Resistência a antibióticos, factores de virulência e grupos filogenéticos em Encherichia coli produtora de ß-lactamases de amplo espectro de frangos para consumo
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2014-01-27
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O desenvolvimento da resistência aos antibióticos tem sido reconhecido como um grave problema de Saúde Pública. A comunidade científica reconhece a necessidade de realizar a avaliação da susceptibilidade aos antibióticos em “bactérias indicadoras” (como Escherichia coli) de diversas origens, tendo em vista o combate o aumento da resistência antimicrobiana. A microflora bacteriana intestinal dos animais de consumo, assim como os seus produtos constituem ambientes favoráveis para a sobrevivência, transferência e disseminação de bactérias resistentes.
O principal objectivo deste trabalho consistiu em estudar a taxa de resistência a antibióticos em isolados de E. coli produtoras de β-lactamases de amplo espectro (BLAE) de carne de frango destinada a consumo humano, detectar factores de virulência e analisar os grupos filogenéticos dos isolados de E. coli.
Um total de 29 amostras de frango (6 de asas, 6 de peito, 6 de coxas 5 de moelas e 6 de pele) foram semeadas em Levine não suplementado com cefotaxima e Levine com suplemento de cefotaxima (2μg/ml). Testou-se a susceptibilidade a 16 antibióticos (ampicilina; amoxicilina + ácido clavulânico; cefotaxima; cefoxitina; ceftazidima; aztreonam; imipenemo; gentamicina; amicacina; tobramicina; estreptomicina; ácido nalidíxico; ciprofloxacina; sulfametoxazol-trimetropim; tetraciclina e cloranfenicol) pelo método da difusão em disco de acordo com as normas do CLSI.
Obtiveram-se 29 isolados (100%) de E. coli das placas de Levine não suplementado com cefotaxima e, 27 isolados (93,1%) de E. coli das placas de Levine suplementadas. Dos 27 isolados obtidos de placas de Levine suplementado com cefotaxima (2μg/ml), verificou-se que 23 dos isolados (85,2%) eram produtores de β-lactamases de amplo espectro (BLAE) e que 4 dos isolados (14,8%) não foram produtores deste tipo de enzimas (negativos para o teste de detecção fenotípica de BLAEs).
Os isolados de E. coli obtidos de meios não suplementados com cefotaxima apresentaram uma elevada percentagem de resistência à tetraciclina (72,4%), à
ampicilina, ao ácido nalidíxico (65,5%), ao cloranfenicol, à ciprofloxacina (34,5%), ao sulfametoxazol-trimetropim (27,6%) e à estreptomicina (27,6%).
Dos 23 isolados BLAE, 13 manifestaram a presença do gene blaTEM-52, 4 a presença do gene blaCTX-M-14a, quatro a presença do gene blaCTX-M-9 e 2 a presença do gene blaCTX-M-1. O gene tetA ou tetB foi encontrado nos 17 isolados resistentes à tetraciclina. Dos 13 isolados resistentes ao sulfametoxazol-trimetropim, 12 manifestaram a presença do gene sul2, 4 a presença do gene sul1 (os quatro em combinação com o gene sul2) e 1 a presença do gene sul3. O gene aadA foi encontrado em oito dos 11 isolados resistentes à estreptomicina. O gene aac(3)-II foi encontrado nos 4 isolados resistentes à gentamicina. No isolado resistente ao cloranfenicol foi detectado o gene cmlA. Os genes gyrA e parC foram amplificados e posteriormente sequenciados nos isolados resistentes às quinolonas. Nos 20 isolados resistentes ao ácido nalidíxico foram identificadas duas alterações aminoacídicas no gene gyrA (S83L+D87N). Os 10 isolados resistentes à ciprofloxacina possuíam o gene parC com uma mutação no tripleto 80 que causa alteração aminoacídica (S80I).
Nos 4 isolados obtidos de placas de Levine com suplemento de cefotaxima, que evidenciaram resultado negativo para o teste de detecção fenotípica de BLAEs, foram detectadas mutações pontuais na região promotora/atenuadora do gene ampC nas posições -42; -18; -1; +58. O gene aadA foi encontrado nos 4 isolados resistentes à estreptomicina. Os 4 isolados resistentes ao sulfametoxazol-trimetropim manifestaram o gene sul1. O gene tetA foi encontrado nos 2 isolados resistentes à tetraciclina. O gene aac(3)-II foi encontrado nos 2 isolados resistentes à gentamicina. Nos 2 isolados resistentes ao ácido nalidíxico foram identificadas duas alterações aminoacídicas no gene gyrA (S83L+D87N).
Dos 27 isolados provenientes de placas de Levine com suplemento de cefotaxima, a pesquisa de factores de virulência evidenciou a presença do gene aer em 74,1% dos isolados, o gene fimA em 40,7% e o gene papC em 7,4% dos isolados. Detectou-se o gene intl1 em 8 isolados e o gene intl2 em 4 isolados. Dos 27 isolados provenientes de placas de Levine com suplemento de cefotaxima, 5 pertenceram ao grupo filogenético A, 11 ao grupo filogenético B1, 3 ao grupo filogenético B2 e 8 ao grupo filogenético D.
