’s intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p < 0.01) increase in heat-resistant spore titers. In C. perfringens, the highest spore germination response observed with the HT-29 cell line, might be in part, due to the expression of germination factor with peptidoglycan cortex hydrolysis activity by HT-29 cells. Collectively, these findings might well have implications in understanding the mechanism of clostridial spore germination in vivo. [Copyright &y& Elsevier]

Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l -glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca2+ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~106-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis. • CspA and cspC genes are expressed as a bi-cistronic operon in strain SM101. • CspA and CspC are required for SM101 spore germination. • CspA and CspC are required for normal DPA release during SM101 spore germination. • CspA and CspC are required for SM101 spore germination with exogenous Ca2+-DPA. • CspA and CspC are required for processing of pro-SleC into active SleC in SM101 spores. [ABSTRACT FROM AUTHOR]

Clostridium difficile is frequently found in meat and meat products. Germination efficiency, defined as colony formation, was previously investigated at temperatures found in meat handling and processing for spores of strain M120 (animal isolate), R20291 (human isolate), and DK1 (beef isolate). In this study, germination efficiency of these spore strains was assessed in phosphate buffered saline (PBS, a w ∼1.00), commercial beef jerky ( a w ∼0.82/0.72), and a w -adjusted PBS ( a w ∼0.82/0.72). Surface hydrophobicity was followed for spores stored in PBS. After three months and for all PBS a w levels tested, M120 and DK1 spores showed a ∼1 decimal reduction in colony formation but this was not the case when kept in beef jerky suggesting a protective food matrix effect. During storage, and with no significant a w effect, an increase in colony formation was observed for R20291 spores kept in PBS (∼2 decimal log increase) and beef jerky (∼1 decimal log increase) suggesting a loss of spore superdormancy. For all strains, no significant changes in spore surface hydrophobicity were observed after storage. Collectively, these results indicate that depending on the germination properties of C . difficile spores and the media properties, their germination efficiency may increase or decrease during long term food storage. [ABSTRACT FROM AUTHOR]

Abstract: The contamination of enterotoxigenic Clostridium perfringens spores on food contact surfaces posses a serious concern to food industry due to their high resistance to various preservation methods typically applied to control foodborne pathogens. In this study, we aimed to develop an strategy to inactivate C. perfringens spores on stainless steel (SS) surfaces by inducing spore germination and killing of germinated spores with commonly used disinfectants. The mixture of l-Asparagine and KCl (AK) induced maximum spore germination for all tested C. perfringens food poisoning (FP) and non-foodborne (NFB) isolates. Incubation temperature had a major impact on C. perfringens spore germination, with 40 °C induced higher germination than room temperature (RT) (20 ± 2 °C). In spore suspension, the implementation of AK-induced germination step prior to treatment with disinfectants significantly (p < 0.05) enhanced the inactivation of spores of FP strain SM101. However, under similar conditions, no significant spore inactivation was observed with NFB strain NB16. Interestingly, while the spores of FP isolates were able to germinate with AK upon their adhesion to SS chips, no significant germination was observed with spores of NFB isolates. Consequently, the incorporation of AK-induced germination step prior to decontamination of SS chips with disinfectants significantly (p < 0.05) inactivated the spores of FP isolates. Collectively, our current results showed that triggering spore germination considerably increased sporicidal activity of the commonly used disinfectants against C. perfringens FP spores attached to SS chips. These findings should help in developing an effective strategy to inactivate C. perfringens spores adhered to food contact surfaces. [Copyright &y& Elsevier]

Abstract: Clostridium perfringens TpeL belongs to the family of large clostridial toxins. Although recent studies demonstrated the expression of tpeL during vegetative growth nothing is known about tpeL expression during sporulation. Our current study demonstrated that tpeL: (i) is also expressed during sporulation; (ii) expression is dependent on the master regulator of sporulation, Spo0A, and the sporulation-specific sigma factor, SigE; and (iii) is localized to the mother cell compartment of the sporulating cell. In summary, these results indicate that TpeL is a sporulation-regulated C. perfringens toxin. [Copyright &y& Elsevier]

