Caracterização genotípica e fenotípica de isolados de Escherichia coli oriundos de animais de produção

Abstract

Escherichia coli is a component of intestinal microbiota of animals and humans, with ability to colonize different niches, causing enteric and extra-enteric infections. Presents genetic plasticity through by acquisition and loss of genes, resulting in genotypic and phenotypic differences among them. Genotypic and phenotypic characterization of E. coli isolates from farm animals (cattle, sheep, horses and broilers) was performed. The genotypic analysis was composed by phylogenetic classification; search for virulence factors – fimbriae (F4, F5, F6, F18 and F41) and toxins (LT, STa, STb and STx2), curli (csgA) and cellulose (bcsA) – from isolates associated with bovine mastitis and feces of calves with and without diarrhea; besides the research of 29 genes related to the virulence of APEC pathotype (avian pathogenic E. coli) on two extraintestinal E. coli isolates from an avian colisepticemia outbreak. Phenotypic analysis was performed by curli and cellulose expression and biofilm formation of E. coli isolates from bovine mastitis and feces of calves with and without diarrhea; and antimicrobial susceptibility testing in all isolates, with evaluation of extended-spectrum beta-lactamase (ESBL) in isolates from bovine mastitis. The majority of E. coli isolates belonged commensal phylo-groups: (i) bovine mastitis: A (50%), B1 (30%), B2 (3.3%), C (6.7%), D (3.3%) and F (6.7%); (ii) feces of calves without diarrhea: B1 (83.3%), E (10%) and unknown (6.7%); (iii) feces of diarrheic calves: B1 (70%), B2 (3.33%), C (3.33%), D (3.33%), E (13.33%) and unknown (6.7%); (iv) E. coli subpopulations of calves without diarrhea: B1 (93.3%), B2 (20%) and E (46.7%); (v) E. coli subpopulations of diarrheic calves: A (6.7%), B1 (80%), D (33.3%) and E (40%); (vi) feces of foals: A (4.62%), B1 (50.77%), C (15.38%), E (21.54%), F (1.54%) and unknown (6.15%); (vii) feces of lambs: A (6.15%), B1 (81.54%), C (10.77%) and E (1.54%); (viii) extraintestinal E. coli isolates: A (100%). No isolates from bovine mastitis were positive for fimbriae and toxins, unrelated to intestinal clinical isolates. E. coli isolates from calves feces presented variable results: (i) without diarrhea: STb (66.7%), LT (56.7%) and STa (33.3%) toxins and no fimbriae; (ii) diarrheic: LT (23.3%), STa (3.3%), STb (3.3%) and STx2 (3.3%) toxins and F5 (16.7 %) and F18 (13.3%) fimbriae. The frequencies of csgA and bcsA genes were: (i) E. coli isolates from bovine mastitis: 48% and 34%, respectively; (ii) isolates from feces of calves without diarrhea: 96.7% and 83.3%, respectively; and (iii) isolates associated with diarrhea: 90% for both genes. The extraintestinal E. coli isolates from avian colisepticemia outbreak, presented several virulence factors important for Avian Pathogenic E. coli (APEC) related to adhesion (fimH, papC, mat), invasion (tia), iron acquisition system (iroN) and serum resistance (iss, ompA, traT), but divergence in others (adhesin: hrlA/hek; invasin: gimB; iron acquisition system: sitD ep. and fyuA; toxins: sat and hylA) characterizing an coinfection. In vitro biofilm formation was observed in 10% of the isolates from bovine mastitis, and curli and cellulose expression was verified in these isolates. Among the isolates from calves without diarrhea, curli and cellulose was expressed in 83.3%, and among isolates of diarrheal calves, there was 100% expression. Biofilm formation was verified in a single isolate from calves without diarrhea. Host factors may be to influence gene expression important for this mechanism. Antimicrobial resistance was observed in isolates from all animal species of this study, with emphasis to bovine mastitis isolates, which showed greater resistance to tetracycline (46%) and ampicillin (40%), with 24% and 10% multidrug resistant and ESBL isolates, respectively; and isolates from diarrhea in calves, with greater resistance to tetracycline (63,3%), ampicillin (50%), streptomycin (50%) and sulfonamide (46,7%), with 53,3% multidrug resistant isolates. The resistance rates of E. coli isolates in the present study following global trends, associated with the careless use of antimicrobials in animal production.