Foi nosso intuito caracterizar, em todas as estirpes provenientes de placas de Levine suplementadas com cefotaxima, os mecanismos envolvidos na resistência
fenotípica aos antibióticos, bem como descrever os possíveis elementos de aquisição e disseminação destes genes.
Por último, referir que os dados obtidos neste estudo são importantes para estabelecer políticas de uso prudente de antibióticos em animais de consumo, bem como em humanos. De salientar a importância de continuar estudos de vigilância da resistência aos antibióticos, tendo em vista a evolução da resistência no tempo, em diferentes nichos, assim como o estudo de factores que influenciam a sua selecção e disseminação.
The development of antibiotic resistance has been recognized as a serious public health problem. The scientific community recognizes the need for the evaluation of susceptibility to antibiotics in “indicator bacteria” (such as Escherichia coli) from various sources in order to combat increasing antimicrobial resistance. The gut micro flora of animals for consumption, as well their products, are favorable environments for survival, transfer and dissemination of resistant bacteria. The main objective of this work was to study the rate of antibiotic resistance in chicken meat extended-spectrum producing beta-lactamases (ESBLs) Escherichia coli isolates, research virulence factors and analyze the phylogenetic groups. A total of 29 samples of chicken products (6 thighs, 6 breasts, 6 wings, 5 gizzard and 6 skin) were seeded in Levine not supplemented with cefotaxime and Levine supplemented with cefotaxime (2μg/ml). Antimicrobial susceptibility was performed by disk diffusion agar method as recommended by the CLSI recommendations. A total of 16 antimicrobial agents were tested: ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, aztreonam, gentamicin, tobramycin, amikacin, streptomycin, tetracycline, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and chloramphenicol. Twenty nine Escherichia coli strains (100%) isolated from plates of Levine not supplemented with cefotaxime and twenty seven E. coli strains (93,1%) isolated from plates supplemented. In relation to the 27 strains of E. coli isolates from plates of Levine supplemented with cefotaxime, 23 isolates (85,2%) were extended-spectrum producing beta-lactamases (ESBLs) and 4 isolates (14,8%) were not extended-spectrum producing beta-lactamases (showed a negative result in the test for detection phenotypic of ESBLs). The E. coli isolates obtained from Levine not supplemented with cefotaxime, showed a high percentage of resistance to tetracycline (72,4%), ampicillin , nalidixic acid (65,5%), chloramphenicol, ciprofloxacin (34,5%), sulfamethoxazole- trimethoprim (27,6%) and streptomycin (27,6%). In relation to the 23 ESBLS isolates, 13 of them manifest the presence of the blaTEM-52 gene, 4 the presence of the blaCTX-M-14a gene, four the presence of the blaCTX-M-9 gene, and two the presence of the blaCTX-M-1 gene. The tetA or tetB genes were found in all 17 tetracycline resistant isolates and the aadA gene was found in eight of 11 streptomycin resistant isolates. In relation to the 13 sulfamethoxazole- trimethoprim resistant isolates, 12 of them manifest the sul2 gene, 4 the sul1 gene (all in combination with sul2 gene) and 1 the sul3 gene. The aac(3)-II gene were founded in the 4 gentamicin resistant isolates. In the chloramphenicol resistant isolated, were detected the cmlA gene. The gyrA and parC genes were amplified and sequenced in the quinolone-resistant isolates. Two amino acid changes in gyrA gene (S83L + A87A) were detected in the 20 nalidixic acid resistant isolates and one in parC gene (S80I) were identified in the ciprofloxacin resistant isolate. In the 4 isolates obtained from plates of Levine with supplement of cefotaxime, that showed a negative result in the test for detection phenotypic of ESBLs, were detected mutations in the promoter/attenuator region of ampC gene in the -42; -18; -1; +58 positions. In relation to the 4 isolates with hyperproduction of chromosomal AmpC β-lactamase, the 4 sulfamethoxazole- trimethoprim resistant isolates manifest the sul1 gene. The tetAgene was detected in the 2 tetracycline resistant isolates, the 2 gentamicin resistant isolates manifest the aac(3)-II gene. Two amino acid changes in gyrA gene (S83L + A87A) were detected in the 2 nalidixic acid resistant isolates. The research of virulence factors, class 1 and 2 integrons and phylogenetic group was performed to the 27 isolates obtained from plates of Levine with cefotaxime supplement. The research of virulence factors showed the presence of the aer gene in 74,1% of the isolates, the fimA gene in 40,7% the isolates and the papC gene in 7,4% of the isolates. The class 1 integron were detected in 8 isolates and class 2 integron in 4 isolates. Four isolates belong to the A phylogenetic group, 11 to the B1 phylogenetic group, 3 to the B2 phylogenetic group and eight to the D phylogenetic group. Our aim was to characterize, in all strains from plates of Levine supplemented with cefotaxime , the mechanisms involved in phenotypic resistance to antibiotics, as well as describing the possible elements of the acquisition and dissemination of these genes. Finally, note that the data obtained in this study are important for establishing policies for prudent use of antibiotics in animal consumption as well as in humans. To emphasize the importance of continuing surveillance studies of antibiotic resistance, in view of the evolution of resistance over time, in different niches, such as the study of factors who influencing the selection and dissemination.
