Cultured-dependent and cultured-independent study of bacteria associated with Thai commercial stingless bee Lepidotrigona terminata

Abstract

This study aimed to investigate the lactic acid bacteria in stingless bees (Lepidotrigona terminata). Stingless bees were randomly collected from five different nests in Chiang Mai University, identified via the COI gene, and compared to those in the NCBI database. The result showed that all samples were L. terminata. Bees were extracted and subjected to 10-fold dilution to screen on MRS agar to estimate the number of lactic acid bacteria (LAB) cells in the samples. The colony forming unit (CFU) was between 1.28 x 10 6 ± 0.02 and 1.37 x 10 6 ± 0.08 CFU/g. Fifty isolates were chosen to perform an agar well assay to quantify the capacity of inhibiting against Paenibacillus larvae (strain LMG9820), a honey bee pathogen that causes American foulbrood. The results showed that bacteria from two nests (selected from those of the five nests) could inhibit the growth of P. larvae with the mean of a clear zone of 12.15 ± 0.2 mm. On the other hand, bacteria from the other three nests could not inhibit the pathogen. Identification of the effective bacteria species using 16S rRNA and the construction of the phylogenetic tree revealed that they were Fructobacillus fructosus of the Bacillus group. The cultured-independent result showed that Betaproteobacteria was the most abundant group (41.6%) of bacteria in stingless bees while Actinobacteria was the lowest in abundance (0.6%). Further experiments will need to be performed to examine the functions of these microorganisms in stingless bees.