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The immunomodulatory potential of probiotics and (poly)phenols (PP) is recognized; however, studies regarding microorganisms-PP synergisms are yet to be explored. Here, we investigated the cooperation between probiotics and berry-derived PP extracts in modulating the cytokine responses in dendritic cells. Bacteria elicited immune responses in a strain-dependent manner. PP extracts showed different modulation of cytokine triggered by bacteria. Also with LPS, used as pro-inflammatory stimulus, PP from blueberry (BB) and cranberry (CB) most efficiently reduced IL12 production. L. paracasei LPC-S01 and B. bifidum MIMBb23sg resulted the best bacterial association in abrogating IL12 and increasing IL10. The use of PP fraction from BB50f and CB1 with the LPC-S01 + MIMBb23sg association resulted the most efficient combinations in terms of anti-inflammatory activity. These results provide bases for further investigation in vivo, in the perspective to develop food supplements that might conceivably deliver the single and combined benefits of probiotics and berry (poly)phenols.

Purpose: Aquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable (VBNC) state, as a consequence of starvation in seawater at low temperatures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resuscitate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress. Methods: Cell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating. Results: The cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide. Conclusions: The timin

Fermented foods are receiving growing attention for their health promoting properties. In particular, there is a growing demand for plant-based fermented foods as dairy alternatives. Considering that soy is a vegetal food rich in nutrients and a source of the phytoestrogen isoflavones, the aim of this study was to select safe food micro-organisms with the ability to ferment a soy drink resulting in a final product with an increased estrogenic activity and improved functional properties. We used milk kefir grains, a dairy source of microorganisms with proven health-promoting properties, as a starting inoculum for a soymilk. After 14 passages of daily inoculum in fresh soy drink, we isolated four lactic acid bacterial strains: Lactotoccus lactis subsp. lactis K03, Leuconostc pseudo-mesenteroides K05, Leuconostc mesenteroides K09 and Lentilactobacillus kefiri K10. Isolated strains were proven to be safe for human consumption according to the assessment of their antibiotic resistance profile and comparative genomics. Furthermore, functional characterization of the bacterial strains demonstrated their ability to ferment sugars naturally present in soybeans and produce a creamy texture. In addition, we demonstrated, by means of a yeast-based bioluminescence reporter system, that the two strains belonging to the genus Leuconostoc increased the estrogenic activity of the soybean drink. In conclusion, the proposed application of the bacterial strains characterized in this study meets the growing demand of consumers for health-promoting vegetal food alter-natives to dairy products.

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota—Faecalibaculum rodentium and its human homologue, Holdemanella biformis—that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

An immunosuppressive microenvironment in lung concurs to pre-malignant lesions progression to cancer. Here, we explore if perturbing lung microbiota, which contribute to immunosuppression, by antibiotics or probiotic aerosol interferes with lung cancer development in a mouse carcinogen-induced tumor model. Urethane-injected mice were vancomycin/neomycin (V/N)-aerosolized or live or dead L. rhamnosus GG (L.RGG)-aerosolized, and tumor development was evaluated. Transcriptional profiling of lungs and IHC were performed. Tumor nodules number, diameter and area were reduced by live or heat-killed L.RGG, while only a decrease in nodule diameter was observed in V/N-treated lungs. Both L.RGG and V/N reduced Tregs in the lung. In L.RGG-treated groups, the gene encoding the joining chain (J chain) of immunoglobulins was increased, and higher J chain protein and IgA levels were observed. An increased infiltration of B, NK and myeloid-derived cells was predicted by TIMER 2.0. The Kaplan–Meier plotter revealed an association between high levels of J chain mRNA and good prognosis in lung adenocarcinoma patients that correlated with increased B and CD4 T cells and reduced Tregs and M2 macrophages. This study highlights L.RGG aerosol efficacy in impairing lung cancer growth by promoting local immunity and points to this non-invasive strategy to treat individuals at risk of lung cancer.

