The membrane integrity and viability of S. Image analysis revealed green fluorescence in cells with intact membrane and red fluorescence in cells with damaged membrane Supplementary Figure 1.
TABLE 2. Monitoring of membrane integrity of S. Untreated cells had a well-defined intact spherical shape with a smooth surface morphology Figure 6A. The cells exposed to CA and DCA had shrunken appearance and indented on the surfaces indicating possible leakage of intracellular contents.
Vesicles and blebs were also present on the surface Figures 6B,C. Surface morphology of S. Transmission electron microscopy demonstrated pronounced ultra-structural defects in bile salts treated cells when compared untreated cells.
The untreated cells revealed intact and well defined cell membrane, cell wall and midline septum with homogenous cytoplasm Figure 7A. Cell wall breaks, thinning and disintegration of cell wall, cell membrane breaks, and abnormal septation were noticed in the cells exposed to all four bile salts studied Figures 7B—E.
Cells exposed to CA and DCA had membrane enclosed and non-membrane enclosed mesosome like structures inside the cytoplasm Supplementary Figure 2 reflecting possible cell membrane damage and increased permeability. Ultra structural morphology of S.
White arrows represent mesosome like structures. White triangles represent ghost cells. Human bile salts in the intestine are an important facet of innate defense against enteric pathogens. They play an important role in maintaining indigenous microbiota and protection against enteric pathogens in the intestine Sung et al.
Reduced levels of bile salts in the intestine correlate with cases of bacterial overgrowth and translocation in the small intestine, resulting in endotoxemia in cirrhotic rats Lorenzo-Zuniga et al. Oral supplementation with bile salts in such rats can prevent small intestinal bacterial overgrowth and translocation Lorenzo-Zuniga et al. If one is to develop bile salts or related molecules as potential antibacterial therapy against pathogens, their mode of action needs to be established.
Up till now, knowledge on bactericidal action of bile salts on S. Here, we propose that bile salts can kill S. The accumulation of weak organic acids in the cytoplasm involves passive diffusion of the protonated form of weak acid across membranes, followed by its intracellular deprotonation and decrease in intracellular pH Salmond et al. Bile salts, due to their structural and chemical properties, are generally considered to be weak acids and were shown to decrease intracellular pH and dissipate transmembrane potential in lactobacilli and bifidobacteria Kurdi et al.
In this study, a dose-dependent decrease in the intracellular pH in the presence of bile salts was observed. This difference could be attributed to the higher pKa values of unconjugated bile salts 6.
Conjugated bile salts are stronger acids having lower pKa values Carey, , and at physiological pH 7 are effectively fully ionized, unable to cross cell membrane unless a specific transporter is available Begley et al.
Many antibacterial agents act by disrupting the cytoplasmic membrane resulting in loss of proton gradients and electrical potential across the membrane leading eventually to cell death Nelson et al.
Our results also showed a dose-dependent decrease in the transmembrane electrical potential of bile salt treated cells. Thus reduction in the intracellular pH and transmembrane potential suggest that a dissipation of proton motive force is involved in bile salt mediated growth inhibition. Our data also showed a significant fold difference between the MICs of CA and DCA, and their effect on internal pH and transmembrane electrical potential, which cannot simply be attributed to a difference in their pKa values Carey, ; Cantafora et al.
The reduction in the membrane integrity demonstrated by fluorescent dye staining of S. This hypothesis was strengthened by detection of significant leakage of intracellular potassium from S.
Previous research demonstrated a similar alteration in the membrane integrity in lactobacilli and bifidobacteria, following exposure to unconjugated bile salts Kurdi et al. Increased uptake of gentamicin in Lactobacillus plantarum Elkins and Mullis, and rifaximin in enterotoxigenic E.
Our study also showed a significant increase in the leakage of macromolecules such as proteins and nucleic acids in the presence of unconjugated bile salts at MIC and conjugated bile salts at 20 mM , suggesting substantial cell membrane damage occurred. Bile salts at higher concentrations rapidly dissolve membrane lipids and cause dissociation of membrane proteins.
