Sequenciação aplicada à microbiologia clínica moderna: pesquisa e identificação de espécies bacterianas multirresistentes de Escherichia coli e Enterococcus spp
Data
2019-12-04
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
O intenso uso de antibióticos tanto na medicina humana como na medicina veterinária, contribui para o desenvolvimento e para a disseminação da resistência em distintos grupos de bactérias. Este é um problema de saúde pública global que requer uma abordagem multidisciplinar e holística, pois a resistência bacteriana aos antibióticos está a disseminar-se e a evoluir de forma semelhante em humanos, animais e no meio ambiente. A Organização Mundial de Saúde considera os Enterococcus spp. e Escherichia coli essenciais para monitorizar o nível de resistência aos antibióticos. Estas bactérias podem atuar como reservatório de genes de resistência a antibióticos que podem ser transferidos para outras bactérias patogénicas representando um problema mundial. Alguns genes de resistência estão localizados em plasmídeos sendo rapidamente disseminados para outras espécies bacterianas, incluindo isolados de populações de animais selvagens. Assim, embora os animais selvagens não estejam em contacto direto com antibióticos, são indiretamente afetados pelo uso geral excessivo de antibióticos noutros contextos. Um total de 21 estirpes de Enterococcus spp., isoladas em carne picada, almôndegas e hambúrgueres, assim como um total de 45 estirpes de Enterobacteriaceae, isoladas em animais selvagens, em animais para consumo e em carne, foram selecionadas da nossa coleção de estirpes bacterianas. Estas foram identificadas usando espetrometria de massa tendo sido identificadas 3 espécies diferentes de Enterococcus spp. [E. faecium (n=16), E. gallinarum (n=3) e E. durans (n=2)] e 45 E. coli. Através do método da difusão em agar pela técnica de Kirby-Bauer, interpretado segundo as normas do EUCAST encontraram-se 19 enterococos resistentes à vancomicina (VREs) e estes isolados apresentaram resistência à teicoplanina (84,2%), eritromicina (84,2%) e ampicilina (68,4%). Através da sequenciação do genoma, usando a tecnologia Illumina, foram detetados, mais frequentemente, os genes vanA, erm(B) e aac(6')-Ii. Por outro lado, em E. coli foram detetados 28 isolados produtores de ESBLs que eram resistentes à ampicilina (100%), ticarcilina (100%), piperacilina (100%), cefuroxima (96,4%) e cefotaxima (96,4%). Relativamente aos genes que codificam para ESBLs o mais frequente foi o blaCTX-M-1 que estava, maioritariamente associado ao plasmídeo IncI1. Outros genes foram detetados, contudo aqueles com maior frequência foram os genes tet(A) (64,3%), tetR (64,3%) e sul2 (53,6%). No que respeita o estudo do background genético, foram detetados 17 tipos de MLST diferentes e um total de 41761 SNP. A informação obtida com a sequenciação do genoma completo das bactérias no decorrer deste trabalho foi essencial para melhorar o conhecimento sobre a disseminação de estirpes resistentes nos animais selvagens, nos animais para consumo, assim como, na carne que deles deriva e as possíveis implicações envolvidas na transferência para outros animais ou humanos. Futuramente, e tendo em conta o conceito One Health, podem ser realizados estudos no sentido de investigar a prevalência de bactérias resistentes a antibióticos tanto em seres humanos como em amostras do meio ambiente, possibilitando a identificação das vias de transmissão de bactérias resistentes a antibióticos e dos seus genes de resistência.
