Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955T = LMG 30142T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956T = LMG 30143T), reference strain XV4

Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment

Abstract BACKGROUND: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.The slightly higher variability of the hsp60 gene sequences with respect to the 16S rRNA sequences offers better opportunities to design or develop molecular assays, allowing identification and differentiation of closely related species. hsp60 can be considered an excellent additional marker for inferring the taxonomy of the members of Bifidobacterium genus. RESULTS: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns. CONCLUSIONS: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis

SPECIATION OF HEAVY METALS AT WATER-SEDIMENT INTERFACE

The objective of the study was to understand the equilibrium relationship between the heavy metals concentrations in superficial water and pore water. At  water-sediment interface, the equilibrium rapidly changed and it is influenced by chemico-physical parameters of aquatic ecosystems. The hydraulic safety of Bologna plain (North Italy) depends on network of artificial canals and they are related with natural rivers of Reno basin (Reno river and its tributaries). The natural and artificial water courses flowed in agricultural, urban and industrial land. The heavy metals concentration in water and sediment discriminated the human pressure on the land and their spatial distribution in sediment could predict the hazard of pollution in aquatic ecosystems. We compared the heavy metals concentrations in pore water and superficial water determined in natural rivers and artificial canals, and more pollution in artificial canals than natural rivers was found. Furthermore, the coefficient of partition (log Kd) between water and sediments was calculated to evaluate the bioavailability of heavy metals adsorbed on the sediments. The heavy metals extracted in deionised water at equilibrium after 16 h showed higher concentrations than those determined directly on water samples

USE OF EPS PRODUCING BIFIDOBACTERIUM AESCULAPII STRAINS TO IMPROVE TEXTURE AND QUALITY OF SOY MILK BASED FERMENTED MILKS

Functional foods represent one of the fastest growing areas in the food industry because considered a dietary strategy to reduce illness. The development of dairy functional products has become an important subject. Consumers have increased the demand for low/fat-free, but also smooth and creamy, functional dairy products. Since milk fat contributes to flavour and texture of the dairy products, its removal leads to textural defects. So several attempts have been made to increase the textural and quality properties of these products. Among the strategies, the use of strains able to produce exopolysaccharides (EPS) has been suggested as an alternative to additives such as xanthan, pectin and carrageenan. So, the use of EPS-producing Bifidobacterium aesculapii strains was proposed for to obatain functional fermented milks from soy milk. Seven strains of B. aesculapii, recently described, able to ferment lactose and producing an exocellular gelling matrix, were employed for the fermentation of soy-milk. The strains were inoculated (7.5 log cfu/ml) and incubated at 37°C. The resulting fermented milks were subjected to EPS and volatile molecule profiles (GC/MS-SPME) analysis and Texture Analyzer. Also the survival of B. aesculapii in the products was checked during the refrigerated storage. The results showed all the strains were able to grow in soymilk and to reach a final concentration of 9.0 log cfu/ml. The strains produced large amount of EPS in products. The fermented milks were characterized by specific volatile molecule profiles. Generally, the strains able to produce the major amount of EPS resulted in more viscous products. The results obtained have highlighted the chance to exploit EPS producing Bifidobacterium aesculapii strains to produce fermented milks characterized by specific “aromagramma and texture properties. These results could be exploited also to expand the market of fermented soy-milk products by using a biotechnological approach enhancing also the overall product quality

In Vitro Activity of Tea Tree Oil Vaginal Suppositories against Candida spp. and Probiotic Vaginal Microbiota

The aim of this work is to evaluate the in vitro microbicidal activity of vaginal suppositories (VS) containing tea tree oil (TTO-VS) towards Candida spp. and vaginal probiotics. A total of 20 Candida spp. strains, taken from patients with vaginitis and from an established type collection, including reference strains, were analysed by using the CLSI microdilution method. To study the action of VS towards the beneficial vaginal microbiota, the sensitivity of Bifidobacterium animalis subsp. lactis (DSM 10140) and Lactobacillus spp. (Lactobacillus casei R-215 and Lactobacillus acidophilus R-52) was tested. Both TTO-VS and TTO showed fungicidal activity against all strains of Candida spp. whereas placebo-VS or the Aloe gel used as controls were ineffective. The study of fractional fungicidal concentrations (FFC) showed synergistic interaction with the association between Amphotericin B and TTO (0.25 to 0.08 µg/ml, respectively) against Candida albicans. Instead, the probiotics were only affected by TTO concentration ≥ 4% v/v, while, at concentrations < 2% v/v, they remained viable. TTO-VS exhibits, in vitro, a selective fungicidal action, slightly affecting only the Bifidobacteriun animalis strain growth belonging to the vaginal microbiota. In vivo studies are needed to confirm the efficacy to prevent acute or recurrent vaginal candidiasis. Copyright © 2015 John Wiley & Sons, Ltd

