title: Effect of ethanol on the growth of Clostridium botulinum.
abstract: Model broth studies were carried out to investigate the effect of ethanol on the growth of proteolytic (group I) strains of Clostridium botulinum. Ethanol extended the pathogen's lag phase, decreased its exponential growth rate, and decreased its final level of growth in the stationary phase. In all cases, botulinum neurotoxin production was associated with growth. Micrographs of C. botulinum cultures grown at 37 degrees C in trypticase peptone glucose yeast extract (TPGY) broths containing 2 and 4% ethanol showed elongation of vegetative cells and interference with cell division. The inhibition of growth and toxin production at the ethanol level predicted (5.5%, wt/wt) was confirmed by microscopy and by the mouse bioassay. A subsequent study was carried out to determine the combined effect of ethanol (0 to 8% [wt/wt]), water activity (aw; 0.953 to 0.997), and pH (6.2 to 8.2) on the probability of the growth of and neurotoxin production by proteolytic strains of C. botulinum (10(3) spores per ml). Growth and neurotoxin production occurred in 1 to 3 days in TPGY broths without ethanol (0%) and in 2 to 4 days in broths containing 2% ethanol regardless of the aw or pH levels (P < 0.005). Growth and neurotoxin production were delayed by an ethanol concentration of 4% ethanol and completely inhibited by a concentration of 6%. At an ethanol concentration of 4%, the probability of growth and toxin production over 365 days (Pt) was influenced by aw and pH. After 365 days, the maximum probability of growth and toxin production (Pmax) was 1 for all but one combination. However, tau, the time it took for 50% of all eventually positive replicates for any given combination of barriers to show growth and/or turbidity, ranged from <3 to 229 days. All tubes of TPGY broths that showed no growth after 365 days were subcultured in fresh TPGY broths. In all cases, growth and toxin production occurred within 24 h at 37 degrees C, indicating the reversible (sporostatic and/or bacteriostatic) effect of ethanol on C. botulinum.
authors.affiliation: Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9.
authors.forename: Daphne Phillips
authors.initials: DP
authors.lastname: Daifas
authors.forename: James P
authors.initials: JP
authors.lastname: Smith
authors.forename: Burke
authors.initials: B
authors.lastname: Blanchfield
authors.forename: Brigitte
authors.initials: B
authors.lastname: Cadieux
authors.forename: Greg
authors.initials: G
authors.lastname: Sanders
authors.forename: John W
authors.initials: JW
authors.lastname: Austin
mesh_terms: D001905:Botulinum Toxins / Q000096:biosynthesis*; D003014:Clostridium botulinum / Q000254:growth & development* / Q000378:metabolism; D003470:Culture Media; D000431:Ethanol / Q000494:pharmacology*; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D013997:Time Factors
chemical_list: D003470:Culture Media; D000431:Ethanol; D001905:Botulinum Toxins