Biosurfactant derived from probiotic Lactobacillus acidophilus exhibits broad-spectrum antibiofilm activity and inhibits the quorum sensing-regulated virulence

Abstract

Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophilus was tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.

Figure 1.

Characterization of L. acidophilus-derived biosurfactant via Fourier-transform infrared spectroscopy (FTIR) analysis.

Figure 2.

Antibacterial activity of L. acidophilus-derived biosurfactant against different Gram-negative pathogenic bacteria. Values are denoted as the mean ± SD of three independent experiments.

Figure 3.

Antibiofilm and EPS inhibition activity of L. acidophilus-derived biosurfactant against different Gram-negative pathogenic bacteria. (A) Quantitative inhibition of biofilm production analysis using L. acidophilus-derived biosurfactant; (B) Quantitative inhibition of EPS production analysis using L. acidophilus-derived biosurfactant. Values are denoted as the mean ± SD of three independent experiments. MIC: Minimum inhibitory concentration; EPS: Exopolysaccharides.

Figure 4.

Illustrative light micrograph of a biofilm showing the effects of L. acidophilus-derived biosurfactant at its highest sub-MICs. (A) Control of P. aeruginosa; (B) Treatment of P. aeruginosa with 1/2 MIC; (C) Control of C. violaceum; (D) Treatment of C. violaceum with 1/2 MIC; (E) Control of S. marcescens; (F) Treatment of S. marcescens with 1/2 MIC. MIC: Minimum inhibitory concentration.

Figure 5.

Anti-QS activity of L. acidophilus-derived biosurfactant against C. violaceum and S. marcescens. (A) Quantitative inhibition of violacein analysis in C. violaceum using L. acidophilus-derived biosurfactant; (B) Quantitative inhibition of prodigiosin analysis in S. marcescens using L. acidophilus-derived biosurfactant. Values are denoted as the mean ± SD of three independent experiments. QS: Quorum sensing; MIC: Minimum inhibitory concentration.

Figure 6.

Anti-QS activity of L. acidophilus-derived biosurfactant against P. aeruginosa. (A) Quantitative inhibition of pyocyanin production analysis in P. aeruginosa using L. acidophilus-derived biosurfactant; (B) Quantitative inhibition of LasA protease production analysis in P. aeruginosa using L. acidophilus-derived biosurfactant against P. aeruginosa. Values are denoted as the mean ± SD of three independent experiments. QS: Quorum sensing; MIC: Minimum inhibitory concentration.

Figure 7.

Anti-QS activity of L. acidophilus-derived biosurfactant against P. aeruginosa. (A) Quantitative inhibition of LasB protease production analysis in P. aeruginosa using L. acidophilus-derived biosurfactant; (B) Quantitative inhibition of total bacterial protease production analysis in P. aeruginosa using L. acidophilus-derived biosurfactant. Values are denoted as the mean ± SD of three independent experiments. QS: Quorum sensing; MIC: Minimum inhibitory concentration.

Figure 8.

Swimming motility inhibition of P. aeruginosa and S. marcescens by L. acidophilus-derived biosurfactant. (A) Control of P. aeruginosa; (B) Treatment of P. aeruginosa with 1/2 MIC; (C) Control of S. marcescens; (D) Treatment of S. marcescens with 1/2 MIC. MIC: Minimum inhibitory concentration.

Figure 9.

Gas chromatography–mass spectrometry-based fatty acids profiles of biosurfactant derived from L. acidophilus.

Figure 10.

Binding affinities of top-rated pose of ligand-receptor complex.

Figure 11.

(A and B) Visualization of docking analysis of EsaI and 2,3-dihydroxypropyl octadecenoate; (C and D) Visualization of docking analysis of LasI and tetradecane; (E and F) Visualization of docking analysis of LasR and 2,3-dihydroxypropyl octadecanoate.

Figure 12.

(A and B) Visualization of docking analysis of PilY1 and 2,3-dihydroxypropyl octadecenoate; (C and D) Visualization of docking analysis of LasA and 2,3-dihydroxypropyl octadecenoate; (E and F) Visualization of docking analysis of PilT and 2,3-dihydroxypropyl octadecanoate.

Figure 13.

(A and B) Visualization of docking analysis of CViR’ and tetradecane; (C and D) Visualization of docking analysis of CViR and hexadecanoic acid; (E and F) Visualization of docking analysis of PqsR and 2,3-dihydroxypropyl octadecenoate.