Genomic and Genetic Characterization of the Bile Stress Response of Probiotic Lactobacillus reuteri ATCC 55730

Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to the low pH in the stomach and bile in the small intestine. The probiotic microorganism Lactobacillus reuteri ATCC 55730 has previously been shown to survive in the human small intestine. To address how L. reuteri can resist bile stress, we performed microarray experiments to determine gene expression changes that occur when the organism is exposed to physiological concentrations of bile. A wide variety of genes that displayed differential expression in the presence of bile indicated that the cells were dealing with several types of stress, including cell envelope stress, protein denaturation, and DNA damage. Mutations in three genes were found to decrease the strain's ability to survive bile exposure: lr1864, a Clp chaperone; lr0085, a gene of unknown function; and lr1516, a putative esterase. Mutations in two genes that form an operon, lr1584 (a multidrug resistance transporter in the major facilitator superfamily) and lr1582 (unknown function), were found to impair the strain's ability to restart growth in the presence of bile. This study provides insight into the possible mechanisms that L. reuteri ATCC 55730 may use to survive and grow in the presence of bile in the small intestine.     

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.     

Cholic Acid Induces a Cftr Dependent Biliary Secretion and Liver Growth Response in Mice

The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr^-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous) or chronic (three weeks via the diet). In Cftr^-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr^-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr^-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr^-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr^-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr^-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr^-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.     

Isolation of Saccharomyces cerevisiae strains from different food matrices and their preliminary selection for a potential use as probiotics

Aims: To isolate acid‐ and bile‐resistant Saccharomyces cerevisiae strains directly from food samples and to preliminarily select them on the basis of fundamental probiotic properties. Methods and Results: A rapid screening method allowed the isolation and selection of 20 acid‐ and bile‐resistant yeasts from foods, avoiding time‐consuming isolation steps. The strains were characterized for their specific survival in simulated gastric juice and in intestinal fluid after pre‐exposure at low pH. Ten isolates demonstrated a satisfactory survival percentage in intestinal fluid after pre‐exposure to gastric juice and appreciable lipolytic and proteolytic properties, as demonstrated by the API‐ZYM test. By using molecular methods five strains were identified as Saccharomyces cerevisiae, three as Candida spp., one as Candida pararugosa and one as Pichia spp. The Saccharomyces cerevisiae strains showed considerable probiotic properties, achieving a 80< % <90 survival through the simulated gastrointestinal tract, as well as interesting glucosidase activities. Conclusions: The research represents an efficient strategy to select and identify Saccharomyces cerevisiae strains with desirable acid and bile resistances. Significance and Impact of the Study: This paper reports the direct selection of potentially probiotic yeasts from foods and provides indications about the ability of Saccharomyces cerevisiae strains to survive conditions simulating the human gastrointestinal tract.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Transfer of heavy metals from sediment to fish,and their biliary excretion

Uptake and biliary excretion of metals were studied in rainbow trout, Oncorhynchus mykiss, exposed through spiked sediment to a mixture of seven heavy metals. Metal concentrations and toxicity of bile and blood plasma were used as indicators of exposure. Among the seven metals (Cd, Cr, Cu, Hg, Ni, Pb, and Zn) only three (Cu, Hg, and Pb) were concentrated in the bile (bile-plasma ratio >1). Bile-plasma ratios in the rainbow trout were similar to those found in rats for Cu and Hg. Daphnia magna bioassays were used to determine toxicity of bile and blood plasma in the same trout. Toxicity of bile and blood plasma increased after treatment with acid. An analysis of variance (ANOVA) showed that toxicity of bile and blood plasma to D. magna in metal-exposed trout was significantly correlated with (1) bile and blood plasma test concentration, (2) acid treatment of bile and blood plasma (hydrolysis of metal-plasma and metal-bile complexes) and (3) sediment concentration of metals during exposure of trout. In order to significantly detect the magnitude of the exposure to a xenobiotic the biomarker must respond in a dose- or time-dependent manner. Therefore, the potential use of bile toxicity as a biomarker of heavy metal exposure in fish is probably limited by the low bioconcentration of many of these toxicants in bile.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Estrogen receptor alpha mediates 17alpha-ethynylestradiol causing hepatotoxicity

Estrogens are known to cause hepatotoxicity such as intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal replacement therapy. Enterohepatic nuclear receptors including farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive active/androstane receptor (CAR) are important in maintaining bile acid homeostasis and protecting the liver from bile acid toxicity. However, no nuclear receptor has been implicated in the mechanism for estrogen-induced hepatotoxicity. Here Era(-/-), Erb(-/-), Fxr(-/-), Pxr(-/-), and Car(-/-) mice were employed to show that Era(-/-) mice were resistant to synthetic estrogen 17alpha-ethynylestradiol (EE2)-induced hepatotoxicity as indicated by the fact that the EE2-treated Era(-/-) mice developed none of the hepatotoxic phenotypes such as hepatomegaly, elevation in serum bile acids, increase of alkaline phosphatase activity, liver degeneration, and inflammation. Upon EE2 treatment, estrogen receptor alpha (ERalpha) repressed the expression of bile acid and cholesterol transporters (bile salt export pump (BSEP), Na(+)/taurocholate cotransporting polypeptide (NTCP), OATP1, OATP2, ABCG5, and ABCG8) in the liver. Consistently, biliary secretions of both bile acids and cholesterol were markedly decreased in EE2-treated wild-type mice but not in the EE2-treated Era(-/-) mice. In addition, ERalpha up-regulated the expression of CYP7B1 and down-regulated the CYP7A1 and CYP8B1, shifting bile acid synthesis toward the acidic pathway to increase the serum level of beta-muricholic acid. ERbeta, FXR, PXR, and CAR were not involved in regulating the expression of bile acid transporter and biosynthesis enzyme genes following EE2 exposure. Taken together, these results suggest that ERalpha-mediated repression of hepatic transporters and alterations of bile acid biosynthesis may contribute to development of the EE2-induced hepatotoxicity.     

Detection of virulence-related genes in <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> and their expression in response to different conditions

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.     

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: Role of the Akt/mTOR survival pathway and Bcl-2 family proteins

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.     

Bile salt-induced calcium fluxes in artificial phospholipid vesicles

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.     

Adaptation and preadaptation of Salmonella enterica to Bile

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS(-) mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10(-6) and 10(-7) per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations.