Tony T. Tran

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.

Initial Cell Concentration and Selective Media Effects on the Isolation of Listeria monocytogenes from Enrichment Cultures of Inoculated Foods

To facilitate rapid quantitation of Listeria monocytogenes in artificially inoculated dairy products, seafoods, and other foods, the recently revised U.S. Food and Drug Administration methodology for Listeria in foods was further abbreviated. The ability to isolate L. monocytogenes was measured in terms of the number of inoculated colony forming units (cfu)/g of food. Isolation values for the foods tested, using modified McBrides agar (MMA), were <1, 1-10, and >11 inoculated cfu/g in 30, 44, and 26% of test samples, respectively. The corresponding percentages with lithium chloride-phenylethanol-moxalactam (LPM) agar were 41, 51, and 8%, respectively. In eight (21%) instances, isolation values on LPM agar were significantly superior to those on MMA. The superiority of LPM was most dramatic when MMA isolation values were >10 initial cfu/g. However, LPM was not an infallible remedy for MMA deficiencies even at >10 cfu/g. Ease of isolation was apparently not related to food type. For 10 different test portions of Brie cheese, isolation values ranged from 0.3 to > 10,000 of inoculated cfu/g with MMA and 0.3 to 2500 of inoculated cfu/g with LPM agar.

Detection of thermophilic Campylobacter spp. in blood-free enriched samples of inoculated foods by the polymerase chain reaction.

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.

Evaluation of a selective enrichment most probable number enumeration method for viable Listeria spp. in dairy products

A most probable number (MPN) method for enumerating low numbers of Listeria spp. in dairy foods was developed by adapting the U.S. Food and Drug Administration (FDA) Listeria isolation methodology. Milk, cheese, and other milk products were diluted and homogenized in enrichment broth (1 g/10 ml). Homogenates were inoculated with L. monocytogenes Lm82, a streptomycin-resistant variant of strain Scott A, at <1 to 320 CFU/g and further diluted in FDA enrichment broth to give 0.1, 0.01, and 0.001 g of food sample per 10 ml. Dilution aliquots (10 ml) in triplicate or quintuplicate were incubated at 30°C for 48 h before being subcultured on Oxford agar at 35°C. Esculin-hydrolyzing colonies on Oxford agar were confirmed as the inoculum strain by their ability to grow on Trypticase soy agar containing streptomycin. Differences between inoculum and MPN values were evaluated by using tabulated 95% confidence limits. The calculated MPNs agreed with the inoculum levels in 91% (58 of 64) of noncheese dairy foods and in 49% (56 of 112) of 15 varieties of ripened cheeses. Competitive microflora affected by cheese age and the kind of milk used may account for the suboptimal performance of the MPN method with the cheeses.

Microbial survey of shared-use cosmetic test kits available to the public

SummarySome people like to try cosmetics before purchasing them. With repeated use by different customers, however, the tester kits provided by many retail outlets can become potential vectors of microbial pathogens. A survey was conducted to assess the health risk from bacteria found on shared-use cosmetics. A total of 3027 shared-use cosmetic product samples were collected from 171 retail establishments throughout the contiguous United States. Eye, face and lip cosmetics were tested within situ nondestructive swabbing and the use of the Transette 3R Modified Amies Charcoal Culture and Transport System. Bacteria were isolated from about 50% of the items for all three categories. Semiquantitatively-estimated mean densities were 2288, 1685 and 1088 CFU g−1 for eye, face and lip products, respectively. Ranges for all categories were 0–155 CFU g−1. About 5% of the items had bacterial counts above 5000 CFU g−1 (eye products) or 10 000 CFU g−1 (other products). More than 60% of isolates were typical of microflora from human skin; the remainder were environmental microbes. About 60% of the isolates were Gram-positive cocci:Staphylococcus spp. (especiallyS. epidermidis) andMicrococcus spp. The Gram-negative pathogenPseudomonas aeruginosa constituted 0.07% of the isolates. The survey results suggest that the preservation systems of some of the cosmetics failed under excessive use (abuse), and indicated a potential for microbiological safety problems with shared-use consmetics.

Adequacy of cosmetic preservation: chemical analysis, microbial challenge and in-use testing.

The adequacy of preservation of seven previously unopened commercial cosmetic products was tested by individual challenges with Aspergillus niger ATCC 9642, Candida albicans ATCC 10231, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 15422, and Staphylococcus aureus ATCC 6538, using the Cosmetic, Toiletry, and Fragrance Association (CTFA) method. Each product was consecutively challenged three times, 28 days apart. Inoculated composite products were counted by conventional techniques at eight prefixed intervals. Six of seven cosmetics passed the CTFA acceptance criteria. On the basis of viable counts seven days after inoculation (CTFA criteria), the products were classified as follows: five products were well preserved, one was marginally preserved, and one was poorly preserved. The poorly preserved product failed the CTFA criteria for all three bacteria tested. Concentrations of preservative ingredients in uninoculated composites were determined by high performance liquid chromatography. All preservatives listed on the labels of the seven cosmetic products were identified by chemical analysis. Tentative in‐use validation of the CTFA criteria was performed for three of the seven cosmetic formulations. The results suggested that some cosmetic products may be underpreserved.

Direct contact membrane inoculation of yeasts and moulds for evaluating preservative efficacy in solid cosmetics

A direct contact membrane inoculation technique for yeasts and moulds was used to evaluate the preservation efficacy and antimicrobial activity of Germall 115 and Germall II in pressed eye shadows. Test organisms on membrane filters were placed in direct contact with cosmetics at room temperature under humid conditions. Growth on membranes was removed daily, or as appropriate, and cultured on potato dextrose agar containing lecithin and Tween 80. Linear regression analysis was used to determine product preservation efficacy. Average D values of 1 and 3 days for Candida albicans American Type Culture Collection (ATCC) 10231 and 17 and 29 days for Aspergillus niger ATCC 16404 were obtained on two eye shadows we prepared (in‐house eye shadows) with parabens and either Germall II or Germall 115 as preservatives. A decimal reduction time (D value) of 6–7 days was calculated for the yeast on a commercial eye shadow preserved with parabens and Germall 115. A. niger multiplied on six of seven replicates of this commercial product to attain a nearly 3 log10 increase in 20 days. On one replicate, A. niger showed a 1 log10 increase in the first 10 days, and then decreased linearly (r =— 0.95) to <10 colony‐forming units per membrane by day 24. The method used with C. albicans and A. niger was then used with bacteria. The method was sensitive enough to differentiate the antimicrobial activity of the Germall 115 and Germall II against fungi but not against bacteria. The in‐house and commercial products were preserved most effectively against the three bacteria tested and least effectively against the mould.