Caracterização Genética da Resistência à Eritromicina em Streptococcus agalactia e Degestantes saudáveis
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2009
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Streptococcus agalactiae (Grupo B de Lancefield, EGB), um microrganismo comensal do homem, reconhecido em 1920 como o agente etiológico da mastite bovina, tem sido associado a infecções em parturientes e recém-nascidos, sendo o principal agente de septicemia e meningite neonatal. Para evitar a infecção perinatal recomenda-se a pesquisa da bactéria na região vagino-perianal durante o terceiro semestre de gravidez, indicando o tratamento antibiótico durante o parto das gestantes colonizadas. O objectivo deste estudo foi verificar a ocorrência de colonização por Streptococcus agalactiae em gestantes saudáveis, avaliar a associação da colonização com variáveis maternas demográficas e clínicas, avaliar a susceptibilidade dos isolados aos antibióticos e determinar os fenótipos de resistência à eritromicina e à clindamicina e os mecanismos genéticos nelas implicados, com utilidade clínica na recomendação da profilaxia e terapêutica empírica. Realizou-se um estudo envolvendo 520 gestantes em acompanhamento pré-natal, atendidas no Centro Hospitalar de Trás-os-Montes e Alto Douro, durante o período de Janeiro a Junho de 2008. Durante o atendimento foram colhidas amostras vaginal e anorrectal para rastrear a presença de colonização por S. agalactiae. As gestantes colonizadas por EGB foram caracterizadas por variáveis demográficas (idade e número de gestações) e clínicas (ocorrência de abortos anteriores e presença ou ausência de diabetes mellitus). As colheitas foram efectuadas com zaragatoas estéreis e colocadas em meio selectivo de Todd Hewitt. Procederam-se às provas presuntivas de identificação de EGB (coloração pelo método de Gram, observação microscópica, prova da catalase), e provas confirmatórias (CAMP e aglutinação em látex). Realizou-se o estudo da resistência aos antibióticos eritromicina, clindamicina, penicilina, cefotaxima, vancomicina, quinupristina-dalfopristina, cloranfenicol, ofloxacina e meropenem. O E-teste para o cálculo das concentrações mínimas inibitórias (mg/l) foi aplicado, adicionalmente, para a eritromicina e clindamicina. Os fenótipos de resistência MLSB induzida e constitutiva (cMLSB e iMLSB, respectivamente), bem como o fenótipo de resistência M da eritromicina e clindamicina foram determinados pelo teste de disco duplo. A presença dos genes que conferem resistência aos macrólidos como ermA, ermB, ermC, ermTR, msrA e mef(A/E) foram estudados através de PCR, com o uso de primers específicos e posterior sequenciação.
A prevalência de colonização materna por este microrganismo foi de 18% na população estudada. Todos os 93 isolados mostraram susceptibilidade à penicillina, cefotaxima, vancomicina, quinupristina-dalfopristina, cloranfenicol, ofloxacina e meropenem. A resistência à eritromicina e clindamicina foi detectada em 15% e 9,6% dos isolados, respectivamente. Os fenótipos de resistência detectados entre os 14 isolados resistentes à eritromicina foram os seguintes (número de isolados): cMLSB (9), iMLSB (3), e de M (2). As CMI (mg/l) para a eritromicina e clindamicina foram: isolados cMLSB (128-B (16-mg/l) e isolados M (2-8 e 1 mg/l). A seguinte combinação de genes foi detectada entre os isolados com fenótipos cMLSB ou iMLSB: ermB (6 isolados), ermA + ermTR (3), ermA + ermB + ermTR (1) e nenhum destes genes (2). Os dois isolados com fenótipo M possuíam o gene mef(A/E) e o gene msrA também foi encontrado num dos isolados de S. agalactiae com fenótipo M.