Genotypic Identification of Yeasts Isolated from Yaghnobi Fermented Milk

The Yaghnob Valley in Tajikistan is inhabited by a small isolated human population. The area is geographically isolated, causing the inhabitant’s culture, food and lifestyle to remain uninfluenced by the rest of the world. Traditional methods of fermenting milk involve the use of indigenous microorganisms, leading to the production of a variety of fermented milk products. The people in Yaghnobi produce a fermented milk from goat, which constitutes one of their main foods. The aim of the current work was to determine which yeasts were present in the original Yaghnobi fermented milk as well as in the same product reproduced at home for three years with monthly reculturing in cow milk. Thirty yeasts have been isolated using different lab media such as M17, MRS, WL,YPD and YPD plus chloramphenicol, and colonies were re-cultivated until pure. Twenty isolates have been obtained from the original product and 10 from fermented milk reproduced at home. They were identified by Sanger sequencing of the amplified ribosomal Internal Transcribed Spacers (ITS1-4). Furthermore, RFLP analyses of the ITS1-4 region were performed when quality, coverage or similarity of ITS1-4sequences did not permitted the unambiguously identification of the yeast isolates. Only for Saccharomyces cerevisiae, identity was also confirmed with GTG5Rep PCR. The results showed the presence of Kluyveromycesmarxianus (11), Pichia fermentans (7) and Saccharomyces cerevisiae (10) in both products. However, Kluyveromyceslactis (1) was found in the original fermented milk sample and Kazachstaniaunispora(1) in home maintained fermented milk. Great attention has nowadays been devoted to microbial resources and the study of traditional fermented products can be a very interesting source of microbial biodiversity. This study provides for the first time data on yeast composition and characteristics in naturally Yaghnobi fermented milk, showing an unexpected richness in yeast communities leading to fermented milk production

Phenotypic Characterization of Yeasts Isolated from Yaghnobi Fermented Milk

Introduction In the Yaghnob Valley in Tajikistan lives an isolated human population, who still utilize ancient fermentation methods to produce fermented milk as one of their main foods. The area is very isolated, so theinhabitants and their diet have remained protected without influence or contamination from the surroundingworld. Thus, it is interesting to study the microbial biodiversity of their fermented food.The aim of the present work is thephenotypic characterization of yeasts isolated from Yaghnobi fermented milk. Materials and Methods Thirty yeast isolates were obtained from the original Yaghnobi fermented milk and from the sameone reproduced at home for three years.The isolation was performed on M17, MRS, WL,YPD and YPD plus chloramphenicol. Phenotypic characterization were assessed by growth i) on different carbon sources, ii) in presence of ox bile, iii) at high temperatures, iv) in acidic condition and v) in presence of hydrogen peroxide. Also the invasiveness in YPD and hyphae formation was studied. Results The 30 yeast strains, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae plus one Kazachstania unispora and one Kluyveromyces lactis. revealed different phenotypes within the same species; e.g. invasiveness from 0 to 3 among the isolates of both S. cerevisiae and P. fermentans, the latter species also showed large variations in thermo-tolerance (inhibition from 40°Cto 46°C), and varying tolerance to acidic conditions. Only the strain of K. lactis and about 50%of K .marxianus strains grew at 46°C. All strains of K. marxianus grew at ox bile up to 2%. Of the S. cerevisiae strains, 50% grew in ox bile up to 2%, while two strains were inhibited already at 0.5%. Conclusions The data obtained suggest the presenceof yeasts with interesting probiotic properties in Yaghnobi fermented milk, such as the ability to survive in harsh gastrointestinal environment. The complete probiotic characterization of these yeasts will be investigated further

Associazione di oli essenziali e idrolati: Studi in vitro per una potenziale loro applicazione in patologie quali l\u2019IBS