The use of seawater and marine microorganisms can represent a sustainable alternative to avoid large consumption of freshwater performing industrial bioprocesses. Debaryomyces hansenii, which is a known halotolerant yeast, possess metabolic traits appealing for developing such processes. For this purpose, we studied salt stress exposure of two D. hansenii strains isolated from marine fauna. We found that the presence of sea salts during the cultivation results in a slight decrease of biomass yields. Nevertheless, higher concentration of NaCl (2 M) negatively affects other growth parameters, like growth rate and glucose consumption rate. To maintain an isosmotic condition, the cells accumulate glycerol as compatible solute. Flow cytometry analysis revealed that the osmotic adaptation causes a reduced cellular permeability to cell-permeant dye SYBR Green I. We demonstrate that this fast and reversible phenomenon is correlated to the induction of membrane depolarization, and occurred even in presence of high concentration of sorbitol. The decrease of membrane permeability induced by osmotic stress confers to D. hansenii resistance to cationic drugs like Hygromycin B. In addition, we describe that also in Saccharomyces cerevisiae the exposure to hyper-osmotic conditions induced membrane depolarization and reduced the membrane permeability. These aspects are very relevant for the optimization of industrial bioprocesses, as in the case of fermentations and bioconversions carried out by using media/buffers containing high nutrients/salts concentrations. Indeed, an efficient transport of molecules (nutrients, substrates, and products) is the prerequisite for an efficient cellular performance, and ultimately for the efficiency of the industrial process.

Purpose: Ability to survive the digestive process is a major factor in determining the effectiveness of a probiotic. In this study, the ability of the probiotic L. casei DG® (Lactobacillus paracasei CNCMI1572) to survive gastrointestinal transit in healthy children was investigated for the first time. Methods: Twenty children aged 3–12 years received L. casei DG® as drinkable solution of 1 × 109 colony forming units (CFU), once daily for 7 consecutive days. Recovery in faecal samples was evaluated at baseline and at different time-points during and after administration. Defecation frequency, faeces consistency, digestive function and product safety were also assessed. Results: Nineteen (95%) of the 20 enrolled children presented viable L. casei DG® cells in their faeces at least once during the study, with a maximum count (mean 4.3 log10 CFU/g ± 2.3) reached between day 4 and 6 from the beginning of consumption. Notably, for 11 (57.9%) of the 19 children with viable cells, L. casei DG® survived in faecal samples up to 3 days after treatment end. Defecation frequency, faeces consistency and digestive function did not change considerably during or after study treatment. Safety of the study product was very good. Conclusions: This study showed for the first time that L. casei DG® survives the gastrointestinal transit when ingested by children with a paediatric probiotic drinkable solution containing 1 × 109 CFU, and persists in the gut up to 3 days after the end of product intake, demonstrating resistance to gastric juices, hydrolytic enzymes and bile acids.

Probiotic microorganisms may benefit the host by influencing diverse physiological processes, whose nature and underlying mechanisms are still largely unexplored. Animal models are a unique tool to understand the complexity of the interactions between probiotic microorganisms, the intestinal microbiota, and the host. In this regard, in this pilot study, we compared the effects of 5-day administration of three different probiotic bacterial strains (Bifidobacterium bifidum MIMBb23sg, Lactobacillus helveticus MIMLh5, and Lacticaseibacillus paracasei DG) on three distinct murine intestinal sites (ileum, cecum, and colon). All probiotics preferentially colonized the cecum and colon. In addition, probiotics reduced in the ileum and increased in the cecum and colon the relative abundance of numerous bacterial taxonomic units. MIMBb23sg and DG increased the inducible nitric oxide synthase (iNOS) in the ileum, which is involved in epithelial homeostasis. In addition, MIMBb23sg upregulated cytokine IL-10 in the ileum and downregulated the cyclooxygenase COX-2 in the colon, suggesting an anti-inflammatory/regulatory activity. MIMBb23sg significantly affected the expression of the main gene involved in serotonin synthesis (TPH1) and the gene coding for the serotonin reuptake protein (SERT) in the ileum and colon, suggesting a potential propulsive effect toward the distal part of the gut, whereas the impact of MIMLh5 and DG on serotonergic genes suggested an effect toward motility control. The three probiotics decreased the expression of the permeability marker zonulin in gut distal sites. This preliminary in vivo study demonstrated the safety of the tested probiotic strains and their common ability to modulate the intestinal microbiota. The probiotics affected host gene expression in a strain-specific manner. Notably, the observed effects in the gut were site dependent. This study provides a rationale for investigating the effects of probiotics on the serotonergic system, which