Abstract: This study evaluated the effects of various polyphosphates (SPP, STPP, SAPP and TSPP) on growth, sporulation and spore germination of Clostridium perfringens, and germination and outgrowth of C. perfrinegns spores in poultry meat. We have found that the requirements of polyP (0.8–1.0%) to inhibit C. perfringens bacterial growth were higher than those reported for other bacteria. Sub-lethal concentrations of polyP significantly (p<0.01) inhibited sporulation of C. perfringens by reducing sporulating cells (heat-resistant cells) ∼5–6log10. While C. perfringens spores were able to germinate in the presence of 1% STPP, their outgrowth was significantly (p<0.01) inhibited. Finally, a significant (p<0.01) reduction of survival of C. perfringens was observed when meat samples contaminated with a cocktail of spores of C. perfringens isolates carrying enterotoxin gene on the chromosome were treated with 1% STPP. Collectively, this study demonstrated the inhibitory effects of polyP on growth, sporulation and spore outgrowth of C. perfringens, and suggests that polyP can be used not only as an enhancer of the functional properties of meat products, but also as a promising C. perfringens antimicrobial agent. [Copyright &y& Elsevier]

Abstract 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. Graphical abstract Image Highlights • An expanded C. perfringens toxinotyping scheme is presented. • Two new toxinotypes are proposed. • C. perfringens type F strains produce CPE, but not β, ε or ι toxins. • C. perfringens type G strains produce NetB. • A mechanism for the introduction of new toxinotypes is presented. [ABSTRACT FROM AUTHOR]

C. perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and non-food-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly heat-resistant spores, it is of great interest to develop strategies alternative to thermal processing to inactivate C. perfrinegens . Thus, in this study we evaluated the inhibitory effects of essential oil constituents ( EOCs ) (cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol) against germination, outgrowth and vegetative growth of spores of C. perfringens FP and NFB disease isolates in laboratory medium and chicken meat. The cinnamaldehyde, eugenol and carvacrol, but not AITC, all at 0.05–0.1%, inhibited the germination of spores of all tested C. perfringens isolates in Tripticase-glucose-yeast extract (TGY) medium. Furthermore, all tested EOCs at 0.05–0.1% arrested the outgrowth and vegetative growth of C. perfringens spores in TGY, with AITC and carvacrol being the most effective. However, among four tested EOCs , only AITC (at 0.5%–2.0%) was able to inhibit the growth of C. perfringens spores in chicken meat and no such inhibitory effect was observed even with a 10-fold higher concentration (5%) of carvacrol. In conclusion, our current work identified AITC as an effective EOC to control spores and vegetative cells of C. perfringens isolates in laboratory medium and chicken meat. Further studies on evaluating the effectiveness of different combination of EOCs against C. perfringens spore growth in different meat products should establish an effective use of EOCs to control the risk of C. perfringens -mediated illnesses. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin ( cpe ) gene (C- cpe ) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P- cpe ) are associated with non-food-borne gastrointestinal diseases. Both C- cpe and P- cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P- cpe isolates (P- cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P- cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P- cpe spores. P- cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA , respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P- cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination. [ABSTRACT FROM AUTHOR]

The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C. perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe . In vitro , the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe -harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe -null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe -complemented strain. In contrast, both wild-type and cpe -null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity. [ABSTRACT FROM AUTHOR]

This study evaluated the inhibitory effects of sorbate and benzoate against Clostridium perfringens type A food poisoning (FP) and non-food-borne (NFB) disease isolates. No significant inhibition of germination of spores of both FP and NFB isolates was observed in rich medium (pH 7.0) supplemented with permissive level of sodium sorbate (0.3% ≈ 0.13 mM undissociated sorbic acid) or sodium benzoate (0.1% ≈ 0.01 mM undissociated benzoic acid) used in foods. However, these levels of sorbate and benzoate effectively arrested outgrowth of germinated C. perfringens spores in rich medium. Lowering the pH of the medium increases the inhibitory effects of sorbate and benzoate against germination of spores of NFB isolates, and outgrowth of spores of both FP and NFB isolates. Furthermore, sorbate and benzoate inhibited vegetative growth of C. perfringens isolates. However, the permissible levels of these organic salts could not control the growth of C. perfringens spores in chicken meat stored under extremely abusive conditions. In summary, although sorbate and benzoate showed inhibitory activities against C. perfringens in the rich medium, no such effect was observed in cooked chicken meat. Therefore, caution should be taken when applying these organic salts into meat products to reduce or eliminate C. perfringens spores. [ABSTRACT FROM AUTHOR]

