A taxonomic study was performed on Gram-stain-positive, catalase-negative and regular rod-shaped bacterial strains R4B^Tand R4C, isolated from the stomachs of honeybees. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strains was within the genus Lactobacillus ; the highest sequence similarity to R4B^Twas shown by Lactobacillus acidophilus BCRC 10695^T(93.6 %). Lower sequence similarities were found to other obligately homofermentative lactobacilli. A PCR–DGGE method could detect the sequence of the 16S rRNA gene of strain R4B^Tat different developmental stages of honeybees occurring in two different locations in the Czech Republic. The distinctiveness of the strains from other lactobacilli was also confirmed by analysis of sequences of other phylogenetic markers applicable to the taxonomy of the genus Lactobacillus , ribotyping and rep-PCR analysis. The DNA G+C content of strain R4B^Twas 41.3 mol%. The predominant cellular fatty acids of strain R4B^Twere C_18 : 1ω9c, summed C_19 : 1ω6c/C_19 : 0cyclo ω10c, C_16 : 0, summed C_18 : 1ω7c/C_18 : 1ω6cand summed C_16 : 1ω7c/C_16 : 1ω6c. The major polar lipids of strain R4B^Twere glycolipids, lipids and phospholipids. Phenotypic and phylogenetic characteristics also confirmed the independent status of the strains at the species level. Interestingly, strain R4B^Twas able to inhibit growthin vitroof Paenibacillus larvaesubsp.larvae (causal agent of American foulbrood in honeybees) and Melissococcus plutonius (causal agent of European foulbrood). The nameLactobacillus apissp. nov. is proposed for this novel taxon; the type strain is R4B^T( = CCM 8403^T = LMG 26964^T).

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.

Members of the Bifidobacteriaceae family represent both dominant microbial groups that colonize the gut of various animals, especially during the suckling stage of their life, while they also occur as pathogenic bacteria of the urogenital tract. The pan-genome of the genus Bifidobacterium has been explored in detail in recent years, though genomics of the Bifidobacteriaceae family has not yet received much attention. Here, a comparative genomic analyses of 67 Bifidobacteriaceae (sub) species including all currently recognized genera of this family, i.e., Aeriscardovia, Alloscardovia, Bifidobacterium, Bombiscardovia, Gardnerella, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia, was performed. Furthermore, in order to include a representative of each of the 67 (currently recognized) (sub) species belonging to the Bifidobacteriaceae family, we sequenced the genomes of an additional 11 species from this family, accomplishing the most extensive comparative genomic analysis performed within this family so far. Phylogenomics-based analyses revealed the deduced evolutionary pathway followed by each member of the Bifidobacteriaceae family, highlighting Aeriscardovia aeriphila LMG 21773 as the deepest branch in the evolutionary tree of this family. Furthermore, functional analyses based on genome content unveil connections between a given member of the family, its carbohydrate utilization abilities and its corresponding host. In this context, bifidobacterial (sub) species isolated from humans and monkeys possess the highest relative number of acquired glycosyl hydrolase-encoding genes, probably in order to enhance their metabolic ability to utilize different carbon sources consumed by the host. Within the Bifidobacteriaceae family, genomics of the genus Bifidobacterium has been extensively investigated. In contrast, very little is known about the genomics of members of the other eight genera of this family. In this study, we decoded the genome sequences of each member of the Bifidobacteriaceae family. Thanks to subsequent comparative genomic and phylogenetic analyses, the deduced pan-genome of this family, as well as the predicted evolutionary development of each taxon belonging to this family was assessed.

Our previous study, based primarily on PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing, focused on the isolation of four bifidobacterial groups from the digestive tract of three bumblebee species. In that study, we proposed that these isolated groups potentially represented novel species of the family Bifidobacteriaceae. One of the four, Bifidobacterium bombi, has been described recently. Strains representing two of the other groups have been classified as members of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA and heat-shock protein 60 (hsp60) gene sequences. Analysis of 16S rRNA gene sequence similarities revealed that the isolates of the first group were affiliated to Bifidobacterium asteroides YIT 11866^T, B. indicum JCM 1302^T and B. coryneforme ATCC 25911^T (96.2, 96.0 and 95.9 % sequence similarity, respectively), together with other bifidobacteria showing lower sequence similarity. Additional representatives of the second group were found to be affiliated to Bifidobacterium minimum YIT 4097^T and B. coryneforme ATCC 25911^T (96.0 and 96.3 % sequence similarity) and also to other bifidobacteria with lower sequence similarity. These results indicate that the isolates of the two groups belong to novel species within the genus Bifidobacterium. This observation was further substantiated by the results of partial sequencing of hsp60. On the basis of phylogenetic and phenotypic analyses and analysis of 16S rRNA and partial hsp60 gene sequences, we propose two novel species, Bifidobacterium actinocoloniiforme sp. nov. (type strain LISLUCIII-P2^T = DSM 22766^T = CCM 7728^T) and Bifidobacterium bohemicum sp. nov. (type strain JEMLUCVIII-4^T = DSM 22767^T = CCM 7729^T).

Gram-positive-staining, anaerobic, non-spore-forming, lactate- and acetate-producing bacterial strains were isolated from the digestive tracts of different bumblebee species (Bombus lucorum, Bombus pascuorum and Bombus lapidarius). All of the isolates produced fructose-6-phosphate phosphoketolase activity. A representative strain, BluCI/TP^T, was characterized further. Cells of strain BluCI/TP^T showed occasional bifurcation and irregular constrictions. The bacterium utilized a wide range of carbohydrates. Glucose was fermented to acetate and lactate. The DNA base composition was 47.2 mol% G+C. Complete 16S rRNA and partial hsp60 gene sequences were obtained and phylogenetic relationships were determined. Strain BluCI/TP^T and related isolates were located in the actinobacterial cluster and were closely related to the genera Bifidobacterium, Scardovia, Aeriscardovia and Parascardovia. The results presented support the proposal of a novel species to accommodate strain BluCI/TP^T, with the name Bifidobacterium bombi sp. nov.; the type strain is BluCI/TP^T (=DSM 19703^T =ATCC BAA-1567^T).