Pulmonary immunological tolerance to inhaled particulates might create a permissive milieu for lung metastasis. Lung microbiota contribute to pulmonary tolerance; here, we explored whether its manipulation via antibiotic or probiotic aerosolization favors immune response against melanoma metastasis. In lungs of vancomycin/neomycin-aerosolized mice, a decrease in bacterial load was associated with reduced regulatory T cells and enhanced T cell and NK cell activation that paralleled a significant reduction of melanoma B16 lung metastases. Reduction of metastases also occurred in lungs transplanted with bacterial isolates from antibiotic-treated lungs. Aerosolized Lactobacillus rhamnosus strongly promoted immunity against B16 lung metastases as well. Furthermore, probiotics or antibiotics improved chemotherapy activity against advanced B16 metastases. Thus, we identify a role for lung microbiota in metastasis and show that its targeting via aerosolization is a therapy that can prevent metastases and enhance responses to chemotherapy. Le Noci et al. reveal that modulation of pulmonary microbiota by antibiotic or probiotic aerosolization decreases tumor growth in the lung. Antibiotic treatment induces a reduction of immunosuppressive cells in the lung, while probiotic administration promotes maturation of resident antigen-presenting cells.

Producers of probiotic products are legally required to indicate on the label only the minimum numbers of viable microorganisms at the end of shelf life expressed as colony-forming units (CFUs). Label specifications, however, describe only a fraction of the actual microbiological content of a probiotic formulation. This paper describes the microbiological components of a probiotic product that are not mentioned on the label, such as the actual number of CFUs, the presence of viable cells that cannot generate colonies on agar plates, and the abundance of dead cells. These “hidden” microbial fractions in probiotic products, the abundance of which may change during the shelf life, can promote biological responses in the host. Therefore, they should not be ignored because they may influence the efficacy and can be relevant for immunocompromised or fragile consumers. In conclusion, we propose the minimum requirements for microbiological characterization of probiotic products to be adopted for label specifications and clinical studies.

Scope: Bifidobacteria play a role in intestinal homeostasis but molecular mechanisms remain underinvestigated. The aim of this study was to assess if probiotic Bifidobacterium strains alter expression of intestinal microRNA and downstream target gene response. Methods and results: The expression of miR-148a and its validated target endothelial PAS domain protein 1 (EPAS1) was analyzed in Caco-2 cells and mice cecum in response to Bifidobacterium bifidum MIMBb75, B. bifidum NCC390, or Bifidobacterium longum NCC2705. In vitro, exposure to B. bifidum MIMBb75, but not to B. bifidum NCC390 or B. longum NCC2705, increased the expression of miR-148a after 1 and 4 h (p < 0.01), but not after 24 h. In vivo, B. bifidum MIMBb75 administration to C57BL/6J mice increased miR-148a expression in the cecum after 2 but not 14 days (p < 0.05). The increase in miR-148a was accompanied by a decrease in EPAS1 expression in Caco-2 cells and cecum (p < 0.05). Silencing of miR-148a reversed B. bifidum MIMBb75 dependent downregulation of EPAS1. Conclusion: This study shows an early response of intestinal cells to B. bifidum MIMBb75 through miR-148a modulation. This brings a new concept of strain- and time-dependent bifidobacteria–host crosstalk via microRNA. Probiotic B. bifidum MIMBb75 may help attenuating EPAS1 overexpression associated with intestinal inflammation.

The proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in the yogurt consortium enhances the growth rate and size of each population. In contrast, the independent growth of the two species in milk leads to a slower growth rate and a smaller population size. In this study, we report the first evidence that the urease activity of S. thermophilus increases the intracellular pH of L. delbrueckii in the absence of carbon source. However, in milk, in the presence of lactose the alkalizing effect of urea-derived ammonia was not detectable. Nevertheless, based on glucose consumption and lactic acid production at different pHin, L. delbrueckii showed an optimum of glycolysis and homolactic fermentation at alkaline pH values. In milk, we observed that ammonia provided by urea hydrolysis boosted lactic acid production in S. thermophilus and in L. delbrueckii when the species were grown alone or in combination. Therefore, we propose that urease activity acts as an altruistic cooperative trait, which is costly for urease-positive individuals but provides a local benefit because other individuals can take advantage of urease-dependent ammonia release.