Streptococcus agalactiae (Lancefield group B, GBS), a microorganism commensal of man, recognized in 1920 as the etiologic agent of bovine mastitis, has been associated with infections in parturients and newly-born, being the main agent of sepsis and neonatal meningitis. To prevent perinatal infection is recommended the search of bacteria in vagino-perianal region in the third trimester of pregnancy, indicating antibiotic treatment during the delivery of the pregnant women colonized. The objective of this study was to verify the occurrence of colonization by Streptococcus agalactiae in healthy pregnant women, evaluate the association of maternal colonization with demographic and clinical variables, evaluate the susceptibility of the isolates to antimicrobial agents and determine the resistance to erythromycin and clindamycin phenotypes, and genetic mechanisms involved, with clinical utility in the recommendation of prophylaxis and empirical therapy. A study was performed involving 520 pregnant women on pre-natal care, at Centro Hospitalar de Trás-os-Montes e AltoDouro, from January to June 2008. During the servicing samples were collected from vaginal and anorrectal areas for tracing the presence of colonization by S. agalactiae. The GBS colonized pregnant women were characterized by demographic variables (age and number of pregnancies) and clinical (occurrence of previous abortions and presence or absence of diabetes mellitus). The samples were obtained with sterile swabs and placed in selective medium of Todd Hewitt. Presumptive test of identification of GBS (color through Gram method, microscopic observation, evidence of catalase) were made and confirmatory tests (CAMP and agglutination in latex). Susceptibility testing for erythromycin, clindamycin, penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin and meropenen was carried out. The E-test for determination of minimal inibitorial concentration (mg/l) was also applied, additionally, for erythromycin and clindamycin. The constitutive and inducible MLSB resistance phenotypes (cMLSB and iMLSB, respectively), as well as the M resistance phenotype were determined by the erythromycin-clindamycin double-disc test. The presence of ermA, ermB, ermC, msrA and mef(A/E) macrolide resistance genes was studied by PCR, with specific primers and subsequent sequencing. The prevalence of colonization by GBS of pregnant women in this study was of 18%. All the 93 isolates showed susceptibility to penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin and meropenen. Resistance to erythromycin and clindamycin was found in 15% and 9.6% of the isolates, respectively. The resistance phenotypes detected among the 14 erythromycin-resistant isolates were as follows (number of isolates): cMLSB (9), iMLSB (3), and M (2). The MIC for erythromycin and clindamycin were: cMLSB isolates (128- 32 mg/l, respectively), iMLSB isolates (16-256 and 1 mg/l), and M isolates (2-The following combination of genes were detected among isolates with cMLSB or iMLSB phenotypes: erm(B) (6 isolates), ermA + ermTR (3), ermA + ermB + ermTR (1), and none of these genes (2). The two isolates with M phenotype harboured the mef(A/E), and msrA gene was also found in one of them.
Streptococcus agalactiae (Lancefield group B, GBS), a microorganism commensal of man, recognized in 1920 as the etiologic agent of bovine mastitis, has been associated with infections in parturients and newly-born, being the main agent of sepsis and neonatal meningitis. To prevent perinatal infection is recommended the search of bacteria in vagino-perianal region in the third trimester of pregnancy, indicating antibiotic treatment during the delivery of the pregnant women colonized. The objective of this study was to verify the occurrence of colonization by Streptococcus agalactiae in healthy pregnant women, evaluate the association of maternal colonization with demographic and clinical variables, evaluate the susceptibility of the isolates to antimicrobial agents and determine the resistance to erythromycin and clindamycin phenotypes, and genetic mechanisms involved, with clinical utility in the recommendation of prophylaxis and empirical therapy. A study was performed involving 520 pregnant women on pre-natal care, at Centro Hospitalar de Trás-os-Montes e AltoDouro, from January to June 2008. During the servicing samples were collected from vaginal and anorrectal areas for tracing the presence of colonization by S. agalactiae. The GBS colonized pregnant women were characterized by demographic variables (age and number of pregnancies) and clinical (occurrence of previous abortions and presence or absence of diabetes mellitus). The samples were obtained with sterile swabs and placed in selective medium of Todd Hewitt. Presumptive test of identification of GBS (color through Gram method, microscopic observation, evidence of catalase) were made and confirmatory tests (CAMP and agglutination in latex). Susceptibility testing for erythromycin, clindamycin, penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin and meropenen was carried out. The E-test for determination of minimal inibitorial concentration (mg/l) was also applied, additionally, for erythromycin and clindamycin. The constitutive and inducible MLSB resistance phenotypes (cMLSB and iMLSB, respectively), as well as the M resistance phenotype were determined by the erythromycin-clindamycin double-disc test. The presence of ermA, ermB, ermC, msrA and mef(A/E) macrolide resistance genes was studied by PCR, with specific primers and subsequent sequencing. The prevalence of colonization by GBS of pregnant women in this study was of 18%. All the 93 isolates showed susceptibility to penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin and meropenen. Resistance to erythromycin and clindamycin was found in 15% and 9.6% of the isolates, respectively. The resistance phenotypes detected among the 14 erythromycin-resistant isolates were as follows (number of isolates): cMLSB (9), iMLSB (3), and M (2). The MIC for erythromycin and clindamycin were: cMLSB isolates (128- 32 mg/l, respectively), iMLSB isolates (16-256 and 1 mg/l), and M isolates (2-The following combination of genes were detected among isolates with cMLSB or iMLSB phenotypes: erm(B) (6 isolates), ermA + ermTR (3), ermA + ermB + ermTR (1), and none of these genes (2). The two isolates with M phenotype harboured the mef(A/E), and msrA gene was also found in one of them.
Descrição
Dissertação de Mestrado em Biologia Clínica Laboratorial
Palavras-chave
Streptococcus agalactiae , gravidez , antibióticos , eritromicina , genótipos