Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Cefovecin is an extended-spectrum long-acting third generation cephalosporin used to treat canine infections. The study objective was to determine the effect of cefovecin on the absolute number and antimicrobial susceptibility of fecal enteric bacteria in healthy dogs. Fourteen Beagles were randomly assigned to a treated (n = 7, 8 mg/kg cefovecin subcutaneously on day 1) or untreated (n = 7) group. LC/MS was used to determine plasma cefovecin concentration on day 14. E. coli, enterococci, and Salmonella were isolated and enumerated from fecal samples collected on days 0, 3, 7, 14, and 28. Antimicrobial resistance was determined using disc diffusion, MIC, and detected using PCR for the bla[subscript CMY-2] gene on select isolates. Mean plasma concentration of cefovecin on day 14 was 9.59 µg/mL in treated dogs; untreated dogs had no measurable plasma cefovecin. The absolute number of E. coli was lower in treated dogs on day 3 (P ≤ 0.0001), and the absolute number of cefovecin-resistant E. coli was higher in treated dogs on days 7 (P = 0.002), 14 (P = 0.004) and 28 (P ≤ 0.0001), compared to untreated dogs. Enterococci increased and were higher in the treatment group on day 7 (P = 0.0226). Isolation of Salmonella was rare. After cefovecin treatment, beta-lactam resistance was more common in fecal E. coli from treated dogs than untreated dogs, while resistance of enterococci was not altered. On day 28, treated dogs were 3.25 times more likely to carry the bla[subscript CMY-2] gene than untreated dogs (95% CI 1.27 – 8.35). The implications of these findings in clinically ill patients require further research.

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 µg/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores. / Demands for sanitary conditions in animal farming have been increasing progressively given the need to combine productivity and high quality products to support increasingly demanding consumer markets. In this context, antimicrobial drugs used in prevention as well as in therapy remain as the control strategy for several pathogenic microorganisms, not important only in animal production but also in human health, although restrictions for the indiscriminate use of these drugs have been intensified. However, the excessive use of these products has been associated to the selection of resistant microorganisms in production areas. On the other hand, investigation on strains of public health importance circulating in animal herds is still limited in Brazil. Enterococcus genus bacteria, usually present in animal and human gastrointestinal microbiota, are environmental indicators of fecal contamination and have become a concerning subject in public and veterinary health given the occurrence of strains resistant to vancomycin (VRE). The present study aimed to isolate, quantify and characterize VRE present in stool samples of sheep and goats from several farms in the center-east and northeast regions of São Paulo State. Swabs collected one hundred and thirty-two stool samples either directly from the animal\'s rectum or from the ground. Samples were plated onto m-Enterococcus agar plates and subcultivated in Bile esculin agar with 6 µg/mL of vancomycin (BEAV) to confirm Enterococcus spp and detect resistant samples. Colonies were identified by colonial morphology, Gram\'s staining, biochemical, and molecular profile. The highest colony count was equal to 2.6 × 105 and 1.7 × 105 CFU/g of feces from environmental and animal samples, respectively. Regarding biochemical characterization, Enterococcus faecalis e Vagococcus fluvialis were the most prevalent species. VRE was detected on BEAV in 33 out of 84 sheep-goat samples and in 21 out of 48 ambient samples, indicating a positivity rate of 46.7% and 29.3% respectively in the investigated samples. Analysis by multiplex PCR of the obtained 54 VRE strains indicated that 23 (43%), 22 (41%) 2 (3.5%) and 2 (3.5%) were positive, respectively, for the vanC2/C3, vanC1, vanA and vanB genes, and no product was amplified for 5.3% of the isolates, suggesting the possible occurrence of other known van gene groups among the isolates. The results obtained in this study indicate, for the first time in the studied areas, the circulation of VRE in sheep and goat farms, with no occurrence of apparent clinical signs in the animals, but with possible health risks to the farmers and workers involved, as well as potential consumers.

