Estudo da prevalência de bactérias gram-negativas com resistência a antimicrobianos e de Escherichia coli portadora de genes de virulência na microbiota intestinal de indivíduos sadios
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Data
2015-12-31
Autores
Patekoski, Katya da Silva [UNIFESP]
Orientadores
Pignatari, Antonio Carlos Campos [UNIFESP]
Tipo
Tese de doutorado
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Objetivos: Esta pesquisa foi desenvolvida com o objetivo de avaliar a prevalência de bactérias Gram-negativas resistentes aos antimicrobianos ?-lactâmicos e às quinolonas nas fezes de indivíduos sadios, de diversas faixas etárias e de ambos os sexos. Material e Métodos: Amostras de fezes foram coletadas de 137 indivíduos (0-90 anos; 67 do sexo masculino, 70 do sexo feminino) residentes na cidade de São Paulo ? SP. O isolamento bacteriano foi realizado em Ágar MacConkey acrescido de ácido nalidíxico (50?g/mL); CHROMagarTM ESBL; CHROMagarTM KPC; Ágar MacConkey suplementado com imipenem (1?g/mL) e Ágar MacConkey adicionado de discos contendo carbapenens (imipenem, meropenem e ertapenem - 10?g). A identificação das colônias e a avaliação do perfil de sensibilidade foram realizadas utilizando-se o sistema BD Phoenix 5.1TM, sendo o perfil de sensibilidade para a ciprofloxacina e para os carbapenens confirmado por microdiluição em caldo. Os isolados positivos para produção de ESBL (?eta-lactamase de espectro estendido) pelo Phoenix e os isolados resistentes aos carbapenens foram submetidos, à pesquisa molecular dos genes que codificam ESBL e carbapenemases (blaTEM, blaSHV e blaCTX-M, blaKPC, blaGES, blaOXA-48, blaIMP, blaVIM, blaSPM, blaGIM, blaNDM). Para os isolados ?possíveis produtores de ESBL? pelo Phoenix, pesquisou-se a presença de blaTEM, blaSHV e blaCTX-M. Os isolados de Escherichia coli e Klebsiella pneumoniae resistentes à cefoxitina foram investigados quanto à presença de CMY-2. Todos os isolados de E. coli foram submetidos à pesquisa de 30 genes codificadores de fatores de virulência, frequentemente pesquisados em E. coli patogênica extraintestinal (ExPEC), e à classificação filogenética. Resultados: Foram encontradas 241 bactérias (considerando-se em quase todos os casos, um isolado de cada espécie bacteriana por amostra de fezes), com prevalência de E. coli e Pseudomonas aeruginosa. Trinta e uma bactérias foram resistentes à ciprofloxacina pela microdiluição em caldo. Destas, 28 eram E. coli, isoladas de 28 (20,4%) amostras de fezes. As bactérias resistentes à ciprofloxacina foram isoladas em alta frequência nas 3 faixas etárias estudadas. Dezesseis enterobactérias (obtidas a partir de 15 amostras de fezes - 10,9%) possuíam genes que codificam ESBL. Estes isolados foram distribuídos semelhantemente nas 3 faixas etárias e em ambos os sexos. Enzimas CTXM foram prevalentes. Vinte bactérias foram resistentes aos carbapenens, mas nenhuma possuía os genes que codificam carbapenemases. Três isolados de E. coli possuíam o gene blaCMY-2. Alta ocorrência de marcadores de virulência, frequentemente associados com ExPEC, foi verificada nos isolados de E. coli, onde 43,3% (26) dos isolados possuíam 10 ou mais genes. O grupo A foi o prevalente (18 isolados - 30%), porém, os grupos B2 e D (associados a ExPEC), ocorreram em alta frequência (21,7 e 15% dos isolados, respectivamente). Isolados de E. coli resistentes à ciprofloxacina e isolados desta espécie associados com a produção de ESBL tiveram menor número médio de fatores de virulência, comparando-se com os isolados que não apresentaram estes tipos de resistência. Conclusões: O intestino de indivíduos sadios é um reservatório de bactérias Gram-negativas resistentes a antimicrobianos de importância clínica, bem como de isolados de E. coli com pleno potencial de virulência para infecções extraintestinais.
