Análise molecular da resistência a antibióticos, factores de virulência e grupos filogenéticos em Escherichia coli e Enterococcus spp. de animais
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2009
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O reconhecimento da resistência antimicrobiana como um fenómeno emergente em saúde pública, tem constituído um problema a nível mundial A comunidade científica constatou a necessidade de realizar a avaliação da susceptibilidade aos antibióticos em “bactérias indicadoras” de diversas origens, como uma medida para combater o aumento da resistência antimicrobiana. As bactérias comensais Escherichia coli e Enterococcusspp. são colonizadoras do tracto gastrintestinal da maioria dos animais e humanos permitindo realizar estudos de emergência, ocorrência e disseminação da resistência aos antibióticos. Este trabalho teve por objectivo estudar a taxa de resistência a antibióticos em isolados fecais de E. coli e Enterococcus spp. de diferentes origens animais, detectar factores de virulência e analisar os grupos filogenéticos dos isolados de E. coli.Um total de 209 amostras fecais (54 de avestruz, 13 de burro, 30 de suíno comercial, 35 de suíno bísaro e 77 de javali) foram semeadas em Slanetz-Bartley (suplementado e não suplementado com vancomicina) e em Levine (suplementado e não suplementado com cefotaxima). Testou-se a susceptibilidade aos antibióticos pelo método da difusão em disco de acordo com as normas do CLSI, usando-se 16 antibióticos em E. coli: ampicilina, amoxicilina+ácido clavulânico, cefoxitina, cefotaxima, ceftazidima, aztreonam, imipenemo, gentamicina, amicacina, tobramicina, estreptomicina, ácido nalidíxico, ciprofloxacina, trimetropim-sulfametoxazol, tetraciclina e cloranfenicol. Em Enterococcus spp. utilizaram-se 11 antibióticos: ampicilina, ciprofloxacina, cloranfenicol, eritromicina, quinupristina-dalfopristina, tetraciclina, teicoplanina, vancomicina, e aminoglicosídeos de elevada carga (estreptomicina, gentamicina e canamicina). Obtiveram-se sete isolados de Enterococcus spp. resistentes à vancomicina (VRE) de amostras fecais de avestruz e 17 isolados de E. coli produtores de -lactamases de amplo espectro (BLAE) de amostras fecais de burro e de suínos (comercial e bísaro). Seis dos sete isolados VRE (quatro isolados da espécie E. duransonde foi detectado o gene vanA e em três isolados onde foi detectado o gene vanC1 que codifica uma resistência intrínseca à vancomicina em E. gallinarum) mostraram resistência à tetraciclina e em todos eles foi detectado o gene tet(M). O gene erm(B) foi detectado nos cinco isolados que mostraram resistência à eritromicina. A pesquisa de factores de virulência permitiu identificar o gene hyl, codificador da hialuronidase, nos três isolados VRE da espécie E. gallinarum.Dos 17 isolados BLAE, 16 manifestaram a presença do gene blaCTX-M1 e oito a presença do gene blaTEM (sete em combinação com o gene blaCTX-M1 e um em combinação com o gene blaCTX-M14).O gene tet(A)outet(B) foi encontrado em 13 de 15 isolados resistentes à tetraciclina. O gene aadA foi encontrado em nove de 10 isolados resistentes à estreptomicina. Em um isolado resistente ao cloranfenicol e um resistente ao sulfametoxazol-trimetropim não foram detectados os genes que codificam a respectiva resistência (cmlA e sul1,sul2 ou sul3, respectivamente). Os genes gyrA e parC foram amplificados e posteriormente sequenciados nos isolados resistentes às quinolonas. Num isolado resistente ao ácido nalidíxico e à ciprofloxacina foram identificadas duas alterações aminoacídicas no gene gyrA (Ser83Leu + Asp87Asn) e uma no gene parC (Ser80Ile). A pesquisa de factores de virulência evidenciou a presença do gene fimA na totalidade dos isolados BLAE e o gene aer em seis destes. Dos 17 isolados BLAE estudados, 14 pertenceram ao grupo filogenético A e três ao grupo filogenético B1. O estudo da relação clonal dos isolados de E. coli produtores de BLAE permitiu identificar nove padrões de restrição diferentes, sendo alguns dos padrões idênticos em isolados provenientes de diferentes animais. Os isolados de E. coli obtidos de meios não suplementados com cefotaxima apresentaram uma elevada percentagem de resistência à tetraciclina (60,7%), à ampicilina (24,8%) e à estreptomicina (21,3%). Os isolados de Enterococcus spp. obtidos de meios não suplementados com vancomicina apresentaram fenótipos de resistência à tetraciclina (68,1%), eritromicina (21%), canamicina (5%), estreptomicina (4,2%), quinupristina-dalfopristina (3,4%), gentamicina (1,7%) e ampicilina (0,8%). Neste estudo, dependendo das diferentes amostras fecais analisadas, foram detectados diferentes níveis de colonização de enterococos com o gene vanA e também diferentes percentagens de isolados de E. coli produtoras de -lactamases de amplo espectro. Uma pequena variedade de genes codificadores de factores de virulência foi encontrada nos isolados BLAE. O estudo da resistência antimicrobiana em E. coli eEnterococcus spp.,isolados a partir de meios não suplementados com antibiótico,permitiu detectar variações na resistência entre os isolados obtidos de diferentes espécies animais. Os dados obtidos neste estudo são importantes para estabelecer políticas de uso prudente de antibióticos tanto em animais como em humanos bem como, advertir a população humana para os riscos do uso inadequado destes agentes.
