Five strains of Lactococcus lactis were inoculated individually into six commercial bulk set growth media, 11% non-fat dry milk (NDM), and Elliker's broth. After growth in each medium the strains were tested for rate of acid production, and activities of proteinase, aminopeptidase, and lipase/esterase. Growth in commercial starter media significantly influenced acid production rate (P = 0.040), aminopeptidase activity (P < 0.0001), and lipase/esterase activity (P < 0.0001) . For selected strain/media combinations, the duration of induced aminopeptidase and lipase/esterase activity was followed. The chosen strains were grown in selected commercial bulk set media, reinoculated into 11% NDM, and enzyme activity was examined for five successive generations. During growth in 11% NDM, aminopeptidase and lipase/esterase activity began high and appeared to decrease after approximately two generations, as compared to the control. This study demonstrated that it is possible to select specific starter and media combinations to produce a bacterial phenotype that might not change before the cheese is pressed, thereby trapping bacteria with an altered phenotype within the cheese matrix.

Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi-antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-13T12:33:03Z No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-13T12:46:33Z (GMT) No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5) / Made available in DSpace on 2016-09-13T12:46:33Z (GMT). No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5) Previous issue date: 2011-11-04 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Concentrações subinibitórias de antimicrobianos (CSI), frequentemente decorrentes de antibioticoterapia, podem resultar em alterações na biologia bacteriana, com implicações no seu potencial agressor. Esse efeito tem considerável importância para as bactérias da microbiota residente, em especial para Fusobacterium nucleatum, um dos mais proeminentes anaeróbios residentes em humanos. Os objetivos deste trabalho foram analisar os efeitos de concentrações subinibitórias (CSI) de antimicrobianos em características morfológicas, bioquímicas, fisiológicas e moleculares de F. nucleatum. A partir da linhagem F. nucleatum ATCC 25586 (denominada FnPAR), foram obtidas 14 linhagens selecionadas por 10 cultivos sucessivos em CSI de ampicilina (FnAMP+), ampicilina/sulbactam (FnAMS+), clindamicina (FnCLI+), cloranfenicol (FnCLO+), levofloxacina (FnLEV+), metronidazol (FnMET+) e piperacilina/tazobactam (FnPTZ+) e, subsequente, 10 cultivos na ausência das mesmas drogas. Foram avaliados o perfil de susceptibilidade aos antimicrobianos, a morfologia bacteriana, o perfil bioquímico, a formação de biofilme e o custo de fitness. Também foram realizados genotipagem e análise dos perfis protéicos. Para avaliação global das alterações fenotípicas e genotípicas foram obtidas matrizes de similaridade. O perfil de susceptibilidade mostrou sensibilidade diminuída para a maioria das linhagens derivadas, mesmo após o cultivo sem drogas. Alterações morfológicas e de complexidade celular foram observadas, principalmente nas linhagens cultivadas em CSI de β-lactâmicos (FnAMP+, FnAMS+ e FnPTZ+), que também expressaram capacidade diminuída para formação de biofilme. Contudo, a morfologia regular e a habilidade de formação de biofilme foram retomadas após o cultivo sem droga. As linhagens FnCLI, FnCLO, FnLEV e FnMET não apresentaram alterações morfológicas evidentes, porém, foi observado aumento na formação de biofilme, com destaque para FnCLI+. As linhagens FnMET+ e FnCLI+ apresentaram um alto custo de fitness. Foram observadas alterações no padrão de metabolismo de carboidratos e na atividade de enzimas microbianas. Comparado com a FnPAR, várias proteínas (de 4.5 a 240 kDa) foram positivamente ou negativamente reguladas nas linhagens derivadas. Observou-se polimorfismo no DNA em todas as linhagens derivadas. As matrizes de similaridade não mostraram relações entre o padrão de polimorfismo de DNA e as outras características. Entretanto, existe uma tendência de que as alterações bioquímicas estejam relacionadas com alterações nos perfis protéicos. CSI de antimicrobianos podem, de fato, induzir alterações em F. nucleatum, com reflexo direto na sua biologia. Estes resultados alertam para o risco de antibioticoterapia inadequada, que podem ter sérias implicações para a microbiologia clinica e doenças infecciosas e, ainda, interferir com a relação bactéria-hospedeiro. / Subinibitory Concentrations (SIC) of antimicrobials, often arising from antibioticotherapy, may result in alterations in bacterial biology with implications for its potential aggressor. This effect has considerable importance for the bacteria of resident microbiota, especially for Fusobacterium nucleatum, one of the most prominent anaerobes in humans. Our aim was to analyze the effects of antimicrobials SIC in morphological, biochemical, physiological and molecular characteristics of F. nucleatum. From the strain F. nucleatum ATCC 25586 (FnWLD) were obtained 14 strains selected by 10 successive culture on SIC of ampicillin (FnAMP+), ampicillin/sulbactam (FnAMS+), clindamycin (FnCLI+), chloramphenicol (FnCLO+), levofloxacin (FnLEV+), metronidazole (FnMET+) and piperacillin/tazobactam (FnPTZ+) and subsequent 10 cultures in the absence of the same drugs. We evaluated the antimicrobial susceptibility patterns, bacterial morphology, biochemical profile, biofilm formation and the fitness cost. We also performed genotyping and analysis of protein profiles. For the global evaluation of phenotypic and genotypic alterations, similarity matrices were obtained. The antimicrobial susceptibility patterns showed decreased sensitivity to most of derived strains, even after culture without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SIC of β-lactam (FnAMP+, FnAMS+ and FnPTZ+), which also expressed decreased ability to biofilm formation. However, the regular morphology and the ability to biofilm formation were restored after culture without drug. The strains FnCLI, FnCLO, FnLEV and FnMET showed no apparent morphological changes, however, there was an increase in biofilm formation, especially for FnCLI+. The strains FnMET+ and FnCLI+ had a high fitness cost. Changes were observed in the carbohydrate metabolism patterns and activity of microbial enzymes. Compared with the FnWLD, several proteins (from 4.5 to 240 kDa) were positively or negatively regulated in the derived strains. It was observed polymorphism in the DNA in all derived strains. The similarity matrices showed no relationship between the DNA polymorphism patterns and other features. However, there is a tendency that the biochemical changes to be related to alterations in protein profiles. SIC of antimicrobials may, indeed, to induce alterations in F. nucleatum with direct impact on its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for the clinical microbiology and infectious diseases and also to interfere with the bacteria-host relationship.