Germination and Growth of Clostridium perfringens Spores in Milk

Abstract

Clostridium perfringens is an anaerobic, Gram-positive, rod-shaped, spore-forming bacterium widely distributed in the environment and recognized as a major cause of foodborne gastrointestinal disease worldwide [15, 47]. Its pathogenicity is largely linked to toxin production, particularly the C. perfringens enterotoxin (CPE), which is produced during sporulation and contributes directly to disease development [26, 12, 59]. This pathogen also poses a substantial food safety concern because it forms highly resistant endospores capable of surviving multiple environmental stresses, including heat treatment, desiccation, and chemical exposure, thereby enabling persistence in food systems [6, 59]. To exert its harmful effects, dormant spores must undergo germination, which is a critical transition step that restores metabolic activity and enables outgrowth into vegetative cells capable of proliferation and toxin production. Spore germination is initiated when spores detect specific environmental signals such as nutrients, amino acids, and physicochemical conditions through germinant receptors located in the spore inner membrane [5, 37]. Previous studies have demonstrated that certain amino acids, including L-lysine and L-cysteine, can stimulate germination in enterotoxigenic strains; however, germination responses may vary depending on strain characteristics and surrounding environmental conditions [4, 60]. Despite extensive investigation of germination mechanisms under controlled laboratory settings, comparatively limited information is available regarding the behavior of C. perfringens spores within complex food matrices, including dairy products. Thermal processing approaches such as pasteurization effectively inactivate vegetative cells but may not fully eliminate spores, allowing their persistence in processed milk and creating potential safety risks if germination and outgrowth occur during handling or storage [6, 59]. Therefore, understanding how factors such as temperature, nutrient cues, and milk composition influence spore germination and outgrowth is essential for improved risk assessment and the development of effective control strategies for this pathogen. The objective of this thesis was to investigate the germination behavior and outgrowth of C. perfringens spores under conditions relevant to dairy environments, with emphasis on environmental parameters including temperature, nutrient signals, and milk composition. The initial phase of this study assessed the ability of pasteurized/UHT liquid milk to facilitate spore germination and outgrowth in comparison to a nutrient-rich laboratory medium. We measured germination and outgrowth by comparing the total number of viable cells obtained by non-heat-treated with the number of viable cells obtained from heat (80 °C for 10 min) surviving spores. This made it possible to tell the difference between newly formed heat-sensitive vegetative cells and dormant spores. In general, milk helped germination and outgrowth in all strains, but the gains in viable counts were always slower and/or lower than those seen in TGY. This study indicates that germination occurs in milk but is not as helpful as rich broth for rapid population growth The second phase of this study looked at how temperature and cold storage affect the speed of germination in milk. At 37 °C, viable counts rose over time in both milk and TGY, with a distinct difference between direct and heat-treated counts. This is in line with the gradual change of latent spores into heat-sensitive vegetative cells. When the incubation temperature was lowered to about 26 °C, germination and outgrowth still happened, although they happened more slowly than at 37 °C. Refrigeration at 4 °C, on the other hand, kept counts steady for 10 days and did not show any difference between direct and heat-treated counts. This shows that heat resistance is still there and that there was no visible germination or outgrowth during cold storage. The third phase of this study looked at whether changing the composition of milk and adding nutrients changed how well seeds germinated and grew. Under the tested conditions, the fat content of milk did not significantly influence 12-hour germination or outgrowth, as both whole and fat-free milk produced equivalent total viable counts and comparable residual spore levels across strains. A separate vegetative-growth assay (6 h at 37 °C) confirmed these results, showing that SM101, E13, and NCTC10239 grew quickly in milk and TGY and had similar growth in milk with 1%, 2%, or 3% fat. This means that the amount of fat did not have a significant effect on vegetative multiplication. The Final phase, the addition of 1% (w/v) L-lysine to ultra-pasteurized milk (pH 6.5) did not enhance total recovery or decrease residual heat-resistant spores after 12 hours, indicating that, although L-lysine is widely regarded as a potent inducer of germination in enterotoxigenic C. perfringens under defined laboratory conditions, it produced no measurable enhancement of germination or outgrowth in the milk matrix under the conditions tested. In summary, our results collectively demonstrate that milk can promote the spore-to-cell transition of C. perfringens under favorable conditions; however, temperature remains the principal factor influencing the advancement to vegetative growth The lack of observable germination or outgrowth at 4 °C over 10 days substantiates cooling as an effective control measure to prevent C. perfringens spore outgrowth during conventional dairy storage. Additional research examining various germinant combinations, strain-specific receptor contributions, and more extensive dairy processing conditions might elucidate the mechanisms regulating spore activation in milk and enhance predictive food safety measures.