The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Cleia Maria Monteiro da Cunha.pdf: 485275 bytes, checksum: a4db2d06d84a6f336975955e4fb1bd45 (MD5) Previous issue date: 2008-10-09 / Streptococcus agalactiae, also referred as group B streptococci (GBS) are commensals microorganisms adapted to asymptomatic colonization of the mammalians gut and genitourinary tract. Initially, this specie was recognized as a major etiologic agent for bovine mastitis but has becoming a leading cause of invasive infections in human neonates. The reasons behind the prompt and persistent emergence of GBS neonatal disease have not been completely elucidated, once human and bovine GBS populations are assumed be distinct and unrelated by divergence on their own physiological characters. Aiming to contribute for the characterization of the two S. agalactiae sub-populations that are in a proximal coexistence in the Rio de Janeiro state, this study evaluated phenotypic and genotypic diversity aspects of regional groups of GBS. The first made up of 50 isolates obtained from human specimens whilst the other group was constituted of 36 isolates from milk of dairy cows presenting clinical or sub clinical mastitis. Phenotypic characterization was based on physiological and serological tests, antimicrobial susceptibility assays were carried out by the disk standard procedure and microdilution method. The genetic aspect was assessed by PCR for detection of genes associated with resistance to tetracycline. According to the results of physiologic tests, β-hemolysis was a faculty shared by about 28% of bovine isolates and 100% of human isolates. GBS bovine isolates also shows different profile of sensitivity to bacitracin, only 33% of them were susceptive to the antibiotic, regardless of the whole human isolates set had demonstrated a 100% susceptive pattern to this substance. A 100% sensitivity percentual to penicillin was shared by all isolates assayed in this study corroborating the general procedure for antibiotic therapy of GBS infection. Otherwise, and in an overall view, bovine isolates showed higher resistance rates to a set of antibiotics, including cephoxitin, erytromicin, clyndamicin, sulphamethoxazole, azithromycin and ciprofloxacin than in their human counterparts. In a similar sense, it was observed in this study that 13,9% of the animal GBS isolates expressed cMLSB and 2,8 % M phenotypes. The M phenotype was expressed in 6% of the human related isolates as the unique MLSB parameter. Genetical assays performed detected 13,8% (5/36) and 14% (7/50) for tet (M), and 30,5% (11/36) and 10% (5/50) for de tet (O), respectively, in bovine and human isolates. These genes are implicated in tetracycline resistance by ribosome protection mechanism through enzymatic structural modification. / Os Streptococcus agalactiae, tamb?m designados como estreptococos do grupo B (EGB), s?o microrganismos comensais adaptados para fazer a coloniza??o assintom?tica do tubo digestivo, e do trato geniturin?rio de mam?feros. Inicialmente, reconhecida como um dos mais importantes agentes etiol?gicos da mastite bovina, esta esp?cie foi tamb?m implicada como uma das principais causas de infec??es invasivas em rec?m-nascidos humanos. As raz?es para a r?pida e consistente evolu??o do EGB como importante agente causal de infec??es neonatais ainda n?o foram completamente elucidadas, uma vez que as subpopula??es de EGB nos isolados de humanos e bovinos s?o independentes e distintas com base no reconhecimento da diversidade de suas caracter?sticas fisiol?gicas. Com o objetivo de contribuir para a caracteriza??o das duas subpopula??es de S. agalactiae que coexistem no Estado do Rio de Janeiro, este estudo avaliou aspectos da diversidade gen?tica e fenot?pica de dois grupos de EGB regionais, sendo o primeiro composto por 50 isolados obtidos a partir de esp?cimes cl?nicos humanos, e o segundo constitu?do por 36 isolados a partir de leite de vacas leiteiras com ind?cios de mastite cl?nica ou subcl?nica. A caracteriza??o fenot?pica dos isolados foi baseada em testes sorol?gicos e fisiol?gicos, testes de suscetibilidade antimicrobiana realizados com t?cnica padronizada para utiliza??o de disco de difus?o e pelo m?todo de microdilui??o. O aspecto gen?tico foi avaliado pela aplica??o de PCR para detec??o de genes associados ? resist?ncia ? tetraciclina. Os testes fisiol?gicos demonstraram que a capacidade de promover β-hem?lise era uma caracter?stica partilhada por cerca de 28% dos isolados a partir do material de