The bacteria antimicrobial resistance is nowadays a public health emerging dilemma. The scientific community recognize the necessity of accomplish the evaluation of this situation with "indicative bacteria" of several origins as a measure to combat the increase of the problem. Escherichia coli and Enterococcus spp. are commensal bacteria from the gastrointestinal tract of the majority of animals and humans that allow to monitories studies in the occurrence and spread of antibiotic resistance. The objective of this work was to study the antibiotic resistance in fecal E. coliand Enterococcus spp. of different animal origins, to detect virulence factors and to analyze the phylogenetic groups of the strains. A total of 209 fecal samples (54 of ostrich, 13 of donkey, 30 of commercial swine, 35 of bísaro swine and 77 of wild boar) were seeded in Slanetz-Bartley (supplemented and not supplemented with vancomycin) and Levine (supplemented and not supplemented with cefotaxime). Antimicrobial susceptibility was performed by disk diffusion agar method as recommended by the CLSI recommendations. A total of 16 antimicrobial agents were tested against E. coli: ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, aztreonam, gentamicin, tobramycin, amikacin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin and chloramphenicol. Antimicrobial susceptibility was tested additionally for 11 antibiotics in enterococcal isolates (ampicillin, vancomycin, teicoplanin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin, ciprofloxacin and high-level resistance for streptomycin, gentamicin, and kanamycin). Seven VRE (vancomycin-resistant-Enterococus) strains isolated from faecal samples of ostrich and 17 extended-spectrumproducing beta-lactamases (ESBLs)-Escherichia coli strains isolated from faecal samples of donkey and swine (commercial and bísaro) were obtained in this study.Six of the seven VRE isolates (4 E. durans with vanA gene and 3 E. gallinarumwithvanC1 gene) were tetracycline resistant and all of them harbored the tet(M) gene. Additionally the five isolates those were erythromycin resistant posses the erm(B) gene. Thehyl virulent factor gene was detected in the three E. gallinarum isolates. In relation to the 17 ESBLs isolates, 16 of them manifest the presence of the blaCTX-M1 gene and eight the presence of the blaTEM gene (seven in combination with the blaCTX-M1gene and one in combination with the blaCTX-M14 gene). The tet(A) or tet(B) genes were found in 13 of 15 tetracycline isolates and the aadA gene was found in nine of 10 streptomycin resistant isolates. However, although one strain waschloramphenicol resistant and one trimethoprim-sulfamethoxazole resistant neither showed the corresponded genes (cmlAandsul1,sul2 or sul3, respectively). The gyrA and parC genes were amplified and sequenced in the quinolone-resistant isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in the nalidixic acid and ciprofloxacin-resistant isolate. Research of virulence factors evidenced the presence of the fimA gene in the totality of the ESBLs isolates and the aergene in six of them. Fourteen ESBL-containing E. coli isolates belonged to the A phylogenetic group and three to the B1 phylogenetic group. The clonal relationship of the ESBL-containing E. coli isolates allowed identifying nine different patterns of restriction, being some of them identically and detected in isolates obtained from different animals. The Enterococcus spp. isolates obtained from Slanetz-Bartley not supplemented with vancomycin showed resistance to tetracycline (68,1%), erythromycin (21%), kanamycin (5%), streptomycin (4,2%), quinupristin-dalfopristin (3,4%), gentamicin (1,7%) and ampicillin (0,8%). On the other hand, the E. coli isolates obtained from Levine not supplemented with cefotaxime showed a high percentage of resistance to tetracycline (60,7%), ampicillin (24,8%) and streptomycin (21,3%). In this study it was detected different levels of colonization by vanAenterococcal strains as well different rates of ESBL-containing E. coli isolates depending of the different faecal samples analyzed. A narrow variety of genes encoding virulence factors were detected in ESBL-containing E. coli isolates. On the other hand, differences in the frequency of antimicrobial resistance have been observed when faecal enterococci and E. coli isolates of different origins, obtained without antibiotic supplementation, have been compared. Data obtained in the present work are important to establish policies of careful use of antibiotics in animals and in humans as well as, to notice the human population for the inadequate use and risks of these agents.
