Phenyl-Lactic Acid Is an Active Ingredient in Bactericidal Supernatants of Lactobacillus crispatus

Abstract

Lactobacillus crispatus is a well-established probiotic with antimicrobial activity against pathogens across several niches of the human body generally attributed to the production of bacteriostatic molecules, including hydrogen peroxide and lactic acid. Here, we show that the cell-free supernatants of clinical isolates of L. crispatus harbor robust bactericidal activity. We further identify phenyl-lactic acid as a bactericidal compound with properties and a susceptibility range nearly identical to that of the cell-free supernatant. As such, we hypothesize that phenyl-lactic acid is a key active ingredient in L. crispatus supernatant. IMPORTANCE Although Lactobacillus crispatus is an established commensal microbe frequently used in probiotics, its protective role in the bladder microbiome has not been clarified. We report here that some urinary isolates of L. crispatus exhibit bactericidal activity, primarily due to its ability to excrete phenyl-lactic acid into its environment. Both cell-free supernatants of L. crispatus isolates and phenyl-lactic acid exhibit bactericidal activity against a wide range of pathogens, including several that are resistant to multiple antibiotics.

Keywords: Lactobacillus; crispatus; microbiome; phenyl-lactic acid; probiotics.

FIG 1

L. crispatus has robustly bactericidal CFS. (A) Lawns of UPEC strain NU14, grown overnight on TSA plates, were spotted with 10 μl of 48-h cultures of L. crispatus or L. gasseri and then photographed to compare zones of clearance. (B) CFS collected from 48-h L. crispatus culture was incubated in an equal volume of an overnight culture of UPEC strain CFT073 (normalized prior to an OD₆₀₀ of 1.0 in tryptic soy broth); this mixture was incubated for the listed x axis time periods (e.g., 2 h, CFS and UPEC incubated together for 2 h), plated onto TSA plates, and incubated at 37°C/aerobic conditions for 24 h, and the CFU per milliliter was enumerated. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay. (C) Shown are 13,000× transmission electron microscopy (TEM) analyses comparing CFT073 E. coli that has been incubated with either MRS medium control (left) or L. crispatus CFS (right) for 24 h. (D) Physiological concentrations of common antimicrobials were quantified and added separately or in combination to CFT073 E. coli, and the impact on viability was assessed at 24 h compared to that of L. crispatus CFS and an MRS medium control. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay.

FIG 2

Analytical chemistry of the bactericidal CFS of L. crispatus. (A) Prior to analytical chemistry, the bactericidal activities of multiple CFS batches from different L. crispatus isolates were tested against E. coli K-12. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay. (B) After ethyl acetate (EtOAc) treatment and chromatography of CFS of L. crispatus isolate LC9873, the resultant A to P fractions were tested for killing activity against E. coli K-12. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay. (C) The chemical diversity of LC9873-H subfractions generated via HPLC represented via chromatogram. (D) Following subfractionation via HPLC, LC9873-H subfractions were tested for killing against E. coli K-12, where they varied in bactericidal activity. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay. (E) Upon further LC9873-H fractionation and killing activity testing, “subfraction subsets” were identified and analyzed further. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay. (F) Comparative LC-MS of a specific LC9873 subfraction (HH1B) compared to that of CFS with strong killing (LC9873, THLC40) and weak killing (OARLC40, LC20). One specific spectrum feature corresponding to a compound with an m/z ratio of 371 (boxed) was present at very high concentrations in subfraction HH1B; it also was present at high concentrations in the strong bactericidal “active” CFS (red spectra, LC9873 and TH-LC40) relative to the weak killing “inactive” CFS (blue spectra, OARLC40 and LC20). This molecule was identified as a candidate bactericidal agent and the subfraction analyzed further.

FIG 3

Phenyl-lactic acid (PLA) is a robust killing agent in the CFS of L. crispatus. (A) H-NMR spectroscopy comparing the profiles of LC9873-HH1B subfraction, PLA-L, and PLA-D. The peaks at δ_H 3.34 represent trace amounts of MeOH in the HH1B subfraction. The peak at δ_H 4.89 corresponds to water. (B) A chemical standard identical to LC9873-HH1B subfraction (and its methylated analogue MR) were solubilized in a 10% DMSO-90% MRS mixture and tested for killing activity against E. coli K-12. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay.

FIG 4

PLA and CFS are highly similar in properties and sensitivities. (A) LC9873 CFS (starting pH 3.7), PLA-L (starting pH 4.2), and methyl-PLA-L (starting pH 5.8) were pH modified and tested for bactericidal activity against E. coli K-12, relative to pH-matched medium controls. MRS (de Man, Rogosa, and Sharpe) liquid medium is the medium control; it results in no reduction in E. coli survival. The dotted line represents the limit of detection (1,000 CFU/ml) for the bactericidal activity assay; this is also the case for subpanels B to D. (B) PLA-L and PLA-D solutions of various concentrations were made by dissolving purified stocks in 10% DMSO-90% MRS mixture and tested for killing against E. coli K-12. (C) LC9873 CFS (left) and PLA-L (right) were incubated for 1 h at either 4°C or 100°C and then stored on benchtop for 24 h or 2 weeks before being tested for killing activity against E. coli CFT073 or E. coli K-12. (D) PLA-L was stored on benchtop for 2 weeks (OLD) or 24 h (NEW) before being tested for killing activity against E. coli K-12.