Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia

Streptococcus agalactiae (Group B Streptococcus - GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus - GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in the dairy industry due to antibiotherapy and loss in milk production. However, molecular characterization of field isolates of Streptococcus spp. occurring in Portugal was not known prior to our studies and is important to improve therapeutic and disease control programs. The aims of this study were the identification of strain molecular features, and the evaluation of antimicrobial drug resistance patterns of S. agalactiae (n=60), S. dysgalactiae subsp. dysgalactiae (n=18) and S. uberis (n=30) collected from bovine subclinical mastitis between 2002/2003 in Portugal. Additionally, two S. dysgalactiae subsp. dysgalactiae strains associated with invasive disease(one collected from cattle and the other from a human), and six Streptococcus dysgalactiae subsp. equisimilis (group C or group G Streptococcus - GCS/GGS) strains from human infection were included in the study, for comparative purposes. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE)/BioNumerics, S. agalactiae and S. uberis multi-locus sequence typing (MLST), macrolide and tetracycline resistance gene profiling, S. agalactiae molecular serotyping, virulence gene profiling, PCR-amplification for screening presence of specific genes and subsequent sequencing for phylogenetic analysis, and reverse transcriptase-PCR (RT-PCR) for gene expression analysis of selected genes. Also, a custom-designed microarray containing 220 virulence genes of the human pathogen Streptococcus pyogenes (Group A Streptococcus - GAS) was used to test bovine GCS S. dysgalactiae subsp. dysgalactiae and human GCS/GGS S.dysgalactiae subsp. equisimilis. Antimicrobial resistance was assessed by disk diffusion against penicillin, gentamicin, streptomycin, amoxicillin-clavulanic acid, cefazolin, cefoperazone,rifaximin, erythromycin, pirlimycin, tetracycline, vancomycin, chloramphenicol and the macrolide lincosamide resistance phenotypes (cMLSB, iMLSB, M, L). Among S. uberis three PFGE clonal groups (defined by at least 80% similarity) comprised almost half of total isolates, and 50% of GBS isolates were included in four major clonal groups (all farm-associated), which is indicative of a contagious route of transmission between animals. The occurrence of PFGE patterns sharing >82.8% and 100% similarity among S. dysgalactiae subsp. dysgalactiae isolates collected from different farms suggests an environmental source for this pathogen in our case. By MLST, we observed that all S. uberis sequence types (ST) were found to be novel (n=14), representing novel genomic backgrounds for this pathogen. Among GBS only three MLST lineages (ST-2, ST-23, and ST-61/ST-554) were detected revealing little heterogeneity among our bovine GBS collection. Five new cpsD-cpsE-cpsF sequences of the cps locus (encoding the capsular polysaccharide) were detected in >70% of the bovine GBS, which may represent new serotypes.

Fundação para a Ciência e a Tecnologia - Doctoral Project (SFRH/BD/32513/2006)