This rapid solubilization effect results in leakage of cellular contents and cell death Noh and Gilliland, Subtle effects on membrane permeability and fluidity, including altered membrane bound enzyme activities and increased transmembrane flux of divalent cations, are found at low or sub-micellar concentrations Coleman et al.
Several electron microscopic studies showed cell surface damage and interior deformities in bacterial cells caused by bile salts Ruiz et al. This study demonstrated severe morphological changes, both at the surface and interior, following exposure to unconjugated bile salts at their MIC, and conjugated bile salts at 20 mM. It is likely that bile salts at sub-inhibitory concentrations exhibit bacteriostatic action through dissipation of the pH gradient and loss of transmembrane electric potential.
At higher concentrations MIC , our results suggest the bactericidal action is caused by membrane damage and leakage of intracellular contents. Thus bile salts have multiple physiological routes by which they can inhibit S. Bile salt based derivatives, specifically a CA analog, has recently been utilized in developing novel cationic steroid antibiotics and can be used to treat topical multidrug resistant bacterial infections Moscoso et al.
This highlights their potential as novel antibacterial agents. Chemical synthesis of various analogs of bile salts utilizing specific physicochemical characteristics in their structure has begun to develop novel antibiotics to combat important bacterial pathogens Moscoso et al.
Performed the experiments: THS. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Felix Trust for providing Ph. Acton, D. Intestinal carriage of Staphylococcus aureus : how does its frequency compare with that of nasal carriage and what is its clinical impact?
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Hepatology 28, — Cheng, J. Effect of membrane composition on antimicrobial peptides aurein 2. Coleman, R. Membrane lipid composition and susceptibility to bile salt damage. Acta , — Darkoh, C. Bile acids improve the antimicrobial effect of rifaximin. Agents Chemother. Elkins, C. Mutations in tol genes destabilize the membrane allowing greater access for bile salts entry.
Loci regulated by the two-component system PhoPQ are also required for bile resistance in S. Reporter gene fusions indicated that the PhoPQ regulon does not sense and respond to bile. These loci have not yet been identified. Mutations in wecD and wecA genes both of which are involved in the biosynthesis and assembly of enterobacterial common antigen ECA render S.
In addition, S. This enzyme may be important in repairing DNA damage induced by exposure to bile. The Salmonella marAB operon, a regulatory locus that controls multiple antibiotic resistance [] , has recently shown to be activated in the presence of bile and this response is concentration-dependent 2.
The bile salt deoxycholate is alone able to activate transcription, while there was no response in the presence of other bile salts tested taurocholate, glycocholate and glycochenodeoxycholate or a non-ionic detergent Triton X Bile resulted in 15 observable changes 14 increased and 1 decreased and deoxycholate resulted in 14 changes 7 increased and 7 decreased in S. A smaller number of alterations were observed in S.
Minimal overlap was observed in the proteins affected for each serovar suggesting that the regulatory factors or the targeted genes differ. Of these genes, were activated in the presence of bile while were repressed.
The majority of bile-sensitive mutants isolated from transposon Tn phoA banks by Thanassi et al. Several of the mutants with changes in their LPS structure isolated by Picken and Beacham [] were bile-sensitive and many of the mutations mapped in or near the rfa locus.
The AcrAB and EmrAB multidrug efflux systems have been shown to actively efflux bile salts chenodeoxycholic acid []. Efflux of bile by an acrAemrB double mutant suggests the involvement of other unknown system s []. Overexpression of the multidrug resistance cluster mdtABCD leads to increased resistance to deoxycholate []. As bile acids can traverse the membrane through porins, it is not surprising that the nature of porins produced influences susceptibility to bile. A strain expressing the OmpF porin that produces a channel with a wider diameter is more susceptible to the inhibitory effect of deoxycholate than a strain which expresses the porin with the smaller diameter channel OmpC [].
