Diversidade e estudo de plasmídeos em estirpes de Escherichia coli produtoras de ESBL isoladas de animais selvagens
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2017
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Os plasmídeos são elementos importantes na mobilidade dos genes de resistência aos antibióticos, como os que codificam beta-lactamases de largo espectro (ESBL), que são de elevada importância clínica. Isolados de E. coli produtoras de ESBL, estão altamente disseminados nos hospitais em todo o mundo e de forma semelhante e já foram detectados em animais selvagens no meio-ambiente. Isolados de E. coli positivos para a produção de ESBL das classes CTX-M, TEM e SHV (n=53) foram recuperados em amostras fecais de animais selvagens em estudos prévios, e o seu fenótipo e genótipo de resistência aos antibióticos foi também determinado previamente nesses estudos. O objectivo do presente estudo foi determinar a sua diversidade clonal através de electroforese em campo pulsado (PFGE) e verificar a transferência de determinantes de resistência entre as estirpes de E. coli produtoras de ESBL e uma estirpe receptora específica, através de conjugação, bem como determinar plasmídeos de resistência nos dadores e transconjugantes obtidos.
Neste estudo foram incluídos cinquenta e três isolados de E. coli de diferentes origens: de lobo (n=14), dourada (n=10), lince ibérico (n=9), javali (n=8), aves de rapina (n=4), raposa (n=3), aves selvagens dos Açores (n=2), veado (n=2) e coruja (n=1), e contendo os genes blaCTX-M-14 (n=18), blaCTX-M-1 (n=12), blaTEM-52 (n=9), blaTEM-1 (n=15) e blaSHV-12 (n=17). As estirpes pertenciam aos grupos filogenéticos A (n=23), B1 (n=20) e D (n=10). Todos os isolados foram submetidos a electroforese em campo pulsado (PFGE) utilizando a enzima XbaI. A maioria apresentou padrões de PFGE diferentes, sendo possível detectar 44 padrões distintos. Vinte e nove isolados foram então seleccionadas com diferente padrão de PFGE, tipo de ESBL e com diferente origem e todas apresentando susceptibilidade à rifampicina. Desta selecção, os isolados pertencentes ao grupo filogenético D (n=8) foram submetidos a tipificação de sequências multilocus (MLST), e foram observados quarto STs distintos: ST69, ST115, ST117 e ST2001. A selecção de 29 isolados possuía os genes blaCTX-M-14 (10 isolados); blaCTX-M-1 (8 isolados); blaTEM-52 (5 isolados); blaTEM-1 (8 isolados); e blaSHV-12 (8 isolados). Vinte e um isolados transferiram os genes ESBL através de conjugação para a estirpe receptora E. coli CSH26 (lactose negative, resistente à rifampicina e sem plasmídeos).
A análise dos plasmídeos foi realizada nos dadores e nas estirpes transconjugantes. Foram observados entre um e seis replicões distintos nos dadores, sendo os mais frequentes, IncK (17 isolados), IncI1 (14 isolados) and IncFIB (12 isolados). O replicão IncI1 foi identificado em 13 transconjugantes obtidos a partir de estirpes dadoras com os genes CTX-M-1, CTX-M-14, TEM-1, TEM-52 e SHV-12. Os genes ESBL são facilmente transferidos através de conjugação para outras estirpes de E. coli e é possível que os plasmídeos IncI1 tenham um papel importante no processo de transferência.
Plasmids are important elements for the mobilization of antibiotic resistance genes, as those encoding extended-spectrum beta-lactamases (ESBL), and of great clinical relevance. ESBL-producing E. coli isolates are largely disseminated in hospitals all over the world and have also been detected in animals and in the environment. ESBL-positive E. coli isolates of the CTX-M, TEM and SHV classes (n=53) have been recovered from wild animals in previous studies and their phenotype and genotype of antibiotic resistance have been previously determined. The purpose of the present study was to determine their clonal diversity by pulsed-field-gel-electrophoresis (PFGE), and to verify the transferability of resistance determinants in these ESBL-positive E. coli isolates to a specific receptor strain by conjugation, as well as to detect R plasmids in donors and transconjugants. Fifty-three ESBL-positive E. coli isolates recovered from Iberian wolf (n=14), gilthead seabream (n=10), Iberian lynx (n=9), wild boar (n=8), birds of prey (n=4), fox (n=3), wild birds from Azores (n= 2), deer (n=2), and owl (n=1), and carrying the genes blaCTX-M-14 (n=18), blaCTX-M-1 (n=12), blaTEM-52 (n=9), blaTEM-1 (n=15) and blaSHV-12 (n=17) were included in this study. Isolates were ascribed to the phylogenetic groups A (n=23), B1 (n=20) and D (n=10). All these isolates were submitted to PFGE analysis with XbaI enzyme. Most of them showed unrelated patterns with the detection of 44 different PFGE patterns. Twenty-nine of these strains were selected as representative, showing different PFGE, type of ESBL and origin, and all of them presented rifampicin susceptibility. From this selection, the isolates ascribed to the phylogenetic group D (n=8) were submitted to multilocus sequence typing (MLST), and four different ST’s were detected: ST69, ST115, ST117 and ST2001. The selection of 29 strains carried the genes blaCTX-M-14 (10 strains); blaCTX-M-1 (8 strains); blaTEM-52 (5 strains); blaTEM-1 (8 strains); and blaSHV-12 (8 strains). Twenty-one isolates transferred by conjugation the ESBL gene to E. coli CSH26 receptor strain (lactose-negative, rifampicin-resistant, non plasmidic). Plasmid replicon typing was performed in donor and transconjugant isolates. From one to six different plasmid replicon types were detected in donor strains being IncK (17 strains), IncI1 (14 strains) and IncFIB (12 strains) the most frequent ones. A IncI1 plasmid was detected in 13 transconjugants obtained from donor strains carrying genes encoding CTX-M-1, CTX-M-14, TEM-52, TEM-1 and SHV-12. ESBL genes can be easily transferred by conjugation to other E. coli and IncI1 plasmids might have an important role in this mobilization process.
Plasmids are important elements for the mobilization of antibiotic resistance genes, as those encoding extended-spectrum beta-lactamases (ESBL), and of great clinical relevance. ESBL-producing E. coli isolates are largely disseminated in hospitals all over the world and have also been detected in animals and in the environment. ESBL-positive E. coli isolates of the CTX-M, TEM and SHV classes (n=53) have been recovered from wild animals in previous studies and their phenotype and genotype of antibiotic resistance have been previously determined. The purpose of the present study was to determine their clonal diversity by pulsed-field-gel-electrophoresis (PFGE), and to verify the transferability of resistance determinants in these ESBL-positive E. coli isolates to a specific receptor strain by conjugation, as well as to detect R plasmids in donors and transconjugants. Fifty-three ESBL-positive E. coli isolates recovered from Iberian wolf (n=14), gilthead seabream (n=10), Iberian lynx (n=9), wild boar (n=8), birds of prey (n=4), fox (n=3), wild birds from Azores (n= 2), deer (n=2), and owl (n=1), and carrying the genes blaCTX-M-14 (n=18), blaCTX-M-1 (n=12), blaTEM-52 (n=9), blaTEM-1 (n=15) and blaSHV-12 (n=17) were included in this study. Isolates were ascribed to the phylogenetic groups A (n=23), B1 (n=20) and D (n=10). All these isolates were submitted to PFGE analysis with XbaI enzyme. Most of them showed unrelated patterns with the detection of 44 different PFGE patterns. Twenty-nine of these strains were selected as representative, showing different PFGE, type of ESBL and origin, and all of them presented rifampicin susceptibility. From this selection, the isolates ascribed to the phylogenetic group D (n=8) were submitted to multilocus sequence typing (MLST), and four different ST’s were detected: ST69, ST115, ST117 and ST2001. The selection of 29 strains carried the genes blaCTX-M-14 (10 strains); blaCTX-M-1 (8 strains); blaTEM-52 (5 strains); blaTEM-1 (8 strains); and blaSHV-12 (8 strains). Twenty-one isolates transferred by conjugation the ESBL gene to E. coli CSH26 receptor strain (lactose-negative, rifampicin-resistant, non plasmidic). Plasmid replicon typing was performed in donor and transconjugant isolates. From one to six different plasmid replicon types were detected in donor strains being IncK (17 strains), IncI1 (14 strains) and IncFIB (12 strains) the most frequent ones. A IncI1 plasmid was detected in 13 transconjugants obtained from donor strains carrying genes encoding CTX-M-1, CTX-M-14, TEM-52, TEM-1 and SHV-12. ESBL genes can be easily transferred by conjugation to other E. coli and IncI1 plasmids might have an important role in this mobilization process.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Animais selvagens , Escherichia coli , Plasmídeos