Effect of vaginal lactobacilli on the growth, adhesion and internalization of uropathogenic Escherichia coli in hela cells

Abstract

Escherichia coli is one of the main causes of uncomplicated urinary tract infections and also responsible of vaginal infections. Lactobacilli can inhibit this pathogen by the production of antimicrobial substances as organic acids, hydrogen peroxide and/or bacteriocins. The aims of this work were to study the effects of beneficial vaginal lactobacilli on the growth of E. coli, and also to determine their ability to inhibit E. coli adhesion and internalization on epithelial cells. Three vaginal Lactobacillus strains isolated from human vagina and previously selected for their beneficial characteristics were used. E. coli strains were isolated from human urine samples in Tucumán, Argentina, or from the kidney of an experimentally intraurethral challenged BALB/c mouse, and identified by standard phenotypic and genotypic techniques. The inhibitory activity of the three vaginal Lactobacillus strains on the E. coli growth was assessed by the plate diffusion agar, with the pathogen in the agar plate, and holes containing Lactobacillus supernatant aliquots (untreated, neutralized with NaOH or neutralized and treated with catalase) to determine the nature of the inhibition. Also, the effect of Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 on the adhesion and internalization capabilities of E. coli was studied on HeLa cells. E. coli 275 suspended in Dulbecco?s modified Eagle?s medium at 2.5 x 107 CFU/ml (colony forming unit) to achieve a 100:1 multiplicity of infection (MOI: ratio of E. coli cells to HeLa cells) were used. Adhesion assays with L. reuteri CRL 1324 were performed by adding 1 ml of L. reuteri at 1 x 108 CFU/ml or 2.5 x 108 CFU/ml to achieve a 400:1 or 2,000:1 ratio of interaction (ROI: ratio of L. reuteri cells to HeLa cells). For the inhibition of adhesion assays, HeLa cells were incubated with L. reuteri CRL 1324 at a ROI of 400:1 or 2,000:1 and E. coli 275 (MOI 100:1) for 1 h at 37°C with 5% CO2. For the inhibition of internalization assays, L. reuteri CRL 1324 and E. coli were simultaneously added to the HeLa cells for 2 h. The results showed that two Lactobacillus strains inhibit the growth of the pathogens by production of organic acids. The adhesion abilities of lactobacilli to HeLa cells were significantly higher at a ROI of 2,000:1 than at a ROI of 400:1. L. reuteri CRL 1324 exhibited poor internalization capabilities into HeLa cells at the two ROI assayed. On the other hand, L. reuteri at a ROI of 2,000:1 showed to reduce the adhesion rate of E. coli at a MOI of 100:1, decreasing 30% the adhesion of E. coli 275. L. reuteri CRL 1324 with a ROI of 400:1 and 2,000:1 led to a significant decrease of E. coli internalization rates by 51% to 57%, respectively. The results obtained suggest that L. reuteri CRL 1324 can be considered as a probiotic candidate for the prevention or treatment of urinary tract infections caused by E. coli.