Enterococcus spp. pertencem ao grupo das bactérias láticas e estão presentes em solos, águas, plantas, microbiota autóctone de vários alimentos e como membros da microbiota intestinal de humanos e animais. Esses microrganismos foram considerados por muito tempo como comensais, mas o aumento da severidade das infecções nosocomiais causadas por enterococos mutirresistentes a antimicrobianos e, a falta de conhecimento sobre seus fatores de virulência geram insegurança na utilização de cepas deste gênero na produção de alimentos como culturas fermentadoras e/ou probióticas. A diferença entre uma cepa de enterococos com potencial patogênico e outra aparentemente segura para uso em processamento de alimentos não é clara, e a probabilidade de que esta última adquira fatores de virulência merece investigação. O objetivo do presente projeto foi determinar características fenotípicas e genotípicas de Enterococcus spp. isolados de amostras de alimentos e águas correlacionando sua presença com indicadores clássicos de higiene e contaminação fecal. De 812 colônias indicativas do gênero enterococos obtidas a partir de 120 amostras de alimentos, 299 isolados (37%) foram presuntivamente caracterizados como Enterococcus spp. Após identificação por PCR, 139 (46,5%) E. faecium, 80 (26,8%) E. faecalis, 36 (12%) E. casseliflavus e 8 (2,7%) E. gallinarum. Produção de gelatinase foi detectada apenas em isolados de E. faecalis (60%). Um isolado de E. faecium (0,7%) e 31 isolados de E. faecalis (38,7%) apresentaram perfil β-hemolítico. Produção de bacteriocina contra Lactobacillus sakei e/ou Listeria monocytogenes foi observada para 10% dos isolados de E. faecalis e 23% dos isolados de E. faecium. Hidrólise de sais biliares foi observada para 100% dos isolados de E. gallinarum, 86% E. casseliflavus, 65% E. faecalis e 62,6% de E. faecium. Alguns isolados de E. faecium apresentaram resistência à vancomicina, eritromicina e tetraciclina. Entre os isolados de E. faecalis não houve resistência à vancomicina, mas foi observada resistência à tetraciclina, eritromicina e alta concentração de gentamicina. Houve uma maior prevalência dos genes de virulência (esp, gel, ace, as, efaA e cylA) entre os isolados de E. faecalis quando comparado a E. faecium. Além disso, os isolados de E. faecalis, resistentes a antibióticos, mostraram forte adesão a células Caco-2 e capacidade de formação de biofilme em superfície abiótica. RAPD-PCR individualizou 14 cepas de E. faecium e 17 cepas de E. faecalis dentre os 52 isolados Enterococcus spp. resistentes a antibióticos. A variabilidade dos resultados impediu o estabelecimento de uma correlação entre a presença ou contagem de coliformes, E. coli e enterococos nas amostras analisadas. Os dados deste trabalho sobre marcadores fenotípicos e genotípicos de virulência, e a presença de cepas resistentes a antibióticos evidenciam a necessidade da avaliação cuidadosa de linhagens de enterococos para aplicações em alimentos. / Enterococcus spp. belong to the group of lactic acid bacteria widely distributed in soil, plants, foods, animals and humans. In the past, these microorganisms were considered commensals but the increase of antibiotic-resistant enterococci and the lack of knowledge about their virulence markers, had raised concerns regarding the safety of using strains of this genus in the food production as fermentative or probiotic cultures. Besides this, literature data suggests the use of enterococci as sanitary indicator for foods. Differences between enterococci strains with pathogenic potential and an apparently safe ones is unclear and there is a concern about virulence markers transfer. The aim of this work was to determine phenotypic and genotypic characteristics of Enterococcus spp. isolated from foods and water and also to correlate their presence with classical indicators of sanitary quality. Out of 812 presumptive enterococci colonies obtained from 120 food samples, 299 isolates (37%) were presuntively characterized as Enterococcus spp. Isolates were identified by PCR: 139 (46.5%) E. faecium, 80 (26.8%) E. faecalis, 36 (12.0%) E. casseliflavus and 8 (2.7%) E. gallinarum. Only E. faecalis isolates (60%) produced gelatinase. One E. faecium (0.7%) and 31 E. faecalis (38.7%) were