In vitro anti-bacterial and anti-adherence effects of Lactobacillus delbrueckii subsp bulgaricus on Escherichia coli

Abstract

Considering the emergence of antibiotic resistance, scientists are interested in using new antimicrobial agents in the treatment of infectious diseases including infections of the enteric systems. Lactic acid bacteria have the great potential to produce antimicrobial compounds that inhibit and control pathogenic bacteria. The aim of this study was to determine the anti-bacterial and anti-adherence properties of Lactobacillus delbrueckii subsp bulgaricus against Escherichia coli. The antibacterial activity of L. delbrueckii was investigated using disc diffusion and spot on lawn methods. In vitro anti-adhesion effect of L. delbrueckii against E. coli was examined using Caco-2 cells. In anti-adhesion assay, three competition conditions including competitive inhibition, adhesion inhibition, and displacement were examined. In spot on lawn method the zone of growth inhibition of E. coli by L. delbrueckii was 21.1 mm. The cell free supernatant of L. delbrueckii showed a good antibacterial activity against E. coli which was mainly related to lactic acid produced by L. delbrueckii. When two bacteria added simultaneously (competitive inhibition) degree of inhibition of E. coli binding by L. delbrueckii was 77%. In adhesion inhibition assay, L. delbrueckii was able to exclude E. coli adherence by around 43.5%. Displacement assay showed that L. delbrueckii had strong displacement ability toward E. coli and reduction of E. coli attachment by bound L. delbrueckii was 81.3%. The results suggest that L. delbrueckii may be able to inhibit E. coli infection in the gut; however more studies including in vivo studies need to be performed.

Keywords: Anti-adherence; Anti-bacterial; Escherichia coli; Lactobacillus delbrueckii.

Fig. 1

Evaluation of antibacterial activity using disk diffusion method. 1: positive control (cephalexin); 2: negative control (PBS); 3: cell free supernatant (CFS) without any treatment; 4: MRS broth whose pH was adjusted to the pH values normally reached by each L. delbrueckii (pH of CFS) by adding enough lactic acid; 5: CFS whose pH was adjusted to 6.5 using 0.1 M NaOH; and 6 : MRS broth.

Fig. 2

Adhesion of L. delbrueckii to Caco-2 cells observed using light microscopy after Gram-staining

Fig. 3

The effect of added bacteria (CFU/ml) on the number of adhered L. delbrueckii to Caco-2 cells. (n=3)

Fig. 4

Adhesion of L. delbrueckii(a) and E. coli(b) to Caco-2 cells. The Caco-2 cell was incubated with DMEM medium containing 0.5×105 CFU/ml of bacteria. L: L. delbrueckiialone; E: E. colialone; L+E: L. delbrueckii and E. coli added simultaneously; L/E: E. coli added after L. delbrueckii; and E/L: L. delbrueckii added after E. coli. Asterisk (FNx01) indicates the means which were significantly different (P<0.05) from the control (Adhesion of L. delbrueckii alone (L) was considered as a control for adhesion assay L. delbrueckii and adhesion of E. coli alone (E) was considered as a control for adhesion assay E. coli. (n=9)