A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non-Yersinia bacteria from a variety of meats, shellfish, and vegetables.
Y. enterocolitica and Y. pseudotuberculosis are emerging foodborne pathogens. Phosphatesaline enrichment of food homogenates at 4°C required about 2 weeks for retrieval of yersinia when present at 10,000/g. Modification by inclusion of 1% sorbitol and 0.15% bile salts increased sensitivity 10-fold and extended the recovery period. MacConkey was superior to bismuth-sulfite, Salmonella-Shigella, Hektoen, and Levine EMB agars for isolation. Incubation at 26°C was more satisfactory for recovery and characterization than 37°C. Isolates were first screened in TSI, mannitol, and lysine which eliminated 75% of interfering cultures. Of 25 biochemicals evaluated, urea, arginine, motility (at 26 and 37C°), and phenylalanine were most useful for confirmation. Cultures isolated from foods were classified into 2 groups (1) giving negative reactions in citrate, raffinose, melibiose, and gelatin; and (2) giving positive reactions in the above biochemicals. The former resembled classical European strains of Y. enterocolitica. The latter were more frequently recovered and designated Y. enterocolitica-like bacteria.
Surveillance for dysentery-related invasive potential in bacteria using the Sereny keratoconjunctivitis test is restricted by expense, time factor, and necessity for confirmation. Primary screening of isolates in a standardized mammalian cell culture system is recommended. Bacteria are grown 20 hr in veal infusion, washed, and resuspended in 20% heat-inactivated fetal bovine serum (FBS) supplemented with 0.12% brain heart infusion and 0.1% bile salts. The HeLa culture is grown 20 hr as a monolayer in chamber slides with 90% minimal essential medium (MEM)-10% FBS. The host culture is infected at a ratio of 10 bacteria/ mammalian cell for 3 hr at 35°C. The infection medium is replaced with MEM-FBS supplemented with 300 μg lysozyme and 5 μg gentamycin/ml. The infected monolayer is incubated 5 hr at 35°C to permit intracellular multiplication. Specimens arc washed, fixed with methanol, and stained successively with May-Grunwald and Giemsa dyes. Bacteria occur within the cytoplasm if invasion has occurred. The criterion for a positive test is that 1% of the host cells possesses at least 5 bacteria in 2 of 3 trials. Invasiveness is correlated with and possibly pre-conditioned by cytotoxic principle(s). Infectivity rates vary from 0 to 30%. The cytopathic effect is noted in 5–50% of HeLa cells. Positive results must be confirmed by the Sereny test.
During a recent outbreak of gastroenteritis associated with serogroup 0124:B17 of Escherichia coli, various problems complicated recovery and identification of the pathogen. Standard methods for E. coli were of limited value because of atypical behavior of isolates. Two modified recovery procedures have been presented. For rapid lactose fermenters, pre-enrichment in MacConkey broth with subsequent transfer to lauryl sulfate tryptose broth and incubation at 44 C is recommended. For slow lactose fermenters, pre-enrichment in nutrient broth with subsequent transfer to Mossel's enteric enrichment broth and incubation at 41.5 C is tentatively proposed. Isolation agars include Levine's eosin methylene blue for lactose fermenters and MacConkey agar for non-lactose fermenters. The merits of a direct streak are considered. To facilitate rapid differentiation of E. coli from closely related Enterobacteriaceae within 3 days a modified Lundbeck procedure is offered. Isolates are first screened for H2S formation, indole production, arabinose fermentation, urease and ONPG-ase. Secondary characterization based on results of the indole and TSI reactions includes Voges-Proskauer test (22 C), lysine decarboxylase activity, KCN tolerance, and fermentation of adonitol, cellobiose, sorbitol, or glucose. Confirmation by gram-reaction nitrate reduction, and cytochrome oxidase activity is required to differentiate from members of other families. Critical factors of serological analysis are stressed. Prospects for future research are discussed.
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