The bacteria antimicrobial resistance is nowadays a public health emerging dilemma. The scientific community recognize the necessity of accomplish the evaluation of this situation with "indicative bacteria" of several origins as a measure to combat the increase of the problem. Escherichia coli and Enterococcus spp. are commensal bacteria from the gastrointestinal tract of the majority of animals and humans that allow to monitories studies in the occurrence and spread of antibiotic resistance. The objective of this work was to study the antibiotic resistance in fecal E. coliand Enterococcus spp. of different animal origins, to detect virulence factors and to analyze the phylogenetic groups of the strains. A total of 209 fecal samples (54 of ostrich, 13 of donkey, 30 of commercial swine, 35 of bísaro swine and 77 of wild boar) were seeded in Slanetz-Bartley (supplemented and not supplemented with vancomycin) and Levine (supplemented and not supplemented with cefotaxime). Antimicrobial susceptibility was performed by disk diffusion agar method as recommended by the CLSI recommendations. A total of 16 antimicrobial agents were tested against E. coli: ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, aztreonam, gentamicin, tobramycin, amikacin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin and chloramphenicol. Antimicrobial susceptibility was tested additionally for 11 antibiotics in enterococcal isolates (ampicillin, vancomycin, teicoplanin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin, ciprofloxacin and high-level resistance for streptomycin, gentamicin, and kanamycin). Seven VRE (vancomycin-resistant-Enterococus) strains isolated from faecal samples of ostrich and 17 extended-spectrumproducing beta-lactamases (ESBLs)-Escherichia coli strains isolated from faecal samples of donkey and swine (commercial and bísaro) were obtained in this study.Six of the seven VRE isolates (4 E. durans with vanA gene and 3 E. gallinarumwithvanC1 gene) were tetracycline resistant and all of them harbored the tet(M) gene. Additionally the five isolates those were erythromycin resistant posses the erm(B) gene. Thehyl virulent factor gene was detected in the three E. gallinarum isolates. In relation to the 17 ESBLs isolates, 16 of them manifest the presence of the blaCTX-M1 gene and eight the presence of the blaTEM gene (seven in combination with the blaCTX-M1gene and one in combination with the blaCTX-M14 gene). The tet(A) or tet(B) genes were found in 13 of 15 tetracycline isolates and the aadA gene was found in nine of 10 streptomycin resistant isolates. However, although one strain waschloramphenicol resistant and one trimethoprim-sulfamethoxazole resistant neither showed the corresponded genes (cmlAandsul1,sul2 or sul3, respectively). The gyrA and parC genes were amplified and sequenced in the quinolone-resistant isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in the nalidixic acid and ciprofloxacin-resistant isolate. Research of virulence factors evidenced the presence of the fimA gene in the totality of the ESBLs isolates and the aergene in six of them. Fourteen ESBL-containing E. coli isolates belonged to the A phylogenetic group and three to the B1 phylogenetic group. The clonal relationship of the ESBL-containing E. coli isolates allowed identifying nine different patterns of restriction, being some of them identically and detected in isolates obtained from different animals. The Enterococcus spp. isolates obtained from Slanetz-Bartley not supplemented with vancomycin showed resistance to tetracycline (68,1%), erythromycin (21%), kanamycin (5%), streptomycin (4,2%), quinupristin-dalfopristin (3,4%), gentamicin (1,7%) and ampicillin (0,8%). On the other hand, the E. coli isolates obtained from Levine not supplemented with cefotaxime showed a high percentage of resistance to tetracycline (60,7%), ampicillin (24,8%) and streptomycin (21,3%). In this study it was detected different levels of colonization by vanAenterococcal strains as well different rates of ESBL-containing E. coli isolates depending of the different faecal samples analyzed. A narrow variety of genes encoding virulence factors were detected in ESBL-containing E. coli isolates. On the other hand, differences in the frequency of antimicrobial resistance have been observed when faecal enterococci and E. coli isolates of different origins, obtained without antibiotic supplementation, have been compared. Data obtained in the present work are important to establish policies of careful use of antibiotics in animals and in humans as well as, to notice the human population for the inadequate use and risks of these agents.
Descrição
Dissertação de Mestrado em Biologia Clínica Laboratorial
Palavras-chave
Escherichia coli , Enterococcus spp. , BLAE , VRE , Antibióticos