Lactobacillus species inhibit the growth and biofilm development by wound's bacterial pathogens
Chronic wounds are characterized as a wound that fails to heal by three months. Microbial colonization of chronic wounds may be responsible for the delayed healing. The most common wound pathogens include: Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is inherently resistant to numerous antibiotics. The multidrug resistance of P. aeruginosa combined with the high cost of producing new antibiotics necessitates the search for other potential therapies to help heal chronic wounds. Lactobacilli, which are the major components in probiotic products, protect the vaginal environment from potentially harmful microorganisms through various mechanisms. Examples of the mechanisms through which lactobacilli keep women healthy include lowering the pH of the environment, secretion of lactic acid, modulation of host cytokines, and production of bacteriocins. The lactobacilli that are most commonly isolated from the vagina are L. gasseri, L. plantarum, and L. crispatus. Based upon known interactions of lactobacilli and other disease-causing bacteria, I hypothesize that certain Lactobacillus strains produce a potential factor(s) that inhibit the growth and/or biofilm development of P. aeruginosa strains. This study aims to provide a potential alternative to treat P. aeruginosa infections through L. gasseri secreted products. Using in-vitro biofilm models, I showed that Lactobacillus gasseri inhibits biofilm development by P. aeruginosa strain PAO1. Zone of inhibition assays showed that 20X-concentrated cell-free supernatant of L. gasseri inhibited the growth of PAO1. I confirmed these results using a quantitative broth assay. I failed to detect colony-forming units (CFU/mL) of PAO1 when the organism was grown in Luria-Bertani broth containing L. gasseri concentrated supernatant. I utilized PAO1/pMP7605, which contains the plasmid pMP7605 that encodes for the red fluorescence protein, to visualize biofilm development. Using confocal microscopy, L. gasseri supernatant was successful in reducing the thickness and growth of PAO1/pMP7605 biofilms as compared to the control. pH alone was not responsible for PAO1 inhibition. While L. gasseri lowered the pH of de Man, Rogosa, and Sharpe (MRS) broth to 3, when MRS broth was adjusted to pH 3 with hydrochloric acid, the lower pH alone had less effect on PAO1 growth as compared to L. gasseri concentrated supernatant. Furthermore, the exact concentration of D- and L-lactic acid that L. gasseri secretes was determined to better understand the ability of L. gasseri to control Pseudomonas biofilm formation. MRS broth was acidified with the same amount of D- and L-lactic acid within the L. gasseri supernatant. The acidified medium reduced PAO1 growth by only 102 CFU/mL confirming that acid alone does not cause the complete inhibition of PAO1 growth. The effect of concentrated supernatant of L. gasseri on the P. aeruginosa strain PA14 and other cystic fibrosis clinical isolates was evaluated and inhibited the growth of all tested P. aeruginosa strains. Conversely, the effects of the concentrated supernatant of L. plantarum and L. crispatus, on PAO1 growth was variable suggesting that the observed inhibition of PAO1 growth is exclusive for L. gasseri. These results confirm that L. gasseri produces an extracellular antimicrobial factor that inhibits P. aeruginosa PAO1 growth and could be a potential candidate for application to chronic wounds.