Studies devoted to evaluating the outcome of different doses of probiotics are very limited, especially for multistrain formulations. In this context, we performed an intervention study that aimed to compare the effect of the administration of two doses (7 billion and 70 billion bacterial cells) of a multistrain probiotic formulation on the persistence of the four probiotic strains that were present in the product in the fecal samples collected from healthy subjects. The overall persistence of the probiotic strains was significantly higher for the 70 billion formulation than for the 7 billion formulation. Furthermore, probiotic strains were detected earlier and for longer for the 70 billion formulation compared to those for the 7 billion formulation. All probiotic strains were recovered alive from the 70 billion preparation, whereas recovery was not possible in a few fecal samples upon administration of the 7 billion preparation. In addition, the overall number of viable probiotic cells recovered on day 14 (i.e., the last day of consumption) was significantly higher for the 70 billion formulation than that for the 7 billion formulation. Finally, we found that the viability of the probiotic cells was stable over the course of the trial independent of volunteers’ handling, demonstrating good manufacturing of the product. In conclusion, this study demonstrated that strains belonging to different taxa may coexist in the human gastrointestinal tract upon ingestion of a multispecies probiotic formulation. Moreover, this study suggests that higher doses of bacterial cells in probiotic formulations may permit a higher, earlier, and longer recovery of the probiotics in the feces of healthy adults.

We report the comprehensive characterization of viral and microbial communities within an aquaculture wastewater sample, by a shotgun sequencing and 16S rRNA gene profiling metagenomic approach. Caudovirales had the largest representation within the sample, with over 50% of the total taxonomic abundance, whereas approximately 30% of the total open reading frames (ORFs) identified were from eukaryotic viruses (Mimiviridae and Phycodnaviridae). Antibiotic resistance genes (ARGs) within the virome accounted for 0.85% of the total viral ORFs and showed a similar distribution both in virome and in microbiome. Among the ARGs, those encoding proteins involved in the modulation of antibiotic efflux pumps were the most abundant. Interestingly, the taxonomy of the bacterial ORFs identified in the viral metagenome did not reflect the microbial taxonomy as deduced by 16S rRNA gene profiling and shotgun metagenomic analysis. A limited number of ARGs appeared to be mobilized from bacteria to phages or vice versa, together with other bacterial genes encoding products involved in general metabolic functions, even in the absence of any antibiotic treatment within the aquaculture plant. Thus, these results confirm the presence of a complex phage-bacterial network in the aquaculture environment.

Probiotics are live microorganisms, and viability after transit through the gastrointestinal tract (GIT) is considered an inherent property of the health benefits of probiotics. The aim of the present study was to quantify the viable and total loads of Lactobacillus paracasei DG cells after passage through the GIT following the consumption of the probiotic product Enterolactis (L. casei DG®; L. paracasei CNCM I-1572; L. paracasei DG) from drinkable vials by healthy adults. We developed a novel method for discriminating and enumerating culturable L. paracasei DG cells based on the unique sticky, filamentous phenotype of this strain on MRS agar containing vancomycin and kanamycin. The identity of DG was also confirmed with strain-specific primers by colony PCR. This method was used for a recovery study of the DG strain to quantify viable cells in the fecal samples of 20 volunteers during a 1-week probiotic consumption period and a 1-week follow-up. We isolated L. paracasei DG from at least one fecal sample from all the volunteers. The highest concentration of viable DG cells [ranging from 3.6 to 6.7 log10 colony-forming unit (CFU) per gram of feces] in the feces was observed between 4 and 8 days from the beginning of Enterolactis intake and for up to 5 days after cessation of intake. As expected, the total DG count determined by real-time quantitative PCR (qPCR) was mostly higher than the viable DG cells recovered. Viable count experiments, carried out by combining ad hoc culture-based discriminative conditions and strain-specific molecular biological protocols, unambiguously demonstrated that L. paracasei DG can survive gastrointestinal transit in healthy adults when ingested as Enterolactis in drinkable vials containing no less than one billion CFU at the end of shelf life.

Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group.