Objectives: This research was developed in order to assess the prevalence of Gram-negative bacteria resistant to antimicrobial β-lactams and quinolones in the feces of healthy individuals of different ages and of both sexes. Materials and Methods: Faecal samples were collected from 137 individuals (0-90 years; 67 males, 70 females) living in the city of São Paulo – SP. The bacterial isolation was carried out in MacConkey agar supplemented with nalidixic acid (50µg/mL); CHROMagarTM ESBL; CHROMagarTM KPC; MacConkey agar supplemented with imipenem (1μg/mL) and on MacConkey agar with 10μg ertapenem, meropenem and imipenem disks. The identification of the colonies and the evaluation of the sensitivity profile were carried out using the BD Phoenix 5.1TM system, and the sensitivity profile for ciprofloxacin and carbapenems confirmed by broth microdilution. Isolates that screened positive for ESBL (extended-spectrum β-lactamase) production by Phoenix and isolates resistant to carbapenems were subjected to molecular screening for ESBL and carbapenemases genes (blaTEM, blaSHV e blaCTX-M, blaKPC, blaGES, blaOXA-48, blaIMP, blaVIM, blaSPM, blaGIM, blaNDM). The "possible ESBL-producing" strains by Phoenix were examined for the presence of blaTEM, blaSHV and blaCTX-M. Cefoxitin resistant strains of Escherichia coli and Klebsiella pneumoniae were examined for the presence of CMY-2. All isolates of Escherichia coli were checked for presence of 30 genes encoding virulence factors, often searched in extraintestinal pathogenic E. coli (ExPEC) and the phylogenetic group was determined. Results: We found 241 bacteria (considering in almost all cases, a single bacterial species for each stool sample), with prevalence of E. coli and Pseudomonas aeruginosa. Thirty-one bacteria were resistant to ciprofloxacin by broth microdilution. Of these, 28 were E. coli isolated from 28 (20.4%) stool samples. The ciprofloxacin-resistant bacteria were isolated at high frequency in the three age groups. Sixteen enterobacteria (obtained from 15 - 10.9% - stool samples) harbored genes encoding ESBL. These isolates were similarly distributed in three age groups and in both sexes. Enzymes CTX-M were prevalent. Twenty bacteria were resistant to carbapenems, but none harbored genes encoding carbapenemases. Three E. coli strains harbored blaCMY-2 gene. High occurrence of virulence markers, often associated with ExPEC, was verified in E. coli isolates, in which 43.3% (26) isolates harbored 10 or more genes. Group A was the most prevalent (18 isolates - 30%), however, the B2 and D groups (associated with ExPEC) occurred in high frequency (21.7 and 15% of the isolates, respectively). E. coli strains resistant to ciprofloxacin and isolates of this species associated with producing the ESBL had lower average number of virulence factors, comparing with the isolates did not present these types of resistance. Conclusions: The intestine of healthy individuals is a reservoir of Gram-negative bacteria resistant to antimicrobial agents of clinical importance, as well as E. coli strains with full potential virulence for extra-intestinal infections.
Objectives: This research was developed in order to assess the prevalence of Gram-negative bacteria resistant to antimicrobial β-lactams and quinolones in the feces of healthy individuals of different ages and of both sexes. Materials and Methods: Faecal samples were collected from 137 individuals (0-90 years; 67 males, 70 females) living in the city of São Paulo – SP. The bacterial isolation was carried out in MacConkey agar supplemented with nalidixic acid (50µg/mL); CHROMagarTM ESBL; CHROMagarTM KPC; MacConkey agar supplemented with imipenem (1μg/mL) and on MacConkey agar with 10μg ertapenem, meropenem and imipenem disks. The identification of the colonies and the evaluation of the sensitivity profile were carried out using the BD Phoenix 5.1TM system, and the sensitivity profile for ciprofloxacin and carbapenems confirmed by broth microdilution. Isolates that screened positive for ESBL (extended-spectrum β-lactamase) production by Phoenix and isolates resistant to carbapenems were subjected to molecular screening for ESBL and carbapenemases genes (blaTEM, blaSHV e blaCTX-M, blaKPC, blaGES, blaOXA-48, blaIMP, blaVIM, blaSPM, blaGIM, blaNDM). The "possible ESBL-producing" strains by Phoenix were examined for the presence of blaTEM, blaSHV and blaCTX-M. Cefoxitin resistant strains of Escherichia coli and Klebsiella pneumoniae were examined for the presence of CMY-2. All isolates of Escherichia coli were checked for presence of 30 genes encoding virulence factors, often searched in extraintestinal pathogenic E. coli (ExPEC) and the phylogenetic group was determined. Results: We found 241 bacteria (considering in almost all cases, a single bacterial species for each stool sample), with prevalence of E. coli and Pseudomonas aeruginosa. Thirty-one bacteria were resistant to ciprofloxacin by broth microdilution. Of these, 28 were E. coli isolated from 28 (20.4%) stool samples. The ciprofloxacin-resistant bacteria were isolated at high frequency in the three age groups. Sixteen enterobacteria (obtained from 15 - 10.9% - stool samples) harbored genes encoding ESBL. These isolates were similarly distributed in three age groups and in both sexes. Enzymes CTX-M were prevalent. Twenty bacteria were resistant to carbapenems, but none harbored genes encoding carbapenemases. Three E. coli strains harbored blaCMY-2 gene. High occurrence of virulence markers, often associated with ExPEC, was verified in E. coli isolates, in which 43.3% (26) isolates harbored 10 or more genes. Group A was the most prevalent (18 isolates - 30%), however, the B2 and D groups (associated with ExPEC) occurred in high frequency (21.7 and 15% of the isolates, respectively). E. coli strains resistant to ciprofloxacin and isolates of this species associated with producing the ESBL had lower average number of virulence factors, comparing with the isolates did not present these types of resistance. Conclusions: The intestine of healthy individuals is a reservoir of Gram-negative bacteria resistant to antimicrobial agents of clinical importance, as well as E. coli strains with full potential virulence for extra-intestinal infections.
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PATEKOSKI, Katya da Silva. Estudo da prevalência de bactérias gram-negativas com resistência a antimicrobianos e de Escherichia coli portadora de genes de virulência na microbiota intestinal de indivíduos sadios. 2015. Tese (Doutorado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.