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Bifidobacterium species are known for their ability to inhabit various habitats and are often regarded as the first colonisers of the human gut. In the present work, we have used comparative genomics to identify conserved genomic signatures specific to Bifidobacterium species associated with the human gut. Our approach discovered five genomic signatures with varying lengths and confidence. Among the predicted five signatures, a 1790 bp multi-drug resistance (MDR) signature was found to be remarkably specific to only those species that can colonise the human gut. The signature codes for a membrane transport protein belonging to the major facilitator superfamily (MFS) generally involved in MDR. Phylogenetic analyses of the MDR signature suggest a lineage-specific evolution of the MDR signature in bifidobacteria colonising the human gut. Functional annotation led to the discovery of two conserved domains in the protein; a catalytic MFS domain involved in the efflux of drugs and toxins, and a regulatory cystathionine-β-synthase (CBS) domain that can interact with adenosyl-carriers. Molecular docking simulation performed with the modelled tertiary structure of the MDR signature revealed the putative functional role of the covalently linked domains. The MFS domain displayed a high affinity towards various protein synthesis inhibitor antibiotics and human bile acids, whereas the C-terminally linked CBS domain exhibited favourable binding with molecular structures of ATP and AMP. Therefore, we believe that the predicted signature represents a niche-specific survival trait involved in bile and antibiotic resistance, imparting an adaptive advantage to the Bifidobacterium species colonising the human gut. [ABSTRACT FROM AUTHOR]

Prior research has suggested that the use of organic acids in the food industry may unintentionally enhance pathogenicity of Listeria monocytogenes strain N1-227 and R2-499. This study explored the connection between habituation to L-lactic acid or acetic acid and virulence in L. monocytogenes strains N1-227 and R2-499 using selected gene expression analysis and the in vivo Galleria mellonella wax worm model for infection. Expression of transcription factors (sigB and prfA) and genes related to acid resistance (gadD2, gadD3, and arcA) and bile resistance (bsh and bilE) or to virulence (inlA, inlB, hly, plcA, plcB, uhpT, and actA) was investigated by quantitative real-time PCR (qRT-PCR), while in vivo virulence was assessed by following the lethal time to 50% population mortality (LT50) of G. mellonella larvae after injection of untreated and habituated L. monocytogenes. Twenty minutes of habituation to the organic acids at pH 6.0 significantly increased expression of key acid and bile stress response genes in both strains, while expression of virulence genes was strain-dependent. The expression of transcription factor sigB was strain-dependent and there was no significant change in the expression of transcription factor prfA in both strains. Habituation to acid increased virulence of both strains as evidenced by decreased LT50 of G. mellonella larvae injected with Listeria habituated to either acid. In summary, habituation of both L. monocytogenes strains to organic acids up-regulated expression of several stress and virulence genes and concurrently increased virulence as measured using the G. mellonella model.

Prior research has suggested that the use of organic acids in the food industry may unintentionally enhance pathogenicity of Listeria monocytogenes strain N1-227 and R2-499. This study explored the connection between habituation to L-lactic acid or acetic acid and virulence in L. monocytogenes strains N1-227 and R2-499 using selected gene expression analysis and the in vivo Galleria mellonella wax worm model for infection. Expression of transcription factors (sigB and prfA) and genes related to acid resistance (gadD2, gadD3 , and arcA) and bile resistance (bsh and bilE) or to virulence (inlA, inlB, hly, plcA, plcB, uhpT , and actA) was investigated by quantitative real-time PCR (qRT-PCR), while in vivo virulence was assessed by following the lethal time to 50% population mortality (LT50) of G. mellonella larvae after injection of untreated and habituated L. monocytogenes. Twenty minutes of habituation to the organic acids at pH 6.0 significantly increased expression of key acid and bile stress response genes in both strains, while expression of virulence genes was strain-dependent. The expression of transcription factor sigB was strain-dependent and there was no significant change in the expression of transcription factor prfA in both strains. Habituation to acid increased virulence of both strains as evidenced by decreased LT50 of G. mellonella larvae injected with Listeria habituated to either acid. In summary, habituation of both L. monocytogenes strains to organic acids up-regulated expression of several stress and virulence genes and concurrently increased virulence as measured using the G. mellonella model. [ABSTRACT FROM AUTHOR]

Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA− and ZapB− mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.

Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA− and ZapB− mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile. [ABSTRACT FROM AUTHOR]

Bile acids (BA), the major components of bile, are biological detergents that facilitate the emulsification and solubilization of dietary lipids and also display potent antimicrobial activity, the bacterial membranes being their main targets. Considering the complicated nature of the stress produced by bile and BA, the microorganism tolerance requires different defence mechanisms including the presence of efflux pumps, bile salt hydrolase (BSH) enzyme, the intrinsic capacity of cells to maintain intracellular homeostasis and modifications in the architecture and composition of the cell membrane. Besides, the expression of proteins involved in carbohydrate and fatty acid metabolism, amino acid and nitrogenous base biosynthesis, and general stress response are commonly affected by the presence of bile. Among the microbial transformations, deconjugation of BA by BSH is the most important. Several studies indicate that BSH activity affects both the host physiology and the microbiota. In fact, it was strongly suggested that BSH could play an important role in the colonization and survival of bacteria in the gut. Also, recent work has shown that BSH and free BA participate in a variety of metabolic processes that include regulation of dietary lipid absorption, cholesterol metabolism, and energy and inflammation homeostasis. In this review we summarize recent advances in the understanding of the mechanisms involved in the tolerance of bacteria to bile, with special emphasis on the contributions of studies applying an omic approach. Besides, the physiological and ecological role of BSH enzyme and its relevance to human health as well as the function of bile acid as metabolic regulator are also discussed. [ABSTRACT FROM AUTHOR]

Probiotics and prebiotics play important role in improvement of the intestinal microbial balance and have a great potential for food industry and public health. Survival of probiotics during gastric stress is influenced by the prebiotic carriers. In order to introduce effective synbiotic pairs, in this study the prebiotic influence of inulin (extracted from chicory and Jerusalem artichoke and also standard lab grade inulin), glucose and non-carbohydrate sample were assessed on bile resistance and inhibition of two Lactobacillus species against E.coli O157:H7. The effect of inulin treatments were studied on growth and viability of Lactobacillus casei PTCC1608 and Lactobacillus rhamnosus PTCC 1637under various bile salts concentrations (0, 0.3, 0.5, 1, 1.5%) in completely randomized factorial design and the inhibition effects were evaluated using well- diffusion technique in completely randomized design, and compared with controls. Results indicated that extracted inulin could be effective on enhancing the bile salts resistance of two lactobacilli. This effect was decreased by increasing in bile concentration while longer inulin chains had less effect compared to glucose. Although the most antimicrobial effects belonged to glucose, standard and chicory inulin showed acceptable antimicrobial effects as well. Inulin from Jerusalem artichoke with the highest degree of polymerization showed the least effects.

Plant extracts with probiotics have been gaining ground in the functional food market. The present study aimed to evaluate the physicochemical, microbiological and sensory characteristics of probiotic fermented drink made of mixed extract of soy and rice byproducts with added waxy corn starch during cold storage (5 °C for 28 days). Furthermore, the probiotic resistance under conditions similar to the digestive tract was evaluated. Slight physicochemical and colour changes were noted in the beverage during the storage period, but these changes did not affect product quality. The sample remained within acceptable microbiological parameters, and sensory scores were higher than 3.0, within the predefined quality threshold. Fermenting microorganisms presented reduced viability during the storage period. Lactobacillus acidophilus and Bifidobacterium spp. resisted the tested gastrointestinal conditions. The product showed a 28-day shelf life, but the probiotic effect lasted only14 days. Further studies should evaluate beverage supplementation with components that could improve the viability of probiotic cultures, in order to maintain their probiotic effect for a longer time. Additionally, consumer studies should be carried out in order to assess current products' acceptance by target consumers. [ABSTRACT FROM AUTHOR]

BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC) importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related compounds have failed to demonstrate transport mediated by BilE. The putative substrate-binding domain (SBD) of BilE was expressed in isolation and the crystal structure solved at 1.5 Å. Although the overall fold is characteristic of SBDs, the binding site varies considerably relative to the well-characterized homologs ProX from Archaeoglobus fulgidus and OpuBC and OpuCC from Bacillus subtilis. This suggests that BilE may bind an as-yet unknown ligand. Elucidation of the natural substrate of BilE could reveal a novel bile resistance mechanism. [ABSTRACT FROM AUTHOR]

