Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce.

Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and without the 0.5 cm mineral oil overlay, for test portions of parsley, cilantro, and celery. When Oxyrase for Broth was used with Shigella broth, strain 357 was isolated at higher rates from all produce types, except cilantro, when 0.5 cm mineral oil overlay was applied to enrichment cultures. The use of mineral oil overlay with Oxyrase for Broth also improved recovery of strain 20143 from test portions of all produce types except green onion and strawberries. These differences were significant (p < 0.05) with parsley, carrots, and cilantro (1 of 2 evaluations). No statistically significant differences (p > 0.05) between the GasPak and Oxyrase for Broth anaerobic methods occurred when mineral oil overlay was used with Oxyrase for Broth. The use of Oxyrase for Broth with a 0.5 cm mineral oil overlay is a practical alternative for anaerobic enrichment with the BAM method in the analysis of some produce types. Differences in recovery among the different produce types and methods occurred between S. sonnei strains 357 and 20143, emphasizing the need for additional S. sonnei strains in future evaluations.

Microbial evaluation of selected fresh produce obtained at retail markets.

The microbial quality of five types of fresh produce obtained at the retail level was determined by standard quantitative techniques. These techniques included aerobic plate count (APC), total coliform counts, Escherichia coli counts, and yeast and mold counts. Three different methods were used to determine total coliform counts, which consisted of MacConkey agar plate counts, Colicomplete most probable number counts, and Petrifilm E. coli (EC) plate counts. The mean APCs for sprouts, lettuce, celery, cauliflower, and broccoli were 8.7, 8.6, 7.5, 7.4. and 6.3 log10 CFU/g, respectively. MacConkey agar counts indicated that 89 to 96% of the APCs consisted of gram-negative bacteria. Yeast and mold counts were in a range expected of fresh produce. Fresh produce was also analyzed for human pathogens. Samples were analyzed for Staphylococcus spp., Bacillus spp., Salmonella spp., Listeria spp., and Campylobacter spp. One isolate of Staphylococcus was found to be enterotoxigenic, and one species of Bacillus was also toxigenic. Neither Salmonella spp. nor Campylobacter spp. were detected in any of the produce samples. A variety of Listeria spp., including Listeria monocytogenes, were found in fresh produce.