Clostridium perfringens spore germination plays a critical role in the pathogenesis of C. perfringens-associated food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Germination is initiated when bacterial spores sense specific nutrient germinants (such as amino acids) through germinant receptors (GRs). In this study, we aimed to identify and characterize amino acid germinants for spores of enterotoxigenic C. perfringens type A. The polar, uncharged amino acids at pH 6.0 efficiently induced germination of C. perfringens spores; l-asparagine, l-cysteine, l-serine, and l-threonine triggered germination of spores of most FP and NFB isolates; whereas, l-glutamine was a unique germinant for FP spores. For cysteine- or glutamine-induced germination, gerKC spores (spores of a gerKC mutant derivative of FP strain SM101) germinated to a significantly lower extent and released less DPA than wild type spores; however, a less defective germination phenotype was observed in gerAA or gerKB spores. The germination defects in gerKC spores were partially restored by complementing the gerKC mutant with a recombinant plasmid carrying wild-type gerKA-KC, indicating that GerKC is an essential GR protein. The gerKA, gerKC, and gerKB spores germinated significantly slower with l-serine and l-threonine than their parental strain, suggesting the requirement for these GR proteins for normal germination of C. perfringens spores. In summary, these results indicate that the polar, uncharged amino acids at pH 6.0 are effective germinants for spores of C. perfringens type A and that GerKC is the main GR protein for germination of spores of FP strain SM101 with l-cysteine, l-glutamine, and l-asparagine. [ABSTRACT FROM AUTHOR]

Abstract: Spores of Clostridium difficile are essential for infection, persistence and transmission of C. difficile infections (CDI). Proteins of the surface of C. difficile spores are thought to be essential for initiation and persistence of CDI. In this work, we demonstrate that three C. difficile collagen-like exosporium proteins (BclA) encoded in the C. difficile 630 genome are expressed during sporulation and localize to the spore via their N-terminal domains. Using polyclonal antibodies against the N- and C-terminal domains and full length BclA1 we demonstrate that BclA1 is likely to be localized to the exosporium layer, presumably undergoes post-translational cleavages and might be cross-linked with other exosporium proteins. The collagen-like region of recombinant BclA1 and BclA2 was susceptible to collagenase degradation. Collagenase digestion assay of C. difficile spores suggests that, similarly as in Bacillus anthracis BclA, the N-terminal domain and the C-terminal domain of BclA1 might be buried in the basal layer and oriented to the exosporium surface, respectively. We also demonstrate that the collagen-like BclAs proteins do not contribute to the spore hydrophobicity and its absence slightly increased the adherence of spores to Caco-2 cells. BclA1 was also shown to have poor immunogenic properties. These results provide the first study on the BclA1 collagen-like proteins of C. difficile spores. [Copyright &y& Elsevier]

Abstract: The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200MPa, 7min) and elevated temperature (80°C, 10min); spore germination at high temperatures (55, 60 or 65°C); and inactivation of germinated spores with elevated temperatures (80 and 90°C, 10 and 20min) and high pressure (586MPa, at 23 and 73°C, 10min). Low pressures (100–200MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80°C, 10min), and germinated at temperatures lethal for vegetative cells (≥55°C) when incubated for 60min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90°C for 20min) required a long germination period (55°C for 60min). However, similar inactivation level was reached with shorter germination period (55°C for 15min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568MPa, 73°C, 10min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50mM AK consisted: (i) a primary heat treatment (80°C, 10min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55°C in about 20min and further incubation at 55°C for about 15min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586MPa at 73°C for 10min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens. [Copyright &y& Elsevier]