Most intervention studies on probiotics or prebiotics consist of several-week feeding periods and focus on specific host dysfunctions. However, probiotics are often consumed by healthy people and time can be limited; the efficacy of such dietary intervention, however, has only rarely been considered. In this paper, we report the results of a short-term (1. week) study, in which 11 healthy subjects consumed a commercial probiotic food supplement, resulting in a daily intake of about 6 billion viable cells of 10 bacterial species. We measured by quantitative real-time PCR (qPCR) the impact that this product had on faecal Bifidobacterium and Lactobacillus spp. Faecal lactobacilli and bifidobacteria, whose number in the gut is a commonly considered parameter to assess the efficacy of a probiotic/prebiotic intervention, were not significantly affected by the intervention. The only significant increase was observed for intestinal Lactobacillus acidophilus group. However, the significance of this change disappeared after only few days, indicating that it was plausibly due to DNA carryover from the ingestion of a large number of L. acidophilus cells. According to this study, short-term intake of a quite high number of live probiotics can be ineffective in healthy humans on the faecal concentration of bifidobacteria and lactobacilli.

Efflux pumps are membrane proteins involved in the active extrusion of a wide range of structurally dissimilar substrates from cells. A multidrug efflux pump named TetA belonging to the major facilitator superfamily (MFS) of transporters was identified in the Streptococcus thermophilus DSM 20617T genome. The tetA-like gene was found in the genomes of a number of S. thermophilus strains sequenced to date and in Streptococcus macedonicus ACA-DC 198, suggesting a possible horizontal gene transfer event between these two Streptococcus species, which are both adapted to the milk environment. Flow cytometry (single-cell) analysis revealed bistable TetA activity in the S. thermophilus population, and tetA-like gene over-expression resulted in a reduced susceptibility to ethidium bromide, tetracycline, and other toxic compounds even when the efflux pump was over-expressed in a strain naturally lacking tetA-like gene. Characterization of a major facilitator superfamily efflux pump in Streptococcus thermophilus involved in detoxification of ethidium bromide, tetracycline, and several antimicrobials.

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the Slayer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook wholegenome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

In order to preserve environmental quality, alternative strategies to chemical-intensive agriculture are strongly needed. In this study, we characterized in vitro the potential plant growth promoting (PGP) properties of a gamma-proteobacterium, named MIMR1, originally isolated from apple shoots in micropropagation. The analysis of the 16S rRNA gene sequence allowed the taxonomic identification of MIMR1 as Luteibacter rhizovicinus. The PGP properties of MIMR1 were compared to Pseudomonas chlororaphis subsp. aurantiaca DSM 19603T, which was selected as a reference PGP bacterium. By means of in vitro experiments, we showed that L. rhizovicinus MIMR1 and P. chlororaphis DSM 19603T have the ability to produce molecules able to chelate ferric ions and solubilize monocalcium phosphate. On the contrary, both strains were apparently unable to solubilize tricalcium phosphate. Furthermore, the ability to produce 3-indol acetic acid by MIMR1 was approximately three times higher than that of DSM 19603T. By using fluorescent recombinants of strains MIMR1 and DSM 19603T, we also demonstrated that both bacteria are able to abundantly proliferate and colonize the barley rhizosphere, preferentially localizing on root tips and in the rhizoplane. Finally, we observed a negative effect of DSM 19603T on barley seed germination and plant growth, whereas MIMR1, compared to the control, determined a significant increase of the weight of aerial part (+22 %), and the weight and length of roots (+53 and +32 %, respectively). The results obtained in this work make L. rhizovicinus MIMR1 a good candidate for possible use in the formulation of bio-fertilizers.

Bifidobacteria are gaining increasing interest as health-promoting bacteria. Nonetheless, the genus comprises several species, which can exert different effects on human host. Previous studies showed that wild blueberry drink consumption could selectively increase intestinal bifidobacteria, suggesting an important role for the polyphenols and fiber present in wild blueberries. This study evaluated the modulation of the most common and abundant bifidobacterial taxonomic groups inhabiting the human gut in the same fecal samples. The analyses carried out showed that B. adolescentis, B. breve, B. catenulatum/pseudocatelulatum, and B. longum subsp. longum were always present in the group of subjects enrolled, whereas B. bifidum and B. longum subsp. infantis were not. Furthermore, it was found that the most predominant bifidobacterial species were B. longum subsp. longum and B. adolescentis. The results obtained revealed a high interindividual variability; however, a significant increase of B. longum subsp. infantis cell concentration was observed in the feces of volunteers after the wild blueberry drink treatment. This bifidobacterial group was shown to possess immunomodulatory abilities and to relieve symptoms and promote the regression of several gastrointestinal disorders. Thus, an increased cell concentration of B. longum subsp. infantis in the human gut could be considered of potential health benefit. In conclusion, wild blueberry consumption resulted in a specific bifidogenic effect that could positively affect certain populations of bifidobacteria with demonstrated health-promoting properties.

