Functional genomic analysis of bile salt resistance in Enterococcus faecium
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 16 January 2013
Accepted: 18 April 2013
Published: 3 May 2013
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.
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.
These findings add to a better understanding of the molecular mechanisms by which E. faecium responds and resists the antimicrobial action of bile salts.
KeywordsEnterococcus faecium Bile resistance Transposon mutant library Transcriptome
Enterococcus faecium is a common inhabitant of the intestines of humans and animals and is present in many different natural environments [1, 2]. However, during the past two decades E. faecium has rapidly emerged as an important multi-drug resistant nosocomial pathogen around the world and is now frequently responsible for hospital-acquired bloodstream, urinary tract and surgical wound infections [3–5]. The establishment of high-level intestinal colonization by enterococci is a crucial step in a process that can finally lead towards nosocomial infections .
Enterococci are known as being highly tolerant to hostile environments including high temperature conditions and high salt concentrations . Enterococci are also relatively resistant to chemical disinfectants like chlorine, glutaraldehyde and alcohol [7–9]. In order to survive in and colonize the human intestinal tract, a bacterium must be able to adapt to the stressful conditions that occur in this environment. Bile represents a major challenge to the intestinal microflora. The human liver daily secretes up to one liter of bile which is stored in the gall bladder and exported into the intestine . Bile is a complex mixture composed mainly of bile salts, phospholipids, cholesterol, proteins and bilirubin . Bile salts are amphipathic molecules that act as detergents, aiding in lipid solubilization and digestion, but they also play a role in host defenses, as bile salts have potent antimicrobial properties that can cause damage to the DNA, proteins and membranes of enteric bacteria [12, 13]. In both Gram-positive and Gram-negative bacteria the disruption of bile tolerance loci often leads to impaired intestinal survival [14–16], while a mutation resulting in high-level bile resistance of Escherichia coli results in a fitness advantage during intestinal colonization .
As a successful colonizer of the intestinal tract, E. faecium must have developed mechanisms to sense, respond to and tolerate bile during its evolution as a gut commensal. Previously, two genetic loci (gls33-glsB and gls20-glsB1) that encode Gls-like proteins in E. faecalis and E. faecium were identified to be involved in bile resistance and pathogenicity in a mouse peritonitis model [18, 19]. E. faecium was also possesses bile salt hydrolase activity , which is conferred by the protein encoded by the bsh gene (accession no. AY260046) . In this study, we performed a genome-wide identification of the genetic loci required for bile salt resistance in E. faecium, using a high-throughput quantitative screening approach of transposon mutant libraries, termed Microarray-based Transposon Mapping (M-TraM) . We also studied the transcriptional response of E. faecium to bile salts-induced stress.
Bacterial strains, plasmids and growth conditions
Strains and plasmids used in this study
Strain or plasmid
Source or reference
E . faecium
Clinical isolate (bloodstream infection), isolated in France, 1996
Markerless deletion mutant of gltK gene of E1162
Markerless deletion mutant of gspA gene of E1162
Complementation strain of ΔgltK; ΔgltK harboring pEF25-gltK
Complementation strain of ΔgspA; ΔgspA harboring pEF25-gspA
E. coli strains
E. coli host strain for routine cloning
MC1000 glgB::repA; host strain for pWS3 derived vectors
Gram-positive thermosensitive origin of replication; Spcr
pWS3 carrying the 5′ and 3′ flanking regions of gene gltK for mutant construction
pWS3 carrying the 5′ and 3′ flanking regions of gspA gene cluster for mutant construction
pDEL3a with a Genr cassette which was flanked by lox66- and lox71-sites cloned between the 5′ and 3′ flanking regions
pDEL4a with a Genr cassette which was flanked by lox66- and lox71-sites cloned between the 5′ and 3′ flanking regions
pWS3 derivative expressing Cre in E. faecium
Shuttle plasmid pAT18 with spectinomycin resistance cassette cloned in the EcoRI site; Spcr Eryr
