Host-specific Lactobacillus and Bifidobacterium species constitute the core microbiota of the honey bee digestive tract and are recognized for their probiotic properties. One of the properties of these bacteria is the inhibition of bacterial pathogens such as Paenibacillus larvae and Melissococcus plutonius, the causative agents of American and European foulbrood, respectively. Additionally, Serratia marcescens has emerged as a relevant opportunistic pathogen. Although several previously published studies have examined the inhibition of selected bacterial pathogens of bees by members of the bee physiological microbiota, none have simultaneously investigated the inhibition of multiple clinical isolates of P. larvae, M. plutonius, and S. marcescens using a wide range of bifidobacterial and lactobacilli strains isolated from various locations within a single country. Thus, this study evaluated the antimicrobial potential of Lactobacillus and Bifidobacterium strains against these pathogens, with a focus on strain-dependent inhibition. A total of 111 bacterial strains (62 Lactobacillus and 49 Bifidobacterium) were isolated from the digestive tracts of honey bees collected from eight sites across the Czech Republic. Using 16S rRNA gene sequencing, the isolates were classified and tested in vitro against four P. larvae isolates, one M. plutonius isolate, and the S. marcescens strain sicaria in modified BHI medium. Twenty-eight strains (~26%) exhibited strong inhibition (≥21 mm) against at least two P. larvae isolates, while 12 strains showed moderate inhibition (16-20 mm) against all four isolates. Inhibition of M. plutonius and S. marcescens was observed in three and twenty strains, respectively. The most effective strains belonged to Bifidobacterium asteroides, B. choladohabitans, B. polysaccharolyticum, Lactobacillus apis, L. helsingborgensis, L. kullabergensis, and L. melliventris. These results underscore the strain-dependent nature of antimicrobial activity and highlight the importance of selecting probiotic strains with broad-spectrum pathogen inhibition to support honey bee health.

• Keywords
• Melissococcus plutonius, Paenibacillus larvae, Serratia marcescens strain sicaria, bifidobacteria, honey bee probiotics, in vitro inhibition, lactobacilli,
• Publication type
• Journal Article MeSH

The order Lactobacillales represents a morphologically, metabolically, and physiologically diverse group of bacteria. Lactic acid bacteria represent the core of this phylogenetic group. They are a part of epiphytic microflora, fermented dairy, meat, fruit and vegetable products, and the digestive tract of humans and animals. Despite the fact that these bacteria form a phenotypically and genotypically heterogeneous group, their phylogenetic relationship enables to propose a common genetic marker usable in classification, typing, and phylogeny. By creation of consensus sequence based on available genomic sequences of some representatives of order Lactobacillales, a specific primer-pair binding variable region of aspS gene (length of 615 nts) encoding the aspartyl-tRNA synthetase was designed. This gene has not yet been used in classification and phylogeny of the order Lactobacillales, although it meets the requirements of molecular markers (distribution and single copy in bacterial genomes, functional constancy and genetic stability, sequence variability among taxonomic units, irreplaceable role in proteosynthesis). Primers were applied on 54 type and wild Lactobacillales strains. Obtained sequences allowed to provide alignments for purpose of phylogenetic tree reconstructions that uncovered particular phylogenetic clusters of vagococci/enterococci, obligately homofermentative and heterofermentative lactobacilli. Although a relatively short fragment of the aspS gene (approximately 33% of the complete gene sequence) was evaluated, much higher sequence variability (61.8% of pairwise identity) among strains examined compared with 16S rRNA gene (90.7%, length of 1318 nt) provides a relatively simple and effective tool for classification and typing of selected representatives of the order Lactobacillales.

• MeSH
• Aspartate-tRNA Ligase genetics   MeSH
• Genes, Bacterial MeSH
• Bacterial Proteins genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Genes, Essential MeSH
• Phylogeny * MeSH
• Genetic Markers MeSH
• Lactobacillales classification   enzymology   genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Aspartate-tRNA Ligase MeSH
• Bacterial Proteins MeSH
• DNA, Bacterial MeSH
• Genetic Markers MeSH
• RNA, Ribosomal, 16S MeSH

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.

• MeSH
• Bacteria classification   genetics   isolation & purification   MeSH
• Denaturing Gradient Gel Electrophoresis MeSH
• DNA, Bacterial genetics   MeSH
• Ecosystem MeSH
• Gastrointestinal Tract microbiology   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Lactobacillaceae genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome * MeSH
• Bees embryology   growth & development   microbiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH
• RNA, Ribosomal, 16S MeSH