Live Lactobacillus brevis KB290 have several probiotic activities, including immune stimulation and modulation of intestinal microbial balance. We investigated the adaptation of L. brevis KB290 to bile as a mechanism of intestinal survival. Strain KB290 was grown for 5 days at 37 °C in tryptone–yeast extract–glucose (TYG) broth supplemented with 0.5% sodium acetate (TYGA) containing 0.15%, 0.3%, or 0.5% bile. Growth was determined by absorbance at 620 nm or by dry weight. Growth was enhanced as the broth's bile concentration increased. Bile-enhanced growth was not observed in TYG broth or with xylose or fructose as the carbon source, although strain KB290 could assimilate these sugars. Compared with cells grown without bile, cells grown with bile had twice the cell yield (dry weight) and higher hydrophobicity, which may improve epithelial adhesion. Metabolite analysis revealed that bile induced more lactate production by glycolysis, thus enhancing growth efficiency. Scanning electron microscopy revealed that cells cultured without bile for 5 days in TYGA broth had a shortened rod shape and showed lysis and aggregation, unlike cells cultured for 1 day; cells grown with bile for 5 days had an intact rod shape and rarely appeared damaged. Cellular material leakage through autolysis was lower in the presence of bile than in its absence. Thus lysis of strain KB290 cells cultured for extended periods was suppressed in the presence of bile. This study provides new role of bile and sodium acetate for retaining an intact cell shape and enhancing cell yield, which are beneficial for intestinal survival. [ABSTRACT FROM AUTHOR]

This study evaluated the effect of soy lecithin on the bile resistance of Lactobacillus plantarum. Six strains were cultured in MRS broth supplemented with soy lecithin at different concentrations. The strains incubated in MRS broth with 1·0% soy lecithin showed no inhibitory effect on cell growth. After culturing in MRS broth with 0·2-1·0% soy lecithin, the survival rate of harvested cells increased significantly ( P < 0·05) in the 0·3% bile challenge compared with the no added soy lecithin group. The cells incubated with 0·6% soy lecithin were able to grow in an MRS broth with a higher bile salt content. The surface hydrophobicity and cell leakage in the bile challenge were assessed to reveal the physical changes caused by the addition of soy lecithin. The cell surface hydrophobicity was enhanced and the membrane integrity in the bile challenge increased after culturing with soy lecithin. A shift in the fatty acid composition was also observed, illustrating the cell membrane change in the soy lecithin culture. Significance and Impact of the Study In this study, we report for the first time the beneficial effect of adding soy lecithin to an MRS broth on subsequent bile tolerance of Lactobacillus plantarum. Soy lecithin had no inhibitory effect on strain viability but significantly enhanced bile resistance. Surface hydrophobicity and cell integrity increased in strains cultured with soy lecithin. The observed shift in the cell fatty acid composition indicated changes to the cell membrane. As soy lecithin is safe for use in the food industry, its protective effects can be harnessed for the development of bile-sensitive strains with health-benefit functions for use in probiotic products. [ABSTRACT FROM AUTHOR]

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Most of the probiotic bacteria currently available in the market belong to the genera Lactobacillus and Bifidobacterium, and specific health-promoting activities, such as treatment of diarrhea or amelioration of gastrointestinal discomfort, have been attributed to them. In order to be able to survive the gastrointestinal transit and transiently colonise our gut, these bacteria must be able to counteract the deleterious action of bile salts, which are the main components of bile. Bile salts are detergent-like biological substances synthesised in the liver from cholesterol. Host enzymes conjugate the newly synthesised free bile acids in the liver with the amino acids glycine or taurine, generating conjugated bile salts. These compounds are stored in the gall bladder and they are released into the duodenum during digestion to perform their physiological function, which is the solubilisation of fat coming from diet. These bile salts possess strong antimicrobial activity, since they are able to disorganize the structure of the cell membrane, as well as trigger DNA damage. This means that bacteria inhabiting our intestinal tract must have intrinsic resistance mechanisms to cope with bile salts. To do that, Lactobacillus and Bifidobacterium display a variety of proteins devoted to the efflux of bile salts or protons, to modify sugar metabolism or to prevent protein misfolding. In this manuscript, we review and discuss specific bile resistance mechanisms, as well as the processes responsible for the adaptation of bifidobacteria and lactobacilli to bile.

