Bifidobacteria in two-toed sloths (Choloepus didactylus): phylogenetic characterization of the novel taxon Bifidobacterium choloepi sp. nov.

Seven bifidobacterial strains were isolated from the faeces of two adult males of the two-toed sloth (Choloepus didactylus) housed in Parco Natura Viva, in Italy. Comparative sequence analysis of 16S rRNA and of five housekeeping (hsp60, rpoB, clpC, dnaJ, dnaG) genes revealed that these strains were classified into two clusters. On the basis of 16S rRNA gene sequence similarity, the type strain of Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854T (95.4 %) was the closest neighbour to strain in Cluster I (BRDM 6T), whereas the type strain of Bifidobacterium dentium DSM 20436T (values were in the range of 98‒99.8 %) was the closest neighbour to the other six strains in Cluster II. The average nucleotide identity (ANI) values of BRDM 6T and of strains in Cluster II with the closely related type strains were 76.0 and 98.9 % (mean value) respectively. Therefore, genotyping based on the genome sequence of the strain BRDM 6T combined with phenotypic analyses clearly revealed that the strain BRDM 6T represents a novel species for which the names Bifidobacterium choloepi sp. nov. (BRDM 6T=NBRC 114053T=BCRC 81222T) is proposed.

Bifidobacterium jacchi sp. nov., isolated from the faeces of a baby common marmoset (Callithrix jacchus).

A novel Bifidobacterium strain, MRM 9.3T, was isolated from a faecal sample of a baby common marmoset (Callithrixjacchus). Cells were Gram-stain-positive, non-motile, non-sporulating, non-haemolytic, facultatively anaerobic and fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA genes as well as multilocus sequences (representing hsp60, rpoB, clpC, dnaJ and dnaG genes) and the core genomes revealed that strain MRM 9.3T exhibited phylogenetic relatedness to Bifidobacterium myosotis DSM 100196T. Comparative analysis of 16S rRNA gene sequences confirmed the phylogenetic results showing the highest gene sequence identity with strain B.ifidobacterium myosotis DSM 100196T (95.6 %). The average nucleotide identity, amino acid average identity and in silico DNA-DNA hybridization values between MRM 9.3T and DSM 100196T were 79.9, 72.1 and 28.5 %, respectively. Phenotypic and genotypic features clearly showed that the strain MRM 9.3T represents a novel species, for which the name Bifidobacterium jacchi sp. nov. is proposed. The type strain is MRM 9.3T (=DSM 103362T =JCM 31788T).

Bifidobacterium primatium sp. nov., Bifidobacterium scaligerum sp. nov., Bifidobacterium felsineum sp. nov. and Bifidobacterium simiarum sp. nov.: Four novel taxa isolated from the faeces of the cotton top tamarin (Saguinus oedipus) and the emperor tamarin (Saguinus imperator).

Four novel Gram-stain-positive, non spore forming and fructose-6-phosphate phosphoketolase-positive strains were isolated from the faeces of a cotton top tamarin (Saguinus oedipus) and an emperor tamarin (Saguinus imperator). Phylogenetic analyses based on 16S rRNA revealed that bifidobacterial strains TRE 1T exhibit close phylogenetic relatedness to Bifidobacterium catulorum DSM 103154 (96.0%) and Bifidobacterium tissieri DSM 100201 (96.0%); TRE DT and TRE HT were closely related to Bifidobacterium longum subsp. longum ATCC 15708T with similarity values of 97.4% and 97.5%, respectively; TRI 7T was closely related to Bifidobacterium tissieri DSM 100201 (96.0%). The Average Nucleotide Identity (ANI) and in silico DDH (isDDH) analysis with closest neighbour supported an independent phylogenetic position of all strains with values ranged from 74 to 85% for ANI and from 24 to 28% for isDDH. DNA base composition of the four strains was in the range of 58.3-63.5mol% G+C. Based on the phylogenetic, genotypic and phenotypic data, the strains TRE 1T, TRE DT, TRE HT and TRI 7T clearly represent four novel taxa within the genus Bifidobacterium for which the names Bifidobacterium primatium sp. nov. (type strain TRE 1T=DSM 100687T=JCM 30945T), Bifidobacterium scaligerum sp. nov. (type strain TRE DT=DSM 103140T=JCM 31792T), Bifidobacterium felsineum sp. nov. (type strain TRE HT=DSM 103139T=JCM 31789T) and Bifidobacterium simiarum sp. nov. (type strain TRI 7T=DSM 103153T=JCM 31793) are proposed.

