Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2003
Publication Date: 4/1/2004
Citation: Novak, J.S., Fratamico, P.M. 2004. An evaluation of ascorbic acid as a quorum sensing analogue to control growth, sporulation, and enterotoxin production in clostridium perfringens. Applied And Environmental Microbiology. Vol. 69. Nr. 3. 2004. p.72-78. Interpretive Summary: The bacterium, Clostridium perfringens is a leading cause of food-borne illness in the United States today. C. perfringens is known to produce small compounds called autoinducers that are involved in cell-to-cell signaling and that regulate the ability of the bacterium to form spores (highly resistant, dormant form of the bacterium) and produce an enterotoxin. This study evaluated the inhibition of this process by compounds that are structurally similar to autoinducers and that are natural food additives, such as vitamin C (ascorbic acid). The results showed that added vitamin C could affect C. perfringens growth and reduce the formation of spores. In contrast, enterotoxin production was enhanced over time. However, pre-formed enterotoxin in foods is not a concern as cooking denatures any toxin activity. These results hold potential for applying vitamin C or other natural inhibitors of cell signaling for controlling pathogens that survive normal food processing treatments.
Technical Abstract: Inhibition of quorum sensing by enterotoxin-producing strains of Clostridium perfringens was investigated. Autoinducer-2 (AI-2) activity was measured in the presence and absence of ascorbic acid (vitamin C; concentrations ranging from 10 to 300 mM), an AI-2 analogue. Subsequent effects on AI-2 production, growth, sporulation, and enterotoxin (CPE) production in C. perfringens were examined. Using a Vibrio harveyi reporter cell coupled assay, AI-2 activity was detected in supernatants of a five-strain cocktail mixture of C. perfringens grown in LB medium and sterile ground beef. Increases in AI-2 activity paralleled increases in CFU from ground beef. Addition of ascorbic acid to supernatants of ground beef that had been inoculated with C. perfringens resulted in decreases in AI-2 activity compared to supernatants with no ascorbic acid; however, supernatants of sterile ground beef, and a control in which supernatant was replaced by sterile water also showed ascorbic acid inhibition of AI-2 activity indicating a negative impact on the reporter strain as well. The addition of sodium ascorbate, a non-acidic salt of ascorbic acid also resulted in AI-2 assay inhibition, despite absence of notable effects on assay medium pH. Addition of ascorbic acid directly to ground beef prior to incubation with C. perfringens, resulted in no notable change in growth prior to 7 days incubation and only ca. 1 log decrease in relative light units (RLUs) after 2 d incubation with 300 mM ascorbic acid. The inhibitory effects of ascorbic acid on AI-2 activity were more apparent in Duncan and Strong (DS) sporulation medium. A 4 h exposure to 100 mM ascorbic acid limited growth of C. perfringens to below 10 CFU/ml in DS medium, likely due to a decrease in medium pH. Culture medium pH, however, did not affect the bioassay pH except at a concentration of 100 mM ascorbic acid. Spore production decreased from 6.83 log spores/ml (no ascorbic acid) to 1.37 and 3.25 log spores/ml in the presence of 10 and 30 mM ascorbic acid, respectively. Western immunoblot analyses showed that CPE levels were highest after 24 h in DS medium without ascorbic acid; however, in the presence of 10 or 30 mM ascorbic acid, levels remained unchanged up to 72 h. Results of this study show that the inhibition of quorum sensing for control of pathogen levels in foods may be possible, however, complex interactions with food components require further study prior to universal application.