Isolation and Preliminary In Vitro Characterization of a Honey-Derived Bacillus licheniformis Isolate

Background/Objectives: Despite its hostile physicochemical environment characterized by high osmotic pressure, acidic pH, and low water activity, honey serves as a unique ecological niche for resilient microorganisms. This study aimed to recover bacterial isolates from honeys of different botanical origins and to characterize the probiotic-associated properties of a honey-derived Bacillus licheniformis isolate. Methods: A total of 20 bacterial isolates were recovered from four honey samples, including two blossoms and two honeydew honeys. 16S rRNA gene sequence analysis showed that the recovered isolates had taxonomic assignments consistent with Bacillus licheniformis. Because 16S rRNA sequencing does not provide sufficient resolution to establish strain-level diversity or clonality, one isolate, designated Blhoney, was selected for subsequent molecular and functional characterization. Results: In vitro assays revealed that Blhoney exhibits robust tolerance to simulated gastrointestinal stress, including extreme acidity (pH 1–3), bile salts (0.3–1%), pepsin, and pancreatin, maintaining viability for up to 24 h. Phenotypic antimicrobial testing demonstrated measurable inhibition zones for erythromycin, kanamycin, chloramphenicol, ampicillin, tetracycline, gentamicin, streptomycin, and spectinomycin, whereas no measurable inhibition zones were observed for penicillin G, rifampicin, ciprofloxacin, or ceftazidime. However, MIC determination and standardized breakpoint interpretation were not performed; therefore, these findings were interpreted solely as preliminary phenotypic inhibition-zone observations. Although the isolate did not display direct antagonistic activity against the tested bacterial pathogens under experimental conditions, its persistence under the tested gastrointestinal stress conditions supports further investigation of its probiotic-associated characteristics. Conclusions: Overall, these findings indicate that the honey-derived Blhoney isolate exhibits preliminary probiotic-associated characteristics under the tested in vitro conditions. However, further strain-level genomic and safety characterization is required before its potential probiotic application can be established.