Bifidobacterium callitrichidarum sp. nov. from the faeces of the emperor tamarin (Saguinus imperator).

Three Gram-stain-positive, non-spore-forming, microaerophilic and fructose-6-phosphate phosphoketolase positive strains were isolated from a faecal sample of an adult subject of the emperor tamarin (Saguinus imperator). Given that the isolates revealed identical BOX PCR profiles, strain TRI 5T was selected as a representative and characterized further. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain TRI 5T was closely related to Bifidobacterium saguini DSM 23967T (96.4 %) and to Bifidobacterium longum subsp. longum ATCC 15708 (96.2 %). Multilocus sequence analyses of five housekeeping genes showed the close phylogenetic relatedness of this strain to Bifidobacterium breve DSM 20213T (hsp60 94.1 %), Bifidobacterium saguini DSM 23967T (clpC 91 %), Bifidobacterium avesanii DSM 100685T (dnaG 80.3 %), Bifidobacterium longumsubsp. infantis ATCC 15697T (dnaJ 85.3 %) and Bifidobacterium longumsubsp. longum ATCC 15708 (rpoB 93 %), respectively. The peptidoglycan type was A3ß, with an interpeptide bridge comprising l-Orn (Lys) - l-Ser - l-Ala - l-Thr - l-Ala. The DNA G+C content of strain TRI 5T was 60.9 mol%. Based on the data provided, strain TRI 5T represents a novel species of the genus Bifidobacterium for which the name Bifidobacteriumcallitrichidarum sp. nov. is proposed. The type strain is TRI 5T (=DSM 103152T=JCM 31790T).

Bifidobacterium eulemuris sp. nov., isolated from faeces of black lemurs (Eulemur macaco).

Forty-three strains of bifidobacteria were isolated from the faeces of two adult black lemurs, Eulemur macaco. Thirty-four were identified as Bifidobacterium lemurum, recently described in Lemur catta. The nine remaining isolates were Gram-positive-staining, non-spore-forming, fructose-6-phosphate phosphoketolase-positive, microaerophilic, irregular rod-shaped bacteria that often presented Y- or V-shaped cells. Typing techniques revealed that these isolates were nearly identical, and strain LMM_E3T was chosen as a representative and characterized further. Phylogenetic analysis based on 16S rRNA gene sequences clustered this isolate inside the genus Bifidobacterium and showed the highest levels of sequence similarity with B. lemurum DSM 28807T (99.3 %), with Bifidobacterium pullorum LMG 21816T and Bifidobacterium longum subsp. infantis ATCC 15697T (96.4 and 96.3 %, respectively) as the next most similar strains. The hsp60 gene sequence of strain LMM_E3T showed the highest similarity to that of Bifidobacterium stellenboschense DSM 23968T (93.3 %), and 91.0 % similarity to that of the type strain of B. lemurum. DNA-DNA reassociation with the closest neighbour B. lemurum DSM 28807T was found to be 65.4 %. The DNA G+C content was 62.3 mol%. Strain LMM_E3T showed a peptidoglycan structure that has not been detected in bifidobacteria so far: A3α l-Lys-l-Ser-l-Thr-l-Ala. Based on the phylogenetic, genotypic and phenotypic data, strain LMM_E3T represents a novel species within the genus Bifidobacterium, for which the name Bifidobacterium eulemuris sp. nov. is proposed; the type strain is LMM_E3T ( = DSM 100216T = JCM 30801T).

Lactobacillus salivarius and L. gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression.

Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans. Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin (LtxA) and cytolethal distending toxin (CdtB) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.

Occurrence of Bifidobacteriaceae in human hypochlorhydria stomach.