BilE has been reported as a bile resistance determinant that plays an important role in colonization of the gastrointestinal tract by Listeria monocytogenes, the causative agent of listeriosis. The mechanism(s) by which BilE mediates bile resistance are unknown. BilE shares significant sequence similarity with ATP-binding cassette (ABC) importers that contribute to virulence and stress responses by importing quaternary ammonium compounds that act as compatible solutes. Assays using related compounds have failed to demonstrate transport mediated by BilE. The putative substrate-binding domain (SBD) of BilE was expressed in isolation and the crystal structure solved at 1.5 Å. Although the overall fold is characteristic of SBDs, the binding site varies considerably relative to the well-characterized homologs ProX from Archaeoglobus fulgidus and OpuBC and OpuCC from Bacillus subtilis. This suggests that BilE may bind an as-yet unknown ligand. Elucidation of the natural substrate of BilE could reveal a novel bile resistance mechanism.

Abstract: We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH7.4, or in media at pH6.0 containing 0 (HCl control) or 4.75mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1h in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p <0.05) and meaningful (≥1log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains suggesting that exposure to organic acid anions may enhance virulence in L. monocytogenes. [Copyright &y& Elsevier]

Background: Enterococcus faecium is a Gram-positive commensal bacterium of the mammalian intestinal tract. In the last two decades it has also emerged as a multi-resistant nosocomial pathogen. In order to survive in and colonize the human intestinal tract E. faecium must resist the deleterious actions of bile. The molecular mechanisms exploited by this bacterium to tolerate bile are as yet unexplored. Results: In this study we used a high-throughput quantitative screening approach of transposon mutant library, termed Microarray-based Transposon Mapping (M-TraM), to identify the genetic determinants required for resistance to bile salts in E. faecium E1162. The gene gltK, which is predicted to encode a glutamate/aspartate transport system permease protein, was identified by M-TraM to be involved in bile resistance. The role of GltK in bile salt resistance was confirmed by the subsequent observation that the deletion of gltK significantly sensitized E. faecium E1162 to bile salts. To further characterize the response of E. faecium E1162 to bile salts, we performed a transcriptome analysis to identify genes that are regulated by exposure to 0.02% bile salts. Exposure to bile salts resulted in major transcriptional rearrangements, predominantly in genes involved in carbohydrate, nucleotide and coenzyme transport and metabolism. Conclusion: These findings add to a better understanding of the molecular mechanisms by which E. faecium responds and resists the antimicrobial action of bile salts. [ABSTRACT FROM AUTHOR]

Little is known about the bacteria that reside in the human gallbladder and the mechanisms that allow them to survive within this harsh environment. Here we describe interactions between two strains from a human bile sample, one Ruminococcus gauvreauii (IPLA60001), belonging to the Lachnospiraceae family, and the other, designated as Ruminococcoides bili (IPLA60002 T ; DSM 110008) most closely related to Ruminococcus bromii within the family Ruminococcaceae. We provide evidence for bile salt resistance and sporulation for these new strains. Both differed markedly in their carbohydrate metabolism. The R. bili strain mainly metabolized resistant starches to form formate, lactate and acetate. R. gauvreauii mainly metabolized sugar alcohols, including inositol and also utilized formate to generate acetate employing the Wood Ljungdahl pathway. Amino acid and vitamin biosynthesis genomic profiles also differed markedly between the two isolates, likely contributing to their synergistic interactions, as revealed by transcriptomic analysis of cocultures. Transcriptome analysis also revealed that R. gauvreauii IPLA60001 is able to grow using the end-products of starch metabolism formed by the R. bili strain such as formate, and potentially other compounds (such as ethanolamine and inositol) possibly provided by the autolytic behavior of R. bili. IMPORTANCE Unique insights into metabolic interaction between two isolates; Ruminococcus gauvreauii IPLA60001 and Ruminococcoides bili IPLA60002, from the human gallbladder, are presented here. The R. bili strain metabolized resistant starches while R. gauvreauii failed to do so but grew well on sugar alcohols. Transcriptomic analysis of cocultures of these strains, provides new data on the physiology and ecology of two bacteria from human bile, with a particular focus on cross-feeding mechanisms. Both biliary strains displayed marked resistance to bile and possess many efflux transporters, potentially involved in bile export. However, they differ markedly in their amino acid catabolism and vitamin synthesis capabilities, a feature that is therefore likely to contribute to the strong synergistic interactions between these strains. This is therefore the first study that provides evidence for syntrophic metabolic cooperation between bacterial strains isolated from human bile.