β-haemolytic. Bacteriocin activity against Lactobacillus sakei and/or Listeria monocytogenes was observed for 10% of the E. faecalis and for 23% of the E. faecium isolates. All E. gallinarum isolates, 86% of the E. casseliflavus, 65% of the E. faecalis and 62.6% of the E. faecium isolates showed bile salt hydrolysis activity. Some E. faecium isolates were resistant to vancomycin, erythromycin and tetracycline. Vancomycin resistance was absent among the E. faecalis but, resistance to tetracycline, erythromycin and highlevel gentamicin was observed. There was a higher prevalence of virulence genes (esp, gel, ace, as, efaA e cylA) among the E. faecalis isolates when compared to the E. faecium. Antibiotic resistant E. faecalis isolates strongly adhered to Caco-2 cells and formed biofilm on abiotic surface. Using RAPDPCR 14 E. faecium and 17 E. faecalis strains could be individualized from the 52 antibiotic resistant enterococci. It was not possible to correlate the presence of total coliforms, E. coli and Enterococcus spp. in the samples of food and water analysed due to results variability. Data obtained regarding phenotypic and genotypic virulence markers and the presence of antibiotic resistant enterococci raise the needs of a carefully evaluation of the Enterococcus spp. strains before future applications in foods.

<p>The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.</p><p>The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).</p><p>Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.</p><p>In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.</p>

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used. The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum). Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces. In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

This thesis presents results from three studies conducted in a veterinary teaching hospital (VTH). Study 1 retrospectively examined a collection of enterococci from clinical infections. Five recurring strains of Enterococcus faecium, and one strain of Enterococcus faecalis were identified using pulsed-field gel electrophoresis (PFGE) as causing clinical infections. Study 2 examined the gastrointestinal tract enterococci as a source of enteroccocal infections in dogs. Enterococcal catheter-associated bacteriuria (CA-bacteriuria) rate was 8%. In 3 of 4 sets of bacteriuria and rectal isolates, CA-bacteriuria isolates were indistinguishable from rectal isolates suggesting that the patient’s fecal enterococci represented the infection source. In all 3 sets of wound and rectal isolates, fecal carriage of the infection isolate was observed. Study 3 examined the prevalence of bacterial species and the overall CA-bacteriuria rate. CA-bacteriuria rate was 24%, with Enterococcus spp., E. coli, Staphylococcus spp., Streptococcus spp. and Enterobacter spp., as the most prevalent bacteria in listed order. / PetTrust

Enterococcus spp. pertencem ao grupo das bactérias láticas e estão presentes em solos, águas, plantas, microbiota autóctone de vários alimentos e como membros da microbiota intestinal de humanos e animais. Esses microrganismos foram considerados por muito tempo como comensais, mas o aumento da severidade das infecções nosocomiais causadas por enterococos mutirresistentes a antimicrobianos e, a falta de conhecimento sobre seus fatores de virulência geram insegurança na utilização de cepas deste gênero na produção de alimentos como culturas fermentadoras e/ou probióticas. A diferença entre uma cepa de enterococos com potencial patogênico e outra aparentemente segura para uso em processamento de alimentos não é clara, e a probabilidade de que esta última adquira fatores de virulência merece investigação. O objetivo do presente projeto foi determinar características fenotípicas e genotípicas de Enterococcus spp. isolados de amostras de alimentos e águas correlacionando sua presença com indicadores clássicos de higiene e contaminação fecal. De 812 colônias indicativas do gênero enterococos obtidas a partir de 120 amostras de alimentos, 299 isolados (37%) foram presuntivamente caracterizados como Enterococcus spp. Após identificação por PCR, 139 (46,5%) E. faecium, 80 (26,8%) E. faecalis, 36 (12%) E. casseliflavus e 8 (2,7%) E. gallinarum. Produção de gelatinase foi detectada apenas em isolados de E. faecalis (60%). Um isolado de E. faecium (0,7%) e 31 isolados de E. faecalis (38,7%) apresentaram perfil &#946;-hemolítico. Produção de bacteriocina contra Lactobacillus sakei e/ou Listeria monocytogenes foi observada para 10% dos isolados de E. faecalis e 23% dos isolados de E. faecium. Hidrólise de sais biliares foi observada para 100% dos isolados de E. gallinarum, 86% E. casseliflavus, 65% E. faecalis e 62,6% de E. faecium. Alguns isolados de E. faecium apresentaram resistência à vancomicina, eritromicina e tetraciclina. Entre os isolados de E. faecalis não houve resistência à vancomicina, mas foi observada resistência à tetraciclina, eritromicina e alta concentração de gentamicina. Houve uma maior prevalência dos genes de virulência (esp, gel, ace, as, efaA e cylA) entre os isolados de E. faecalis quando comparado a E. faecium. Além disso, os isolados de E. faecalis, resistentes a antibióticos, mostraram forte adesão a células Caco-2 e capacidade de formação de biofilme em superfície abiótica. RAPD-PCR individualizou 14 cepas de E. faecium e 17 cepas de E. faecalis dentre os 52 isolados Enterococcus spp. resistentes a antibióticos. A variabilidade dos resultados impediu o estabelecimento de uma correlação entre a presença ou contagem de coliformes, E. coli e enterococos nas amostras analisadas. Os dados deste trabalho sobre marcadores fenotípicos e genotípicos de virulência, e a presença de cepas resistentes a antibióticos evidenciam a necessidade da avaliação cuidadosa de linhagens de enterococos para aplicações em alimentos. / Enterococcus spp. belong to the group of lactic acid bacteria widely distributed in soil, plants, foods, animals and humans. In the past, these microorganisms were considered commensals but the increase of antibiotic-resistant enterococci and the lack of knowledge about their virulence markers, had raised concerns regarding the safety of using strains of this genus in the food production as fermentative or probiotic cultures. Besides this, literature data suggests the use of enterococci as sanitary indicator for foods. Differences between enterococci strains with pathogenic potential and an apparently safe ones is unclear and there is a concern about virulence markers transfer. The aim of this work was to determine phenotypic and genotypic characteristics of Enterococcus spp. isolated from foods and water and also to correlate their presence with classical indicators of sanitary quality. Out of 812 presumptive enterococci colonies obtained from 120 food samples, 299 isolates (37%) were presuntively characterized as Enterococcus spp. Isolates were identified by PCR: 139 (46.5%) E. faecium, 80 (26.8%) E. faecalis, 36 (12.0%) E. casseliflavus and 8 (2.7%) E. gallinarum. Only E. faecalis isolates (60%) produced gelatinase. One E. faecium (0.7%) and 31 E. faecalis (38.7%) were &#946;-haemolytic. Bacteriocin activity against Lactobacillus sakei and/or Listeria monocytogenes was observed for 10% of the E. faecalis and for 23% of the E. faecium isolates. All E. gallinarum isolates, 86% of the E. casseliflavus, 65% of the E. faecalis and 62.6% of the E. faecium isolates showed bile salt hydrolysis activity. Some E. faecium isolates were resistant to vancomycin, erythromycin and tetracycline. Vancomycin resistance was absent among the E. faecalis but, resistance to tetracycline, erythromycin and highlevel gentamicin was observed. There was a higher prevalence of virulence genes (esp, gel, ace, as, efaA e cylA) among the E. faecalis isolates when compared to the E. faecium. Antibiotic resistant E. faecalis isolates strongly adhered to Caco-2 cells and formed biofilm on abiotic surface. Using RAPDPCR 14 E. faecium and 17 E. faecalis strains could be individualized from the 52 antibiotic resistant enterococci. It was not possible to correlate the presence of total coliforms, E. coli and Enterococcus spp. in the samples of food and water analysed due to results variability. Data obtained regarding phenotypic and genotypic virulence markers and the presence of antibiotic resistant enterococci raise the needs of a carefully evaluation of the Enterococcus spp. strains before future applications in foods.