natureza bovina, mas que manifestava-se em todos os isolados de origem humana. Os isolados de EGB bovinos tamb?m mostraram um perfil diferente quanto ? sensibilidade ? bacitracina, uma vez que apenas 33% delas se revelaram suscet?veis a esse antibi?tico contra 100% de sensibilidade para os isolados de origem humana. O percentual de 100% de sensibilidade ? penicilina demonstrado por todos os isolados analisados neste estudo, tamb?m corrobora a import?ncia do uso desse antibi?tico como procedimento geral na terapia de infec??es por EGB. De uma forma geral, neste estudo foi observado que os isolados originados de material bovino demonstraram percentuais de resist?ncia ao conjunto de antibi?ticos analisados (cefoxitina, eritromicina, clindamicina, sulfametoxazol, azitromicina e ciprofloxacina) superiores aos observados em isolados de material humano. Foi tamb?m observado que 13,9% dos isolados de EGB animais examinados expressaram o fen?tipo cMLSB e 2,8% o fen?tipo M. O fen?tipo M foi o ?nico par?metro MLSB expresso entre os isolados de S. agalactiae humanos, com um percentual de 6%. Quanto ? presen?a de genes de resist?ncia a tetraciclina entre as subpopula??es de EGB, detectou-se percentuais de 13,8% (5/36) e 14% (7/50) para tet(M), e 30,5% (11/36) e 10% (5/50) para tet(O), respectivamente, nos isolados bovinos e humanos avaliados. Estes genes est?o implicados na resist?ncia ? tetraciclina por um mecanismo prote??o ribossomal, pela altera??o estrutural mediada por a??o enzim?tica. Palavras-chave: Streptococcus agalactiae, caracteriza??o fenot?pica, gene tet

S. agalactiae (SGB), première cause d'infections néonatales, est aussi associé à des infections chez l'animal. Or, c'est avant tout une bactérie commensale du tube digestif chez l'homme. Son génome est riche en gènes codant pour des systèmes de régulation qui participent à l'adaptation de la bactérie à différents environnements. En partant de l'analyse de données de transcriptomes, j'ai caractérisé la réponse transcriptionnelle associée à plusieurs de ces systèmes de régulation. J'ai pu mettre en évidence que le processus de D-alanylation, responsable de l'ajustement de la charge nette à la surface de la bactérie, est contrôlé par deux boucles de rétrorégulation faisant intervenir deux TCS CiaRH et DltRS. J'ai également étudié la régulation de l'expression de deux facteurs de virulence, la protéine Srr et le pilus PI-2a, qui fait intervenir une régulation croisée de régulateurs de la famille RofA-like et un contrôle indirect par le régulateur CovRS appartenant au core génome. Les EGMs jouent un rôle majeur dans l'évolution des génomes par transfert horizontal. Chez SGB, une nouvelle famille d'ICEs, les TnGBS, est impliquée dans des phénomènes de transfert de type Hfr. Nous avons montré qu'ils constituent la première famille d'ICE codant pour une transposase à motif DDE. Ils possèdent aussi une spécificité d'insertion originale au niveau des régions promotrices. L'analyse comparative des séquences de ces ICE a permis de prédire les protéines impliquées dans les différentes phases du processus de conjugaison. Les TnGBS forment deux sous-familles qui associent une même transposase avec deux modules de conjugaison , de réplication distincts

Physicians frequently face challenges in predicting which bacterial subpopulations are likely to cause severe infections. A more accurate prediction of virulence would improve diagnostics and limit the extent of antibiotic resistance. Nowadays, bacterial pathogens can be typed with high accuracy with advanced genotyping technologies. However, effective translation of bacterial genotyping data into assessments of clinical risk remains largely unexplored. The discovery of unknown virulence genes is another key determinant of successful prediction of infectious disease outcomes. The trial-and-error method for virulence gene discovery is time-consuming and resource-intensive. Selecting candidate genes with higher precision can thus reduce the number of futile trials. Several in silico candidate gene prioritisation (CGP) methods have been proposed to aid the search for genes responsible for inherited diseases in human. It remains uninvestigated as to how the CGP concept can assist with virulence gene discovery in bacterial pathogens. The main contribution of this thesis is to demonstrate the value of translational bioinformatics methods to address challenges in virulence prediction and virulence gene discovery. This thesis studied an important perinatal bacterial