The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335-and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.Listeria monocytogenes is a food-borne pathogen that causes approximately 2,500 cases of human illness and 500 deaths annually in the United States (21). Bacterial subtyping methods have improved our ability to detect and track human listeriosis outbreaks and have also provided tools for tracking sources of L. monocytogenes contamination throughout food systems. Application of subtyping methods also provides insight into the population genetics, epidemiology, ecology, and evolution of L. monocytogenes. A variety of conventional, phenotypic, and DNA-based subtyping methods have been described for differentiation of L. monocytogenes beyond the species and subspecies levels (14). While phenotype-based methods hav...

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.

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Microbiological analyses of workers' hands were made for the common indicators, including aerobic mesophilic plate counts (APC), as well as the common food pathogens. Opportunities were observed for cross-contamination of roast beef by workers' hands during slicing operations. Workers' hands showed APC counts of up to 10(7) CFU/hand and the presence of S. aureus and C. perfringens. Salmonella spp were not isolated from hands. These results show that handling of these foods by such workers would be a risk in transmitting pathogenic microorganisms to the foods and is apparent that it is necessary for these workers to take care of personal hygiene. Decimal reductions obtained in the microbiological counts after washing and antisepsis of workers' hands were at 2,6 logs cycles and still demonstrated the importance of this practice in food services by the fact that pathogens such as S. aureus and C. perfringens were inhibited or killed.

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RESUMO Avaliou-se o perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorou-se as condições de higiene em uma linha de produção de queijo de coalho por meio da técnica de bioluminescência (ATP).

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Esta revisão de literatura teve como objetivo relatar estudos sobre a ocorrência de Staphylococcus enterotoxigênicos e suas enterotoxinas em leite e produtos lácteos, surtos e casos esporádicos de intoxicação, tipos de enterotoxinas estafilocócicas e seus genes codificadores. Dentre o gênero Staphylococcus, S. aureus é a espécie contaminante de maior prevalência em leite e queijos, principalmente queijos artesanais e está freqüentemente associada com surtos de intoxicação alimentar. As vias de contaminação de queijos podem ser o leite, o manipulador e o ambiente de processamento. Causa relevante de contaminação do leite cru é a mastite bovina, que tem S. aureus como o principal agente etiológico. Já foram identificados 18 tipos de enterotoxinas (A, B, C1, 2, 3, D, E, G, H, I, J, K, L, M, N, O, P, Q, R e U) e descritos seus respectivos genes. Relatos de ocorrência de cepas coagulase negativa com potencial enterotoxigênico em leite e produtos lácteos indicam a necessidade de reavaliação dos padrões microbiológicos estabelecidos pela legislação brasileira.