The effect of Lactobacillus reuteri cell free supernatant on growth and biofilm formation of Paenibacillus larvae

Background: Paenibacillus larvae is the etiological agent of American foulbrood (AFB) disease, the most lethal disease in honeybee (Apis mellifera) larvae. Aims: The aim of the present work was to study the antimicrobial effect of cell free supernatant (CFS) of probiotics on an Iranian isolate’s biofilm formation. Methods: A local strain was identified by 16S rRNA sequencing. The antibacterial effect of some probiotics was evaluated through drop plate method, minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and time-kill assay. The biofilm formation ability of P. larvae and the inhibition of biofilm formation by CFS were studied by microplate and scanning electron microscopy (SEM). The nature of the secondary metabolites in CFS was examined by microscale optical density assay (MODA). Results: Alignment of the results of P. larvae KB10 (GenBank accession number MH000685.1) 16S rRNA with the database revealed more than 97% identity with P. larvae. The most antibacterial effect was observed in the CFS of Lactobacillus reuteri ATCC23272 with 12.75 ± 3.2 mm for zone of inhibition (ZOI) at 1000 µL/ml for MIC and MBC. Time-kill assay revealed that CFS eliminated 1.5 × 108 CFU/ml P. larvae KB10 at 2 h of exposure. Microtitre plate and SEM results revealed that CFS (at sub-MIC concentration) was able to inhibit biofilm formation by P. larvae. The results of MODA assay showed that antimicrobial activity were related to the production of organic acids. Conclusion: Cell free supernatant from L. reuteri ATCC 23272 had inhibitory effects on P. larvae KB10 growth and biofilm production due to its acidic nature. The obtained results can be used for antibiotic substitution in AFB control and treatment.