Bernstein et al. The most consistently activated promoters were those for genes that protect against DNA damage and oxidative stress micF which regulates the outer membrane OmpF and is induced by oxidative stress; osmY a periplasmic protein that is osmotically inducible and is also induced by oxidative stress; and dinD which is induced by DNA damage as part of the SOS response. Loci important in V. Analysis of a mutant that lacks the VceAB efflux pump demonstrated a significant role for this system in tolerance of deoxycholate [].
In addition, mutation of the V. Furthermore, OmpU expression is stimulated by bile and is dependent upon ToxR the central regulator of virulence factor expression in V. However, virulence studies have ruled out a role for OmpU in colonisation of the infant mouse intestine []. Lin et al. The MICs of selected bile salts were decreased fold chenodeoxycholic acid , fold deoxycholic acid and fold cholic acid and taurocholic acid , and when grown in sodium choleate crude oxbile extract a cmeB mutant was not detected after 3 h whereas the wildtype demonstrated normal growth over the h period [ , ].
Seven of these proteins were induced by several stress conditions and were designated general stress proteins. Three were identified as the molecular chaperones DnaK and GroEL and Ohr an organic hydroperoxide resistance protein [ 69 , , ]. To identify genes involved in bile resistance a plasmid pORI19 integration bank was screened for mutants sensitive to 0. Leverrier et al. Six of these proteins were common to all of the stresses examined acid, heat and bile and included the molecular chaperones DnaK and ClpB, and BCCP a biotin containing carboxyl carrier protein.
Bile salts specific proteins included a putative two-component sensor kinase, a homolog of a sigma factor AlgU and proteins that play roles in DNA damage repair RecR or oxidative damage remediation SodA, cysteine synthase and oxidoreductase [ 44 , ]. Phan-Thanh and Gormon [] examined protein expression in L. Addition of 0. In support of previous findings in E. Dussurget et al. It was observed that the MICs for porcine bile and purified bile salts are 2-fold lower than the wild-type when the gene is inactivated 0.
We have recently confirmed their data in L. In addition, we have recently employed a transposon-based approach to identify and characterize loci that contribute to inherent bile tolerance. Disrupted loci in bile-sensitive mutants included genes putatively involved in membrane biogenesis lmo and lmo , membrane transport btlA, lmo, lmo, lmo and lmo , anaerobic metabolism pflB , macromolecule stability lmo and transcriptional regulation lmo and zurR [ 36 , , ].
Physiological analyses revealed that growth of specific bile-sensitive mutants was significantly affected under a variety of other stress conditions acid — pH 5. Recently, bile-inducible genes were identified in Lactobacillus plantarum WCFS1 by screening an alr alanine racemase complementation library []. Forty-six plasmid derivatives containing promoter elements conditionally activated by 0. The partial sequence of chromosomal inserts present in 41 of these clones was determined and they corresponded to 31 unique loci of the L.
In addition, expression of these loci was also increased in RNA extracted from the duodenum of mice infected with L. Clone-based DNA microarrays were employed by the same laboratory to investigate the global transcriptional response of L. Comparison of the differential transcript profiles obtained during growth on plates with and without 0.
Up-regulated genes included those that encode proteins involved in stress responses e. Six bile-repressed genes encode cell membrane located transporters. The authors suggest that bile stress-induced loss of membrane integrity can partially be compensated by the down-regulation of genes encoding non-essential membrane proteins.
Grill et al. Growth rates of this mutant were significantly lower than the parent when grown in the presence of a glycoconjugated or a tauroconjugated bile salt glycodeoxycholic acid and taurodeoxycholic acid, respectively. In humans, approximately 0. Transformations include epimerisation inversion of the stereochemistry of the hydroxyl groups at C-3, C-7 and C , deconjugation removal of the amino acid side chain , oxidation expulsion of H 2 , reduction insertion of H 2 , hydroxylation replacement of a hydrogen with a hydroxyl group and dehydroxylation replacement of a hydroxyl group with a hydrogen.
Only deconjugation and 7-dehydroxylation are discussed in this review, the other transformations are reviewed by Bortolini et al. BSHs belong to the choloylglycine hydrolase family of enzymes that also contains penicillin amidases EC 3.