Complementation plasmid for ΔgltK; pEF25 carrying gltK
Complementation plasmid for ΔgspA; pEF25 carrying gspA
Screening for genes involved in bile salt resistance using M-TraM
M-TraM, a high throughput screening technique of transposon mutant libraries has previously been described in detail . Here we use this technique to perform a genome-wide identification of genes involved in bile salt resistance in E. faecium. Briefly, aliquots containing approximately 107 colony-forming units (CFU) from the mutant pool were used to inoculate 20 ml of BHI broth or BHI broth supplemented with 0.02% bile salts (sodium cholate:sodium deoxycholate 1:1, Sigma-Aldrich). Cells were grown at 37°C for 20 hours, after which 1 ml of the cultures were spun down and used for the extraction of genomic DNA, which was then further processed as described previously . Statistical differences in hybridization signals between the conditions were analyzed using Cyber-T  (http://cybert.microarray.ics.uci.edu/). Probes exhibiting a Bayesian P-value <0.005 were deemed statistically significant. A gene of which at least two identical probes (two different probes per gene were spotted in duplicate on the microarray ) passed this threshold were classified as significantly selected during exposure to bile salts. In an addition, genes which were selected between 0.5- and 2-fold were deemed biologically insignificant and were filtered out. This experiment was performed with four biological replicates.
The microarray data generated in the M-TraM screening have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-3797.
E. faecium E1162 was grown in 3 ml BHI broth at 37°C for 18 hours. Cultures were then diluted 100 fold in 20 ml of prewarmed BHI broth (in a 50-ml Falcon tube) and grown until OD660 0.3. Two ml aliquots of the cultures were centrifuged for 12 seconds at 16900 g at room temperature, and pellets were flash frozen in liquid N2 prior to RNA extraction. This sample served as the negative control (t = 0 min) prior to the addition of bile salts. Bile salts (final concentration 0.02%, w/v) were added into the remaining 18 ml of culture. After 5 and 15 minutes of incubation at 37°C, 2 ml aliquots of the cultures were centrifuged and flash frozen as described above. RNA isolation, cDNA synthesis and hybridization were performed as described in our previous work . In this experiment, the expression of genes at t = 5 min and t = 15 min were compared to t = 0 min. Analysis for statistical significance was performed using the Web-based VAMPIRE microarray suite (http://sasquatch.ucsd.edu/vampire/) as described previously [26, 27]. A gene of which all four probes (two different probes were spotted in duplicate on the microarray ) were identified as differentially expressed with a false discovery rate <0.001, were classified as significantly different between samples. Genes with an expression ratio between 0.5- and 2-fold were deemed biologically insignificant and were filtered out. This experiment was performed with two biological replicates.
The microarray data generated in the transcriptome analysis have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-3796.
Construction of markerless deletion mutants and in transcomplementation
Primers used in this study
In trans complemented strains of gltK and gspA gene deletion mutants were generated as described previously [22, 30]. The gltK and gspA genes were PCR amplified, respectively, from the genomic DNA of E1162 using the primers listed in Table 2. The PCR products were cloned into the shuttle vector pEF25 . The resulting plasmids, pEF25-gltK and pEF25-gspA, were introduced into the corresponding mutant strains by electroporation as described above.
Determination of growth curves
A BioScreen C instrument (Oy Growth Curves AB, Helsinki, Finland) was used to determine the effects of bile salts on bacterial growth. Wild type, mutants and the in trans complemented strains were grown overnight in BHI (containing appropriate antibiotics for the in trans complemented strains). Cells were inoculated at an initial OD660 of 0.0025 into 300 μl BHI and BHI with 0.02%, 0.04%, 0.08% and 0.16% of bile salts, respectively. The cultures were incubated in the Bioscreen C system at 37°C with continuous shaking, and absorbance of 600 nm (A600) was recorded every 15 min for 12 hours. Each experiment was performed in triplicate.
