Análise genômica e fenotípica da virulência de cepas de Escherichia coli isoladas da microbiota intestinal de pacientes hospitalizados com e sem infecção urinária
Data
2021
Autores
Santos-Neto, José Francisco
Orientadores
Gomes, Tânia Aparecida Tardelli [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Avaliar cepas de E. coli isoladas da microbiota intestinal de pacientes hospitalizados com e sem infecção do trato urinário (ITU). Métodos: Dezoito amostras de swab retal foram coletadas e semeadas em ágar MacConkey. Ao menos 10 colônias fermentadoras de lactose ou não fermentadoras, por paciente, foram selecionadas e avaliadas. A identificação de espécie foi realizada utilizando provas bioquímicas e a confirmação foi feita pela técnica de MALDI-TOF. Os isolados de cada paciente foram avaliados quanto a sua clonalidade pela técnica de RAPD. Foi realizada avaliação por PCR de um clone de cada tipo, por paciente, para determinar a sua origem filogenética e avaliar a presença dos marcadores moleculares relacionados a ExPEC (pap, iuc/iut, afa/dra, sfa, kpsMTII) e UPEC (vat, chuA, yfcV, fyuA). A susceptibilidade a 13 antimicrobianos, de diferentes classes, foi avaliada pelo método de disco-difusão, seguindo as recomendações do EUCAST. Outras avaliações fenotípicas, como capacidade das cepas de resistirem ao soro humano, curva de crescimento, adesão a células eucarióticas e patogenicidade in vivo em modelo de Galleria mellonella também foram realizadas. Por fim, as cepas foram sequenciadas pelo método de Illumina. Resultados: Entre 180 isolados de E. coli estudados, foram identificados 34 clones e 10 variantes (média de 2 clones por paciente). Após a análise do genoma, cepas classificadas como ExPEC ou UPEC foram identificadas na microbiota intestinal de 13 (~72%) e 8 (~44%) pacientes, respectivamente. A maioria das cepas pertencia aos filogrupos B2 e D, estando em 8 pacientes cada. Cepas fecais produtoras de ESBL foram detectadas em dois pacientes e resistência a ciprofloxacino em oito. Cepas dos STs 59, 69, 131 e 1193, normalmente associadas a infecções extra intestinais, foram identificadas em diversos pacientes, incluindo aqueles que não tinham ITU. Genes móveis de resistência foram identificados em quatro cepas (2 com blaCTX-M-15, blaOXA-1, e aac(6’)-lb-cr; 1 com mcr-1 e qnrB19; e 1 com blaDHA-1 e qnrB4) isoladas de quatro pacientes distintos. A análise genômica comparativa entre as cepas de ExPEC e os isolados da ITU permitiu a identificação de três pacientes com ITU cuja cepa isolada da urina não foi detectada em sua microbiota intestinal, podendo a infecção ter tido origem externa. Conclusão: A elevada incidência de cepas de ExPEC e a presença de clones de alto risco, como do ST131, além da detecção de cepas de E. coli multirresistentes na microbiota intestinal de pacientes, confirma que a vigilância de patógenos na microbiota intestinal é essencial para a prevenção de infecções e possíveis surtos no ambiente hospitalar.