Amino acids that are thought to play a role in catalysis Cys-1, Asp, Tyr, Asn and Arg are conserved in both [ , ]. Penicillin amidases hydrolyse penicillin to yield 6-aminopenicillinic acid 6-APA , which is widely used in the industrial production of semi-synthetic antibiotics with a wide range of antimicrobial activities []. The microbial role of penicillin amidases has not yet been elucidated.
It has been suggested that they may hydrolyse bile acids and that these enzymes and BSHs share substrates [ 15 , ], but this has not yet been investigated. Alternatively, as they hydrolyse phenylacetylated compounds, penicillin amidases may have a role in the non-parasitic environment in generating carbon sources [ , ].
BSHs are generally intracellular enzymes that are oxygen insensitive, have a slightly acidic optimal pH usually between pH 5 and 6 , their activity is coupled to biomass production and they are not regulated by bile salts [ 15—18 , — ]. Indeed, BSH activity of L. This increase is due to induction of BSH by an uncharacterized extracellular factor [].
Finally, BSH activity in L. Hydrolysis of bile salts is mediated by various genera of the resident intestinal microflora such as Clostridium [ , ], Bacteroides [ , ], Lactobacillus [ 15 , 16 , 18 , 56 , , — ], Bifidobacterium [ 17 , , ] and Enterococcus [ , ]. The recent identification of a BSH in L. The ecological significance of microbial BSH activity is not yet fully understood, although three major hypotheses have been advanced.
Firstly, it has been proposed that deconjugation may confer a nutritional advantage on hydrolytic strains as liberated amino acids could potentially be used as carbon, nitrogen and energy sources. Glycine may be metabolised to ammonia and carbon dioxide, and taurine to ammonia, carbon dioxide and sulphate, that could then be incorporated into bacterial metabolites.
To support this theory, Van Eldere et al. In addition, transcription of the B. However, other studies have shown that lactobacilli do not utilize the steroid moiety of bile salts as cellular precursors suggesting that this is not a universal function of BSHs [ , ]. Secondly, it has been proposed that BSHs facilitate incorporation of cholesterol or bile into bacterial membranes [ — ]. Pridmore et al.
Finally, it is possible that deconjugation of bile salts may be a detoxification mechanism and BSH enzymes may play a role in bile tolerance and consequently in survival in the gastrointestinal tract. Numerous investigators have refuted this hypothesis. Taranto et al. Similar studies by Gopal et al. However, studies by three independent groups using wild-type and bsh mutant pairs provide a link between bile salt hydrolysis and bile tolerance [ 16 , 56 , ].
As previously mentioned the Lactobacillus amylovorus mutant with a partial decrease in BSH activity isolated by Grill et al. Indeed, we have recently independently verified their results [ , Begley et al. Finally, a L. The authors suggest that, as the protonated non-dissociated form of bile salts are thought to exhibit toxicity through intracellular acidification in a manner similar to organic acids, BSH positive cells may protect themselves through the formation of the weaker unconjugated counterparts.
This could help negate the drop in pH by recapturing and exporting the co-transported proton Fig. Taken together, these three studies with bsh mutants strongly suggest a role for BSH in bile tolerance, and particularly tolerance to glycoconjugated bile salts.
It is possible that the contradictory studies may have used inappropriate experimental conditions; for example, using tauroconjugated bile acids to detect BSH activity even though the majority of BSHs show a preference for glycoconjugated bile acids. In addition, the bile tolerance of strains was often assayed by growth in low levels of bile where BSH activity may not be important.
Schematic representation of the hypothesis that bile salt hydrolysis is a detoxification mechanism. Bile salt hydrolases convert the protonated-conjugated bile salts that enter cells to their weaker unconjugated counterparts which may recapture co-transported protons thereby preventing the excessive expenditure of ATP to maintain pH homeostasis. A large energy burden may be put on BSH negative cells since they cannot form unconjugated bile salts that trap the protons.
The unconjugated bile acids resulting from bile salt hydrolysis have greater inhibitory effects on bacteria than conjugated bile acids in vitro.