Bile salt adaptation and challenge assays
To compare the sensitivity to bile salts of the parental strain E1162, the mutant strains and in trans complemented strains, overnight cultures were diluted 100 fold in fresh BHI and grown to OD660 0.3. One ml of the cell cultures were harvested by centrifugation at 12500 g for 1 minute and adapted to bile salts by resuspending the cells in BHI containing sub-lethal levels of bile salts (0.02%) or in BHI without any additions. After a 15-minute adaptation period, viable counts were determined by serial dilution and plating on BHI agar plates (time point 0). Adapted and non-adapted cells were spun down as described above and resuspended in BHI containing 0.3% bile salts, which corresponds to a concentration that is commonly reached in the human small intestine after ingestion of a meal . After 5, 30 and 60 minutes of incubation at 37°C, aliquots of cells were washed with PBS and viable counts were determined following serial dilution and plating on BHI agar plates. The experiment was performed in triplicate and statistical analysis of the data was performed using an unpaired two-tailed Student’s t-test.
Results and discussion
Identification of genetic determinants involved in bile salt resistance in E. faeciumby M-TraM
To identify genes that are required for bile salt resistance in E. faecium E1162, we grew the pool of mutants in the presence or absence of a sub-lethal concentration (0.02%) of bile salts for 20 hours, and used M-TraM to determine which mutants were less resistant to bile salts and therefore are selectively lost during culturing in the presence of bile salts. Seventy-five genes belonging to a variety of functional categories were identified to be involved in bile resistance (Additional file 1 and 2). A single gene, gltK (locus tag EfmE1162_1760), encoding a putative glutamate/aspartate transport system permease protein, was identified by M-TraM with the highest fold change (11.5 fold, which was notably higher than the other identified genes), indicating that this gene may contribute considerably to bile resistance in E. faecium. Consequently, we decided to further study the function of this gene in bile resistance (further described below). We were unable to find previous studies that linked GltK and its homologues in other microorganisms to bile resistance. BLAST analysis showed that GltK is conserved (with amino acid identities >97%) in all of the 69 E. faecium genomes available (on 30 October 2012) at NCBI Genomes, indicating that the gltK gene is part of the E. faecium core genome. Another gene that was identified as contributing to bile resistance by M-TraM analysis was a gene (locus tag: EfmE1162_2043) encoding a putative cardiolipin synthetase, which functions as an enzyme in phospholipid metabolism and is involved in enterococcal daptomycin resistance [32, 33]. It possibly acts by protecting the cells from membrane-associated damage induced by bile. In E. faecalis, the sagA gene was previously shown to be important in maintaining cell wall integrity and resistance to bile . The E. faecium homolog (locus tag: EfmE1162_2437) of the sagA gene was also identified by M-TraM as potentially contributing to bile resistance. The bsh gene (locus tag: EfmE1162_2656) which encodes a bile salt hydrolase (BSH)  is conserved in all the 69 publicly available E. faecium genomes, including E1162. However this gene was not identified by M-TraM screening, presumably because BSH does not provide protection despite its predicted activity in the hydrolysis of bile salts. It is also possible that in the M-TraM screening, during which many different transposon insertion mutants are pooled together, the minor proportion of BSH-deficient mutants could be compensated by the extracellular bile salt hydrolase activity that is produced by cells that carry other mutations. We did not identify the two Gls-like protein-encoding loci which were shown to be involved in bile resistance in a previous study . However, single deletions of either locus only resulted in a minor effect on bile salt resistance possibly due to mutual compensation of the two loci , which may also explain why we did not identify these loci in the M-TraM screening, as the mutant library only contains mutants that are inactivated in a single locus by transposon insertion .
Transcriptional responses of E. faeciumto bile salt-induced stress
A microarray-based transcriptome analysis was used to identify genes that are regulated by exposure to bile salts. Compared to the untreated control, 214 (175 up-regulated and 39 down-regulated) and 190 (119 up-regulated and 71 down-regulated) genes were identified to be differentially expressed at 5 min and 15 min incubation with bile salts, respectively (Additional files 2 and 3). The data of the transcriptional analyses at the two different time points (t = 5 min and t = 15 min) exhibited a correlation (R2 of log2-transformed values) of 0.44 with each other (Additional file 4A). However the transcriptome data are completely uncorrlated with the M-TraM analysis (R2 of log2-transformed values ≤ 0.001) (Additional file 4BC), which is consistent with previous observations that gene expression poorly correlates with mutant fitness measurements [22, 35].
