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A quantitative survey of Clostridium perfringens in typical foods served at local restaurants was conducted for 18 months in Guadalajara, Mexico. A total of 151 samples, including goat's birria (50), pozole (50), and beef tamales (51), were collected from small restaurants in Guadalajara. Samples were tested for C. perfringens by the most probable number (MPN) method and for mesophilic aerobic plate counts (MAPCs) and coliform, yeast, and mold counts by plate count methods. Isolates confirmed as C. perfringens were further sporulated and tested for cytotoxic or cytotonic effect against Vero cells as an indication of enterotoxin production. C. perfringens was detected in 78 (52%) of all samples at concentrations that ranged from 2.3 to 5.4 log MPN/g. Average MAPCs were 1.3 to 2.7 log CFU/g, depending on the type of dish. Coliform counts ranged from less than 1.0 to 1.5 CFU/g, and yeast and mold counts were less than 1.0 log CFU/g in all cases. A total of 118 isolates of C. perfringens were tested for enterotoxic effect on Vero cells; 82 (70%) showed activity against Vero cells. Of them, 31 isolates induced cell lysis, indicating cytotoxic effect; 41 induced cell elongation, indicating cytotonic effect; and 10 produced both cytotoxic and cytotonic effect. Dilution of the bacterial filtrates that were still producing an effect on Vero cells ranged from 1:80 to 1:5,120. These results underscore the importance of determining enterotoxigenicity when testing for C. perfringens in foods.

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A survey of Arcobacter spp. was conducted over a 12-month period in Guadalajara, Mexico. A total of 135 samples (45 lean ground beef samples, 45 lean ground pork samples, and 45 chicken samples, including drumsticks, gizzards, and ground or chopped breast) were collected from local butcheries. The samples were enriched in Johnson-Murano enrichment medium and then streaked onto Johnson-Murano agar plates. Typical colonies were subjected to microscopic and biochemical identification followed by polymerase chain reaction confirmation of the genus Arcobacter. All isolates confirmed to be Arcobacter isolates were then inoculated into Eagle's minimum essential medium to determine their cytotoxicity against Vero cells. Arcobacter spp. were detected in 28.8, 51.1, and 40.0% of beef, pork, and chicken samples, respectively. From these samples, 101 isolates were confirmed to be Arcobacter spp. by polymerase chain reaction. Overall, the species most frequently identified was A. butzleri, followed by A. skirrowii. A. cryaerophilus was isolated only from pork meat. Ninety-five (95%) of the Arcobacter isolates produced a virulence mechanism against Vero cells, and 38 of them induced cell elongation, indicating enterotoxin production. Eighteen isolates produced the formation of vacuoles, and 39 produced both vacuolization and elongation. The vacuolization effect may be related to a vacuolizing toxin. The production of a vacuolizing toxin by Arcobacter spp. has not previously been reported. Results obtained in this study indicate that Arcobacter spp. may show cytotoxic effects other than the recognized enterotoxin production.

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To study the potential of three bacterial pathogens to cross-contaminate orange juice during extraction, normal operation conditions during juice preparation at food service establishments were simulated. The spread of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes from inoculated oranges to work surfaces and to the final product was determined. The transference of these three bacterial pathogens to orange juice made from uninoculated oranges with the use of contaminated utensils was also studied. Fresh oranges were inoculated with a marker strain of rifampicin-resistant Salmonella Typhimurium, E. coli O157:H7, or L. monocytogenes. Final pathogen levels in juice were compared as a function of the use of electric or mechanical juice extractors to squeeze orange juice from inoculated oranges. Pathogen populations on different contact surfaces during orange juice extraction were determined on sulfite-phenol red-rifampicin plates for Salmonella Typhimurium and E. coli O157:H7 and on tryptic soy agar supplemented with 0.1 g of rifampicin per liter for L. monocytogenes. After inoculation, the average pathogen counts for the orange rind surface were 2.3 log10 CFU/cm2 for Salmonella Typhimurium, 3.6 log10 CFU/cm2 for E. coli O157:H7, and 4.4 log10 CFU/cm2 for L. monocytogenes. This contamination was spread over all utensils used in orange juice squeezing. Mean pathogen counts for the cutting board, the knife, and the extractor ranged from -0.3 to 2.1 log10 CFU/cm2, and the juice contained 1.0 log10 CFU of Salmonella Typhimurium per ml, 2.3 log10 CFU of E. coli O157:H7 per ml, and 2.7 log10 CFU of L. monocytogenes per ml. Contact with contaminated surfaces resulted in the presence of all pathogens in orange juice made from uninoculated oranges. These results give emphasis to the importance of fresh oranges as a source of pathogens in orange juice.

