Vojtěch Rada

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees

One hundred and eighty-seven fructose-6-phosphate phosphoketolase positive strains were isolated from the digestive tract of three different bumblebee species. Analyses of the partial 16S rRNA gene sequences of the representative strains showed only 92.8% and 92.5% similarity to Bifidobacterium coryneforme YIT 4092(T) and Bifidobacterium indicum JCM 1302(T), 92.2% similarity to Alloscardovia omnicolens CCUG 18650 and slightly reduced similarity of 91% to other members of the family Bifidobacteriaceae. On the other hand, analyses of the partial heat-shock protein 60 (hsp60) gene sequence revealed that the proposed type strain BLAPIII-AGV(T) was affiliated only to the 60 kDa chaperonin sequence of uncultured bacteria from human vagina (79-80%) and the hsp60 gene sequence of A. omnicolens CCUG 31649(T) (75.5%). The peptidoglycan type was A4α with an l-Lys-d-Asp interpeptide bridge. The polar lipids contained diphosphatidylglycerol, an unknown phospholipid, six glycolipids and two phosphoglycolipids. The major fatty acids were C(18:1), C(20:0) and C(18:0). These and other analyses indicated that the isolates represented a new genus within the family Bifidobacteriaceae. This observation was further substantiated by determination of the DNA G+C contents (46.1-47.1 mol%). Affinity of the strains to some scardovial genera (Aeriscardovia, Alloscardovia and Metascardovia) was also confirmed by their ability to grow under aerobic conditions. Besides the above mentioned differences, Bombiscardovia coagulans was found to differ from all scardovial genera in the ability to grow at temperatures as low as 5°C, which was another major phenotypically different characteristic of this new member of the family Bifidobacteriaceae. Hence, on the basis of phylogenetic analyses using partial 16S rRNA and hsp60 gene sequence data, and the temperature related phenotypic difference, we propose a novel taxa, B. coagulans gen. nov., sp. nov. (type strain=BLAPIII-AGV(T)=DSM 22924(T)=ATCC BAA-1568(T)).

Bifidobacteria in the digestive tract of bumblebees.

Bifidobacteria and other bacterial groups (lactobacilli, facultative anaerobes, anaerobes) from the digestive tract of three bumblebee species (Bombus lucorum (34 samples), Bombus pascuorum (18 samples) and Bombus lapidarius (9 samples)) were enumerated and characterised. Counts of facultative anaerobic bacteria and lactobacilli (5.41+/-2.92 and 2.69+/-3.02 log CFU/g of digestive tract content) were lower than those of anaerobes (7.66+/-0.86 log CFU/g). Counts of bifidobacteria were determined using two selective media: MTPY (Modified Trypticase Phytone Yeast extract agar) and a new medium with pollen extract. There was no significant difference between the counts of bifidobacteria from both media, 5.00+/-2.92 log CFU/g on MTPY and 5.00+/-2.87 on the pollen medium. Subsequently, 187 bacterial strains of the family Bifidobacteriaceae (fructose-6-phosphate phosphoketolase-positive) were isolated from three different localities and from all three species of bumblebees. Bifidobacteria were found in 42 out of 61 specimens (69%). Twenty-three (38%) specimens had counts of bifidobacteria higher than 7.0 log CFU/g. Bifidobacteria represented the dominant group of anaerobes (>70% of total anaerobes), i.e., the principal group of bacteria in the bumblebee digestive tract, in only fourteen specimens (23% of total). For the first time, bifidobacteria were isolated from the digestive tract of bumblebees. In addition, we suggest, on the basis of biochemical tests (API 50 CHL and RAPID ID 32) and genetic methods (PCR and DGGE), that these bacteria may represent new species within the family of Bifidobacteriaceae.

Alloscardovia macacae sp. nov., isolated from the milk of a macaque (Macaca mulatta), emended description of the genus Alloscardovia and proposal of Alloscardovia criceti comb. nov.

