Synergistic anti-Helicobacter pylori efficacy of a molecularly identified Limosilactobacillus fermentum isolate in...

Helicobacter pylori (H. pylori) is a Gram-negative gastric pathogen resistant to the acidic stomach environment through urease-mediated neutralization, enabling colonization of the gastric mucosa. It is a major cause of gastritis, peptic ulcer disease, and gastric cancer, and was classified as a Class I carcinogen by the International Agency for Research on Cancer (IARC) in 1994. Rising antibiotic resistance has limited the efficacy of conventional therapies, highlighting the need for alternative or adjunct antimicrobial strategies.

Forty lactic acid bacteria (LAB) isolates were screened for anti-H. pylori activity against twenty clinical H. pylori isolates previously recovered from gastric biopsies of Egyptian patients using neutralized cell-free supernatants (CFSs). The CFSs were evaluated alone and in combination with standard therapeutic antibiotics. The most potent CFS was subsequently evaluated for cytotoxic activity against colorectal adenocarcinoma (Caco-2) cells as a preliminary assessment of its potential anticancer-related properties. The CFS was then subjected to fast protein liquid chromatography (FPLC), and the active fraction was further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate its molecular weight. Following verification of its probiotic characteristics, the producer strain was identified by 16 S rRNA gene sequencing.

Nine out of forty LAB isolates demonstrated anti-H. pylori activity, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging from 93.7 to 750 µg/mL. Combination assays demonstrated isolate-dependent interactions, with synergistic effects observed with clarithromycin (CLR), amoxicillin (AX), metronidazole (MET), tetracycline (TE), rifampicin (RA), and levofloxacin (LEV) against certain H. pylori isolate groups. L. fermentum strain M98, identified as the most potent isolate, exhibited the highest inhibitory activity both alone and in combination with antibiotics. The protein-enriched precipitate containing a putative bacteriocin exhibited cytotoxic activity against Caco-2 cells, with an IC₅₀ value of 19.73 ± 0.16 µg/mL. FPLC of its CFS yielded 22 protein fractions, three of which exhibited anti-H. pylori activity, suggesting the presence of bacteriocin-like compounds. Subsequent SDS-PAGE analysis of the active fraction revealed a prominent protein band of approximately 34 kDa.

The CFS of L. fermentum strain M98 exhibits significant in vitro anti-H. pylori activity and enhances the efficacy of tested antibiotics, supporting its potential as an adjunct therapeutic agent against antibiotic-resistant H. pylori. In addition, LAB-precipitated proteins containing a putative bacteriocin demonstrated cytotoxic activity against Caco-2 cells, suggesting the presence of bioactive compounds with potential therapeutic value. Further in vivo investigations are warranted to confirm their safety, efficacy, and potential biomedical applications.