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Introdução - A crescente resistência antimicrobiana em bactérias responsáveis por infecções hospitalares é um grande desafio à Saúde Pública. as B-lactamases de espectro estendido (ESBL), que hidrolisam a maioria dos compostos B-lactâmicos, são reconhecidas mundialmente como um grande problema para pacientes hospitalizados, devido à localização de seus genes em elementos transferíveis, facilitando sua disseminação. Objetivo - Caracterizar geneticamente cepas de Enterobactérias produtoras de ESBL isoladas de pacientes de um hospital público da cidade de São Paulo. Material e métodos - Todas as cepas de enterobactérias produtoras de ESBL isoladas em um ano foram submetidas a análises moleculares pela PCR, com iniciadores específicos para oito genes bla, e as cepas de Klebsiella pneumoniae ESBL positivas (ESBL-Kp) identificadas nesse período foram comparadas pela técnica de PFGE.Resultados - Os genes, bla(tem), bla(shv), bla(ctx-m), bla(per-2) bla(veb) and bla(ges) foram identificados em 9 espécies: Klebsiella pneumoniae (71,5 por cento), Escherichia coli (13,5 por cento), Morganella morganii (6 por cento), Proteus mirabilis (3 por cento), Klebsiella oxytoca (1,5 por cento), Providencia rettgeri (1,5 por cento), Providencia stuartii (1,5 por cento), Enterobacter aerogenes (0,75 por cento). Os genes bla(per-1) e bla(oxa) não foram detectados. O PFGE revelou 8 perfis moleculares principais em 68,4 por cento das ESBL-Kp, e 31,6 por cento das cepas não estavam relacionadas. Conclusões - Os resultados de PCR revelaram uma grande variedade de grupos de ESBL, e aparentemente este é o primeiro relato de grupos GES e VEB em enterobactérias no Brasil. / Introduction - The increasing antimicrobial resistance in pathogenic bacteria causing nosocomial infections is a major public health challenge. The extended-spectrum β-lactamases (ESBL), which hydrolyze most of β-lactams, are recognized worldwide as a great problem to hospitalized patients, due to the transferable location of their genes, which facilitates their spreading. Objective - Genetically characterize ESBL-producing Enterobacteriaceae strains isolated from patients of a Public Hospital in the city of São Paulo. Material and Methods - All Enterobacteriaceae ESBL-producing strains isolated in an 1-year period were submitted to molecular analysis by PCR with specific primers for eight bla genes, and all ESBL Klebsiella pneumoniae (ESBL-Kp) identified in this period were compared by the PFGE technique. Results - Genes blaTEM, blaSHV, blaCTX-M, blaPER-2, blaVEB and blaGES were identified in 9 species: Klebsiella pneumoniae (71,5%), Escherichia coli (13,5%), Morganella morganii (6%), Proteus mirabilis (3%), Klebsiella oxytoca (1,5%), Providencia rettgeri (1,5%), Providencia stuartii (1,5%), Enterobacter aerogenes (0,75%) and Enterobacter cloacae (0,75%). Genes blaPER-1 and blaOXA were not detected in any strain. PFGE revealed 8 distinct main molecular patterns in 68,4% of ESBL-Kp, and 31,6% of the strains were totally unrelated. Conclusions - PCR results showed a great variety of ESBL groups in the institution, and apparently this is the first report of GES- and VEB-ESBL groups in enterobacteria in Brazil. The results suggest the spread of resistance genes in different strains of ESBL-Kp in some hospital wards, and also that some strongly related clones of these bacteria colonized patients from a neonatal ward in a 3-month period.