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 &micro;g/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores. / Demands for sanitary conditions in animal farming have been increasing progressively given the need to combine productivity and high quality products to support increasingly demanding consumer markets. In this context, antimicrobial drugs used in prevention as well as in therapy remain as the control strategy for several pathogenic microorganisms, not important only in animal production but also in human health, although restrictions for the indiscriminate use of these drugs have been intensified. However, the excessive use of these products has been associated to the selection of resistant microorganisms in production areas. On the other hand, investigation on strains of public health importance circulating in animal herds is still limited in Brazil. Enterococcus genus bacteria, usually present in animal and human gastrointestinal microbiota, are environmental indicators of fecal contamination and have become a concerning subject in public and veterinary health given the occurrence of strains resistant to vancomycin (VRE). The present study aimed to isolate, quantify and characterize VRE present in stool samples of sheep and goats from several farms in the center-east and northeast regions of São Paulo State. Swabs collected one hundred and thirty-two stool samples either directly from the animal\'s rectum or from the ground. Samples were plated onto m-Enterococcus agar plates and subcultivated in Bile esculin agar with 6 &micro;g/mL of vancomycin (BEAV) to confirm Enterococcus spp and detect resistant samples. Colonies were identified by colonial morphology, Gram\'s staining, biochemical, and molecular profile. The highest colony count was equal to 2.6 × 105 and 1.7 × 105 CFU/g of feces from environmental and animal samples, respectively. Regarding biochemical characterization, Enterococcus faecalis e Vagococcus fluvialis were the most prevalent species. VRE was detected on BEAV in 33 out of 84 sheep-goat samples and in 21 out of 48 ambient samples, indicating a positivity rate of 46.7% and 29.3% respectively in the investigated samples. Analysis by multiplex PCR of the obtained 54 VRE strains indicated that 23 (43%), 22 (41%) 2 (3.5%) and 2 (3.5%) were positive, respectively, for the vanC2/C3, vanC1, vanA and vanB genes, and no product was amplified for 5.3% of the isolates, suggesting the possible occurrence of other known van gene groups among the isolates. The results obtained in this study indicate, for the first time in the studied areas, the circulation of VRE in sheep and goat farms, with no occurrence of apparent clinical signs in the animals, but with possible health risks to the farmers and workers involved, as well as potential consumers.

Enterococci levels are measured to assess water safety in recreational beaches through a state surveillance program. This surveillance informs the public of beach safety, yet the sampling methodology is limited to only making an advisory posting one sample at a time. This methodology poses a challenge for managers such as: 24 hour advisory waiting period, untested days and extreme variability of enterococci levels in the environment. Therefore, there is a need to integrate adaptive management methodologies that can assist managers to proactively assess beach water safety. This study explored the utility of a historical analysis and logistic regression modeling as a method and as an advisory tool. The analysis utilized 10 years of enterococci surveillance data (7,422 samples) from 15 sub-tropical beaches in Miami-Dade County, Florida. It was determined that Miami beaches have historical low enterococci exceedance counts (3% of total data), that there are some beaches that are more propense to higher exceedance counts than others and that the wet season overall did not readily appear to affect exceedances counts. The logistic regression model utilized an exceedance/ non-exceedance dichotomy and spatial, temporal and annual variables. The model indicated that the overall range of probability of having an exceedance for the sampled beaches under each variable was less than 10%. The ability to use this model and get probability results showed that logistic regression is an accurate statistical tool that provides the historical probabilities of an exceedance on a beach and can complement a random sampling methodology. Furthermore it’s a simple and inexpensive methodology that provides the ability to categorize and recognize patterns estimating the surveillance-managed sample sites probabilities that provides foresight as to where to focus resources in order to reduce risk and facilitating beach management. Through the use of a historical analysis and a logistic regression model, it is possible to address dynamic recreational beach environments with a large-scale view and in a historically comprehensive manner, instead of only making management choices sample by sample.