We further focused on a gene EfmE1162_1186 (gspA) which is predicted to encode a general stress protein A. This gene was identified by both transcriptome analysis (4.6 and 47.0 fold up-regulated at 5 min and 15 min of bile salts treatment, respectively) and M-TraM (2.8 fold less signal in bile-exposed library compared to the control). GspA is also highly conserved (with amino acid identities >98%) in 66 of the 69 E. faecium genomes. We observed that both of the two Gls-like protein-encoding loci (EfmE1162_1192-EfmE1162_1193 and EfmE1162_1201-EfmE1162_1202) were induced over eight-fold during exposure to bile salts, although they were not identified by M-TraM screening. However, the bsh gene was not identified to be differentially expressed in BHI with bile salts, indicating that the expression of this gene is not regulated by bile salts despite its predicted role in bile salt hydrolysis.
The transcriptional responses of E. faecalis to bovine bile has been investigated in a previous study . A striking common finding of this study and our work is that a large gene cluster (locus tags EfmE1162_0724-EfmE1162_0731 in E. faecium E1162 and EF1492-EF1500 in E. faecalis V583), which putatively encodes a V-type ATPase, exhibits upregulated expression during exposure to bile salts. V-type ATPases are membrane proteins that function as proton- or sodium ion pumps that build up ion gradients at the expense of ATP . Induction of this gene cluster suggested that E. faecium may generate a proton gradient to respond to bile mediated stress. The link between bile mediated stress and maintenance of the proton motive force (PMF) was previously demonstrated in the Gram-positive bacteria Lactobacillus plantarum, Bifidobacterium longum and B. animalis. Bile salts can induce DNA damage in bacteria, and consequently DNA mismatch repair proteins are important for bacterial bile resistance [12, 41, 42]. In this study we identified a gene (locus tag: EfmE1162_1335), encoding the DNA mismatch repair protein MutS, that was higher expressed (23.0 fold at 5 min and 9.5 fold at 15 min) after addition of bile salts to the culture medium.
Effect of bile salts on growth of E. faecium E1162 wild-type and gltK and gspAmutants
Bile salt adaptation and challenge assays
Our data suggest that bile salt-regulated genes do not necessarily contribute to bile resistance. Previous studies indicated that the protective adaptation to bile salts mainly results from changes in membrane composition and architecture that are independent of de novo protein synthesis [43, 44]. Flahaut et al. showed that a 5 second-adaptation of E. faecalis to low level bile salts could provide substantial protection against challenge with lethal bile salt concentrations, and the addition of chloramphenicol during the adaptation period did not prevent development of acquired tolerance . A similar result was also observed in L. monocytogenes. However, the bile salt-regulated genes, rather than directly contributing to bile resistance, could be involved in other functions including virulence and carbohydrate metabolism . It has previously been established in Salmonella[45, 46] and Vibrio[47–49] that bile can be used as an environmental cue to influence the regulation genes involved in intestinal colonization and virulence. We identified many genes involved in carbohydrate metabolism that exhibited upregulated expression upon exposure to bile salts, e.g. a gene cluster (locus tags: EfmE1162_1484 - EfmE1162_1489) putatively involved in maltose utilization (Zhang et al., unpublished work). This may suggest that E. faecium senses bile as an environmental signal indicating that it has entered the host gut, leading to a rapid adjustment of the cell’s carbohydrate metabolism.
Responding and being resistant to bile are important features of bacteria that inhabit the gut . In the present work, we have identified a genetic determinant in E. faecium that contributes to bile salt resistance, and studied the transcriptional response of E. faecium to bile salts. These findings add to a better understanding of the molecular mechanisms that lead to bile resistance in E. faecium.
This work was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek [NWO-VENI grant 916.86.044] and the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage) Evolution and Transfer of Antibiotic Resistance (EvoTAR) [grant agreement number 282004].