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The ability of a yogurt starter culture formed by Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp bulgaricus to inhibit the growth of four enterotoxin type A and B producers Staphylococcus aureus strains (ATCC 6538, S6, FRI-100 and a strain isolated from milk) during fermentation of milk and subsequent storage was investigated. Sterile skim milk was inoculated with about 10(6) CFU/ml of S. aureus and with about 10(6) CFU of starter culture, and incubated at 42 degrees C during 8 h, followed by refrigeration at 4 degrees C. Samples were taken every 2 h during fermentation and every 2 days during storage. Viable count of lactic acid bacteria and S. aureus as well as pH, acidity, thermostable deoxyribonuclease (TNase) and staphylococcal enterotoxin A (SEA) production were evaluated. Behavior of four strains was similar; S. aureus survived the 8 h fermentation with LAB, and its population began to decrease from the first day of storage, being completely inhibited at 9-10 days. TNase and SEA production were positive in all samples taken along the study. It was demonstrated that enterotoxigenic strains of S. aureus were able to survive the fermentation of milk with a yogurt starter culture and they were inhibited after several days during storage of the fermented product, contrary to the general belief which considered it very difficult due to the low pH. Even though S. aureus was inhibited, TNase and SEA were demonstrable along the storage. Therefore, fermented milks may play an important role in the transmission of this organism.

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Fifty-one cooked ham samples were analized by three methods for investigation of Staphylococcus aureus; Modified Van Doorne and American Public Health Association; The Official Mexican Method. The first two are enrichment methods and in all three a comparison between Baird Parker agar and salt milk agar was done. The modified Van Doorne technique was the best for isolation of S. aureus from cooked ham. In Baird Parker agar it was possible to demonstrate the presence of S. aureus in all positive samples. The study shows the importance of using Baird Parker broth as an enrichment medium for the isolation of S. aureus from products in which a thermal treatment and addition of salts as sodium chloride and nitrites, inhibit the growth of this microorganism.

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There are many media recommended for the isolation of Staphylococcus aureus from foods, but only with some media one can obtain a good growth started with stressed cells. The Baird Parker (BP) medium is considered the best choice to recover stressed cells, however, it is not as good a medium to isolate Staphylococcus aureus from powder milk. Therefore, it is important to count with alternative media to enhance the chance for Staphylococcus aureus to grow from dehydrated products. Thirty-one powder milk samples contaminated with Staphylococcus aureus were analysed by Baird Parker method, employing four culture media: Baird Parker (BP), Baird Parker + tween + MgCl2 (BPTM), Pork plasma with bovine fibrinogen agar (PPF) and Salt Milk agar (SL). Staphylococcus aureus was isolated in SL, 38.7%; in BP, 3.2%; in BPTM, 6.4%; and PPF, 0%.

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Demonstration of Staphylococcal Thermonuclease (TNase) from Powder Milk. Some authors have reported that the number of Staphylococcus aureus needed to produce a food-poisoning is 10(6) CFU per gram of food, however, other authors have reported foods without microorganisms but these have produced food-poisoning. Because the methods for staphylococcal enterotoxins demonstration in foods are laborious and expensive procedures, in this paper we tried to demonstrate the thermonuclease (TNase) presence in foods directly, as a helper test for screening foods suspected to be contaminated with this microorganism. To 112 powder milk samples were determined TNase presence by Tatini's et al (1976) and Lachica's (1972) technics, 31 of this samples had S. aureus. Only with Lachica's technique it could be possible to demonstrate TNase in 17 of 112 analyzed samples, 14 of these had viable S. aureus.

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