The aim of this study was to examine whether Gram-positive cocci isolated from processed poultry in Western Australia provided a potential risk for the transfer of antimicrobial-resistant organisms to humans via commercially prepared ready-to-eat chicken. Research in this study was conducted in three phases: the characterisation of Gram-positive cocci isolated from poultry, an assessment of the isolates' thermal tolerance and the development of a Hazard Analysis Critical Control Points (HACCP) based food-safety program. In the first phase of the study, three specific objectives were investigated. The first determined the presence of Gram-positive cocci on poultry and on processing equipment from poultry-processing plants. The findings confirm the presence of staphylococci and enterococci on incoming live and slaughtered birds and processed carcasses. The data also indicate that carcasses probably become cross-contaminated during processing, when these bacteria are present on the incoming live birds and equipment. The second objective was to characterise staphylococcal isolates by antimicrobial susceptibility testing, and chromosomal and plasmid DNA analysis. The susceptibility of isolates to antimicrobial agents was tested by the disk diffusion method according to the NCCLS (National Committee for Clinical Laboratory Standards) guidelines. Isolates were typed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis of SmaI digested chromosomal DNA, and plasmids were isolated by the cetyltrimethylammonium bromide (CTAB) method. Approximately 37% of Staphylococcus aureus and 16% of coagulase-negative staphylococcal (CNS) isolates were resistant to six or more of the antimicrobial agents tested. Many isolates exhibited resistance to antibiotics that are commonly used in human medicine and registered for veterinary use in Australia. / Among the S. aureus isolates there were twenty-four epidemiologically unrelated SmaI CHEF groups. All staphylococcal isolates, except three CNS, were found to harbour from one to seven plasmids. Some staphylococcal isolates with epidemiologically related CHEF patterns had similar plasmid profiles and resistance patterns. The third objective was to determine the antimicrobial susceptibility of enterococci isolates to the glycopeptide antibiotics. The isolation of two vancomycin-resistant E. faecalis isolates is the first report of VRE outside the health-care setting in Western Australia. Additionally the detection of the vanA gene in an E. gallinarum isolate, a motile enterococcus, has potentially important implications for infection control practices in hospitals. In the second phase of the study, three specific objectives were established to investigate the practical implications of these findings for the chicken industry. The first objective of this phase of the study was to determine the thermal tolerance (D and Z-values) of antimicrobial-resistant, Gram-positive cocci in ground chicken meat. The results indicate that these isolates do not exhibit enhanced thermal-resistance characteristics compared to antimicrobial-susceptible bacteria. The second objective established the internal time-temperature profiles for cooking commercially prepared chicken and estimated the process lethality (F-values). / From three cooking trials, it was confirmed that the internal temperature of at least 70°C was achieved for at least thirty-eight minutes. The third objective of this phase assessed the effectiveness of the thermal process in reducing the risk of the transfer of antimicrobial-resistant cocci via the food chain. The data confirm that the lethal effect (F-values) of the thermal process destroyed these antimicrobial-resistant cocci in commercially prepared ready-to-eat chicken. In the third phase of the study, the data obtained in the earlier parts of the study was incorporated into a model food-safety program for a fast-food chicken chain. The model was based upon the internationally accepted HACCP system, adopted by the Codex Alimentarius Commission. Mindful that the thermal-process step represents only one critical control point in the safe preparation of chicken, this preventative approach ensures that all hazards are controlled at every other step of the process. The data suggest that antimicrobial-resistant, Gram-positive cocci will be present on some ready-to-cook poultry meat processed in Western Australia. This creates opportunities for the potential spread of resistant strains or resistance genes to humans via the food chain. The information from this study will be useful in providing background data and direction for future planning in preventing antimicrobial-resistant bacteria from poultry meat being transmitted through the food chain. The full implementation of the HACCP program would offer substantial benefits and protection to consumers.

