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Balsalobre LC. Tetracycline resistance in clinical and environmental isolates of Escherichia coli, Klebsiella pneumoniae and Aeromonas spp.: identification and mapping of tet genes genetic context [Thesis]. São Paulo (BR): Faculdade de Saúde Pública da USP; 2014. Introduction. The antibiotic resistance is accepted as one of the major problems for public health. Tetracyclines are broad spectrum antibiotics, and its indiscriminate use promoted the emergence of resistant bacteria, leading physicians and veterinarians to decrease its use. Objectives. Verify the susceptibility of clinical and environmental isolates of Escherichia coli, Klebsiella pneumoniae and Aeromonas spp. to tetracyclines, and also search for the main tet genes associated with resistance to these antibiotics and determine the potential mechanism of tet genes dissemination by characterizing their genetic context. Material and Methods. Disk-Diffusion and Minimum Inhibitory Concentration tests were carried out in 572 isolates using tetracycline (TET), doxycycline (DOX), minocycline (MIN) and tigecycline (TGC). PCR was carried out in TET non-susceptible isolates for the detection of Inc groups, tet genes and its genetic context determination through the search of classes 1, 2, 3, and 4 integrases, and Tn1721, Tn10, IS26 and ISAS5 mobile genetic elements. Genetic similarities patterns were determined by ERIC-PCR and PFGE techniques. After analyzing the results 33 strains were selected for the S1-PFGE and transformation experiments. Results. From 572 isolates, 18.5% were TET-resistant, 13.5% DOX-resistant, 8% MIN-resistant and none resistant to TGC. Twenty-two per cent and 16.3% of clinical and environmental isolates were TET-resistant, in that order. Genes tet(A), tet(B), tet(C), tet(D) and tet(E), coding for efflux pump mechanism, were found in 25.5%, 33%, 6.5%, 18.9% and 23.5% of the isolates, respectively. Ninety-five per cent, 100%, 100% and 4.5% of the isolates carrying tet(A), tet(B), tet(D) and tet(E) were non-susceptible to DOX, respectively. Resistance to MIN was observed in 4.2%, 78.8% and 100% of isolates carrying tet(A), tet(B) and tet(D), in that order. The gene tet(A) was associated with Tn1721, tet(B) with Tn10, and tet(C) and (D) with IS26. None of the searched integrases were associated with the tet genes detected. Groups IncF, IncFIB and IncA/C were respectively observed in 54.8%, 41.1% and 28.7% of the isolates. One Aeromonas spp. was carrying an IncP plasmid. The genetic similarities patterns demonstrated that there were identical genetic patterns among the hospital K. pneumoniae isolates, however all the remaining isolates possessed distinct genetic patterns. Of the 33 strains selected for plasmid linearization and transformation experiments, 8 were successfully transformed, in which the presence of tet genes in plasmids were observed. Conclusions. A low level of tetracycline resistance was detected. TGC was the most active tested antibiotic, followed by MIN. Genes tet(A) and tet(B) were the most prevalent among the isolates. All strains carrying tet(B) and tet(D) were non-susceptible to DOX and MIN. Groups IncF, IncFIB and IncA/C were the most detected in this study. The results suggest that tet(A), (B), (C) and (D) are disseminated by plasmids and are associated with Tn1721, Tn10 and IS26. Additional studies assembling recent isolates and other genera are necessary in order to contribute with information about the bacteria resistance to tetracyclines. Descriptors: Public Health; Antimicrobial resistance; Horizontal gene transfer; tetracyclines; Plasmids; Integrons; Transposons. ÍNDICE 1. INTRODUÇÃO 21 1.1 Considerações gerais 21 1.2 Antibióticos e o panorama atual 25 1.3 A resistência bacteriana na área da Saúde Pública 28 1.4 O papel do ambiente na resistência bacteriana 29 1.5 Classes de antibióticos 32 1.6 Tetraciclinas 33 1.6.1 Mecanismos de ação das tetraciclinas  37 1.6.2 Mecanismos de resistência à tetraciclinas 39 1.7 Aquisição genética e a resistência bacteriana 43 1.7.1 Plasmídios 45 1.7.2 Elementos genéticos mobilizáveis: transposons e integrons 49 1.7.3 A disseminação de genes tet 57 1.8. Relevância do estudo em Saúde Pública 62 2. OBJETIVOS 65 2.1 Objetivo Geral 65 2.2 Objetivos Específicos 65 3. MATERIAL E MÉTODOS 66 3.1 Seleção dos isolados 66 3.2 Testes fenotípicos para detecção de resistência a tetraciclinas 67 3.2.1 Detecção de genes tet 67 3.2.1.1 Extração de DNA total 67 3.2.1.2 Detecção de genes tet pela PCR 68 3.3 Caracterização do ambiente genético dos genes tet 69 3.3.1 Pesquisa de integrases de classes 1, 2, 3 e 4 69 3.3.2 Mapeamento de integrons 70 3.3.3 Pesquisa e mapeamento de transposons 71 3.4 Tipagem plasmidial 78 3.5 Tipagem molecular 79 3.5.1 Extração do DNA genômico 79 3.5.2 ERIC-PCR 80 3.5.3 PFGE para DNA cromossômico 81 3.6 Critérios para seleção de cepas para metodologias posteriores 81 3.7 Determinação da localização dos genes 82 3.7.1 Eletroforese em Campo Pulsado (PFGE) – Preparo dos Plugs 82 3.7.2 Linearização e determinação do tamanho dos plasmídios 82 3.7.3 Transformação de plasmídios 82 3.7.3.1 Preparação de células competentes 82 4. ANÁLISE DOS DADOS 84 5. RESULTADOS 86 5.1 Resistência fenotípica a tetraciclinas 86 5.2 Detecção de genes tet 91 5.3 Pesquisa de transposons 96 5.3.1 Caracterização do ambiente genético dos genes tet 100 5.4 Integrons 102 5.5 Detecção de grupos de incompatibilidade (Inc) 104 5.6 Genotipagem 106 5.7 Seleção das cepas para localização dos genes tet 107 6. DISCUSSÃO 112 6.1 Resistência fenotípica a tetraciclinas 114 6.2 Genes tet e determinação de seu ambiente genético 119 6.3 Integrons 129 6.4 Grupos Inc 130 6.5 Limitações do estudo 133 6.6 Considerações Finais 134 7. CONCLUSÕES 138 8. REFERÊNCIAS BIBLIOGRÁFICAS 140 LISTA DE QUADROS Quadro 1. Desenvolvimento da resistência aos antibióticos introduzidos na prática clínica comparados com o ano de aprovação pelo FDA (Food Drug Administration) (adaptado de: BUSH, 2004)

Resistência a tetraciclinas em isolados clínicos e ambientais de Escherichia coli, Klebsiella pneumoniae e Aeromonas spp.: identificação e mapeamento do ambiente genético de genes tet