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.

• Klíčová slova
• Melissococcus plutonius, Paenibacillus larvae, Serratia marcescens strain sicaria, bifidobacteria, honey bee probiotics, in vitro inhibition, lactobacilli,
• Publikační typ
• časopisecké články MeSH

The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.

• MeSH
• bakteriální geny MeSH
• Bifidobacterium klasifikace   enzymologie   MeSH
• DNA bakterií genetika   MeSH
• DNA primery MeSH
• fylogeneze * MeSH
• genetické markery MeSH
• genotyp MeSH
• glutaminsynthetasa genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• techniky typizace bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA primery MeSH
• genetické markery MeSH
• glutamine synthetase I MeSH Prohlížeč
• glutaminsynthetasa MeSH
• RNA ribozomální 16S MeSH

In the modern era, molecular genetic techniques are crucial in ecological studies, as well as in the classification, typing, and phylogenetic analysis of prokaryotes. These techniques are mainly aimed at whole genome comparisons and PCR-derived experiments, including amplifying the 16S rRNA and other various housekeeping genes used in taxonomy, as well as MLST (multilocus sequence typing) and MLSA (multilocus sequence analysis) of different taxonomic bacterial groups. The gene encoding threonine-tRNA ligase (thrS) is a gene potentially applicable as an identification and phylogenetic marker in bacteria. It is widely distributed in bacterial genomes and is subject to evolutionary selection pressure due to its important function in protein synthesis. In this study, specific primers were used to amplify a thrS gene fragment (~740 bp) in 36 type and 30 wild strains classified under family Bifidobacteriaceae. The full-length gene has not yet been considered as a possible identification, classification, and phylogenetic marker in bifidobacteria. The thrS sequences revealed higher sequence variability (82.7% of pairwise identities) among members of the family than that shown by 16S rRNA gene sequences (96.0%). Although discrepancies were found between the thrS-derived and previously reported whole genome phylogenetic analyses, the main phylogenetic groups of bifidobacteria were properly assigned. Most wild strains of bifidobacteria were better differentiated based on their thrS sequences than on their 16S rRNA gene identities. Phylogenetic confidence of the evaluated gene with respect to other alternative genetic markers widely used in taxonomy of bifidobacteria (fusA, GroELhsp60, pyrG, and rplB genes) was confirmed using the localized incongruence difference - Templeton analysis.

• Klíčová slova
• Bifidobacterium, classification, genetic marker, phylogenetics, thrS gene,
• MeSH
• bakteriální geny MeSH
• bakteriální proteiny genetika   MeSH
• Bifidobacterium klasifikace   enzymologie   genetika   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze * MeSH
• multilokusová sekvenční typizace MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• techniky typizace bakterií MeSH
• threonin-tRNA-ligasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH
• threonin-tRNA-ligasa MeSH

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P- and K-distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health-promoting probiotic bacteria.

• Klíčová slova
• Bifidobacteriaceae, Bifidobacterium, CTP synthetase, classification, phylogenetics, scardovia,
• MeSH
• Actinobacteria klasifikace   enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• DNA primery genetika   MeSH
• fylogeneze * MeSH
• ligasy tvořící vazby C-N chemie   genetika   metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• techniky typizace bakterií metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• CTP synthetase MeSH Prohlížeč
• DNA bakterií MeSH
• DNA primery MeSH
• ligasy tvořící vazby C-N MeSH
• RNA ribozomální 16S MeSH

