Molecular Identification and In Vitro Probiotic-Associated Characterization of Indigenous Lactic Acid Bacteria from Raw Honey and the Honey Bee (Apis mellifera) Gut in Punjab, Pakistan.

The honey bee (Apis mellifera) and raw honey constitute ecologically distinct, acidic and osmotically stressful niches that harbour autochthonous lactic acid bacteria (LAB) with promising probiotic-associated properties. This study isolated and molecularly characterized Lactobacillus-related isolates from raw honey and honey bee gut samples collected from Chichawatni Reserved Forest, Punjab, Pakistan, and further identified selected strains by 16 S rRNA gene sequencing. From 30 samples (10 raw honey samples and 20 honey bee gut pools, each collected from an independent Apis mellifera colony), 18 presumptive Lactobacillus isolates were recovered on de Man, Rogosa and Sharpe (MRS) agar and confirmed at the genus level by Lactobacillus-specific 16 S rRNA-23 S rRNA intergene amplification. In vitro probiotic-associated properties were evaluated, including tolerance to simulated gastric juice, bile salt tolerance, auto-aggregation, co-aggregation with four reference enteric pathogens, antimicrobial activity of cell-free supernatants (CFS), and antibiotic susceptibility. Honey-derived isolates showed higher values for acid tolerance and auto-aggregation parameters in the evaluated assays than gut derived isolates. Isolate H1 showed the highest survival at pH 2, the highest auto-aggregation, and produced the largest inhibition zones against Escherichia coli, Salmonella enterica serovars Typhimurium and Enteritidis, and Campylobacter jejuni. Neutralization of the CFS to pH 7 abolished the antimicrobial activity, and heat treatment reduced it only partially, a pattern suggesting that organic acids may contribute substantially to the observed antimicrobial activity. All isolates were intrinsically non-susceptible to vancomycin and polymyxin B, which is consistent with the intrinsic resistance profile of the genus. Three isolates showing complementary in vitro probiotic-associated phenotypes (H1, B4 and B13) were tentatively identified by partial 16 S rRNA gene sequencing as best matches to Lactiplantibacillus plantarum, and the sequences were deposited in NCBI GenBank under accession numbers PZ694971 (H1), PZ676443 (B4) and PZ676444 (B13). These isolates are proposed as candidates for further probiotic evaluation, pending multi-locus or whole-genome confirmation of species identity, formal safety testing (haemolytic activity, gelatinase production, biogenic amine synthesis and DNase activity), targeted characterization of the antimicrobial metabolites and in vivo evaluation. These findings identify Chichawatni honey and honey bee gut as an underexplored source of indigenous LAB and highlight H1, B4 and B13 as potential candidates for further probiotic evaluation, pending safety assessment, whole-genome characterization, adhesion assays and in vivo validation.