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Autochthonous microbial populations from urogenital tract of gilts and pregnant sows. Isolation and characterization of native Escherichia coli
Foz do iguaçu
Congreso; Vii Congreso Internacional de Suinocultura; 2016
Institución organizadora:
Embrapa Suinos e AVES
INTRODUCTIONThe vaginal microbial composition has been widely studied and associated with reproductive tract health and fertility in mammals (9, 13) and has been reported as an equilibrated and dynamic ecosystem which consists of a combination of aerobic, facultative anaerobic, and obligate anaerobic microorganisms. The post-farrowing dis-balances in the microbiota of both genital and urinary tracts affect the reproductive performance of sows (4, 7). Escherichia coli is a common inhabitant of the female genital tract in farms conditions (1, 13). However pathogenic strains often cause many extraintestinal infections including important postpartum diseases and urinary tract infections (UTIs) in sows (4, 7). Uropathogenic E. coli (UPEC) possess characteristic virulence factors, such as hemolysin, cytotoxic necrotizing factors, siderophores and adhesins (P fimbriae, S and F1C fimbriae, and type 1 pili) (10). Nevertheless, there is scarce information about commensal E. coli from the urogenital tract of sows. Thus, the aim of this study was to evaluate some bacterial populations that usually colonize the urogenital tract of gilts and pregnant sows, and to characterize commensal E. coli.MATERIALS AND METHODSSamples: 10 pregnant sows and 10 young gilts, housed in an outdoor hatchery were used for sampling. The vulvar area was washed before inserting into the vagina a stainless steel vaginoscope moistened with a small amount of antiseptic-free lubricant gel (0.8% w/v carbopol, 1% v/v triethanolamine, 10% v/v glycerin, Sigma-Aldrich). Once the vaginoscope was in place, long-handled sterile cytobrushes were disposed to scrape the vaginal wall close to the cervix. Urethral samples were obtained using a stainless steel speculum to access the meatus; sterile cytobrushes were settled and rotated on the urethral wall. Each loaded cytobrush was collected in 1 mL of phosphate buffered saline solution (PBS) pH 7.0 and kept refrigerated until processing. Bacterial isolation and E. coli characterization. All samples were inoculated onto blood agar (AS), LAPTg and Mac Conkey?s agar; while vaginal samples were also cultured on Manitol Salt agar, Bile Esculin, MRS and Sabourau agar. Plates were incubated at 37°C for 24?48 h; also the AS plates were incubated with 5%CO2. For spore-forming isolation, samples-aliquots were previously heated (80°C, 15 min) and plated on LAPTg agar. From MacConkey´s agar, 3 to 4 lactose positive colonies were selected and examined for colony morphology, Gram staining and standard biochemical tests. Indol-positive and citrate-negative isolates were presumptively identified as E. coli and confirmed by PCR detection of the D-glucuronidase gene (uidA [specific E. coli]) (14). All E. coli isolates were evaluated for phylogenetic grouping (A, B1, B2, and D) by a multiplex PCR (2), using three sets of primers (for chuA, yjaA, and the DNA fragment TspE4.C2).E. coli isolates evaluation. The expression of type 1 pili and P fimbriae were performed according to Krag et al. (7), with some modifications: bacterial suspensions were prepared from a single colony (over-night on LB agar) in PBS (OD540nm= 1,2), with 1012 cfu/mL, approximately. a) Mannose-sensitive yeast agglutination for type 1 pili expression: Saccharomyces cerevisiae cells were suspended on PBS and equal aliquots of bacterial and yeast suspensions were mixed on a glass slide. For positive cases, agglutination was also tested with 1% D-mannose. b) haemagglutination assay for P fimbriae expression was performed with type A human erythrocytes (5% RBC). c) Virulence factors related to adherence in extraintestinal pathogenic E. coli (ExPEC): fimH (type1 pili), papC (P fimbriae) and sfa/focDE (F1C fimbriae) genes were examined by PCR (6).RESULTS AND DISCUSSIONSignificant differences in the mean values of total microaerophilic microorganisms were found between urethra and vagina (4.61±0.37 and 3.17±0.37, respectively, Fisher test p