However, it is possible that they are precipitated at the low pHs in the intestine caused by the fermentation products of lactic acid bacteria. In fact, this localized precipitation phenomenon is the basis for the agar plate assay used to detect BSH activity []. De Smet et al. Dehydroxylated molecules have low solubility and precipitate at moderately acidic pH. A role for BSH in colonization of the gastrointestinal tract was investigated in L.
A bsh mutant demonstrated reduced bacterial faecal carriage after oral infection of guinea pigs. It was also observed that intestinal multiplication of the parent could be increased by supplying cells with an extra copy of the gene on a plasmid, and also that BSH activity was exclusive to pathogenic species of Listeria , further confirming the role of BSH in persistence of L. It is likely that future investigations will reveal a similar role for BSHs of other organisms.
In fact, it has been noted that BSH activity is found primarily in organisms isolated from the gastrointestinal tracts of mammals Bifidobacterium spp. Interestingly, the complete genome sequences of intestinal probiotic bacteria have revealed that some strains may possess more than one bsh gene e.
Deconjugation has important consequences for the physicochemical properties of bile acids. As unconjugated bile acids are less efficient than conjugated molecules in the emulsification of dietary lipids and the formation of micelles, lipid digestion and absorption of fatty acids and monoglycerides could be impaired []. Microbial BSH activity has been related to growth depression in chickens [ , ] but not in mice []. Efficient enterohepatic recirculation of bile acids is partially dependent on their recognition in the conjugated form by active transport sites in the terminal ileum.
Unconjugated bile acids bind with a lower affinity to the transport sites and thus may pass into the large intestine or caecum. This may result in enhanced faecal loss of bile salts that would increase the demand for cholesterol for de novo bile salt synthesis that may in turn lower serum cholesterol levels.
In fact, a reduction in serum cholesterol levels has been demonstrated in pigs, minipigs, and germfree and conventional mice administered with BSH-active bacteria [ , — ]. However, according to some authors, BSH activity of probiotics would not be favourable as subsequent modification of unconjugated bile salts could generate toxic compounds, which could disturb the normal microbiota of the gut leading to diarrhoea, mucosal inflammation or activation of carcinogens in the intestinal contents [ 29 , 31 , 32 , 66 , ].
In this multistep transformation, primary bile acids are actively transported into the cell and conjugated to coenzyme A. A bile acid inducible bai operon that contains at least nine open reading frames, encodes the enzymes required for the pathway [ 19—21 , , ].
The mechanisms employed by bacteria to sense and respond to bile are currently unknown but are likely to be similar to those used for other stress responses. Bile may be sensed directly by two-component systems consisting of a membrane-associated histidine kinase and a cytoplasmic response regulator.
The histidine kinase generally monitors an environmental parameter and when it senses a change e. The direct sensing of bile may also involve transcriptional activators.
Usually the binding of signal molecules e. Cells may sense bile indirectly by responding to the consequences of exposure, e. Known bacterial membrane damage sensors include the E. Bile-induced alterations in the states of macromolecules may also function as sensors and signal transducers. It is possible that exposure to bile may influence DNA supercoiling levels in a manner similar to other stresses pH and osmolarity where variations act as a sensor of external change and co-ordinate gene expression patterns [ — ].
In fact, many supercoiling-sensitive promoters are induced by bile in E. In addition to activating certain promoters, increasing or decreasing DNA supercoiling levels can have consequences for other topological perturbations of DNA, e. It is also possible that accumulation of proteins damaged or denatured by bile may trigger induction of a general stress response. Transcription of genes in response to bile is likely to be controlled by transcriptional regulators including sigma factors.
In fact, we have recently demonstrated that mutation of sigB impairs the ability of L. Bacteria continuously monitor environmental parameters during colonization or infection of the host, and subsequently express genes that assist in survival and repress those which are unnecessary.
It is therefore not surprising that virulence factors are co-ordinately regulated by a variety of environmental signals such as acid, temperature and osmolarity []. It is becoming increasingly obvious that enteric pathogens can also use bile as an environmental cue to establish location and influence the regulation of virulence genes. However, in addition to using bile to control established virulence determinants, it is likely that some of the gene products involved in bile tolerance will contribute to survival and colonization of the intestinal tract and thus, in their own right, function as virulence factors.