The feasibility of in situ bioremediation of Cr(VI) in groundwater and aquifer media was investigated using microcosm and mesocosm reactors inoculated with indigenous species of bacteria from dry sludge. Microcosm cores were used to simulate contaminant movement in the vadose and aquifer zones of the aquifer system. Cr(VI) breakthrough analysis through the experimental cores demonstrated successful Cr(VI) immobilisation in simulated barrier systems. Cr(VI) reduction was continuously monitored and microbial culture dynamics were evaluated using 16S rRNA genomic fingerprinting. A culture shift was observed in the microcosm cores with the emerging predominance of known Cr(VI) reducers - Enterococci from soil and Lysinibacilli from sludge - after operation for 45 days. The Cr(VI) reduction process in the columns was determined to be enzyme mediated and non-competitively inhibited by Cr(VI). The microbial cultures under microaerobic conditions depicted a threshold Cr(VI) concentration (Cr) of approximately 100 mg/L which was much higher than the target operation concentration of 40 mg/L at the proposed remediation sites. Using the Computer Program for the Identification and Simulation of Aquatic Systems (Aquasim), it was possible to predict Cr(VI) removal efficiency and the impact of Cr(VI) toxicity on culture dynamics in the barrier. The study demonstrates the potential of applying selected Cr(VI) reducing bacteria in biological permeable reactive barrier systems in preventing the spread of the pollutant into adjacent water supply aquifers. The impact of the presence of natural carbon sources was also evaluated by filtering the feed water through a saw dust bed. Reactors without added carbon source removed up to 70% Cr(VI), and no removal was observed in sterile controls. In the packed mesocosm reactor, the areas before the reactive barrier had no chromium reduction whereas most of the areas after the barrier achieved near 100% reduction. The microbial dynamics were monitored by the 16S rRNA fingerprinting after exposure to Cr(VI). After operating the microcosm reactors under oxygen stressed conditions in the presence of other soil bacteria, a community shift was expected. The soil from inoculated reactors contained a wide range of soil dwelling species of bacteria as well as the newly introduced bacteria from the dried sludge. There was a noted presence of Cr(VI) reducing bacteria, Microbacterium, Acinetobacter, Arthrobacter, Brevibacterium, Rumen bacteria, and several Enterococci in the sludge culture and Arthrobacter spp., Clostridium spp., and Klebsella spp. were amongst the evident among identified species. A non-competitive inhibition model was used for the evaluation of aerobic performances in batch experimental studies, whereas the inhibition threshold term C0-Cr/C0, was introduced for the anaerobic model performance for the reduction of chromium in batch studies. In sterile packed soil columns a model for saturated soil column with dispersion was adopted from AQUASIM 2.0. This model was used in combination with the chromium reduction rate adopted from the anaerobic batch modelling for most non sterile reactors in the microcosm performance. The study demonstrates the potential of applying selected Cr(VI) reducing bacteria in biological permeable reactive barrier systems in restraining the spread of the pollutant into adjacent water supply aquifers. The outcome of this exercise could be useful in the formulation of biological permeable barriers for protection against the spread of the pollutant from hot spots in the area. This is serves as a significant step towards a pilot study. / Thesis (PhD)--University of Pretoria, 2010. / Chemical Engineering / unrestricted