toxR, a conserved virulence-associated gene in vibrios, is identified in Vibrio alginolyticus ZJ51-O, a pathogenic strain isolated from diseased fish. To reveal the role of ToxR in the pathogenicity of V. alginolyticus, a deletion mutant was constructed by allelic exchange. The mutant showed the same level of growth in trypticase soy broth (TSB) and iron-limiting condition, as the wild type strain. However, deletion of toxR severely reduced resistance against bile salts and the capability of biofilm formation. Outer-membrane protein (OMP) analysis showed that a 37-kD protein was absent and a 43-kD protein was decreased in the mutant. By MS/MS, the two proteins are identified as the homologues of OmpT and OmpN, respectively. These data suggest that ToxR might have enhanced the bile resistance and biofilm formation through modulating the production of OMP without affecting the ability of iron acquisition and the virulence to the fish via injection. These results indicate that ToxR may assist V. alginolyticus to colonize on the surface of the fish intestine which is crucial for the initiation of the infection, though it may not be involved in the proliferation of the bacteria in the host tissue. [ABSTRACT FROM AUTHOR]

Abstract: Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics were complemented with physiological studies on both strains focusing on glycolytic end-products, the ability to adhere to the human intestinal epithelial cell line HT29-MTX and survival to simulated gastrointestinal conditions. Protein pattern comparison of strains grown with and without bile allowed us to identify 9 different proteins whose production was regulated by bile in both strains, and 17 proteins that showed differences in their levels between the parental and the bile-resistant derivative. These included general stress response chaperones, proteins involved in transcription and translation, in peptidoglycan/exopolysaccharide biosynthesis, in the lipid and nucleotide metabolism and several glycolytic and pyruvate catabolism enzymes. Differences in the level of metabolic end-products of the sugar catabolism were found between the strains 200 and 200+. A decrease in the adhesion of both strains to the intestinal cell line was detected in the presence of bile. In simulated gastric and intestinal juices, a protective effect was exerted by milk improving the survival of both microorganisms. These results indicate that bile tolerance in L. delbrueckii subsp. lactis involves several mechanisms responding to the deleterious impact of bile salts on bacterial physiology. [ABSTRACT FROM AUTHOR]

Abstract: Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that altering their fatty acid composition (i.e. increased hexadecanoic acid and octadecenoic acid and decreased hexadecenoic acid and C-19 cyclopropane fatty acid) by changing growth conditions may be a useful way to enhance their bile resistance in lactococci. [Copyright &y& Elsevier]

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na+/H+ antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na+ and Li+ as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li+, Na+, and K+ ions in pH-dependent manner with maximal activity at pH 9.0–9.5. Exchange was electrogenic (more than one H+ translocated per cation moved in opposite direction). The apparent Km at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li+, Na+, and K+, respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Our aim was to obtain derivatives of non-intestinal lactobacilli—commonly used in the dairy industry—able to grow under physiological concentrations of bile salts. Six resistant derivatives (five from Lactobacillus delbrueckii subsp. lactis and one from L. acidophilus) growing in the presence of 0.5% bile salts were obtained from 24 strains of L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis and L. helveticus, and also one strain of L. acidophilus with low resistance to bile salts. Carbohydrate fermentation profiles remained unchanged but the rate of fermentation of several sugars was generally slower in bile-resistant derivatives. No significant differences with respect to the parent strains were observed for the cell-wall hydrophobicity, capacity to grow in milk and survival during frozen storage. Some bile-resistant derivatives were also resistant to low pH, but in other strains this capacity was acquired following progressive adaptation to low pH. Adaptation to bile salts and low pH might be valuable tools for increasing the survival under gastrointestinal conditions of non-intestinal lactobacilli used for the manufacture of dairy products. [Copyright &y& Elsevier]

Abstract: Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab1, YSD V and Ib3) and four spontaneous phage-resistant mutants (strains A7, A17, V2 and I39) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab1, as well as from its spontaneous phage-resistant mutants A7 and A17. Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A7). After progressive ox gall adaptation, the phage-resistant mutant A7 also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A7 0.9) became sensitive to the phage, but derivatives with low (A7 0.3) and intermediate (A7 0.6) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three (L. delbrueckii Ab1 and their phage-resistant mutants A7 and A17) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods. [Copyright &y& Elsevier]