The development of antibiotic resistance has been recognized as a serious public health problem. The scientific community recognizes the need for the evaluation of susceptibility to antibiotics in “indicator bacteria” (such as Escherichia coli) from various sources in order to combat increasing antimicrobial resistance. The gut micro flora of animals for consumption, as well their products, are favorable environments for survival, transfer and dissemination of resistant bacteria. The main objective of this work was to study the rate of antibiotic resistance in chicken meat extended-spectrum producing beta-lactamases (ESBLs) Escherichia coli isolates, research virulence factors and analyze the phylogenetic groups. A total of 29 samples of chicken products (6 thighs, 6 breasts, 6 wings, 5 gizzard and 6 skin) were seeded in Levine not supplemented with cefotaxime and Levine supplemented with cefotaxime (2μg/ml). Antimicrobial susceptibility was performed by disk diffusion agar method as recommended by the CLSI recommendations. A total of 16 antimicrobial agents were tested: ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, imipenem, aztreonam, gentamicin, tobramycin, amikacin, streptomycin, tetracycline, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and chloramphenicol. Twenty nine Escherichia coli strains (100%) isolated from plates of Levine not supplemented with cefotaxime and twenty seven E. coli strains (93,1%) isolated from plates supplemented. In relation to the 27 strains of E. coli isolates from plates of Levine supplemented with cefotaxime, 23 isolates (85,2%) were extended-spectrum producing beta-lactamases (ESBLs) and 4 isolates (14,8%) were not extended-spectrum producing beta-lactamases (showed a negative result in the test for detection phenotypic of ESBLs). The E. coli isolates obtained from Levine not supplemented with cefotaxime, showed a high percentage of resistance to tetracycline (72,4%), ampicillin , nalidixic acid (65,5%), chloramphenicol, ciprofloxacin (34,5%), sulfamethoxazole- trimethoprim (27,6%) and streptomycin (27,6%). In relation to the 23 ESBLS isolates, 13 of them manifest the presence of the blaTEM-52 gene, 4 the presence of the blaCTX-M-14a gene, four the presence of the blaCTX-M-9 gene, and two the presence of the blaCTX-M-1 gene. The tetA or tetB genes were found in all 17 tetracycline resistant isolates and the aadA gene was found in eight of 11 streptomycin resistant isolates. In relation to the 13 sulfamethoxazole- trimethoprim resistant isolates, 12 of them manifest the sul2 gene, 4 the sul1 gene (all in combination with sul2 gene) and 1 the sul3 gene. The aac(3)-II gene were founded in the 4 gentamicin resistant isolates. In the chloramphenicol resistant isolated, were detected the cmlA gene. The gyrA and parC genes were amplified and sequenced in the quinolone-resistant isolates. Two amino acid changes in gyrA gene (S83L + A87A) were detected in the 20 nalidixic acid resistant isolates and one in parC gene (S80I) were identified in the ciprofloxacin resistant isolate. In the 4 isolates obtained from plates of Levine with supplement of cefotaxime, that showed a negative result in the test for detection phenotypic of ESBLs, were detected mutations in the promoter/attenuator region of ampC gene in the -42; -18; -1; +58 positions. In relation to the 4 isolates with hyperproduction of chromosomal AmpC β-lactamase, the 4 sulfamethoxazole- trimethoprim resistant isolates manifest the sul1 gene. The tetAgene was detected in the 2 tetracycline resistant isolates, the 2 gentamicin resistant isolates manifest the aac(3)-II gene. Two amino acid changes in gyrA gene (S83L + A87A) were detected in the 2 nalidixic acid resistant isolates. The research of virulence factors, class 1 and 2 integrons and phylogenetic group was performed to the 27 isolates obtained from plates of Levine with cefotaxime supplement. The research of virulence factors showed the presence of the aer gene in 74,1% of the isolates, the fimA gene in 40,7% the isolates and the papC gene in 7,4% of the isolates. The class 1 integron were detected in 8 isolates and class 2 integron in 4 isolates. Four isolates belong to the A phylogenetic group, 11 to the B1 phylogenetic group, 3 to the B2 phylogenetic group and eight to the D phylogenetic group. Our aim was to characterize, in all strains from plates of Levine supplemented with cefotaxime , the mechanisms involved in phenotypic resistance to antibiotics, as well as describing the possible elements of the acquisition and dissemination of these genes. Finally, note that the data obtained in this study are important for establishing policies for prudent use of antibiotics in animal consumption as well as in humans. To emphasize the importance of continuing surveillance studies of antibiotic resistance, in view of the evolution of resistance over time, in different niches, such as the study of factors who influencing the selection and dissemination.
Descrição
Dissertação de Mestrado em Segurança Alimentar
Palavras-chave
Escherichia coli , Resistência , Antibióticos , BLAE , Carne de frango