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Authors
Abstract(s)
Nas últimas décadas, o papel do ambiente na disseminação de resistência a
antibióticos de relevância clínica tem recebido especial atenção. Neste âmbito, as águas
residuais hospitalares e municipais, onde resíduos de antibióticos e bactérias resistentes são
descarregados, estão entre os mais importantes reservatórios ambientais. Porém, o seu
papel na disseminação da resistência ainda está pouco compreendido. O principal objectivo
deste estudo foi o de avaliar a relevância dos efluentes hospitalares na disseminação de
bactérias resistentes a antibióticos e de genes de resistência no ambiente. O estudo focou-se
num efluente hospitalar e nas águas residuais da estação de tratamento receptora (ETAR).
Através da análise multivariada baseada nas variações da composição das
comunidades bacterianas e das populações resistentes a antibióticos cultiváveis em função
das variações da concentração de resíduos de antibióticos e metais, procurou-se
compreender como se interligam estas variáveis. As correlações positivas significativas
observadas sugeriram que a presença dos contaminantes químicos analisados poderia estar
associada a rearranjos nas comunidades bacterianas ou à selecção de populações resistentes
em águas residuais, principalmente em efluentes hospitalares. O potencial das águas
residuais hospitalares e municipais como reservatórios de resistência a antibióticos foi de
seguida investigado, usando como modelo membros da espécie Escherichia coli resistentes
a quinolonas.
Foi seleccionado um grupo de estirpes de E.coli resistentes a quinolonas,
representativo de amostragens de águas residuais municipais e de ribeiras urbanas
abrangendo um período de nove anos. A diversidade genética destes isolados foi avaliada
com base em multilocus sequence typing (MLST), e esta informação foi comparada com
dados dos isolados sobre a presença de determinantes genéticos associados com resistência
a quinolonas e com outras características associadas a resistência. Assim, foi possível
inferir sobre a ocorrência de disseminação vertical e/ou horizontal de resistência. A análise
baseada em MLST demonstrou que isolados de diferentes tipos de água e datas de
isolamento se agrupavam, sugerindo não só o carácter ubiquista de algumas linhagens, mas
também a sua persistência em ambientes aquáticos. A indicação de disseminação vertical
de resistência sugerida por estes dados foi confirmada pelo facto de que a resistência a
quinolonas era essencialmente de base cromossómica. Apesar disso, a presença dos
mesmos determinantes adquiridos de resistência a quinolonas e beta-lactâmicos e de replicões plasmídicos em E.coli de diferentes linhagens e origens sugeriu que estas
bactérias têm um papel importante na transferência horizontal da resistência em ambientes
aquáticos. Esta ideia foi reforçada pela observação de que alguns destes determinantes se
encontravam em elementos genéticos conjugativos. Nesta fase, a questão era se as mesmas
linhagens e genes encontrados em E.coli de ambientes aquáticos ocorriam em outros
habitats. A mesma abordagem aplicada a um grupo alargado de isolados de origens
diferentes mostrou que as mesmas linhagens podiam ser encontradas em pacientes
hospitalares e em gaivotas, mas não em aves de rapina de uma reserva natural, sugerindo
que algumas linhagens de E.coli podem ser disseminadas entre ambientes com influência
humana. Sendo E.coli uma espécie comensal em animais e humanos, o estudo de um grupo
bacteriano ambiental não associado com humanos era também de interesse. A escolha
recaiu sobre o género Aeromonas e, numa perspectiva de continuidade com o trabalho
prévio, foram seleccionadas estirpes resistentes a quinolonas. Surpreendentemente, apesar
da fraca associação com humanos, observou-se que entre as Aeromonas spp. de efluente
hospitalar se encontrava uma prevalência de resistência a antibióticos superior às da
ETAR. O papel desta espécie como vector de disseminação de resistência foi evidenciado
de várias formas. Primeiro, foi possível identificar em Aeromonas spp. do efluente
hospitalar um gene de beta-lactamase previamente não descrito em Aeromonas spp. nem
fora do contexto clínico. Segundo, viu-se que possuíam dois genes de resistência adquirida
a quinolonas, um claramente ubíquo e outro de origem não clínica, o que pode ser
interessante em estudos de fontes de dispersão de resistência. Terceiro, alguns dos genes de
resistência a antibióticos podiam ser transferidos para E.coli por conjugação. Confirmou-se
que Aeromonas spp. podem ser veículos relevantes de resistência, e que o hospital pode ser
uma fonte importante de membros deste género.