Introduzione. Numerose evidenze scientifiche mostrano come il microbiota sia in grado di influenzare e mantenere l\u2019omeostasi del sistema immunitario, fungere da barriera per i microrganismi patogeni e supportare l\u2019ospite grazie alla produzione di composti nutrizionali. La variazione della composizione del microbiota, generalmente nota come disbiosi, pu\uf2 compromettere gravemente questa interazione mutualistica e influenzare negativamente la fisiologia dell\u2019ospite alterandone lo stato di salute. Disbiosi intestinali sono strettamente legate ad importanti patologie umane incluso quelle infiammatorie e autoimmuni, come IBD (Intestinal Bowel Disease) e IBS (Intestinal Bowel Syndrome), metaboliche, come l\u2019obesit\ue0 e il diabete, e neurologiche. L\u2019IBS \ue8 un disordine gastrointestinale associato a dolore addominale, cambiamenti dell\u2019habitat intestinale, manifestazioni ricorrenti/remittenti. Analogamente, l\u2019IBD \ue8 associata all\u2019alterazione dell\u2019asse intestino-cervello nonch\ue9 a microbiota e sistema immunitario alterati. Recenti studi mostrano come pazienti con IBD presentano un microbiota differente rispetto ai soggetti sani. In particolare, Sokol et al (2016) descrivono che in pazienti con IBD alcune specie fungine (Ascomycetes, specialmente Saccharomyces cerevisiae) diminuiscono in percentuale, mentre altre aumentano (Basidiomycetes, come Mallasezia sympodialis). Inoltre, nei soggetti malati, specie appartenenti al genere Candida incrementano in percentuale pur non causando patologie infettive. Scopi. In questo lavoro si \ue8 cercato di (i) Individuare oli essenziali (OE) che siano pi\uf9 attivi su ceppi di Candida spp piuttosto che su ceppi di Saccharomyces cerevisiae, (ii) Valutare gli effetti degli OE selezionati, su ceppi probiotici comunemente usati nelle formulazioni commerciali, (iii) Studiare l\u2019effetto immunomodulante di alcuni idrolati (Id) su polimorfonucleati (PBMC) isolati da donatori sani Materiali e Metodi. Per lo studio sono stati usati 7 OE e 4 Id. Sono stati allestiti esperimenti di micro-brodo diluizione vs 54 ceppi fungini (38 Candida spp., 8 Galactomyces spp., 4 S. cerevisiae) e 7 ceppi probiotici. Lo stesso test, combinato con la conta delle cfu/ml, \ue8 stato usato per studiare l\u2019effetto modulante di 4 OE selezionati vs 5 ceppi di Candida spp. e 4 di S. cerevisiae e per valutarne l\u2019effetto degli stessi su mix caratterizzati da un ceppo di C. albicans e uno di S. cerevisiae. Infine, per valutare l\u2019effetto immunomodulante degli Id, concentrazioni scalari (da 1/4 a 1/32) sono state incubate con PBMC ottenuti da due donatori, e i terreni di coltura sono stati analizzati per l\u2019espressione di 4 citochine pro e anti-infiammatorie con test ELISA. Resultati. I nostri risultati preliminari mostrano che ceppi di Candida spp. e S. cerevisiae sono ugualmente modulati dagli OE di Monarda didyma, Citrus aurantium var amara e C. zeylanicum. Tra questi, quello di C. aurantium var amara, quando usato in sub-MIC e testato su miscele contenenti da ceppi appartenenti ad entrambi i generi, \ue8 capace di inibire maggiormente C. albicans rispetto a S. cerevisiae. Inoltre, le concentrazioni attive sui ceppi fungini sono innocue sui ceppi probiotici usati generalmente nelle formulazioni commerciali. Infine, lo studio eseguito con gli Id mostra come quelli di Vitis vinifera siano in grado di stimolare maggiormente la produzione di citochine anti-infiammatorie rispetto a quelle pro-infiammatorie. Conclusioni. I nostri dati preliminari, se confermati, potranno essere di grande interesse nel trattamento di patologie quali l\u2019IBS al fine di ripristinare il corretto equilibrio dei ceppi microbici caratterizzanti il microbiota normale. Ulteriori indagini dovranno essere sviluppate affinch\ue8, in futuro, possa essere applicata l\u2019azione antimicrobica degli OE selezionati in associazione con quella immunomodulante degli Id nei trattamenti delle IB