Determinação do tipo capsular e do perfil de resistência aos antimicrobianos em linhagens de Streptococcus agalactiae isoladas de amostras de leite bovino

Abstract

The success of Streptococcus agalactiae infections can be attributed to the polysaccharide capsule (CPS), which is one of the virulence factors, capable of enabling evasion of the immune system and the persistence of the pathogen within the host. Furthermore, the different types of anticrobials resistance mechanisms and the ease of acquiring genetic material (genes) from other microorganisms contribute to its survival, even in the presence of certain antimicrobials used for the treatment of bovine mastitis, one of the most prevalent diseases in dairy herds in Brazil, with S. agalactiae being one of the main causes. Nevertheless, information about the characteristics of S. agalactiae affecting cattle in the country is scarce and the prevalence of this pathogen in Brazilian herds is high. Thus, the aim of the present study was to determine the capsular serotyping of S. agalactiae strains and establish the antimicrobial resistance profile used for mastitis treatment in S. agalactiae isolates from dairy cows in the Zona da Mata Mineira and Campo das Vertentes regions. One thousand one hundred and seventy (n= 1170) milk samples were collected during four consecutive semesters, between the years 2009 and 2011, on 11 dairy farms located in Zona da Mata Mineira and Campo das Vertentes regions. Among the bacterial isolates, 284 strains were identified as S. agalactiae through biochemical methods and PCR, with 141 isolates from collects 1 and 4 (20 months interval) used in the present study. The genetic diversity of this subpopulation was assessed by comparing restriction profiles of DNA macrofragments generated by the SmaI enzyme separated by pulsed-field gel electrophoresis (PFGE). The epidemiological distribution of S. agalactiae was inferred by capsular serotyping of isolates using multiplex PCR. Antibiotic susceptibility tests were performed by disk diffusion for the antimicrobials ampicillin, penicillin, oxacillin, cephalothin, ceftiofur, clindamycin, gentamicin, erythromycin, enrofloxacin, sulfamethoxazole, sulfamethoxazole+trimethoprim and tetracycline. Resistance genes to the most prevalent antimicrobials (gentamicin, erythromycin and tetracycline) were evaluated by PCR, using the genetic markers ermA, ermB, tetO, tetk, tetL, tetM and aacA-aphD. Among the 97 DNA profiles that were digested by SmaI restriction enzyme, 95 strains of S. agalactiae presented genetically distinct DNA profiles in PFGE. Seventeen isolates were considered genetically redundant and 78 were genetically non-redundant, revealing great genetic diversity between S. agalactiae isolates from the first and fourth collection. Serotype II was the most prevalent (n= 34), followed by serotypes III (n= 24) and Ia (n= 10). Among the 78 S. agalactiae isolates subjected to antibiogram, 74 were considered resistant to at least one antibiotic, and one strain (628) was resistant to nine antimicrobials. Fiftyfour (69,2%) isolates showed resistance to tetracycline in the phenotypic test. Fortysix (58,9%) isolates showed high resistance to tetracycline and carried the tetM and tetO. The distribution of pulsotypes among S. agalactiae isolates suggests that, in some cases, the same microorganisms remained in the herd over a period of twentymonths. When associated with serotype/antimicrobial resistance, serotype II showed high resistance to the antibiotics erythromycin (65,8%), tetracycline (59,2%) and gentamicin (45,1%). Studies on the assessment of genetic diversity, capsular serotyping and antimicrobial susceptibility of S. agalactiae strains isolated from dairy cattle in different regions of Brazil are necessary in order to better understand the biology of this pathogen with a view to developing and implementing new prevention and treatment options for mastitis caused by S. agalactiae.