Antimicrobial resistance is rising as a major threat to public health and food safety, prompting evaluation of alternative biologically derived agents. One promising approach uses postbiotics derived from lactic acid bacteria, notably Lactobacillus cell-free supernatants (CFS). CFS are non-viable secreted mixtures that can include organic acids, proteins and peptides, hydrogen peroxide, and bacteriocins, and their combined activities may reduce the likelihood of resistance emergence compared with single-agent antimicrobials.
This study evaluated the antimicrobial potential of CFS produced by 30 Lactobacillus strains isolated from vaginal microbiota. The antimicrobial panel comprised 15 bacterial and yeast pathogens, which included multidrug-resistant clinical isolates. The experimental strategy combined phenotypic screening, determination of inhibitory and bactericidal/fungicidal concentrations, viability assays, mechanistic perturbations, cytotoxicity testing, and exploratory chemical characterization.
A species-independent pattern of activity was observed: CFS activity did not strictly follow species identity, and strains of the same Lactobacillus species could produce either effective or ineffective supernatants. From the 30 CFS tested, the authors classified 10 as effective and 5 as ineffective; details for the remaining CFS classifications were reported in the full manuscript. Effective CFS consistently produced lower MIC and MBC/MFC values relative to ineffective ones.
The predominant inhibitory and killing concentrations for effective CFS were reported as 20 mg/mL for MIC and MBC/MFC in many cases. Treatment with effective CFS caused substantial reductions in viable pathogen counts, typically in the range of 2–4 log (CFU/mL). Fluorescence microscopy-based viability assessments indicated residual viable cell densities after treatment on the order of 10^4 to 10^5 cells/cm2. These quantitative outcomes support broad-spectrum bactericidal and fungicidal potential for selected Lactobacillus-derived CFS against both susceptible and multidrug-resistant targets.
To examine contributors to antimicrobial activity, the study applied pH neutralization and enzymatic treatments to CFS preparations. Neutralizing CFS pH and treating with proteolytic enzymes produced a partial loss of antimicrobial activity, indicating that both acidic metabolites (for example, organic acids) and non-acidic components (for example, peptides or bacteriocins) participate in the observed effects. The partial rather than complete loss implies a multifactorial mechanism in which complementary metabolites act together.
CFS safety for mammalian cells was explored using two cell lines. Exposure of HepG2 cells (human hepatocyte-derived) to CFS did not produce a significant reduction in cell viability. In contrast, RAW 264.7 macrophages displayed a reduced MTT signal after exposure. The authors interpret the RAW 264.7 finding cautiously, noting it could reflect altered cellular metabolic activity or increased sensitivity rather than definitive cytotoxicity. No evidence of overt HepG2 toxicity was reported in the source material.
The authors report that Lactobacillus CFS are chemically diverse and that this complexity likely underlies variable activity across strains and isolates. Because activity was strain-dependent and driven by multiple metabolite classes, the authors recommend prioritizing effective CFS for detailed chemical characterization to identify active components and define structure–function relationships. Such chemical and mechanistic follow-up would inform translational development of CFS-based antimicrobials for applications in clinical or food-safety contexts.
In summary, selected Lactobacillus cell-free supernatants demonstrated broad-spectrum antimicrobial activity against a panel of 15 pathogens, including multidrug-resistant isolates, with many effective CFS exhibiting MIC/MBC/MFC values around 20 mg/mL and producing 2–4 log reductions in viability. Mechanistic experiments indicate contributions from both acidic and non-acidic metabolites. HepG2 cytotoxicity was not observed, while an altered MTT signal in RAW 264.7 macrophages warrants cautious interpretation. The authors conclude that Lactobacillus-derived CFS merit further chemical characterization and translational evaluation. Specific experimental parameters, full datasets, and detailed chemical profiling are provided in the original manuscript and its supporting information; any additional specifics not summarized here were not reported in the excerpt provided.