BACKGROUND: The human stomach, when healthy, is not a suitable host for microorganisms, but in pathological conditions such as gastritis, when gastric acid secretion is impaired, microbial overgrowth can be observed. Apart from Helicobacter pylori, the composition of microbiota, resident or exogenously introduced during neutral/high pH conditions, has not been investigated thoroughly. Thus, it is possible that Bifidobacteriaceae, important autochthonous and beneficial bacteria of human gastrointestinal microbiota, could over-colonize the stomach of hypochlorhydria patients suffering from autoimmune atrophic gastritis (AAG) or omeprazole-treated (OME) gastritis. This prompted us to characterize the Bifidobacteriaceae in such patients' gastric microbiota and to study its abnormal colonization. METHODS: Samples of gastric juices, and antrum and corpus mucosa from 23 hypochlorhydria patients (13 AAG and 10 OME) and from 10 control volunteers with base-line normochlorhydria, were cultivated in Brain Heart Infusion (BHI) and selective Bifidobacterium-Tryptone-Phytone-Yeast extract (Bif-TPY) media. The isolates were characterized by the fructose-6-phosphate phosphoketolase (F6PPK) test, electrophoresis of cellular proteins, the fermentation test, guanine-cytosine% DNA content, and DNA-DNA hybridization. Negative F6PPK isolates were characterized by order-specific polymerase chain reaction (PCR). RESULTS: A total of 125 isolates, assigned to the Bifidobacteriaceae family on the basis of their morphology, were obtained from AAG and OME patients, but not from normal subjects. Of these isolates, 55 were assigned to the Bifidobacteriaceae family on the basis of their fructose-6-phosphoketolase (PPK) activity, PPK being the key taxonomic enzyme of this family. The remaining 70 isolates, which were PPK-negative, were attributed to the Actinomycetales order following specific primer PCR analysis. We observed a significantly higher abundance of Bifidobacteriaceae (Bifidobacterium dentium, Scardovia inopinata, and Parascardovia denticolens) in OME group than the AAG group. Furthermore, the Actinomycetales distribution was homogeneous for both hypochlorhydria patient groups. CONCLUSIONS: This study suggests that the Bifidobacteriaceae species, typically found in the oral cavity, readily colonizes the hypochlorhydria stomach of OME patients. The clinical relevance and the mechanism underlying this Bifidobacteriaceae presence in OME gastritis requires further functional studies.

Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.).

The present study focused on inhibitory activity of freshly extracted essential oils from three legal (THC<0.2% w/v) hemp varieties (Carmagnola, Fibranova and Futura) on microbial growth. The effect of different sowing times on oil composition and biological activity was also evaluated. Essential oils were distilled and then characterized through the gas chromatography and gas chromatography-mass spectrometry. Thereafter, the oils were compared to standard reagents on a broad range inhibition of microbial growth via minimum inhibitory concentration (MIC) assay. Microbial strains were divided into three groups: i) Gram (+) bacteria, which regard to food-borne pathogens or gastrointestinal bacteria, ii) Gram (-) bacteria and iii) yeasts, both being involved in plant interactions. The results showed that essential oils of industrial hemp can significantly inhibit the microbial growth, to an extent depending on variety and sowing time. It can be concluded that essential oils of industrial hemp, especially those of Futura, may have interesting applications to control spoilage and food-borne pathogens and phytopathogens microorganisms.

Gut health promoting activity of new putative probiotic/protective Lactobacillus spp. strains: a functional study in the small intestinal cell model.

In interaction studies with the host intestine, the use of the appropriate gut functional cell model is essential. Therefore, we examined the protective properties of selected lactobacilli in a newly established intestinal cell model. Bacteria were cocultured with the pig small intestinal epithelial cells (PSIc1) and pig blood monocytes (PoM2) in a functional intestinal cell model. Intercellular intestinal integrity was measured by transepithelial electrical resistance (TER), before and after coculture with selected bacterial strains. All selected bacterial strains showed important gut health promoting activity by: enhancing the intestinal integrity and increasing metabolic activity of intestinal cells. Stimulation of immune response was strain specific. The best stimulants were unidentified lactobacillus strains obtained from fermented food in Africa (PCK87 and 66), followed by Lactobacillus plantarum (PCS26). Their activity was significantly higher (p<0.05) than that of the commercial Lactobacillus casei Shirota strain.