Eva Vlková

Auto-Aggregation and Co-Aggregation Ability in Bifidobacteria and Clostridia

A total of 142 human and 88 calf bifidobacteria were isolated and identified; ≈12 % of all isolated strains exhibited auto-aggregation (Agg) phenotype (Agg+). Properties considered to be predicting for their adhesion to intestine, i.e. auto-aggregation, and hydrophobicity were determined by xylene extraction in 18 human and 8 calf origin bifidobacteria. Co-aggregation of 8 human bifidobacteria with 8 clostridia was also evaluated. Agg varied between 16.3 and 96.4 %, hydrophobicity values ranged from 0 to 82.8 %. The strongest Agg and hydrophobicity were observed in B. bifidum and B. merycicum isolates. However, there were no statistically significant correlations between these two properties. Variability in the percentage of Agg and hydrophobicity was observed after cultivation of bifidobacteria on different carbon sources. All bifidobacteria showed co-aggregation ability with clostridia tested but there were remarkable differences depending on specific combinations of strains. The bifidobacterial strains with the highest ability to co-aggregate with clostridia were B. bifidum I4 and B. longum I10 isolated from infants; these strains gave also high values of Agg. Agg properties together with co-aggregation ability with potential pathogen can be used for preliminary selection of probiotic bacteria.

Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa)

Seventeen fructose-6-phosphate phosphoketolase-positive bacterial strains were isolated from the digestive tract of wild pigs (Sus scrofa). Most of them were identified as Bifidobacterium boum according to sequences of 16S rRNA gene. Two strains isolated from the small intestine content had unusual morphology of cells in comparison with bifidobacteria. Cells growing in liquid anaerobic media were regular shaped rods arranged mostly in pairs. These isolates showed relatively low 16S rRNA gene sequence similarities (maximum identity of 94%) to members of the family Bifidobacteriaceae. Nevertheless, phylogenetic analyses of 16S rRNA, hsp60 and xfp gene sequences revealed that these strains are more related to recently described Neoscardovia, Aeriscardovia and other scardovial genera, than to Bifidobacterium species. Partial gene sequences of other phylogenetic markers showed low (65.8-89.5%) similarities to genome sequences of bifidobacteria and Gardnerella vaginalis. The major fatty acids detected in cells of the representative strain DPTE4(T) were C(16:0), C(18:1), C(14:0). The peptidoglycan type of the DPTE4(T) strain was A3βl-Orn(l-Lys)-l-Ser(l-Ala)-l-Ala(2). Polar lipid analysis revealed two phosphoglycolipids and phospholipids, a glycolipid and diphosphatidylglycerol. The results of phylogenetic, genotypic and phenotypic analyses support the proposal of a novel taxa, Pseudoscardovia suis gen. nov., sp. nov. (type strain=DPTE4(T)=DSM 24744(T)=CCM 7942(T)).

Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella Typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine

The colonization, translocation and protective effect of two intestinal bacteria – PR4 (pig commensal strain of Bifidobacterium choerinum) or EcN (probiotic Escherichia coli strain Nissle 1917) – against subsequent infection with a virulent LT2 strain of Salmonella enterica serovar Typhimurium were studied in gnotobiotic pigs after oral association. The clinical state of experimental animals correlated with bacterial translocation and levels of inflammatory cytokines [a chemokine, interleukin (IL)‐8, a proinflammatory cytokine, tumour necrosis factor (TNF)‐α and an anti‐inflammatory cytokine, IL‐10] in plasma and intestinal lavages. Gnotobiotic pigs orally mono‐associated with either PR4 or EcN thrived, and bacteria were not found in their blood. No significant inflammatory cytokine response was observed. Mono‐association with Salmonella caused devastating septicaemia characterized by high levels of IL‐10 and TNF‐α in plasma and TNF‐α in the intestine. Di‐associated gnotobiotic pigs were given PR4 or EcN for 24 h. Subsequently, they were infected orally with Salmonella and euthanized 24 h later. Pigs associated with bifidobacteria before Salmonella infection suffered from severe systemic infection and mounted similar cytokine responses as pigs infected with Salmonella alone. In contrast, EcN interfered with translocation of Salmonella into mesenteric lymph nodes and systemic circulation. Pigs pre‐associated with EcN thrived and their clinical condition correlated with the absence of IL‐10 in their plasma and a decrease of TNF‐α in plasma and ileum.

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.

Growth of bifidobacteria and clostridia on human and cow milk saccharides.

For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.

Selective growth inhibitory effect of biochanin A against intestinal tract colonizing bacteria.

Both bifidobacteria and clostridia are part of the natural gut microflora and while clostridia may be responsible for severe intestinal infections, bifidobacteria are probiotic microorganisms belonging to the most important prospective bacteria in the bowel. The antimicrobial activity of biochanin A was tested in vitro against six Bifidobacterium spp., and eight Clostridium spp. using the broth microdilution method. Biochanin A showed an inhibition against all clostridia in the range of minimum inhibitory concentrations (MIC) from 64 μg/mL (for Cl. clostridioforme, strains DSM 933 and I3) to 1,024 μg/mL (for Cl. perfringens, DSM 11778). No bifidobacteria were suppressed at four-fold higher concentration (MICs > 4,096) than MIC of Cl. perfringens. These results indicate selective growth inhibition of biochanin A and its potential use in antimicrobial prevention and/or protection.

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)).

Pseudoscardovia radai sp. nov., a representative of the family Bifidobacteriaceae isolated from the digestive tract of a wild pig (Sus scrofa scrofa)

Presence of bifidobacteria and representatives of the new genus Pseudoscardovia within the family Bifidobacteriaceae in the digestive tract of wild pigs has been reported recently. Results based on comparative 16S rRNA gene sequence analysis of a new fructose-6-phosphate phosphoketolase-positive bacterial isolate originated from the small intestine of a wild pig revealed a relationship to Pseudoscardovia suis DPTE4T (96.8% sequence similarity). Phylogenetic and comparative analyses based on 16S rRNA, hsp60, xfp, fusA, tuf and rpoC partial gene sequences confirmed relationship of the new bacterial strain to Pseudoscardovia suis compared with bifidobacteria species occurring in the digestive tract of domestic and wild pigs. Differences in utilization of various substrates, production of enzymes, cell morphology, peptidoglycan structure, profile of cellular fatty acids and polar lipids between the new bacterial isolate designated as DPVI-TET3T and P. suis DPTE4T allow to establish a new bacterial taxon for which the name Pseudoscardovia radai sp. nov. (= DPVI/TET3T = CCM 7943T = DSM 24742T) was proposed.

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.

Bifidobacteria from the gastrointestinal tract of animals: differences and similarities

At present, the genus Bifidobacterium includes 48 species and subspecies, and this number is expected to increase. Bifidobacteria are found in different ecological niches. However, most were originally isolated from animals, mainly mammals, especially during the milk feeding period of life. Their presence in high numbers is associated with good health of the host. Moreover, bifidobacteria are often found in poultry and insects that exhibit a social mode of life (honeybees and bumblebees). This review is designed as a summary of currently known species of the genus Bifidobacterium, especially focused on their difference and similarities. The primary focus is on their occurrence in the digestive tract of animals, as well as the specificities of animal strains, with regard to their potential use as probiotics.