- Top J, Willems R, Bonten M: Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen. FEMS Immunol Med Microbiol. 2008, 52 (3): 297-308.View ArticlePubMedGoogle Scholar
- Sghir A, Gramet G, Suau A, Rochet V, Pochart P, Dore J: Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl Environ Microbiol. 2000, 66 (5): 2263-2266.PubMed CentralView ArticlePubMedGoogle Scholar
- Willems RJ, Top J, Van Schaik W, Leavis H, Bonten M, Siren J, Hanage WP, Corander J: Restricted gene flow among hospital subpopulations of Enterococcus faecium. MBio. 2012, 3 (4):
- Willems RJ, van Schaik W: Transition of Enterococcus faecium from commensal organism to nosocomial pathogen. Future Microbiol. 2009, 4 (9): 1125-1135.View ArticlePubMedGoogle Scholar
- Arias CA, Murray BE: The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol. 2012, 10 (4): 266-278.PubMed CentralView ArticlePubMedGoogle Scholar
- Facklam RR, Collins MD: Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol. 1989, 27 (4): 731-734.PubMed CentralPubMedGoogle Scholar
- Bradley CR, Fraise AP: Heat and chemical resistance of enterococci. J Hosp Infect. 1996, 34 (3): 191-196.View ArticlePubMedGoogle Scholar
- Freeman R, Kearns AM, Lightfoot NF: Heat resistance of nosocomial enterococci. Lancet. 1994, 344 (8914): 64-65.View ArticlePubMedGoogle Scholar
- Kearns AM, Freeman R, Lightfoot NF: Nosocomial enterococci: resistance to heat and sodium hypochlorite. J Hosp Infect. 1995, 30 (3): 193-199.View ArticlePubMedGoogle Scholar
- Begley M, Gahan CG, Hill C: The interaction between bacteria and bile. FEMS Microbiol Rev. 2005, 29 (4): 625-651.View ArticlePubMedGoogle Scholar
- Esteller A: Physiology of bile secretion. World J Gastroenterol. 2008, 14 (37): 5641-5649.PubMed CentralView ArticlePubMedGoogle Scholar
- Merritt ME, Donaldson JR: Effect of bile salts on the DNA and membrane integrity of enteric bacteria. J Med Microbiol. 2009, 58 (Pt 12): 1533-1541.View ArticlePubMedGoogle Scholar
- Hofmann AF, Hagey LR: Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol Life Sci. 2008, 65 (16): 2461-2483.View ArticlePubMedGoogle Scholar
- Begley M, Sleator RD, Gahan CG, Hill C: Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes. Infect Immun. 2005, 73 (2): 894-904.PubMed CentralView ArticlePubMedGoogle Scholar
- Kristich CJ, Wells CL, Dunny GM: A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence. Proc Natl Acad Sci USA. 2007, 104 (9): 3508-3513.PubMed CentralView ArticlePubMedGoogle Scholar
- Reynolds MM, Bogomolnaya L, Guo J, Aldrich L, Bokhari D, Santiviago CA, McClelland M, Andrews-Polymenis H: Abrogation of the twin arginine transport system in Salmonella enterica serovar Typhimurium leads to colonization defects during infection. PLoS One. 2011, 6 (1): e15800-PubMed CentralView ArticlePubMedGoogle Scholar
- De Paepe M, Gaboriau-Routhiau V, Rainteau D, Rakotobe S, Taddei F, Cerf-Bensussan N: Trade-off between bile resistance and nutritional competence drives Escherichia coli diversification in the mouse gut. PLoS Genet. 2011, 7 (6): e1002107-PubMed CentralView ArticlePubMedGoogle Scholar
- Teng F, Nannini EC, Murray BE: Importance of gls24 in virulence and stress response of Enterococcus faecalis and use of the Gls24 protein as a possible immunotherapy target. J Infect Dis. 2005, 191 (3): 472-480.View ArticlePubMedGoogle Scholar