The role of bile in Salmonellae pathogenesis is well recognised. Transcription of S. As a result, bacteria grown in the presence of bile are less able to invade epithelial cells than those grown without bile [ , ]. The bacterium therefore uses bile as an environmental signal to repress its invasive capacity in the intestinal lumen where bile concentrations are high, while invasion may then be initiated when bile concentrations have decreased, e.
In addition, Salmonellae show reduced expression of flagellar biosynthesis genes including flhC, flgC and fliC , and reduced motility in the presence of bile []. When the bacterium is within the intestinal lumen bile inhibits transcription of invasion gene regulators. This results in a decrease in SPI-1 genes involved in epithelial cell entry.
After transit of the mucus layer where bile concentrations decrease, invasion genes are derepressed Adapted from []. Bile present in the lumen results in decreased virulence gene expression perhaps via inactivation of ToxT and increased motility. When the bacterium swims into the mucus gel bile concentrations decrease, motility is blocked and virulence gene expression is induced Adapted from []. Bile has also been shown to promote the formation of Salmonellae S.
If biofilm formation was to occur in vivo not only would it protect bacteria from the high levels of bile in the gallbladder but it would also limit the effectiveness of certain antibiotics. Inactivation of the S. Furthermore, the S. Mutation of S. As mutation of these loci renders Salmonella bile-sensitive, Ramos-Morales et al.
Contrary to the negative effects of bile on Salmonella invasion, Pope et al. Growth in the presence of other structurally similar bile salts or detergents had little or no effect suggesting a specific interaction between certain bile salts and virulence gene expression in this organism.
Bile acids play a key role in the pathogenicity of Vibrio species. Conjugated bile acids enhance production of V. In addition, bile acids enhance the expression of other attributes associated with virulence such as Congo red binding ability, capsule production and adherence to epithelial cells []. Pace et al. The authors suggest that as bile acids are calcium chelators, bile may trigger a low calcium response, which is known to regulate virulence genes in other pathogens [ , ].
In contrast to V. It was also noted that addition of bile resulted in increased motility. This may assist bacteria to penetrate the mucosal layer and gain access to the underlying epithelial cells []. A study by Schuhmacher and Klose [] demonstrated that bile inhibits the activity of the transcription factor ToxT that activates numerous promoters required for colonization and disease in the host including those for CT and the toxin co-regulated pilus TCP genes. The authors propose that once bacteria swim to a location where bile concentrations are low, inhibition of ToxT activity will be relieved resulting in expression of genes required for effective colonization Fig.
Mutation of the V. Mutation of these loci also results in colonization defects in the infant mouse model of infection [ , ]. Bile salts sodium deoxycholate, cholate and chenodeoxycholate affect invasiveness by stimulating the synthesis of Campylobacter invasion Cia proteins []. It was observed that synthesis was specific for bile as altering pH, calcium concentration, osmolarity or temperature did not induce their synthesis.
One of these invasion proteins, CiaB has been shown to play a role in chicken cecal colonization []. Bile upregulates the C. An increase in FlaA synthesis and hence chemotaxis likely maximizes the likelihood of colonizing the mucus layer and pathogenesis. It has also been reported that bile deoxycholate stimulated the production of Campylobacter pili, which are known to be important virulence determinants in many pathogenic bacteria []. However, further investigations revealed that these appendages were not pili but were bacteria-independent morphological artefacts of the growth medium [].
In controlled experiments, the wild-type strain colonized chickens as early as day 2 postinoculation whereas the cmeABC mutants failed to colonize any of the inoculated chickens throughout the day study []. It has been shown that growth of E. Since biomaterial centered infections with E. Recent in vivo bioluminescence imaging experiments performed by Hardy and co-workers [] revealed that L.
