Non-typhoidal Salmonella serovars are worldwide spread foodborne pathogens that cause diarrhea in humans and animals. Colonization of gnotobiotic piglet intestine with porcine indigenous mucinolytic Bifidobacterium boum RP36 strain and non-mucinolytic strain RP37 and their interference with Salmonella Typhimurium infection were compared. Bacterial interferences and impact on the host were evaluated by clinical signs of salmonellosis, bacterial translocation, goblet cell count, mRNA expression of mucin 2, villin, claudin-1, claudin-2, and occludin in the ileum and colon, and plasmatic levels of inflammatory cytokines IL-8, TNF-α, and IL-10. Both bifidobacterial strains colonized the intestine comparably. Neither RP36 nor RP37 B. boum strains effectively suppressed signs of salmonellosis. Both B. boum strains suppressed the growth of S. Typhimurium in the ileum and colon. The mucinolytic RP36 strain increased the translocation of S. Typhimurium into the blood, liver, and spleen.

• Klíčová slova
• Bifidobacterium boum, Salmonella Typhimurium, germ-free, gnotobiotic, goblet cells, mucin, mucinolytic, piglet,
• 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