pathogen, group B streptococcus (GBS), the leading cause of neonatal sepsis and meningitis in developed countries. While several antibiotic prophylactic programs have successfully reduced the number of early-onset neonatal diseases (infections that occur within 7 days of life), the prevalence of late-onset infections (infections that occur between 7??30 days of life) remained constant. In addition, the widespread use of intrapartum prophylactic antibiotics may introduce undue risk of penicillin allergy and may trigger the development of antibiotic-resistant microorganisms. To minimising such potential harm, a more targeted approach of antibiotic use is required. Distinguish virulent GBS strains from colonising counterparts thus lays the cornerstone of achieving the goal of tailored therapy. There are three aims of this thesis: 1. Prediction of virulence by analysis of bacterial genotype data: To identify markers that may be associated with GBS virulence, statistical analysis was performed on GBS genotype data consisting of 780 invasive and 132 colonising S. agalactiae isolates. From a panel of 18 molecular markers studied, only alp3 gene (which encodes a surface protein antigen commonly associated with serotype V) showed an increased association with invasive diseases (OR=2.93, p=0.0003, Fisher??s exact test). Molecular serotype II (OR=10.0, p=0.0007) was found to have a significant association with early-onset neonatal disease when compared with late-onset diseases. To investigate whether clinical outcomes can be predicted by the panel of genotype markers, logistic regression and machine learning algorithms were applied to distinguish invasive isolates from colonising isolates. Nevertheless, the predictive analysis only yielded weak predictive power (area under ROC curve, AUC: 0.56??0.71, stratified 10-fold cross-validation). It was concluded that a definitive predictive relationship between the molecular markers and clinical outcomes may be lacking, and more discriminative markers of GBS virulence are needed to be investigated. 2. Development of two computational CGP methods to assist with functional discovery of prokaryotic genes: Two in silico CGP methods were developed based on comparative genomics: statistical CGP exploits the differences in gene frequency against phenotypic groups, while inductive CGP applies supervised machine learning to identify genes with similar occurrence patterns across a range of bacterial genomes. Three rediscovery experiments were carried out to evaluate the CGP methods: a) Rediscovery of peptidoglycan genes was attempted with 417 published bacterial genome sequences. Both CGP methods achieved their best AUC >0.911 in Escherichia coli K-12 and >0.978 Streptococcus agalactiae 2603 (SA-2603) genomes, with an average improvement in precision of >3.2-fold and a maximum of >27-fold using statistical CGP. A median AUC of >0.95 could still be achieved with as few as 10 genome examples in each group in the rediscovery of the peptidoglycan metabolism genes. b) A maximum of 109-fold improvement in precision was achieved in the rediscovery of anaerobic fermentation genes. c) In the rediscovery experiment with genes of 31 metabolic pathways in SA-2603, 14 pathways achieved an AUC >0.9 and 28 pathways achieved AUC >0.8 with the best inductive CGP algorithms. The results from the rediscovery experiments demonstrated that the two CGP methods can assist with the study of functionally uncategorised genomic regions and the discovery of bacterial gene-function relationships. 3. Application of the CGP methods to discover GBS virulence genes: Both statistical and inductive CGP were applied to assist with the discovery of unknown GBS virulence factors. Among a list of hypothetical protein genes, several highly-ranked genes were plausibly involved in molecular mechanisms in GBS pathogenesis, including several genes encoding family 8 glycosyltransferase, family 1 and family 2 glycosyltransferase, multiple adhesins, streptococcal neuraminidase, staphylokinase, and other factors that may have roles in contributing to GBS virulence. Such genes may be candidates for further biological validation. In addition, the co-occurrence of these genes with currently known virulence factors suggested that the virulence mechanisms of GBS in causing perinatal diseases are multifactorial. The procedure demonstrated in this prioritisation task should assist with the discovery of virulence genes in other pathogenic bacteria.

Techn. Universiẗat, Diss., 2004--Berlin.