Disinfectants have been used in a variety of environmental applications, in products for personal care and in the food industry. The food industry has increased the use of biocides and chemical-based disinfectants to control microbial ecology at production sites in an effort to improve hygiene measures and food safety. However, the susceptibility profile of micro-organisms to disinfectants has been largely neglected. This study therefore aimed to provide this type of information by focusing on the four most commonly used biocides in the food industry, determining their minimum inhibitory concentrations (MICs) and analysing the distribution of MICs across a variety of micro-organisms. In total, 99 different strains of Bifidobacterium spp. were studied. Results showed a unimodal distribution of MICs for chlorhexidine, triclosan (Irgasan) and sodium hypochlorite with no apparent species-specific correlation. Conversely, part of the tested bifidobacteria population (20%) showed reduced susceptibility to benzalkonium chloride compared with the susceptibility exhibited by the majority of the tested bacterial community. The highest MICs were distributed among almost all of the considered Bifidobacterium spp. In generally, the sensitivity of the studied strains to the four tested biocides appeared to be a genus-related trait.

Bifidobacterium bifidum MIMBb75 is a recently identified probiotic. However, its distribution along the intestine and impact on resident microbiota is unknown. Herein, we established a quantitative real-time polymerase chain reaction assay targeting the B. bifidum-specific BopA region for the quantification of B. bifidum in feces and used this assay to investigate transit of B. bifidum MIMBb75 through the murine intestine. We also analyzed the consequential impact on resident microbial cohorts. C57BL/6J mice were daily gavaged with 0.2 mL of either sterile PBS or PBS containing 108 colony-forming units of B. bifidum MIMBb75 for 2 weeks, after which intestinal contents and fecal samples were analyzed for microbial compositional changes. Bifidobacterium bifidum MIMBb75 was able to transiently colonize the murine intestine, with the predominant niche being the ceco-proximal colonic region. Region-specific effects on host microbiota were observed including decreased levels of Clostridium coccoides in the cecum, increased levels of bifidobacteria in the proximal and distal colon, total bacteria and Clostridium leptum in the proximal colon, and of C. coccoides in the feces. These findings suggest that probiotic properties of B. bifidum MIMBb75 may partially depend on its ability to at least transiently colonize the intestine and impact on the resident microbial communities at various intestinal loci.

We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982-7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3×102CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance.

Wild blueberries are a rich source of polyphenols and other compounds that are highly metabolized by the intestinal microbiota and may, at the same time, affect the intestinal environment itself. A repeated-measure, crossover dietary intervention on human volunteers was designed to study the effect of six week consumption of a wild blueberry (Vaccinium angustifolium) drink, versus a placebo drink, in modulating the intestinal microbiota. Relative to total eubacteria, Bifidobacterium spp. significantly increased following blueberry treatment (P ≥ 0.05), while Lactobacillus acidophilus increased after both treatments (P ≥ 0.05). No significant differences were observed for Bacteroides spp., Prevotella spp., Enterococcus spp., and Clostridium coccoides. Bifidobacteria, which have been largely proposed to be of benefit for the host, appeared to be selectively favored suggesting an important role for the polyphenols and fiber present in wild blueberries. Results obtained suggest that regular consumption of a wild blueberry drink can positively modulate the composition of the intestinal microbiota.

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B. bifidum to only restricted distal sites of the gut.

Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO(2) metabolism of S. thermophilus DSM 20617(T), grown in either a N(2) atmosphere or an enriched CO(2) atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO(2) depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO(2) depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO(2) availability. In agreement with proteomic data, cells grown under N(2) showed a significantly higher milk acidification rate compared with those grown in an enriched CO(2) atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO(2) to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role Of CO(2) in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.