Clostridium perfringens spores : inactivation, germination, and formation Public Deposited

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  • The enterotoxin-producing Clostridium perfringens type A isolates are responsible for the third most common foodborne illness in the United States and can also cause non-foodborne human gastrointestinal (GI) diseases such as antibiotic- associated and sporadic diarrheas. Three important factors contribute to the ability of C. perfringens to cause GI diseases, including its extremely rapid growth rate, its ubiquitous distribution in foods and environments, and its capability to form highly resistant endospores. In the first study, the antimicrobial peptide nisin was evaluated for its antimicrobial effect against enterotoxigenic C. perfringens food poisoning (FP) and non-foodborne (NFB) GI disease isolates. Nisin did not affect spore germination, whereas germinated spores were very susceptible to low concentration of nisin and thus spores outgrowth were arrested. Nisin also exerted its inhibitory effect against vegetative growth of C. perfringens FP and NFB isolates in rich medium; however, FP cells were less resistant to nisin than NFB cells. Nevertheless, nisin was not effective in controlling germination and outgrowth of C. perfringens spores in cooked meat products during storage at abusive temperature, even at ~ 4 times elevated concentration than the regulatory approved level. Strikingly, spores of NFB isolates also exhibited higher resistance to nisin than that of FP isolates in both laboratory medium as well as in meat systems. Collectively, despite its effectiveness in controlling spore outgrowth and vegetative cell growth in laboratory conditions, nisin showed no antimicrobial activity against C. perfringens spores inoculated into meat model systems. The main focus of the second study was to develop an effective spore inactivation strategy on food contact surfaces by inducing spore germination prior to inactivation of the more susceptible spores with commonly used surface disinfecting agents. The mixture of L-asparagine and KCl (AK) was the most effective germinant for spores of enterotoxigenic C. perfringens type A. Germination temperature had a significant influence on the germination extent and subsequent inactivation by variety of surface disinfectants. Implementation of germination step significantly increased the inhibitory effect of all tested disinfecting agents against spores of C. perfringens FP strain SM101 with lower efficacy against the spores of NFB strain NB16. Furthermore, spores of C. perfringens FP isolates could germinate with AK upon their adhesion onto stainless steel chips and were subsequently inactivated with disinfectant agents by i.e. 1.53 to 2.70 log reductions of colony forming units per chip. Overall, AK-induced germination followed by treatment with iodophore represents a promising strategy to inactivate spores of C. perfringens FP isolates on food contact surfaces. Spore germination is initiated upon sensing a variety of compounds, termed germinants, via the cognate germinant receptors. In the third study, we identified sodium ions and inorganic phosphate (NaPi) at pH ~ 6.0 as a novel germinant for spores of enterotoxin-producing C. perfringens FP isolates. The spores lacking germination proteins GerAA and GerKA-KC were severely impaired in their ability to germinate with NaPi, whereas GerKB-negative spores germinated to a similar extent as wild type spores with NaPi, but their initial rate of germination was lesser. Spores lacking GerO or GerO GerQ germinated to a lower extent and with a significantly slower rate than wild type spores. In contrast, gerQ spores exhibited only a slightly slower and lesser extent of germination with NaPi than its parent strains. Therefore, the germinant receptor proteins GerKA-KC, GerAA, and the putative antiporter GerO are essential for normal germination of C. perfringens spores with NaPi. In the fourth study, we demonstrated that polar, uncharged amino acids at pH 6.0 could efficiently trigger germination of spores of enterotoxigenic C. perfringens. While L-glutamine is a unique nutrient germinant for spores of C. perfringens FP isolates, L-asparagine, L-cysteine, L-serine, and L-threonine can induce germination of both FP and NFB spores. The germinant receptor GerKC is the major receptor involved in cysteine- and glutamine-induced germination and release of dipicolinic acid (DPA) from the spore’s core, whereas less pronounced germination defects were observed in gerAA and gerKB spores. GerKC also has a key role in L-asparagine germination. For serine and threonine (pH 6.0)-induced germination, GerKA is the dominant receptor and GerKC and GerKB are also required for efficient germination of FP spores. The objectives of the fifth study were to identify and characterize the putative sensor histidine kinases of C. perfringens. We identified six genes encoding putative sporulation-associated sensor histidine kinases in the genome of C. perfringens SM101. These putative kinase genes were highly expressed under sporulation- stimulating conditions. Two genes encoding putative orphan sensor histidine kinases, cpr1728 and cpr1055, were inactivated and roles of each putative kinase on various aspects in the life cycle of C. perfringens had been characterized. Inactivation of cpr1728 and cpr1055 significantly lowered C. perfringens sporulation capacity in two sporulation-inducing conditions. Moreover, sporulation delayed phenotype was also observed in strain lacking CPR1055. Inactivation of either cpr1728 or cpr1055 led to a marked defect in C. perfringens spore germination with all known germinants. Spores of two kinase mutants also exhibited slower outgrowth than their parental strain; however, no difference in colony forming efficiency was observed among tested strains. Additionally, mutations in cpr1728 and cpr1055 did not affect vegetative growth; however, both mutants grew at higher rate under sporulation-inducing conditions. In conclusion, this dissertation reports the experimental results that are relevant to various aspects of C. perfringens spores. These include the development of spore inactivation strategies in food products as well as on food contact surfaces, the identification of compounds triggering germination of spores of CPE-producing C. perfringens, and the insights into the roles of putative sensor histidine kinases in the process of spore formation and spore germination under a variety of conditions.
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