Antibiotic-resistant enterococci are major causes of hospital- acquired infections. The emergence of Enterococcus faecalis as a significant nosocomial pathogen is a consequence of its inherent resistance to certain antibiotics and of its ability to survive and proliferate in the intestinal tract. Genetic determinants of E. faecalis conferring these properties are largely unknown. Here we show that PrkC, a one-component signaling protein containing a eukaryotic-type Ser/Thr kinase domain, modulates inherent anti-microbial resistance and intestinal persistence of E. faecalis. An E. faecalis mutant lacking PrkC grows at a wild-type rate in the absence of antimicrobial stress but exhibits enhanced sensitivity to cell-envelope-active compounds, including antibiotics that target cell-wall biogenesis and bile detergents. Consistent with its bile sensitivity, the mutant was also impaired at persistence in the intestine of mice. Thus, PrkC regulates key physiological processes in E. faecalis associated with its success as a nosocomial pathogen. The predicted domain architecture of PrkC comprises a cytoplasmic kinase domain separated by a transmembrane segment from extracellular domains thought to bind uncross-linked peptidoglycan, suggesting that PrkC is a transmembrane receptor that monitors the integrity of the E. faecalis cell wall and mediates adaptive responses to maintain cell-wall integrity. Given its role in modulating traits of E. faecalis important for its ability to cause nosocomial infections, we suggest that the one-component signaling protein PrkC represents an attractive target for the development of novel therapies to prevent infections by antibiotic-resistant enterococci. [ABSTRACT FROM AUTHOR]

Abstract: The ability to inhibit the adhesion and to displace selected pathogens from human intestinal mucus of two Bifidobacterium strains with acquired resistance to bile, were assessed and compared with those of their bile sensitive original strains. A preliminary characterization of the macromolecules involved in the adhesion was also carried out. The inhibition of adhesion and the displacement of enteropathogens previously adhered were found to be specific, depending on the strains used. The cholate-resistant strain Bifidobacterium bifidum M6dCo, that adhered more to mucus than its original, was able to inhibit the adhesion and to displace pathogens from mucus significantly more than its original cholate-sensitive strain B. bifidum M6. Contrary to this, two strains showing similar adhesion levels, B. bifidum A1 and its bile resistant derivative B. bifidum A1dOx, did not display any differences. Different molecules appear to be involved in the adhesion of the strains B. bifidum M6 and B. bifidum M6dCo. These differences in the cellular surface may explain the differences in competitive exclusion observed between both strains. [Copyright &y& Elsevier]

Abstract: The hydrophobicity and capacity to adhere to human intestinal mucus of Bifidobacterium strains with acquired resistance to bile were assessed and compared with those of their more sensitive original strains. The resistant variants used were previously obtained [Int. J. Food Microbiol. 82 (2003) 191; Int. J. Food Microbiol. 94 (2004) 79] by progressive adaptation of originally more sensitive strains to gradually increasing concentrations of bile. In five out of the seven groups of original and bile-resistant variants tested the resistant strains showed higher adhesion levels to human mucus (range between 1.4- and 4-fold) than their corresponding original strains. However, in the presence of physiologic concentrations of bile (0.3%, w/v) the adhesion level of all Bifidobacterium strains dropped between 7% and 74%, depending on the strain. In spite of this, the adhesion capability of three bile-resistant variants remained higher than that of their originals. Hydrophobicity evidenced considerable variability; in four out of the seven bile-resistant strains it was higher than in the original strains, although no direct correlation between adhesion and hydrophobicity could be established. It was concluded that the acquisition of bile resistance by our Bifidobacterium strains promoted changes in hydrophobicity and in the adhesion of these microorganisms to human intestinal mucus. [Copyright &y& Elsevier]