A novel bacterial strain, designated M8(T), was isolated from milk of a female macaque bred in captivity. The strain was Gram-stain-positive, anaerobic, irregular coccoid-rod-shaped without catalase activity. Analysis of 16S rRNA gene sequence similarity revealed that the isolate was most closely related to Alloscardovia omnicolens CCUG 31649(T) (96.4%) and Metascardovia criceti OMB105(T) (96.6%). Sequences of hsp60, fusA, and xfp genes also confirmed that the strain was most closely related to the type strains of A. omnicolens and M. criceti. The isolate produced fructose-6-phosphate phosphoketolase which is in agreement with classification within the family Bifidobacteriaceae. The major fatty acids were C18 : 1ω9c (35.8%), C16 : 1 (6.2 %) and C14 : 0 (5.7 %). Polar lipid analysis revealed five different glycolipids, two unidentified phospholipids and diphosphatidylglycerol. The peptidoglycan was of the type A4α l-Lys-d-Asp with the presence of d(l)-alanine, d-glutamine, d-asparagine and l-lysine. The DNA G+C content of strain M8(T) was 50.1 mol%. On the basis of genetic, phylogenetic and phenotypic data, strain M8(T) represents a novel species of the genus Alloscardovia for which the name Alloscardovia macacae sp. nov. is proposed. The type strain is M8(T) ( = DSM 24762(T) = CCM 7944(T)). In addition, our results also revealed that Alloscardovia omnicolens DSM 21503(T) and Metascardovia criceti DSM 17774(T) do not belong to different genera within the family Bifidobacteriaceae. We therefore propose to reclassify Metascardovia criceti as Alloscardovia criceti comb. nov. An emended description of the genus Alloscardovia is also provided.

Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris).

Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive tracts of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under anaerobic conditions. The isolates were identified according to 16S rRNA gene sequence analysis as undescribed members of the genus Lactobacillus, with the highest 16S rRNA gene sequence similarity (96.9 %) to the uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest related species with a validly published name, with 92.9 % 16S rRNA gene sequence similarity to the type strain. However, phylogenetic analyses based on different markers revealed that this species is phylogenetically very distant from the novel strains. The DNA G+C content of the proposed type strain BTLCH M1/2(T) is 37.8 mol%. The fatty acids C(19 : 1)ω6c and/or C(19 : 0) cyclo ω10c/19ω6, C(18 : 1)ω9c and C(16 : 0) were predominant in all strains. Diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids were detected in the novel strains. Growth was observed at 47 °C. The peptidoglycan type A4α L-Lys-D-Asp was determined for strain BTLCH M1/2(T). Genotypic characteristics and phylogenetic analyses based on the phylogenetic markers hsp60, pheS, rpoA and tuf as well as phenotypic characteristics and the results of chemotaxonomic analyses confirmed that the new isolates belong to a novel species of the genus Lactobacillus, for which the name Lactobacillus bombi sp. nov. is proposed. The type strain is BTLCH M1/2(T) ( = DSM 26517(T) = CCM 8440(T)).

Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis

The taxonomic position of Bifidobacterium stercoris Eg1(T) ( = JCM 15918(T)) based on comparative 16S rRNA gene and hsp60 sequence analyses was found to be controversial, as the strain showed high similarity to the type strain of Bifidobacterium adolescentis, CCUG 18363(T). Therefore, the relationship between the two species was investigated by a taxonomic study that included, in addition to re-evaluation of the 16S rRNA gene sequence, determination of DNA-DNA binding and multilocus sequence analysis (MLSA) of housekeeping genes encoding the DNA-directed RNA polymerase B subunit (rpoC), putative xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp), elongation factor EF-G (fusA), 50S ribosomal protein L2 (rplB) and DNA gyrase B subunit (gyrB). Comparative 16S rRNA gene sequence analysis showed relatively high similarity (98.9 %) between B. stercoris KCTC 5756(T) and B. adolescentis ATCC 15703(T). MLSA revealed close relatedness between B. stercoris KCTC 5756(T) and B. adolescentis CCUG 18363(T), with 99.3-100 % similarity between the rpoC, xfp, fusA, rplB and gyrB gene sequences. In addition, relatively high dnaJ1 gene sequence similarity of 97.7 % was found between the strains. Similar phenotypes and a high DNA-DNA binding value (78.9 %) confirmed that B. stercoris and B. adolescentis are synonymous. Based on these results, it is proposed that the species Bifidobacterium stercoris Kim et al. 2010 should be reclassified as a later heterotypic synonym of Bifidobacterium adolescentis Reuter 1963 (Approved Lists 1980).