Introdução - A crescente resistência antimicrobiana em bactérias responsáveis por infecções hospitalares é um grande desafio à Saúde Pública. as B-lactamases de espectro estendido (ESBL), que hidrolisam a maioria dos compostos B-lactâmicos, são reconhecidas mundialmente como um grande problema para pacientes hospitalizados, devido à localização de seus genes em elementos transferíveis, facilitando sua disseminação. Objetivo - Caracterizar geneticamente cepas de Enterobactérias produtoras de ESBL isoladas de pacientes de um hospital público da cidade de São Paulo. Material e métodos - Todas as cepas de enterobactérias produtoras de ESBL isoladas em um ano foram submetidas a análises moleculares pela PCR, com iniciadores específicos para oito genes bla, e as cepas de Klebsiella pneumoniae ESBL positivas (ESBL-Kp) identificadas nesse período foram comparadas pela técnica de PFGE.Resultados - Os genes, bla(tem), bla(shv), bla(ctx-m), bla(per-2) bla(veb) and bla(ges) foram identificados em 9 espécies: Klebsiella pneumoniae (71,5 por cento), Escherichia coli (13,5 por cento), Morganella morganii (6 por cento), Proteus mirabilis (3 por cento), Klebsiella oxytoca (1,5 por cento), Providencia rettgeri (1,5 por cento), Providencia stuartii (1,5 por cento), Enterobacter aerogenes (0,75 por cento). Os genes bla(per-1) e bla(oxa) não foram detectados. O PFGE revelou 8 perfis moleculares principais em 68,4 por cento das ESBL-Kp, e 31,6 por cento das cepas não estavam relacionadas. Conclusões - Os resultados de PCR revelaram uma grande variedade de grupos de ESBL, e aparentemente este é o primeiro relato de grupos GES e VEB em enterobactérias no Brasil. / Introduction - The increasing antimicrobial resistance in pathogenic bacteria causing nosocomial infections is a major public health challenge. The extended-spectrum β-lactamases (ESBL), which hydrolyze most of β-lactams, are recognized worldwide as a great problem to hospitalized patients, due to the transferable location of their genes, which facilitates their spreading. Objective - Genetically characterize ESBL-producing Enterobacteriaceae strains isolated from patients of a Public Hospital in the city of São Paulo. Material and Methods - All Enterobacteriaceae ESBL-producing strains isolated in an 1-year period were submitted to molecular analysis by PCR with specific primers for eight bla genes, and all ESBL Klebsiella pneumoniae (ESBL-Kp) identified in this period were compared by the PFGE technique. Results - Genes blaTEM, blaSHV, blaCTX-M, blaPER-2, blaVEB and blaGES were identified in 9 species: Klebsiella pneumoniae (71,5%), Escherichia coli (13,5%), Morganella morganii (6%), Proteus mirabilis (3%), Klebsiella oxytoca (1,5%), Providencia rettgeri (1,5%), Providencia stuartii (1,5%), Enterobacter aerogenes (0,75%) and Enterobacter cloacae (0,75%). Genes blaPER-1 and blaOXA were not detected in any strain. PFGE revealed 8 distinct main molecular patterns in 68,4% of ESBL-Kp, and 31,6% of the strains were totally unrelated. Conclusions - PCR results showed a great variety of ESBL groups in the institution, and apparently this is the first report of GES- and VEB-ESBL groups in enterobacteria in Brazil. The results suggest the spread of resistance genes in different strains of ESBL-Kp in some hospital wards, and also that some strongly related clones of these bacteria colonized patients from a neonatal ward in a 3-month period.