Six derivatives with increased resistance to ox gall (MIC: ≥1% w/v) and one derivative resistant to sodium cholate (MIC: 0.8% w/v) were obtained from more sensitive original Bifidobacterium strains. These microorganisms, and two additional cholate resistant derivatives obtained in a previous study (Int. J. Food Microbiol. 82 (2003) 191), were partially characterised in this study. Acquisition of resistance against a given bile salt, also conferred cross-resistance to other bile salts, and promoted an increase in the survival of these microorganisms at low pH. Bile resistance levels of derivatives were dependent on the external pH so that the resistance was lower at neutral pH values than in acidic environments. In addition, the acquisition of bile resistance induced changes on glycoside-hydrolysing activities of derivatives obtained from five out of eight original strains, with certain activities such as β-glucosidase showing more than tenfold increases in some of these microorganisms. These data suggest that the exposure to high bile salts concentrations may have induced a synergic response on Bifidobacterium for the adaptation to the conditions of the gastrointestinal tract. This could have improved the survival at low pH in these microorganisms, the resistance to high bile salts concentrations, and the assimilation of non-digestible carbohydrates by the enhancement of some glycoside-hydrolysing activities. [Copyright &y& Elsevier]

The susceptibility levels against bile salts (ox gall) and sodium cholate of 19 Bifidobacterium strains from different origins ranged between 0.125% and 2.0% and 0.05% and 0.4%, respectively. Seven of these strains were subsequently selected for the isolation of sodium cholate resistant derivatives by exposure to gradually increasing concentrations of this compound. Derivatives resistant to at least 1.2% of cholate were obtained from Bifidobacterium bifidum CECT 4549 and B. bifidum M6. Further analysis of the CECT 4549 derivative indicated that the phenotype induced was stable; the enzymatic activities analysed remained unchanged and no major rearrangements of chromosomal DNA were produced. However, the cells of the resistant derivative were smaller and more regular in average size than those of the original strain. In addition, the derivative had lost the ability to use lactose, fructose and galactose, and showed reduced expression levels of two membrane proteins of 78 and 114 kDa. Finally, the resistant strain displayed higher survival and lower growth inhibition in the presence of cholate than the original strain. [Copyright &y& Elsevier]

Introduction. Salmonella enterica serovar Typhi ( S . Typhi) is the etiological agent of typhoid fever. To establish an infection in the human host, this pathogen must survive the presence of bile salts in the gut and gallbladder. Hypothesis. S . Typhi uses multiple genetic elements to resist the presence of human bile. Aims. To determine the genetic elements that S . Typhi utilizes to tolerate the human bile salt sodium deoxycholate. Methodology. A collection of S . Typhi mutant strains was evaluated for their ability to growth in the presence of sodium deoxycholate and ox-bile. Additionally, transcriptomic and proteomic responses elicited by sodium deoxycholate on S . Typhi cultures were also analysed. Results. Multiple transcriptional factors and some of their dependent genes involved in central metabolism, as well as in cell envelope, are required for deoxycholate resistance. Conclusion. These findings suggest that metabolic adaptation to bile is focused on enhancing energy production to sustain synthesis of cell envelope components exposed to damage by bile salts.

Simple Summary: Ligilactobcillus salivarius is a Gram-positive bacterium that commonly colonizes the mucous membranes of the digestive tracts of humans and animals, including birds. It belongs to the group of lactic acid bacteria which, by producing lactic acid, acidify the intestinal environment and limit the development of undesirable intestinal microflora. In addition, L. salivarius can produce other antimicrobial substances, such as bacteriocins and hydrogen peroxide. Due to limiting the development of unfavourable microflora and other health-promoting effects, L. salivarius bacteria are considered as potential probiotics that may increase animal health, and thus animal production indicators. In this work, we undertook research on the characteristics of L. salivarius strains from chickens, geese, turkeys and domestic pigeons. We showed great variation in phenotypic and genotypic traits between strains and the evolutionary adaptation of L. salivarius strains to the colonization of a specific host. The results of the study contribute to knowledge of the characteristics of the species L. salivarius and may be useful in the selection of probiotic strains. Ligilactobacillus salivarius is an important member of the human and animal gut microbiota, and selected strains are promising probiotics, but knowledge of the characteristics of avian isolates is still limited. In this study, we examined selected phenotypic and genotypic traits of 33 L. salivarius strains from geese, chickens, turkeys and pigeons. The strains varied in terms of cell size, colony morphology, broth growth characteristics, biofilm formation, tolerance to bile, hydrophobicity and phenotypic and genotypic antibiotic resistance profiles. Large variation among strains was noted for the utilization of sorbitol, salicin, trehalose, rhamnose, inulin and N-acetyl-D-glucosamine. The presence of genes related to sugar metabolism, i.e., mipB, tktA, rhaB and LSL_1894, was not always correlated with the biochemical phenotypic profile. Correlations were recorded between the host and utilization of certain sugars as well as tolerance to bile. The repA-type megaplasmid and genes coding for Abp118 bacteriocin were detected in 94% and 51.5% of L. salivarius strains, respectively. Phylogeny based on groEL gene sequences was partly correlated with the origin of the strains and revealed an evolutionary distance between L. salivarius strains from humans and birds. The results of the study contribute to knowledge of the characteristics of the species L. salivarius. Intraspecies variations of L. salivarius strains may affect their ability to colonize specific niches and utilize nutrients and reveal potential strain-dependent effects on host health. [ABSTRACT FROM AUTHOR]