A transferência horizontal de genes por conjugação e o efeito de concentrações subinibitórias
de antibióticos são temas chave para a compreensão da disseminação da
resistência a antibióticos. Este tema foi investigado com base numa estirpe multiresistente
de E.coli de efluente hospitalar. Observou-se que enquanto uma concentração subinibitória
de ceftazidime aumentava significativamente a taxa de conjugação, tetraciclina
produzia o efeito oposto. O plasmidoma conjugativo, que em média foi transferido em
mais de 90% dos ensaios, incluía possíveis determinantes de persistência, de resistência a
antibióticos e a metais, sugerindo a importância de fenómenos de co-selecção.
Confirmou-se portanto que o efluente hospitalar é uma fonte relevante de bactérias
resistentes a antibióticos e de determinantes de resistência para o ambiente.
Over the last decades the environment has received particular attention regarding its role on the dissemination of antibiotic resistance of clinical relevance. Hospital and municipal wastewater, where antibiotic residues and resistant bacteria are discharged, are among the most obvious reservoirs. However, their role on the resistance dissemination is still poorly understood. The primary goal of this study was to assess the role of hospital effluent in the dissemination of antibiotic resistant bacteria and antibiotic resistance genes in the environment. The focuses of the study were a hospital effluent and the wastewater of the receiving urban wastewater treatment plant. Multivariate analyses based on the variation of the composition of the bacterial communities and prevalence of antibiotic resistant culturable populations in function of variations on the concentration of antibiotic and metal residues were performed in order to search for possible correlations among these variables. The significant positive correlations observed were a possible indication that the presence of the analyzed chemical contaminants could be associated with rearrangements of the bacterial communities or selection of resistant populations in wastewater, mainly in hospital effluent. The potential of hospital and municipal wastewater as environmental reservoirs of antibiotic resistance was, thus, further investigated, using quinolone resistant Escherichia coli as model. A group of quinolone resistant E. coli strains representative of sampling events from municipal and hospital wastewater and urban streams, spanning a period of nine years, was selected. The genetic diversity of these strains was assessed based on multilocus sequence typing (MLST) and this information was compared with data onthe isolates regarding the presence of genetic determinants associated with quinolone and with other resistanceassociated traits. In this way, it was possible to infer about the occurrence of vertical and/or horizontal resistance dissemination. The MLST-based studies showed that strains from different types of water and isolation dates clustered together, suggesting not only the ubiquitous character of some lineages but also their persistence in aquatic environments. The indication of vertical resistance dissemination that these data suggested was confirmed by the fact that quinolone resistance was mostly chromosome-based. However, acquired genetic determinants of resistance to quinolones, to beta-lactams and plasmid replicons, found in the different E. coli lineages and origins, suggested that these bacteria play an important role on the horizontal transfer of resistance in aquatic environments. Indeed, some of these determinants were observed to be located in conjugative mobile genetic elements. At this stage, the question was if the same lineages and genes found in E. coli from aquatic environments could be found in other habitats. The same approach, applied to an extended set of isolates from different origins, showed that the same lineages could be found also in hospital patients and urban gulls, although not in birds of the prey from a natural reserve, suggesting that at least some E. coli bacterial lineages can be disseminated among the human-impacted environments. While E. coli is an human and animal commensal species, the study of an environmental bacterial group not associated with humans was also of interest and Aeromonas spp. were the choice, and, as a common point, quinolone resistant strains were selected. Surprisingly, in spite of the loose association with humans, Aeromonas spp. from hospital effluent showed higher antibiotic resistance prevalence among them than those from urban wastewater. The role of members of this species as vectors of resistance dissemination was evidenced in different ways. First, Aeromonas spp. from hospital effluent were found to harbor a beta-lactamase gene not previously described in Aeromonas spp. or outside the clinical settings. Second, they harbored acquired quinolone resistance genes that showed either ubiquitous distribution or a non-clinical origin, both interesting tools for source tracking surveys. Third, some of their antibiotic resistance genes could be transferred to an E. coli receptor via conjugation. Aeromonas spp. were confirmed as relevant carries of resistance and the hospital can be an important source for members of this genus. Conjugation and the effect of sub-inhibitory concentrations of antibiotics are two key issues in the understanding of the dissemination of antibiotic resistance, herein investigated based on a multidrug resistant E.coli isolate from hospital effluent. While a sub-inhibitory concentration of ceftazidime was observed to significantly increase the conjugation rate, tetracycline had the opposite effect. The conjugative plasmidome, which on average was transferred in more than 90% of the assays, included genes putatively associated with persistence, resistance to antibiotics and to metals, suggesting the importance co-selection phenomena. The hospital effluent was therefore confirmed as a significant source of antibiotic resistant bacteria and antibiotic resistance determinants to the environment.