- Choudhury T, Singh KV, Sillanpaa J, Nallapareddy SR, Murray BE: Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence. J Infect Dis. 2011, 203 (8): 1147-1154.PubMed CentralView ArticlePubMedGoogle Scholar
- Franz CM, Specht I, Haberer P, Holzapfel WH: Bile salt hydrolase activity of Enterococci isolated from food: screening and quantitative determination. J Food Prot. 2001, 64 (5): 725-729.PubMedGoogle Scholar
- Wijaya A, Hermann A, Abriouel H, Specht I, Yousif NM, Holzapfel WH, Franz CM: Cloning of the bile salt hydrolase (bsh) gene from Enterococcus faecium FAIR-E 345 and chromosomal location of bsh genes in food enterococci. J Food Prot. 2004, 67 (12): 2772-2778.PubMedGoogle Scholar
- Zhang X, Paganelli FL, Bierschenk D, Kuipers A, Bonten MJ, Willems RJ, van Schaik W: Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium. PLoS Genet. 2012, 8 (6): e1002804-PubMed CentralView ArticlePubMedGoogle Scholar
- van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JE, Schapendonk CM, Hendrickx AP, Nijman IJ, Bonten MJ, Tettelin H: Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island. BMC Genomics. 2010, 11: 239-PubMed CentralView ArticlePubMedGoogle Scholar
- Leenhouts K, Buist G, Bolhuis A, ten Berge A, Kiel J, Mierau I, Dabrowska M, Venema G, Kok J: A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet. 1996, 253 (1–2): 217-224.View ArticlePubMedGoogle Scholar
- Baldi P, Long AD: A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes. Bioinformatics. 2001, 17 (6): 509-519.View ArticlePubMedGoogle Scholar
- Lebreton F, van Schaik W, Sanguinetti M, Posteraro B, Torelli R, Le Bras F, Verneuil N, Zhang X, Giard JC, Dhalluin A: AsrR is an oxidative stress sensing regulator modulating Enterococcus faecium opportunistic traits, antimicrobial resistance, and pathogenicity. PLoS Pathog. 2012, 8 (8): e1002834-PubMed CentralView ArticlePubMedGoogle Scholar
- Hsiao A, Ideker T, Olefsky JM, Subramaniam S: VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data. Nucleic Acids Res. 2005, 33 (Web Server issue): W627-632.PubMed CentralView ArticlePubMedGoogle Scholar
- Sauer B: Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987, 7 (6): 2087-2096.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Vrijenhoek JE, Bonten MJ, Willems RJ, van Schaik W: A genetic element present on megaplasmids allows Enterococcus faecium to use raffinose as carbon source. Environ Microbiol. 2011, 13 (2): 518-528.View ArticlePubMedGoogle Scholar
- Top J, Sinnige JC, Majoor EA, Bonten MJ, Willems RJ, van Schaik W: The recombinase IntA is required for excision of esp-containing ICEEfm1 in Enterococcus faecium. J Bacteriol. 2011, 193 (4): 1003-1006.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Deest BW, Fordtran JS, Morawski SG, Wilson JD: Bile salt and micellar fat concentration in proximal small bowel contents of ileectomy patients. J Clin Invest. 1968, 47 (6): 1314-1324.View ArticlePubMedGoogle Scholar
- Palmer KL, Daniel A, Hardy C, Silverman J, Gilmore MS: Genetic basis for daptomycin resistance in enterococci. Antimicrob Agents Chemother. 2011, 55 (7): 3345-3356.PubMed CentralView ArticlePubMedGoogle Scholar
- Arias CA, Panesso D, McGrath DM, Qin X, Mojica MF, Miller C, Diaz L, Tran TT, Rincon S, Barbu EM: Genetic basis for in vivo daptomycin resistance in enterococci. N Engl J Med. 2011, 365 (10): 892-900.PubMed CentralView ArticlePubMedGoogle Scholar