Gram-stain-positive, catalase and oxidase-negative and short rod-shaped bacterium C10 with occasional branching was isolated under strictly anaerobic conditions from the rumen fluid of a red deer (Cervus elaphus) in the course of study attempting to uncover new xylanolytic and cellulolytic rumen bacteria inhabiting the digestive tract of wild ruminants in the Czech Republic. The anaerobic M10 medium containing bovine rumen fluid and carboxymethylcellulose as a defined source of organic carbon was used in the process of bacterial isolation. The 16S rRNA gene similarity revealed recently characterized new species Actinomyces succiniciruminis Am4T (GenBank accession number of the gene retrieved from the complete genome: LK995506) and Actinomyces glycerinitolerans G10T (GenBank accession number from the complete genome: NZFQTT01000017) as the closest relatives (99.7 and 99.6% gene pairwise identity, respectively), followed by the Actinomyces ruminicola DSM 27982T (97.2%, in all compared fragment of 41468 pb). Due to the taxonomic affinity of the examined strain to both species A. succiniciruminis and A. glycerinitolerans, its taxonomic status towards these species was evaluated using variable regions of rpsA (length of 519 bp) and rplB (597 bp) gene sequences amplified based on specific primers designed so as to be applicable in differentiation, classification, and phylogeny of Actinomyces species/strains. Comparative analyses using rpsA and rplB showed 98.5 and 97.9% similarities of C10 to A. succiniciruminis, respectively, and 97.5 and 97.6% similarities to A. glycerinitolerans, respectively. Thus, gene identities revealed that the evaluated isolate C10 (=DSM 100236 = LMG 28777) is a little more related to the species A. succiniciruminis isolated from the rumen of a Holstein-Friesian cow than A. glycerinitolerans. Phylogenetic analyses confirmed affinity of strain C10 to both recently characterized species. Unfortunately, they did not allow the bacterial strain to be classified into a particular species. Phenotypic characterization suggested similar conclusions. This brief contribution is aimed at classification and detailed phenotypic characterization of bacterial strain C10 isolated from the rumen of a wild red deer exhibiting, from the point of view of Actinomyces species, noteworthy cellulolytic and xylanolytic activities.

• MeSH
• Actinomyces klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• bachor mikrobiologie   MeSH
• bakteriální geny genetika   MeSH
• celulosa metabolismus   MeSH
• DNA bakterií genetika   MeSH
• fenotyp MeSH
• fylogeneze MeSH
• mastné kyseliny analýza   MeSH
• peptidoglykan analýza   MeSH
• RNA ribozomální 16S genetika   MeSH
• vysoká zvěř mikrobiologie   MeSH
• xylany metabolismus   MeSH
• zastoupení bazí MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• celulosa MeSH
• DNA bakterií MeSH
• mastné kyseliny MeSH
• peptidoglykan MeSH
• RNA ribozomální 16S MeSH
• xylany 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 klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA bakterií genetika   MeSH
• ekosystém MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• Lactobacillaceae genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra * MeSH
• včely embryologie   růst a vývoj   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

Bifidobacteria are considered as one of the key genera in intestinal tracts of animals, and their species composition vary depending on the host. The aim of this study was to identify faecal bifidobacteria from Asian elephants (Elephas maximus), housed in Zoological gardens (Ostrava, Czech Republic). Using culturing, bifidobacteria were found in counts 7.60+/-0.56 log CFU/g. Twenty-six pure strains were isolated from faeces of Asian elephant. The isolates were clustered into two groups according to fingerprinting profiles and fermentation characteristic. Bacteria were identified by a combination of MALDI-TOF MS, PCR methods and sequencing as B. boum (12 isolates) and B. adolescentis (14 isolates). Elephant strains showed different fingerprinting profiles than type and collection strains. Since these two species are frequently isolated from gastrointestinal tract of herbivores, they seem to be typical of animals fed plant diets.

• MeSH
• bakteriální RNA genetika   MeSH
• Bifidobacterium genetika   růst a vývoj   izolace a purifikace   MeSH
• DNA fingerprinting MeSH
• feces mikrobiologie   MeSH
• fenotyp MeSH
• fermentace MeSH
• molekulární typizace MeSH
• polymerázová řetězová reakce MeSH
• RNA ribozomální 16S genetika   MeSH
• sloni mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• RNA ribozomální 16S MeSH