Bifidobacterium animalis subsp. lactis strains isolated from dog faeces

The aim of the study was to identify and characterize dog bifidobacterial isolates and compare them with commercial probiotic strains. Sixteen isolates of Bifidobacterium animalis ssp. lactis from dog faeces (German Shepherd Dog) were identified by subspecies-specific PCR, MALDI-TOF MS and sequencing. This study is the first describing B. animalis ssp. lactis occurring within the intestinal tract of dogs. Our dog isolates showed slightly different fingerprinting profiles obtained by RAPD-PCR and REP-PCR from those isolated from yogurt and type strains of B. animalis ssp. lactis. Both, dog and yogurt origin strains indicated survival in the simulated in vitro digestion assay and were resistant to low pH and bile salts. Moreover, strong auto-aggregation activity was observed only in dog origin B. animalis ssp. lactis strains. Dog strains showed good properties predicting their survival ability in GIT and could be tested as a potential new probiotics for dogs or other hosts.

Occurrence of bifidobacteria in faeces of calves fed milk or a combined diet

The development of faecal bacteria composition in calves fed milk or a combined diet was investigated from 4 to 21 days of age. On day 7, bifidobacteria in faeces of milk-fed calves already increased from about 7.6 to 9.2 log CFU/g and did not change until the end of the study, whereas in calves fed the combined diet bifidobacteria only moderately increased to 7.9 log CFU/g and decreased slowly until day 21. The counts of bifidobacteria in calves on a combined diet were significantly (p < 0.01) lower compared to those in milk-fed calves. Bifidobacterial counts determined by cultivation or by fluorescence in situ hybridisation (FISH) did not differ significantly. Our results showed that the occurrence of bifidobacteria in calf faeces is highly dependent on the diet composition. Faecal bacteria flora of calves fed exclusively by milk is rich in bifidobacteria, but in calves on a combined diet coliforms dominated.

Occurrence of bifidobacteria and lactobacilli in digestive tract of some freshwater fishes

Distal parts of the fish intestine were analyzed for presence of bifidobacteria and lactobacilli using selective agars. Seventy seven samples from Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, Tinca tinca, Perca fluviatilis, Rutilus rutilus, Scardinius erythrophthalmus, Oreochromis niloticus, and Squalius cephalus were collected randomly throughout years 2008 and 2009. Bifidobacteria were detected in 5 samples from 4 fish species at counts 2.18–4.29 log CFU/g, lactobacilli were present in 6 fish species at counts 1.21–3.65 log CFU/g. Seven bifidobacterial isolates were identified to the species level using biochemical tests and by sequencing of 16S rRNA gene. Three strains belonged to species B. longum, two isolates were identified as B. dentium, one strain as B. asteroides and one isolate was not determined to the species level by employed methods. As identified bifidobacterial species are considered to be of human, animal or honeybee origin, they probably derived as contamination from sewage or other sources. After further more detail testing, the possible use of isolated bifidobacteria as probiotics is promising since they were able to pass through the digestive tract successfully.

Characterization of bifidobacteria suitable for probiotic use in calves

In our previous experiment, the ten calves originated bifidobacterial strains were administered to calves and re-isolated. Fingerprinting techniques used in this study enabled us to distinguish the surviving and non-surviving strains. Only the species Bifidobacterium animalis ssp. animalis and Bifidobacterium longum ssp. suis were found to survive in the intestine.

Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris)

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).