Characterization of native Escherichia coli populations from bovine vagina of healthy heifers and cows with postpartum uterine disease

Abstract

Even though Escherichia coli are common bacteria of the bovine vaginal microbiota, they represent an important pathogen that causes diseases in the reproductive tract and subfertility. However, the actual endometrial virulence profile of E. coli is poorly understood. The present study aims to characterize the phylogenetic structure and virulence potential of native vaginal populations of E. coli from healthy heifers (H), and cows with postpartum uterine diseases (PUD), such as metritis/endometritis (MT) or repeat breeder cows (RB). To this end, the virulence repertoire of 97 E. coli isolates was genotypically and phenotypically assessed. Most of them were assigned to phylogenetic group A (74%), followed by B1 (17%) and D (9%); RB strains were significantly (p < 0.05) more represented by B1. Seven of the 15 evaluated virulence genes (VFG) were detected and the most prevalent were fimH (87%), agn43 (41%) and csgA (35%); while traT (27%), fyuA (11%), hlyA (5%) and kpsMT II (5%) were observed in a lower proportion. Particularly, fyuA was significantly higher (p < 0.05) in MT cows whereas csgA showed the same behavior in PUD animals (p < 0.05). When comparing H and PUD strains, these last ones were associated to positive expression of biofilm, fimbriae curli/cellulose and motility; yet RB strains did not show motility. Vaginal B1 E. coli populations, that possess VFG (fyuA and csgA) as well as the expression of motility, curli fimbriae/cellulose and biofilm, may represent risk factors for endometrial disorders; specifically, those that also, have kpsMT II may have a pathogenic potential for causing the RB syndrome. Future research focusing on the detection of these strains in the vaginal microbiota of cows with postpartum uterine diseases should be done since the control of their presence in vagina could reduce the risk that they access the uterus during the postpartum period.

Fig 1. Dendrograms generated from fingerprints (ERIC-PCR) of bovine vaginal E. coli.

The degree of similarity (%) between fingerprints is given at the top by the Pearson coefficient. The cut-off level ≥ 90% (red line) defined common clonal origin. Reference of isolates and animal origin are indicated on the right. Colored boxes indicate isolates from the same animal that presented patterns of similarity ≥ 90% have a common clonal origin. H: healthy heifers, PUD: cows with postpartum uterine diseases.

Fig 2. Phylogenetic classification of vaginal E. coli isolated from healthy heifers (H), cows with metritis or endometritis (MT) and repeat breeders (RB).

**: indicate significant differences (p < 0.05 Fisher's exact test).

Fig 3. Fifteen VFG were evaluated in vaginal E. coli from healthy heifers (H), cows with metritis or endometritis (MT) and repeat breeders (RB).

The matrix on the left indicates positive or negative detection; **: indicate significant differences (p < 0.05, Kruskal Wallis test).

Fig 4. VF score of vaginal E. coli from (H) healthy heifers, (MT) cows with metritis or endometritis and (RB) repeat breeders.

* General media, ^# Decision limits. ▀: indicate significant differences (p < 0.0001, ANOM).

Fig 5. Principal component analysis based on the VFG (virulence factor genes) detection in vaginal E. coli isolated from healthy heifers (H), cows with metritis or endometritis (MT) and repeat breeders (RB).

Fig 6

A. Biofilm formation of vaginal E. coli isolates on polystyrene and glass. B. Distribution of biofilm production values on glass: median value = 0.42; quartiles: Q1 = 0.24 and Q3 = 0.82, which define the IQ (interquartile interval) of the values of average capacity of biofilm formation. **: indicate significant differences (p < 0.0001, Student's t-test).

Fig 7. Analysis of correspondence: Biofilm formation, expression of curli fimbriae/cellulose and motility of vaginal E. coli isolated from (H) healthy heifers, (MT) cows with metritis or endometritis and (RB) repeat breeders.

The contribution to Chi-square is indicated in brackets.