Inhibition of Clostridium perfringens by Natural Products

Abstract

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that produces up to 20 distinct toxins and is responsible for various gastrointestinal (GI) and histotoxic diseases in humans and animals. C. perfringens strains may be categorized into seven toxinotypes (A-G) based on the toxins they generate. C. perfringens type F strains that generate C. perfringens enterotoxin (CPE) induce food poisoning (FP) and non-foodborne (NFB) illnesses in humans and livestock animals, leading to significant annual economic losses in the United States. C. perfringens displays several distinctive traits that allow to induce a wide array of diseases in various hosts: i) it is anaerobic organism that can grow in circumstances with relatively limited exposure to air or low oxygen levels, such as soil, sewage, and the GI system of humans and animals; ii) it owns the ability to form spores that resist heat and other harsh stresses in environment; iii) over favorable conditions, C. perfringens spores can quickly turn to vegetative cells through a procedure known as germination; and iv) it can release a range of toxins under different settings. In addition to these intrinsic virulence properties, external factors such as improper cooking, improper chilling, and storing meat and poultry products at abusive temperatures may significantly contribute to the incidence of C. perfringens type F food poisoning outbreaks. The possibility of obtaining foodborne infections is typically minimized by widely employed food preservation techniques, such as heat processing. Even so, inhibiting highly heat-resistant spores is a significant issue; consequently, alternative solutions to traditional thermal processing technologies must be implemented immediately. The initial study investigated the inactivation ability of several natural products as antimicrobial agents against the spore growth of C. perfringens type F (FP) isolates. Our findings revealed that garlic, onion juice, and undiluted essential oil constituents (EOCs) of clove, rosemary, and peppermint exhibited the highest activity. Therefore, we examined the inhibitory effects of these products on all forms in the life cycle of C. perfringens FP strains, including spore germination, spore outgrowth, and vegetative growth, in laboratory media and chicken meat. Clove and peppermint oils demonstrated efficacy against C. perfringens spore germination in a nutrient-rich medium. Furthermore, EOCs, but not garlic and onion juice, blocked the spore outgrowth of C. perfringens in laboratory medium. Interestingly, EOCs at 0.5% completely prevented the vegetative growth of FP isolates during a 6 h incubation under laboratory medium. However, even at 4-fold higher concentrations (2%), EOCs could not inactivate C. perfringens spore growth in contaminated chicken meat stored under abusive conditions. Although some natural products inhibited C. perfringens spore germination, outgrowth, and vegetative growth under laboratory-controlled conditions, no such inhibitory activity was observed when these products were applied to C. perfringens spore-inoculated chicken meat. Our earlier study has shown considerable inhibitory effects of medicinal plants, including essential oils of clove, rosemary, and peppermint, against vegetative cells of C. perfringens, hence raising their application in traditional medicine and industrial practices in developing countries. EOs have gained increased attention across multiple sectors, particularly as natural substances, with their value as food preservatives having been previously proven. Their distinctive chemical structure facilitates a broad spectrum of biological and antimicrobial activity. In the second study, we assessed the possible effect of EOs, including clove, rosemary, and peppermint, which had previously been shown to have activity against selected FP strains of C. perfringens in laboratory media. The three evaluated oils markedly lowered intracellular ATP levels during the initial 3 h, as estimated using the BacTiter-Glo reagent, and damaged the cellular membrane, as illustrated by the release of compounds absorbing at 260 nm, protein leakage, and the uptake of crystal violet dye. Subsequent research using a scanning electron microscope (SEM) revealed cellular membrane disruption in oil-treated cells. In conclusion, as natural products, essential oils may utilize their anti-C. perfringens activities by disrupting the cytoplasmic membrane, hence modifying bacterial cell membrane permeability and resulting in the release of certain cellular components. Recent research has elucidated potential mechanisms of action of essential oils against C. perfringens; however, their in vivo efficacy has yet to be determined. In the third study, a mixture of lactic acid (lactate) and the antimicrobial peptide nisin was evaluated to assess their combined inactivation effect against a collection of FP strains of C. perfringens, with a focus on spore germination and outgrowth in cooked meat products. Results showed that, among the tested concentrations, a mixture comprising 0.02% nisin, which is the acceptable level, and (0.5-1) % calcium lactate was determined to be the most effective combination for inhibiting spore germination and outgrowth in meat products, depending on the meat product under examination. Our findings suggest that the combination of nisin and calcium lactate may offer a promising alternative approach for controlling C. perfringens spore growth in meat products. Consequently, the objective of this work is to integrate low concentrations of antimicrobials to ensure the safety and efficacy of meat products and to inhibit C. perfringens proliferation under adverse conditions. Together, this dissertation presents observations on several inactivation techniques designed for controlling spore growth of C. perfringens FP isolates in both laboratory medium and cooked meat as a model system. These include methodologies for applying natural antimicrobials recognized as generally recognized as safe (GRAS)-listed antimicrobial agents in food products, as well as the identification of their target sites within the cells of C. perfringens, which provides insights into developing new treatment options to inactivate spore formation. The integration of these practices should successfully reduce the risk of C. perfringens related to foodborne diseases.