Made available in DSpace on 2014-06-11T19:30:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-01Bitstream added on 2014-06-13T19:19:24Z : No. of bitstreams: 1 salvador_r_dr_jabo.pdf: 343849 bytes, checksum: 71707b8f3990268b373dfeaa6953f1d9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve por objetivos avaliar a inter-relação entre a suplementação alimentar com 0,3% de parede celular de levedura e vacinação com extrato oleoso de Streptococcus agalactiae sobre o desempenho produtivo, parâmetros fisiopatológicos e componente celular inflamatório em tilápia do Nilo. Oitenta e quatro tilápia com peso médio inicial de 125,0 + 1,5g foram distribuídas em 12 caixas de fibra, seguindo o esquema fatorial 2x2x3, correspondente a dois níveis de parede celular de levedura (0,0 e 0,3% parede celular), dois tratamentos (solução salina e vacina) e três coletas após o desafio com a bactéria viva (seis, 24 e 48h) com sete repetições. Os peixes foram alimentados durante 77 dias. A vacinação foi realizada 60 dias após o início da alimentação, por meio da inoculação intraperitoneal de 0,5 mL da vacina contendo 108 UFC/mL. Após 15 dias da vacinação, todos os peixes foram submetidos ao desafio com Streptococcus agalactiae vivo, por meio da inoculação intraperitoneal de 108 UFC/mL, veiculadas em 0,5 mL de solução salina (0,85%). As análises do desempenho produtivo mostraram que a suplementação dietética com parede celular de levedura associada à vacinação não influenciou o desempenho produtivo da tilápia do Nilo e o melhor desempenho ocorreu na utilização da parede celular de levedura. Os parâmetros hematológicos mostraram que a suplementação com 0,3% de parede celular de levedura associada à vacinação contra Streptococcus agalactiae em tilápia do Nilo foi essencial para incrementar a hematopoiese. A suplementação alimentar com 0,3% de parede celular de levedura associada à vacinação melhorou a resposta de defesa dos peixes, no que se refere à inflamação aguda e destaca a importância da vacinação. / The work evaluates the interrelation between supplementation diets with 0,3% of cellular wall of yeast and vaccination with oily extract of Streptococcus agalactiae in the growth performance, phisiopathological parameters and inflamatory cellular component of Nile tilapia (Oreochromis niloticus). Eighty four tilapia with initial average weight of 125,0 ± 1,5g were distributed in 12 fiber aquaria, following the factorial design 2x2x3, corresponding to two levels of cellular wall of yeast (0,0 and 0,3% cellular wall), two treatments (saline solution and vaccine) and three collections, after challenge with bacteria (six, 24 and 48h) with seven repetitions. Fish were fed during 77 days. The vaccination was accomplished 60 days after the beginning of the feeding, through inoculation intraperitoneal of 0,5 mL of the vaccine containing 108 UFC/mL. After 15 days of the vaccination, all fishes were challenged with Streptococcus agalactiae, through inoculation intraperitoneal of 108 UFC/ml, diluted in 0,5 mL of saline solution (0,85%). The analyses of the pattern performance showed that supplementation diets with cellular wall of yeast associated to vaccination did not influence the growth performance of Nile tilapia and the best growth performance was obtained using cellular wall of yeast. The phisiopathological parameters showed supplementation diets with 0,3% of cellular wall of yeast associated to the vaccination against Streptococcus agalactiae in Nile tilapia were essential to increase the hematopoiesis. The supplementation diets with 0,3% of cellular wall of yeast associated to the vaccination improved the immune response of fish, in relation to the sharp inflammation and it detaches the importance of the vaccination.