Streptococcus agalactiae is a pathogen of multiple hosts. The bacterium, an aetiological agent of septicaemia and meningo-encephalitis in freshwater and saltwater fish species, is considered a major threat to the aquaculture industry, particularly for tilapia. Cattle and humans are however the main known reservoirs for S. agalactiae. In humans, the bacterium (commonly referred to as Group B Streptococcus or GBS) is a member of the commensal microflora of the intestinal and genito-urinary tracts, but it is also a major cause of neonatal invasive disease and an emerging pathogen in adults. In cattle, S. agalactiae is a well-recognized causative agent of mastitis. Numerous studies focusing on S. agalactiae from human and bovine origins have provided insight into the population structure of the bacterium, as well as the genome content and pathogenic mechanisms through identification of virulence determinants. Concerning S. agalactiae from aquatic origins, scientific information mainly focused on case reporting and/or experimental challenges, with a limited or absence of information in terms of pathogenesis, virulence determinants and genotypes of the strains involved. The objective of this study was to enhance our understanding of the molecular epidemiology, host-adaptation and pathogenicity of S. agalactiae in aquatic species, with particular emphasis on tilapia. Firstly, a collection of 33 piscine, amphibian and sea mammal isolates originating from several countries and continents was assembled, with the aim of exploring the population structure and potential host specificity of aquatic S. agalactiae. Isolates were characterised using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and a standardised 3-set genotyping system comprising molecular serotypes, surface protein gene profiles and mobile genetic element profiles. Two major subpopulations were identified in fish. The first subpopulation consisted of non-haemolytic isolates that belonged to sequence type (ST) 260 or 261, which are STs that have been reported only from teleosts. These isolates exhibited a low level of genetic diversity by PFGE and clustered with other STs that have been reported only in fish. Another common feature was the absence of all surface protein genes or mobile genetic elements targeted as part of the 3-set genotyping and that are usually found in human or bovine isolates. The second subpopulation consisted of β-haemolytic isolates recovered from fish, frogs and sea mammals, and that exhibited medium to high genetic diversity by PFGE. STs identified among these isolates have previously been identified from strains associated with asymptomatic carriage and invasive disease in humans. The human pathogenic strain ST7 serotype Ia was detected in fish from Asia. Moreover, ST283 serotype III-4 and its novel single locus variant ST491 detected in fish from Southeast Asia shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. These observations suggested that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. STs found among the seal isolates (ST23) have also been reported from humans and numerous other host species, but never from teleosts. This work provided an excellent basis for exploration of the virulence of selected strains in experimental challenges. The virulence of two strains of S. agalactiae was experimentally investigated by intra-peritoneal infection of Nile tilapia (Oreochromis niloticus), using an isolate originally recovered from fish and belonging to ST260, and an isolate originating from a grey seal and belonging to ST23. The clinical signs, the in vivo distribution of viable bacteria and bacterial antigens, and the gross and histopathological lesions that developed during the time course of the infection were investigated. The ST260 strain was highly virulent, whereas no major clinical sign or mortalities occurred in the fish challenged with the ST23 strain. After injection, both strains however gained access to the bloodstream and viable bacteria were recovered from all organs under investigation. During the early stages of infection, bacteria were mostly found within the reticulo-endothelial system of the spleen and kidney. Thereafter, the ST260 demonstrated a particular tropism for the brain and the heart, but granulomatous inflammation and associated necrotic lesions were observed in all organs. ST23 was responsible for a mixed inflammatory response associated with the presence of bacteria in the choroid rete and in the pancreatic tissue only. After 7 days post-challenge and for both strain, the formation or containment of bacteria within granulomata or other encapsulated structures appeared to be a major component of the fish response. However, the load of viable bacteria remained high within organs of fish infected with ST260, suggesting that, unlike ST23, this strain is able to survive within macrophages and/or to evade the immune system of the fish. This work demonstrates that the lack of report of ST23 strains in fish is possibly not due to a lack of exposure but to a lack of virulence in this host. The two strains, which differ in prevalence and virulence in fish, provide an excellent basis to investigate genomic differences underlying the host-association of distinct S. agalactiae subpopulations. The genome of