Over the last decades the environment has received particular attention regarding its role on the dissemination of antibiotic resistance of clinical relevance. Hospital and municipal wastewater, where antibiotic residues and resistant bacteria are discharged, are among the most obvious reservoirs. However, their role on the resistance dissemination is still poorly understood. The primary goal of this study was to assess the role of hospital effluent in the dissemination of antibiotic resistant bacteria and antibiotic resistance genes in the environment. The focuses of the study were a hospital effluent and the wastewater of the receiving urban wastewater treatment plant. Multivariate analyses based on the variation of the composition of the bacterial communities and prevalence of antibiotic resistant culturable populations in function of variations on the concentration of antibiotic and metal residues were performed in order to search for possible correlations among these variables. The significant positive correlations observed were a possible indication that the presence of the analyzed chemical contaminants could be associated with rearrangements of the bacterial communities or selection of resistant populations in wastewater, mainly in hospital effluent. The potential of hospital and municipal wastewater as environmental reservoirs of antibiotic resistance was, thus, further investigated, using quinolone resistant Escherichia coli as model. A group of quinolone resistant E. coli strains representative of sampling events from municipal and hospital wastewater and urban streams, spanning a period of nine years, was selected. The genetic diversity of these strains was assessed based on multilocus sequence typing (MLST) and this information was compared with data onthe isolates regarding the presence of genetic determinants associated with quinolone and with other resistanceassociated traits. In this way, it was possible to infer about the occurrence of vertical and/or horizontal resistance dissemination. The MLST-based studies showed that strains from different types of water and isolation dates clustered together, suggesting not only the ubiquitous character of some lineages but also their persistence in aquatic environments. The indication of vertical resistance dissemination that these data suggested was confirmed by the fact that quinolone resistance was mostly chromosome-based. However, acquired genetic determinants of resistance to quinolones, to beta-lactams and plasmid replicons, found in the different E. coli lineages and origins, suggested that these bacteria play an important role on the horizontal transfer of resistance in aquatic environments. Indeed, some of these determinants were observed to be located in conjugative mobile genetic elements. At this stage, the question was if the same lineages and genes found in E. coli from aquatic environments could be found in other habitats. The same approach, applied to an extended set of isolates from different origins, showed that the same lineages could be found also in hospital patients and urban gulls, although not in birds of the prey from a natural reserve, suggesting that at least some E. coli bacterial lineages can be disseminated among the human-impacted environments. While E. coli is an human and animal commensal species, the study of an environmental bacterial group not associated with humans was also of interest and Aeromonas spp. were the choice, and, as a common point, quinolone resistant strains were selected. Surprisingly, in spite of the loose association with humans, Aeromonas spp. from hospital effluent showed higher antibiotic resistance prevalence among them than those from urban wastewater. The role of members of this species as vectors of resistance dissemination was evidenced in different ways. First, Aeromonas spp. from hospital effluent were found to harbor a beta-lactamase gene not previously described in Aeromonas spp. or outside the clinical settings. Second, they harbored acquired quinolone resistance genes that showed either ubiquitous distribution or a non-clinical origin, both interesting tools for source tracking surveys. Third, some of their antibiotic resistance genes could be transferred to an E. coli receptor via conjugation. Aeromonas spp. were confirmed as relevant carries of resistance and the hospital can be an important source for members of this genus. Conjugation and the effect of sub-inhibitory concentrations of antibiotics are two key issues in the understanding of the dissemination of antibiotic resistance, herein investigated based on a multidrug resistant E.coli isolate from hospital effluent. While a sub-inhibitory concentration of ceftazidime was observed to significantly increase the conjugation rate, tetracycline had the opposite effect. The conjugative plasmidome, which on average was transferred in more than 90% of the assays, included genes putatively associated with persistence, resistance to antibiotics and to metals, suggesting the importance co-selection phenomena. The hospital effluent was therefore confirmed as a significant source of antibiotic resistant bacteria and antibiotic resistance determinants to the environment.