Orientador: José Carlos de Figueiredo Pantoja / Resumo: O objetivo deste estudo foi avaliar novas estratégias de identificação e tratamento de mastite subclínica causada por Streptococcus agalactiae. Dois estudos foram conduzidos para alcançar os objetivos propostos: 1) um ensaio clínico randomizado, para avaliar a eficácia do tratamento de S. agalactiae com cloxacilina intramamária (CLOXIMM), cefquinoma intramamária (CEFIMM) e cefquinoma intramuscular (CEFIM); e avaliar a não-inferioridade da CLOXIMM em relação a CEFIMM. E, 2) um estudo de acurácia diagnóstica, para estimar a acurácia do Somaticell, California Mastitis Test (CMT) e do exame microbiológico do leite composto (leite dos quatro quartos) na detecção de quartos e animais infectados com S. agalactiae. Os resultados indicaram que quartos tratados com CEFIM apresentaram menor taxa de cura bacteriológica (55%) do que aqueles tratados com CLOXIMM (86%) ou CEFIMM (98%). A diferença na proporção de cura bacteriológica entre CEFIMM e CLOXIMM foi de 0,121 (intervalo de confiança de 95%: 0,056 - 0,184). A CLOXIMM foi considerada não inferior a CEFIMM quando margens de não inferioridade de 0,20 e 0,25 foram utilizadas. Contudo, a determinação da não inferioridade foi inconclusiva para margens de 0,10 e 0,15. A cultura do leite composto apresentou acurácia diagnóstica satisfatória (95,7%) quando comparada a cultura individual por quarto. Para identificação dos quartos infectados com S. agalactiae, o ponto de corte considerado mais adequado foi de 205.000 células/mL para o teste So... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the present study was to assess new strategies to identify and treat Streptococcus agalactiae subclinical mastitis. Two studies were conducted to achieve the proposed objectives: 1) a randomized clinical trial, to assess the efficacy of intramammary cloxacillin (CLOXIMM), intramammary cefquinome (CEFIMM), and intramuscular cefquinome (CEFIM), to treat S. agalactiae intramammary infections (IMI); and assess whether CLOXIMM was non-inferior to CEFIMM to treat S. agalactiae IMI. And, 2) a diagnostic accuracy study, to estimate the accuracy of the Somaticell, California Mastitis Test (CMT), and the composite milk microbiological examination of milk to detect S. agalactiae IMI. Results indicated that the bacteriological cure rate was lower for quarters treated with CEFIM (55%), as compared with CLOXIMM (86%) or CEFIMM (98%). The bacteriological cure difference between CEFIMM and CLOXIMM was 0.121 (95% confidence interval: 0.056 - 0.184). The CLOXIMM was considered non-inferior to CEFIMM when the non-inferiority margins of 0.20 and 0.25 were considered. Nevertheless, determination of non-inferiority was inconclusive for margins of 0.10 and 0.15. Microbiological examination of composite milk was of high accuracy (95.7%), as compared with microbiological examination of quarter milk samples. The thresholds of 205,000 cells/mL for the Somaticell and of score 1 for the CMT can be considered the most appropriate for diagnosing S. agalactiae IMI. The higher sensitivity an... (Complete abstract click electronic access below) / Mestre

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-03-28T14:59:45Z No. of bitstreams: 1 texto completo.pdf: 1474259 bytes, checksum: 056642d909ee7e78e9dc9b22ef3349f8 (MD5) / Made available in DSpace on 2016-03-28T14:59:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1474259 bytes, checksum: 056642d909ee7e78e9dc9b22ef3349f8 (MD5) Previous issue date: 2015-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Staphylococcus aureus e Streptococcus agalactiae são patógenos contagiosos associados à forma subclínica e persistente da mastite bovina, principal doença da pecuária leiteira. Neste trabalho avaliou-se a presença de dois patógenos em amostras de leite CMT positivas coletadas durante seis meses de animais pertencentes a um rebanho da Zona da Mata de Minas Gerais. Foi encontrada uma alta prevalência dos patógenos contagiosos o que indica possível falta de higiene nos procedimentos de rotina de ordenha ou falha no tratamento da doença. A análise de multilocus de repetições em tandem de número variável (MLVA) revelou apenas um genótipo de S. aureus e seis genótipos de S. agalactiae, alguns dos quais foram associados a infecções persistentes e ao número de parições do animal. A maioria das bactérias isoladas foi considerada forte produtora de biofilme e no caso de S. aureus, os isolados apresentaram uma variada atividade hemolítica. É possível que