A strictly anaerobic, resistant starch-degrading, bile-tolerant, autolytic strain, IPLA60002 T , belonging to the family Ruminococcaceae , was isolated from a human bile sample of a liver donor without hepatobiliary disease. Cells were Gram-stain-positive cocci, and 16S rRNA gene and whole genome analyses showed that Ruminococcus bromii was the phylogenetically closest related species to the novel strain IPLA60002 T , though with average nucleotide identity values below 90 %. Biochemically, the new isolate has metabolic features similar to those described previously for gut R. bromii strains, including the ability to degrade a range of different starches. The new isolate, however, produces lactate and shows distinct resistance to the presence of bile salts. Additionally, the novel bile isolate displays an autolytic phenotype after growing in different media. Strain IPLA60002 T is phylogenetically distinct from other species within the genus Ruminococcus . Therefore, we propose on the basis of phylogenetic, genomic and metabolic data that the novel IPLA60002 T strain isolated from human bile be given the name Ruminococcoides bili gen. nov., sp. nov., within the new proposed genus Ruminococcoides and the family Ruminococcaceae . Strain IPLA60002 T (=DSM 110008 T =LMG 31505 T ) is proposed as the type strain of Ruminococcoides bili .

• The abundance of 129 genes and 34 proteins was significantly changed by bile. • The barrier function of cells and the active extrusion of bile were enhanced. • Enzymes responsible for protein refolding and antioxidant defense were induced. • Bile-adapted cells expanded the utilization profile of carbon sources in gut. Tolerance to bile stress is a crucial property for probiotics to survive in the gastrointestinal tract and exert their beneficial effects. In this work, transcriptomic analysis combined with two-dimensional electrophoresis revealed that the transcript levels of 129 genes and the abundance of 34 proteins were significantly changed in Lactobacillus salivarius Ren when exposed to 0.75 g/L ox-bile. Notably, carbohydrate metabolism shifted to the utilization of maltose and glycerol for energy production, suggesting that L. salivarius Ren expanded carbon sources profile for gut adaptation in response to bile. Moreover, the enzymes involved in cell surface charge modification and the cell envelope-located hemolysin-like protein were overproduced, which was supposed to hinder the penetration of bile. Then, the up-regulated ABC transporters could contribute to the extrusion of bile accumulated in the cytoplasm. Additionally, proteolytic system was activated to provide more amino acids for the synthesis and repair of proteins damaged by bile. Finally, γ-glutamylcysteine with antioxidant activity and oxidoreductases for redox homeostasis were increased to cope with the bile-induced oxidative stress. These findings provide new insights into the molecular mechanisms involved in bile stress response and adaptation in L. salivarius. [ABSTRACT FROM AUTHOR]

The milk fat globule membrane (MFGM) is a complex, highly conserved structure surrounding fat droplets secreted into mammalian milk. This study evaluated the impact of MFGM on Lactobacillus rhamnosus GG (LGG). MFGM-10 (2.5 g/L, 5 g/L, and 10 g/L) did not affect LGG growth in MRS medium but enhanced the ability of LGG to survive in the presence of 0.5% porcine bile. In the presence of MFGM-10 (5 g/L) and bile (0.5%), there were less complex polysaccharides in the media and less capsular polysaccharides associated with the LGG cells compared to the bile exposure alone ( p < 0.05). The expression of four EPS genes was modulated by bile stress and MFGM. Biofilm thickness was increased ( p < 0.05) during bile stress with MFGM compared to other treatments. Furthermore, MFGM increased LGG survival during transit in the murine GI tract. Future experiments will determine the impact of MFGM on LGG probiotic functionality.