Objective: to evaluate E. coli isolates from the microbiota of inpatients with and without urinary tract infection (UTI). Methods: Eighteen rectal swab samples were collected and seeded onto MacConkey Agar. At least 10 lactose-positive or lactose-negative colonies per patient were selected and evaluated. Species identification was performed by biochemical assays and confirmed by MALDI-TOF. The isolates from each patient were screened for clonal identification by RAPD. A clone of each type was assessed by PCR for phylogenetic origin determination and molecular markers related to ExPEC (pap, iuc/iut, afa/dra, sfa, kpsMTII) and UPEC (vat, chuA, yfcV, fyuA) classification. Antimicrobial susceptibility to 13 antibiotics was determined by disk-diffusion according to EUCAST, including different classes of antibiotics. Other phenotypic evaluations, such as the ability of strains to resist human serum, growth curve, adhesion to eukaryotic cells and in vivo pathogenicity in a Galleria mellonella model were also performed. Lastly, the strains’ genomes were sequenced using Illumina. Results: Among the 180 E. coli isolates studied, 34 clones and 10 variants were identified (mean of 2 clones per patient). After the genome analysis, it was identified that strains classified as ExPEC or UPEC occurred in the gut of 13 (~72%) and 8 (~44%) patients, respectively. They belonged mainly to phylogroups B2 and D, occurring in 8 patients each. ESBL-producing fecal strains were detected in two patients and ciprofloxacin resistance in eight. Clones from the ST59, ST69, ST131, and ST1193, usually associated with extra intestinal infections, were identified even in patients without UTI. Mobile drug-resistance genes were identified in the genome of four strains (2 with blaCTX-M-15, blaOXA-1, and aac(6’)-lb-cr; 1 with mcr-1 and qnrB19; and 1 with blaDHA-1 and qnrB4) from four different patients. Comparative genomic analysis between ExPEC strains and UTI isolates allowed the identification of three patients with UTI in whom the strain isolated from urine was not identified in their intestinal microbiota, and the infection may have had an external origin. Conclusion: The elevated incidence of ExPEC, and the presence of high-risk clones such as ST131 and multidrug-resistant E. coli in the gut of inpatients, confirm that surveillance of pathogens in the gut microbiota is essential for the prevention of infections and outbreaks in the hospital environment.
Objective: to evaluate E. coli isolates from the microbiota of inpatients with and without urinary tract infection (UTI). Methods: Eighteen rectal swab samples were collected and seeded onto MacConkey Agar. At least 10 lactose-positive or lactose-negative colonies per patient were selected and evaluated. Species identification was performed by biochemical assays and confirmed by MALDI-TOF. The isolates from each patient were screened for clonal identification by RAPD. A clone of each type was assessed by PCR for phylogenetic origin determination and molecular markers related to ExPEC (pap, iuc/iut, afa/dra, sfa, kpsMTII) and UPEC (vat, chuA, yfcV, fyuA) classification. Antimicrobial susceptibility to 13 antibiotics was determined by disk-diffusion according to EUCAST, including different classes of antibiotics. Other phenotypic evaluations, such as the ability of strains to resist human serum, growth curve, adhesion to eukaryotic cells and in vivo pathogenicity in a Galleria mellonella model were also performed. Lastly, the strains’ genomes were sequenced using Illumina. Results: Among the 180 E. coli isolates studied, 34 clones and 10 variants were identified (mean of 2 clones per patient). After the genome analysis, it was identified that strains classified as ExPEC or UPEC occurred in the gut of 13 (~72%) and 8 (~44%) patients, respectively. They belonged mainly to phylogroups B2 and D, occurring in 8 patients each. ESBL-producing fecal strains were detected in two patients and ciprofloxacin resistance in eight. Clones from the ST59, ST69, ST131, and ST1193, usually associated with extra intestinal infections, were identified even in patients without UTI. Mobile drug-resistance genes were identified in the genome of four strains (2 with blaCTX-M-15, blaOXA-1, and aac(6’)-lb-cr; 1 with mcr-1 and qnrB19; and 1 with blaDHA-1 and qnrB4) from four different patients. Comparative genomic analysis between ExPEC strains and UTI isolates allowed the identification of three patients with UTI in whom the strain isolated from urine was not identified in their intestinal microbiota, and the infection may have had an external origin. Conclusion: The elevated incidence of ExPEC, and the presence of high-risk clones such as ST131 and multidrug-resistant E. coli in the gut of inpatients, confirm that surveillance of pathogens in the gut microbiota is essential for the prevention of infections and outbreaks in the hospital environment.