esses fenótipos contribuam com a persistência da infecção. No capítulo 2, estirpes de S. aureus isoladas de animal com manifestação de mastite persistente ou não persistente foram contrastadas quanto a características importantes para a patogênese bacteriana e virulência in vivo. Genotipagem por eletroforese em campo pulsado (PFGE) revelou que as bactérias possuíam uma similaridade de 90%. As duas estirpes contrastadas apresentaram os mesmos genes de virulência. Porém, elas diferiram significativamente na produção de hemolisina e biofilme, na capacidade de invasão e persistência em células do epitélio mamário bovino (MAC-T). Nos ensaios in vivo realizados com o modelo Galleria mellonella, a estirpe SAU 302 revelou-se mais virulenta do que a SAU 322. Uma forte reação do sistema imune do inseto foi observada mediante infecção com as bactérias. Os resultados mostraram variações fenotípicas entre as estirpes geneticamente relacionadas que podem contribuir positivamente para o desenvolvimento da doença e devem ser cuidadosamente exploradas. Conclui-se que bactérias geneticamente relacionadas podem apresentar uma variação fenotípica não revelada por técnicas de fingerprint do DNA e que esta variação pode ter uma implicação na progressão da mastite bovina. Sugere-se a definição de um conjunto de ensaios fenotípicos relevantes para a doença a fim de caracterizar bactérias isoladas de animais com manifestação conhecida. / Staphylococcus aureus and Streptococcus agalactiae are contagious pathogens associated with subclinical and persistent bovine mastitis, the main disease of dairy cattle. In this work we investigated the presence of these pathogens in samples of milk CMT-positive collected during six months from animals belonging to a herd from the Zona da Mata of Minas Gerais. A high prevalence of the contagious pathogens was found indicating a possible lack of hygiene in the milking routine procedures or failure in the treatment of the disease. The multilocus variable-number tandem repeat analysis of (MLVA) revealed one genotype of S. aureus and six genotypes of S. agalactiae, some of them associated with persistent infections and calving number. Most bacterial isolates was considered strong biofilm producer and for S. aureus the isolates showed a variation in hemolytic activity. These phenotypes may contribute to the persistence of the infection. In Chapter 2, we analyzed the in vivo virulence and phenotypic traits believed to be important for the pathogenicity of bovine S. aureus. In this study the strains SAU 302 and SAU 322 isolated from animals with persistent or non-persistent mastitis, respectively, were contrasted. Genotyping by pulsed field gel electrophoresis (PFGE) revealed a genetic relatedness of 90% between bacteria. The two strains showed the same set of virulence genes investigated. However, significant differences were seen in the production of hemolysin and biofilm, in invasiveness and persistence in the bovine mammary epithelial cells (MAC-T). In vivo tests carried out with the Galleria mellonella model showed that SAU 302 was more virulent than the SAU 322. A strong reaction of the insect immune system was observed upon infection with both bacteria. Despite their genetic proximity, the strains showed a phenotypic variation undetected by DNA-based methods that can contribute to the development of the disease and should be explored further. Our present study suggests that a set of phenotypic assays relevant to bovine mastitis should be defined and used to analyze a collection of bacteria for which information on the nature of the clinical manifestation is available.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-07T15:27:28Z No. of bitstreams: 1 texto completo.pdf: 1815676 bytes, checksum: 0d62c2dc57ee0ade1ba91497365e0867 (MD5) / Made available in DSpace on 2016-06-07T15:27:28Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1815676 bytes, checksum: 0d62c2dc57ee0ade1ba91497365e0867 (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A mastite bovina é considerada por muitos autores a principal doença do rebanho leiteiro. Staphylococcus aureus e Streptococcus agalactiae são patógenos contagiosos comumente associados à forma subclínica e persistente da doença. A caracterização de estirpes em circulação nos rebanhos é de extrema importância na definição de estratégias de manejo para o controle da doença e de marcadores de prognóstico da mastite. No capítulo 1, 175 amostras positivas para o teste California Mastitis (CMT) foram coletadas