Strong signals were observed from the gallbladder in asymptomatic as well as diseased animals. The ability to grow transiently in this location may not only allow possible escaping the immune system but also permit secretion via bile into the intestine to reinfect the same animal or be transmitted. Similar growth in human gallbladders may allow human listeriosis to spread unknowingly in a manner similar to that of typhoid fever.
The L. Deletion of bsh results in reduced faecal carriage after oral infection of guinea pigs, and reduced virulence and liver colonization after intravenous inoculation of mice []. We have also shown that mutation of either L. Interestingly, the gene encoding a putative penicillin V amidase lmo contributes to bile tolerance in vitro but not to murine colonization and is absent from certain pathogenic strains of L. The ability to respond to environmental signals present in the host and to modulate virulence gene expression accordingly is a requirement of a successful enteric pathogen.
It is now clear that bile can act as an environmental signal in the same way as classical signals such as pH, osmolarity and temperature.
In addition, pathogens can respond to bile in contradictory fashions to facilitate their own pathogenic mechanisms, e. Bile represents a major challenge to the survival and subsequent colonization of microorganisms in the gastrointestinal tract.
It is evident that certain bacteria have evolved to resist its actions and pathogens can even use bile to their advantage to regulate virulence determinants. However, compared to other physiological stresses, bile stress is as yet largely unexplored. Detailed physiological analyses are required to ascertain both the limits and comparative bile tolerance of various bacteria.
Future genetic analyses should concentrate on expanding the information available on the molecular mechanisms governing bile tolerance and investigate how bacteria sense bile and regulate the responses it induces. Research should also focus on the effect of bile on virulence factor production and the impact this has on pathogenesis. Undoubtedly, knowledge gained through bile research will provide further insight into the survival of pathogens in vivo.
It is possible that information obtained from bile research may be exploited in antibacterial and preventive therapies. Future antimicrobial treatments may involve bile salts and in fact recent studies have demonstrated their effectiveness as topical microbicidal agent for sexually transmitted diseases [].
Bile-mediated pathogenic mechanisms may also be targeted, however care must be taken not to alter the indigenous gastrointestinal microflora. Compounds could potentially be designed to inhibit the action of bile efflux pumps or target the gallbladder to eliminate carriage of organisms able to cause cholecystitis. The realisation that pathogens use bile to regulate virulence properties highlights the fact that an adequate concentration of bile in the intestine is of utmost importance to human health.
Individuals with low levels of bile such as those with biliary or liver abnormalities, deficiencies in enterohepatic circulation, obstructions in intestinal absorption or malnourished individuals can be given bile acids orally conjugated bile acid replacement therapy or given hormones to increase bile acid synthesis in the liver. The connection between bile tolerance and other stresses should be borne in mind by food processors, especially when designing safe, minimally processed products.
Knowledge gained from investigating the interplay between bile stress and other stresses may however aid in the development of better probiotics. Exposing strains to sublethal levels of bile or heterologous stresses before consumption may increase their bile tolerance and thereby improve their survival in the intestine.
It may also be possible to manipulate bile-resistance genes associated with colonization or persistence in the intestinal tract. One possible candidate is the bile salt hydrolase gene discussed in Section 6. As hydrolysis of bile salts results in increased usage of cholesterol to synthesize new bile salts, serum cholesterol levels are lowered. Therefore, in addition to gaining a better understanding of the disease processes of pathogens, knowledge gained from bile research may ultimately lead to the design of new antimicrobial treatments or assist in the development of improved probiotic strains.
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Yamoto A. Takumi K. These include Escherichia coli, Salmonella typhi and enterica , and Vibrio cholerae. Resistance of these bacteria to bile is actually exploited for laboratory diagnosis in that they can be isolated away from other bacteria by growth on medium containing bile salts e. At least three mechanisms have been shown to impart resistance of bacteria to the deliterious effects of bile salts:. Another property of many enteric bacteria is the ability to form biofilms in which groups of bacteria secrete and become encased in an exopolysaccharide that decreases access to bile salts.
In addition to resisting the toxic effects of bile, there are numerous indications that bile salts actually stimulate expression of virulence factors in some pathogenic enteric bacteria.
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