- Breton YL, Maze A, Hartke A, Lemarinier S, Auffray Y, Rince A: Isolation and characterization of bile salts-sensitive mutants of Enterococcus faecalis. Curr Microbiol. 2002, 45 (6): 434-439.View ArticlePubMedGoogle Scholar
- Deutschbauer A, Price MN, Wetmore KM, Shao W, Baumohl JK, Xu Z, Nguyen M, Tamse R, Davis RW, Arkin AP: Evidence-based annotation of gene function in Shewanella oneidensis MR-1 using genome-wide fitness profiling across 121 conditions. PLoS Genet. 2011, 7 (11): e1002385-PubMed CentralView ArticlePubMedGoogle Scholar
- Solheim M, Aakra A, Vebo H, Snipen L, Nes IF: Transcriptional responses of Enterococcus faecalis V583 to bovine bile and sodium dodecyl sulfate. Appl Environ Microbiol. 2007, 73 (18): 5767-5774.PubMed CentralView ArticlePubMedGoogle Scholar
- Senior AE: The proton-translocating ATPase of Escherichia coli. Annu Rev Biophys Biophys Chem. 1990, 19: 7-41.View ArticlePubMedGoogle Scholar
- Bron PA, Molenaar D, de Vos WM, Kleerebezem M: DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum. J Appl Microbiol. 2006, 100 (4): 728-738.View ArticlePubMedGoogle Scholar
- Sanchez B, Champomier-Verges MC, Anglade P, Baraige F, de Los Reyes-Gavilan CG, Margolles A, Zagorec M: Proteomic analysis of global changes in protein expression during bile salt exposure of Bifidobacterium longum NCIMB 8809. J Bacteriol. 2005, 187 (16): 5799-5808.PubMed CentralView ArticlePubMedGoogle Scholar
- Sanchez B, Reyes-Gavilan CG Dl, Margolles A: The F1F0-ATPase of Bifidobacterium animalis is involved in bile tolerance. Environ Microbiol. 2006, 8 (10): 1825-1833.View ArticlePubMedGoogle Scholar
- Payne A, Schmidt TB, Nanduri B, Pendarvis K, Pittman JR, Thornton JA, Grissett J, Donaldson JR: Proteomic analysis of the response of Listeria monocytogenes to bile salts under anaerobic conditions. J Med Microbiol. 2013, 62 (Pt 1): 25-35.View ArticlePubMedGoogle Scholar
- Prieto AI, Ramos-Morales F, Casadesus J: Bile-induced DNA damage in Salmonella enterica. Genetics. 2004, 168 (4): 1787-1794.PubMed CentralView ArticlePubMedGoogle Scholar
- Begley M, Gahan CG, Hill C: Bile stress response in Listeria monocytogenes LO28: adaptation, cross-protection, and identification of genetic loci involved in bile resistance. Appl Environ Microbiol. 2002, 68 (12): 6005-6012.PubMed CentralView ArticlePubMedGoogle Scholar
- Flahaut S, Hartke A, Giard JC, Benachour A, Boutibonnes P, Auffray Y: Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis. FEMS Microbiol Lett. 1996, 138 (1): 49-54.View ArticlePubMedGoogle Scholar
- Prouty AM, Brodsky IE, Manos J, Belas R, Falkow S, Gunn JS: Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. FEMS Immunol Med Microbiol. 2004, 41 (2): 177-185.View ArticlePubMedGoogle Scholar
- Prouty AM, Gunn JS: Salmonella enterica serovar typhimurium invasion is repressed in the presence of bile. Infect Immun. 2000, 68 (12): 6763-6769.PubMed CentralView ArticlePubMedGoogle Scholar
- Krukonis ES, DiRita VJ: From motility to virulence: Sensing and responding to environmental signals in Vibrio cholerae. Curr Opin Microbiol. 2003, 6 (2): 186-190.View ArticlePubMedGoogle Scholar
- Schuhmacher DA, Klose KE: Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae. J Bacteriol. 1999, 181 (5): 1508-1514.PubMed CentralPubMedGoogle Scholar
- Gupta S, Chowdhury R: Bile affects production of virulence factors and motility of Vibrio cholerae. Infect Immun. 1997, 65 (3): 1131-1134.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.