Thirteen bacterial cultures were isolated from the North arm of Great Salt Lake during the months of January and February of 1973. Eleven isolates were gram-negative pleomorphic rods which lysed in hypotonic solution. The remaining two were gram-positive cocci. All isolates and one known strain of Halobacterium salinarium were subjected to examination of morphological, cultural, physiological, and biochemical characteristics. A numerical taxonomic analysis was applied to the compiled characters to compute a coefficient of similarity for each individual isolate as compared to all other isolates. A comparative analysis was included in the similarity computation using characters assembled from those reported in the literature for six taxonomically accepted species of halophilic bacteria. The lake isolates proved to be extreme halophiles with relative high levels of similarity between each other and the known bacteria included in the numerical analysis.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:30Z No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:42Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:53Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Made available in DSpace on 2015-06-30T13:13:53Z (GMT). No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os produtos farmacêuticos que requerem a característica de esterilidade devem ser submetidos ao Ensaio de Esterilidade que deve ser realizado em áreas limpas, a fim de evitar resultados falso-positivos. A legislação brasileira recomenda a identificação de microrganismos provenientes dos Ensaios e do ambiente onde estes foram realizados. A dificuldade da identificação de vários gêneros bacterianos por metodologias fenotípicas têm sido relatada em vários estudos e mostram a necessidade da utilização de metodologias moleculares para esta finalidade. Neste estudo foi realizada a caracterização fenotípica (API e VITEK BioMerieux) e genotípica (análise da sequência do gene 16S rRNA) de 58 estirpes de cocos Gram positivos não fermentadores da glicose, provenientes de produtos farmacêuticos estéreis e ambiente controlado. O resultado da caracterização fenotípica realizada utilizando o sistema VITEK demonstrou que 100% das identificações foram equivocadas quanto ao gênero e espécie bacteriana. O sistema API identificou corretamente 69% das estirpes quanto ao gênero bacteriano quando comparado com a análise da sequência do gene 16S rRNA. Vinte e cinco estirpes foram submetidas ao sistema VITEK 2 e 68% dessas foram identificados corretamente quanto ao gênero bacteriano. A análise da sequência do gene 16S rRNA mostrou-se eficiente na determinação do gênero e mostrou a diversidade bacteriana deste grupo de organismos. Entre os cocos Gram positivos não fermentadores da glicose analisados foram identificados os gêneros Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter e Brachybacterium. Essa análise também mostrou que 8,6% das estirpes avaliadas podem representar espécies ainda não descritas. Esta metodologia possibilita a diferenciação de quase todas as espécies do gênero encontrado com mais frequência, o Micrococcus, exceto o Micrococcus yunnanesis e Micrococcus luteus. Essas espécies também não puderam ser diferenciadas pela análise da sequência de segmentos de genes conservados (rpoB, gyrB, groEL and recA). Os equívocos das identificações fenotípicas alertam para a necessidade da implementação de metodologias moleculares para concluir a identificação correta de estirpes bacterianas provenientes de testes de esterilidade e ambientes controlados. / Sterile pharmaceutical products must be submitted to sterility testing to be carried out in clean rooms, in order to avoid false positive results. Brazilian law recommends the identification of microorganisms from sterility tests and the environment where these tests were performed. It has been reported in several studies difficulty in identifying various genera using phenotypic methods. This suggests the need of molecular methods which are more suitable for this purpose. In this study we performed phenotypic (API and VITEK systems (BioMerieux)) and genotypic (sequence analysis of 16S rRNA) characterization of 58 strains of Gram positive cocci non-fermenting glucose, from pharmaceuticals sterile and controlled environment. The results of phenotypic characterization performed using the VITEK system showed that 100% of the identifications of bacterial genus and species were misleading. The API system correctly identified the bacterial genus of 69% of the strains compared with the sequence analysis of 16S rRNA. Twenty-five strains were identified with the Vitek 2 system and 68% of the strains were identified with the correct bacterial genus. Sequence analysis of 16S rRNA gene was effective in determining the bacterial genus and also showed bacterial diversity of this group of organisms. Among the glucose non-fermenting Gram-positive cocci analyzed, the identified genera were: Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter and, Brachybacterium. This analysis also showed that 8.6% of the strains tested may represent species not yet described. This methodology allowed the differentiation of almost all species of the genus Micrococcus, except Micrococcus yunnanesis and Micrococcus luteus. These species were also not differentiated by sequence analysis of fragments of housekeeping genes (rpoB, gyrB, groEL and recA). The mistake phenotypic identifications highlight the need of the implementation of molecular methods to achieve the correct identification of bacterial strains from sterility testing and controlled environments.