de vacas holandesas e análises microbiológicas revelaram a presença de S. agalactiae em 34,2% das amostras. Em 36% das amostras foi observado o crescimento de bactérias pertencentes a outros grupos, em especial com S. aureus. A tipagem de S. agalactiae pela metodologia multilocus de repetições em tandem de número variável (MLVA) revelou seis genótipos (SagT1-T6), sendo SagT1 o mais prevalente no rebanho e também o mais frequentemente isolado de infecções mistas com S. aureus. Os isolados de S. agalactiae foram fortes produtores de biofilme in vitro e produziram hemólise do tipo beta. A análise de virulência dos isolados em larvas de Galleria mellonella definiu os tipos SagT3 e SagT1 como os mais e menos virulentos, respectivamente. Porém, dois isolados pertencentes a esses tipos não invadiram nem apresentaram efeito citotóxico em células epiteliais bovinas MAC-T. No capítulo 2, genomas de quatro estirpes de S. aureus causadoras de mastite subclínica, sendo SAU302 e SAU1364 isoladas de infecções persistentes e SAU170 e SAU1269 de infecções não persistentes, foram sequenciados. Uma grande conservação foi encontrada entre os genomas, como tamanho médio de 2,6 Mb, 32,8% de conteúdo GC e 2589 CDS. A tipagem por MLST classificou SAU170, SAU302, e SAU1364 como ST126, um tipo frequentemente encontrado em rebanhos brasileiros, e SAU1269, como ST1, associado a infecções humanas e bovinas. A comparação entre os genomas sequenciados e uma referência construída com genomas de dois isolados de mastite bovina, revelou uma identidade maior que 90% para a maioria dos genes anotados. A análise filogenética dos isolados sequenciados agrupou em ramos separados isolados de diferentes manifestações, sugerindo que algumas das particularidades compartilhadas entre isolados podem estar relacionados à persistência das infecções causadas por eles. A análise de polimorfismos de nucleotídeo único (SNPs) mostrou SNPs em genes relacionados à adesão das células bacterianas ao hospedeiro e na sequência de agrC, proteína receptora do sinal de quorum-sensing em S. aureus, alguns deles importantes para estrutura da proteína. / Bovine mastitis is considered the main disease of the dairy herd. Staphylococcus aureus and Streptococcus agalactiae are contagious pathogens commonly associated with subclinical and persistent form of the disease. The characterization of circulating strains in herds is of utmost importance to define management strategies to the control of the disease and to identify mastitis prognostic markers. In the first chapter, 175 milk samples positive for the California Mastitis Test (CMT) were collected from Holstein cows and microbiological analyzes revealed the presence of S. agalactiae in 34.2% of samples. The growth of bacteria belonging to other groups, in particular S. aureus, was seen in 36% of the milk samples. Multilocus variable-number tandem repeat analysis (MLVA) revealed the presence of six genotypes (SagT1-T6) of S. agalactiae in the herd with SagT1 being the most prevalent and also the most frequently isolated from mixed infections with S. aureus. S. agalactiae isolates were beta-hemolytic and strong in vitro biofilm producers. Virulence analysis in Galleria mellonella larvae defined the SagT3 and SagT1 types as the most and the less virulent, respectively. However, invasion and cytotoxicity in MAC-T cells did not reveal any difference between them. In Chapter 2, the genomes of four strains of Staphylococcus aureus causing subclinical mastitis, SAU302 and SAU1364 isolated from persistent infections and SAU170 and SAU1269 from non-persistent infections, were sequenced. A major conservation was found among them like the average size (2.6 Mb), the GC content (32.8%), and the CDS (2589). Multilocus Sequencing typing classified SAU170, SAU302 and SAU1364 as ST126, a type often found in Brazilian herds, while SAU1269 was classified as ST1, commonly associated with human and bovine infections. Comparative analyses with the genome of S. aureus RF122, a strain that causes severe mastitis, showed more than 90% of identity with annotated genes. Phylogenetic analysis grouped the sequenced genomes into separate branches suggesting that some of the characteristics shared between the isolates can be related to the persistence of infections. Single nucleotide polymorphisms (SNP) were found in genes related to bacterial adhesion and in agrC that codes for a receptor protein involved in quorum-sensing signaling in S. aureus, some of them important for protein structure.