the ST260 strain used in challenge studies was sequenced. We therefore provided the first description for the genome sequence of a non-haemolytic S. agalactiae isolated from tilapia (strain STIR-CD-17) and that belongs by multi-locus sequence typing (MLST) to clonal complex (CC) 552, which corresponds to a presumptive fish-adapted subgroup of S. agalactiae. The genome was compared to 13 S. agalactiae genomes of human (n=7), bovine (n=2), fish (n=3) and unknown (n=1) origins. Phylogenetic analysis based on the core genome identified isolates of CC552 as the most diverged of all S. agalactiae studied. Conversely, genomes from β-haemolytic isolates of CC7 recovered from fish were found to cluster with human isolates of CC7, further supporting the possibility that some strains may represent a zoonotic or anthroponotic hazard. Comparative analysis of the accessory genome enabled the identification of a cluster of genes uniquely shared between CC7 and CC552, which encode proteins that may provide enhanced fitness in specific niches. Other genes identified were specific to STIR-CD-17 or to CC552 based on genomic comparisons; however the extension of this analysis through the PCR screening of a larger population of S. agalactiae suggested that some of these genes may occasionally be present in isolates belonging to CC7. Some of these genes, occurring in clusters, exhibited typical signatures of mobile genetic elements, suggesting their acquisition through horizontal gene transfer. It is not possible to date to determine whether these genes were acquired through intraspecies transfer or through interspecies transfer from the aquatic environment. Finally, general features of STIR-CD-17 highlighted a distinctive genome characterised by an absence of well conserved insertion sequences, an abundance of pseudogenes, a smaller genomic size than normally observed among human or bovine S. agalactiae, and an apparent loss of metabolic functions considered conserved within the bacterial species, indicating that the fish-adapted subgroup of isolates (CC552) has undergone niche restriction. Finally, genes encoding recognised virulence factors in human S. agalactiae were selected and their presence and structural conservation was evaluated within the genome of STIR-CD-17.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: A alta frequência de Estafilococos Coagulase-Negativa (CoNS) na pele de indivíduos saudáveis e em doenças associadas ao sangue, associados à seleção de cepas resistentes devido a uso indiscriminado de antimicrobianos, tornou mais estreitos os limites entre o ambiente hospitalar e o comunitário quanto à distribuição de cepas. Objetivou-se, com este estudo, caracterizar isolados de CoNS de origem hospitalar e comunitária da cidade de Botucatu-SP quanto ao perfil clonal, analisar os aspectos de resistência à oxacilina pela aferição de metodologia de detecção, e investigar os determinantes de heterorresistência à vancomicina nessas cepas. As espécies estudadas incluíram S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans e S. xylosus. O teste de disco-difusão (TDD) com discos de oxacilina e cefoxitina, fitas de Etest impregnadas com oxacilina e pesquisa do gene mecA por PCR em tempo real foram realizadas. A triagem em ágar com 6 e 8 µg/ml de vancomicina, microdiluição em caldo para aferição da Concentração Inibirtória Mínima (MIC), microscopia eletrônica de transmissão para verificar espessamento da parede celular e alterações fenotípicas por testes bioquímicos foram realizadas. O perfil clonal foi determinado por PFGE (Pulsed-Field Gel Eletrophoresis) e para clones de S. epidermidis, o MLST (Multilocus Sequence Typing). S. epidermidis apresentou alta diversidade clonal, mas presença de clusters no ambien... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The high frequency of Coagulase-Negative Staphylococci (CoNS) on the skin of healthy individuals and in bloodstream infections, together with the selection of resistant strains, has narrowed the boundaries between the hospital and the community environment for the distribution of strains. This study aimed to characterize CoNS isolated from clinical and colonization specimens of patients and individuals from Botucatu-SP, to compare their clonal profile, to analyze the determination of oxacillin resistance by the evaluation of the methodology of detection, and to investigate the determinants of reduced susceptibility to vancomycin in those strains. CoNS species included S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans and S. xylosus. The disc diffusion test (DDT) using oxacillin and cefoxitin discs was employed, Etest strips impregnated with oxacillin and mecA gene detection by real-time PCR were used. An agar screening with 6 and 8 µg/ml of vancomycin, the broth microdiluition method for the Minimal Inhibitory Concentration (MIC), the transmission eletronic microscopy for evaluation of cellwall thickening and phenotypic modifications by biochemical tests were performed. Clonal profile was determined by PFGE (Pulsed-Field Gel Eletrophoresis) and, for S. epidermidis clones, MLST (Multilocus Sequence Typing). S. epidermidis presented high clonal diversity, despite some clusters circulating within hospi... (Complete abstract click electronic access below) / Doutor