The intense use of antibiotics in both human and veterinary medicine contributes to the development and spread of resistance in different groups of bacteria. This is a global public health problem which requires a multidisciplinary and holistic approach because bacterial resistance to antibiotics is spreading and evolving in similar ways in humans, in animals and in the environment. Escherichia coli and Enterococcus spp. are considered by the World Health Organization as essential microorganisms for monitoring the level of antibiotic resistance. These bacteria may act as reservoir of antibiotic resistance genes that can be transferred to other pathogenic bacteria and represent a worldwide problem. Some resistance genes are located on plasmids that are rapidly disseminated to other bacterial species, including isolates from wild animal populations. Thus, even though wild animals are not in direct contact with antibiotics, they are indirectly affected by the excessive general use of antibiotics in other settings. A total of 21 Enterococcus spp. strains isolated from minced meat, meatballs and hamburgers, as well as a total of 45 Enterobacteriaceae strains isolated from wildlife, animals for consumption and meat were selected from our previously studied collection of bacterial strains. These strains were identified using mass spectrometry and 3 different species of Enterococcus spp. [E. faecium (n = 16), E. gallinarum (n = 3) and E. durans (n = 2)] and 45 E. coli were identified. Using the Kirby-Bauer agar diffusion method, interpreted according to EUCAST standards, 19 vancomycin resistant enterococci (VREs) were found and these isolates showed resistance to teicoplanin (84.2%), erythromycin (84.2%) and ampicillin. (68.4%). Whole-genome sequencing of bacteria using Illumina technology detected most frequently the vanA, erm(B) and aac(6 ')-Ii genes. On the other hand, in E. coli, were detected 28 ESBL-producing isolates that were resistant to ampicillin (100%), ticarcillin (100%), piperacillin (100%), cefuroxime (96.4%) and cefotaxime (96.4%). Regarding ESBL-coding genes, the most frequent was blaCTX-M-1, which was mostly associated with IncI1 plasmid. Other genes were detected but the most frequent were tet(A) (64.3%), tetR (64.3%) and sul2 (53.6%) genes. Regarding the genetic background study, were detected 17 different MLST types and a total of 41761 SNP. The data obtained from whole-genome sequencing of bacteria is essential to improve the knowledge about the spread of resistant strains in wild animals, animals for consumption, as well as in meat derived from them and the possible implications involved in transfer to other animals or humans. In the future, and considering the One Health concept, studies may be conducted to investigate the prevalence of antibiotic resistant bacteria both in humans and in environmental samples enabling the identification of antibiotic resistant bacteria as well antibiotic resistant genes and their transmission pathways.
The intense use of antibiotics in both human and veterinary medicine contributes to the development and spread of resistance in different groups of bacteria. This is a global public health problem which requires a multidisciplinary and holistic approach because bacterial resistance to antibiotics is spreading and evolving in similar ways in humans, in animals and in the environment. Escherichia coli and Enterococcus spp. are considered by the World Health Organization as essential microorganisms for monitoring the level of antibiotic resistance. These bacteria may act as reservoir of antibiotic resistance genes that can be transferred to other pathogenic bacteria and represent a worldwide problem. Some resistance genes are located on plasmids that are rapidly disseminated to other bacterial species, including isolates from wild animal populations. Thus, even though wild animals are not in direct contact with antibiotics, they are indirectly affected by the excessive general use of antibiotics in other settings. A total of 21 Enterococcus spp. strains isolated from minced meat, meatballs and hamburgers, as well as a total of 45 Enterobacteriaceae strains isolated from wildlife, animals for consumption and meat were selected from our previously studied collection of bacterial strains. These strains were identified using mass spectrometry and 3 different species of Enterococcus spp. [E. faecium (n = 16), E. gallinarum (n = 3) and E. durans (n = 2)] and 45 E. coli were identified. Using the Kirby-Bauer agar diffusion method, interpreted according to EUCAST standards, 19 vancomycin resistant enterococci (VREs) were found and these isolates showed resistance to teicoplanin (84.2%), erythromycin (84.2%) and ampicillin. (68.4%). Whole-genome sequencing of bacteria using Illumina technology detected most frequently the vanA, erm(B) and aac(6 ')-Ii genes. On the other hand, in E. coli, were detected 28 ESBL-producing isolates that were resistant to ampicillin (100%), ticarcillin (100%), piperacillin (100%), cefuroxime (96.4%) and cefotaxime (96.4%). Regarding ESBL-coding genes, the most frequent was blaCTX-M-1, which was mostly associated with IncI1 plasmid. Other genes were detected but the most frequent were tet(A) (64.3%), tetR (64.3%) and sul2 (53.6%) genes. Regarding the genetic background study, were detected 17 different MLST types and a total of 41761 SNP. The data obtained from whole-genome sequencing of bacteria is essential to improve the knowledge about the spread of resistant strains in wild animals, animals for consumption, as well as in meat derived from them and the possible implications involved in transfer to other animals or humans. In the future, and considering the One Health concept, studies may be conducted to investigate the prevalence of antibiotic resistant bacteria both in humans and in environmental samples enabling the identification of antibiotic resistant bacteria as well antibiotic resistant genes and their transmission pathways.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Escherichia coli , Enterococcus spp