Orientador: Jose Antonio Simões / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T04:28:03Z (GMT). No. of bitstreams: 1 Alves_ValeriaMoraesNeder_M.pdf: 694618 bytes, checksum: 74c214a9027dcefb819c6b963eba6be0 (MD5) Previous issue date: 2005 / Resumo: Introdução - A colonização pelo estreptococo do Grupo B (EGB) é, em nível mundial, altamente prevalente entre as gestantes, variando de 4% a 30%. Estes microorganismos podem ser transmitidos verticalmente e causar sérias conseqüências neonatais. No Brasil ainda não foram adotadas estratégias de prevenção e tratamento para reduzir a incidência de infecção neonatal pelo EGB, as quais, para serem efetivas, devem ser elaboradas com base em conhecimentos sobre a prevalência, aos fatores associados ao maior risco de colonização e às características fenotípicas do estreptococo do Grupo B. Objetivo: estabelecer a prevalência da colonização pelo EGB no trato genital de parturientes, identificar os fatores associados à essa colonização e as características fenotípicas destes estreptococos. Método: foi realizado um estudo de corte transversal, no período de 11 de novembro de 2003 a 14 de maio de 2004. No momento da admissão para o parto, uma amostra de 316 parturientes do Hospital Universitário de Jundiaí foi submetida à coleta, com swab estéril, de material das regiões retal e vaginal, para detecção do EGB, por cultura seletiva no meio de Todd-Hewitt. Dados referentes aos fatores associados à colonização foram obtidos dos prontuários rotineiramente preenchidos, ou perguntados às parturientes e transcritos para a ficha de dados. A susceptibilidade a sete antimicrobianos (penicilina, ampicilina, eritromicina, nitrofurantoina, clindamicina, cefalotina e gentamicina) foi obtida através da técnica de disco difusão dos antibióticos. As amostras foram diferenciadas pela tipagem sorológica e anti-soros específicos para os tipos sorológicos Ia, Ib, II, III, IV, V). Resultados: A prevalência da colonização pelo EGB na amostra estudada foi de 14,6%. Trabalhar fora de casa foi um fator significativamente associado à colonização. Nenhuma cepa foi resistente à penicilina, ampicilina, eritromicina e nitrofurantoína. A maior resistência foi para a gentamicina (76,1%), seguida pela clindamicina (17,4%). O sorotipo mais freqüente foi o Ib (23,9%), seguido pelos sorotipos II e Ia (19,6% e 17,4%, respectivamente). Não houve correlação entre o sorotipo e a maior resistência antimicrobiana. CONCLUSÃO: a prevalência da colonização pelo EGB em parturientes do Hospital Universitário de Jundiaí foi alta. Não houve fatores associados à colonização, exceto ao que se refere ao fato de trabalhar fora de casa. A penicilina continua sendo a droga de escolha para a profilaxia intraparto, porém a clindamicina como alternativa em mulheres alérgicas à penicilina deverá ser melhor avaliada por antibiograma ou substituída pela cefalotina. O sorotipo mais freqüente (Ib) diferiu da maioria dos estudos em outros países, demonstrando a necessidade da identificação da sorotipagem em cada região, a fim de uma futura elaboração de vacinas específicas para as gestantes brasileiras / Abstract: Introduction - Genital colonization with Group B Streptococcus(GBS) occurs in 4% to 30% of pregnant women. GBS can be vertically transmitted and cause significant morbidity and mortality in newborns. Strategies for prevention and treatment to reduce the incidence of the newborn infection have not been established yet in Brazil. In order to be effective, these strategies must be made based on the knowledge on prevalence, risk factors for GBS colonization and on GBS phenotypic characteristics. Objectives: To establish the prevalence of GBS genital colonization, to identify the factors associated with this colonization and the GBS phenotypic characteristics (antibiotic susceptibility and serotypes). Methodology: The study was carried out from November 11, 2003 to May 14, 2004. A total of 316 pregnant women of the University Hospital of Jundiaí was sampled for GBS cultures at the admission for delivery. Samples were collected using sterile swab from the vagina and rectum, which were immediately inoculated onto Todd Hewitt¿s selective medium. Data regarding to the factors associated with the colonization were obtained from patient¿s clinical record files. GBS antibiotic susceptibilities were determined using disk diffusion to seven antibiotics (penicillin, ampicillin, erythromycin, clindamycin, cefalotin, gentamycin and nitrofurantoin) according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). Serotypes were determined using specific anti-serum Ia, Ib, II, III, IV and V. Results: GBS colonization rate was 14.6%. The only factor significantly associated with GBS colonization was to have a job out of home. None isolate was resistant to penicillin, ampicillin, erythromycin or nitrofurantoin. The highest resistance rate was to gentamycin (76.1%), followed by clindamycin (17.4%). The most frequent serotype was Ib (23.9%) followed by serotypes II and Ia (19.6% and 17.4%, respectively). No correlations were found between serotype and antibiotic resistance. Conclusion: GBS colonization rate was high in pregnant women attending the University Hospital of Jundiaí for delivery. No risk factors were associated with the colonization except to have a job out of home. Penicillin continues to be the drug of choice for intrapartum antibiotic prophylaxis. However, clindamycin as an alternative in women allergic to penicillin should be better evaluated using antibiotic sensitivity tests. The most frequent serotype found in this study (Ib) was not the same found in most of studies from another countries. This result highlights the importance of the knowledge of the most prevalent serotypes in each region to further development of an appropriate vaccine for GBS / Mestrado / Tocoginecologia / Mestre em Tocoginecologia