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• W2940808813 abstract "•VRE forms long chains and biofilms in physiological concentrations of bile acids•This morphotype switch is reversed by cations•Selection against chaining is linked to sensitivity to the antibiotic daptomycin•Chaining-deficient VRE mutants exhibit reduced persistence in the gut Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation. Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation. Single bacterial cells exist in a range of sizes from near the 200 nm limit of resolution of light microscopy (Luef et al., 2015Luef B. Frischkorn K.R. Wrighton K.C. Holman H.Y. Birarda G. Thomas B.C. Singh A. Williams K.H. Siegerist C.E. Tringe S.G. et al.Diverse uncultivated ultra-small bacterial cells in groundwater.Nat. Commun. 2015; 6: 6372Crossref PubMed Scopus (238) Google Scholar), up to the hundreds of microns resolvable by the human eye (Schulz et al., 1999Schulz H.N. Brinkhoff T. Ferdelman T.G. Mariné M.H. Teske A. Jorgensen B.B. Dense populations of a giant sulfur bacterium in Namibian shelf sediments.Science. 1999; 284: 493-495Crossref PubMed Scopus (345) Google Scholar). Bacterial cell size, however, is not static, and it varies depending on the conditions of the local environment (Schaechter et al., 1958Schaechter M. Maaloe O. Kjeldgaard N.O. Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium.J. Gen. Microbiol. 1958; 19: 592-606Crossref PubMed Google Scholar, Weart et al., 2007Weart R.B. Lee A.H. Chien A.C. Haeusser D.P. Hill N.S. Levin P.A. A metabolic sensor governing cell size in bacteria.Cell. 2007; 130: 335-347Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Some bacteria switch between distinct growth modes called morphotypes that alter cell size and shape in a predictable and regulated manner. Transitions between morphotypes are important characters in bacterial evolution, pathogenesis, and resistance to antibiotics (Deforet et al., 2015Deforet M. van Ditmarsch D. Xavier J.B. Cell-size homeostasis and the incremental rule in a bacterial pathogen.Biophys. J. 2015; 109: 521-528Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, Justice et al., 2008Justice S.S. Hunstad D.A. Cegelski L. Hultgren S.J. Morphological plasticity as a bacterial survival strategy.Nat. Rev. Microbiol. 2008; 6: 162-168Crossref PubMed Scopus (405) Google Scholar, Kysela et al., 2016Kysela D.T. Randich A.M. Caccamo P.D. Brun Y.V. Diversity takes shape: understanding the mechanistic and adaptive basis of bacterial morphology.PLoS Biol. 2016; 14: e1002565Crossref PubMed Scopus (63) Google Scholar, Weiser, 2013Weiser J.N. The battle with the host over microbial size.Curr. Opin. Microbiol. 2013; 16: 59-62Crossref PubMed Scopus (20) Google Scholar). An example of a morphotype switch in Gram-positive bacteria occurs in liquid cultures of Bacillus subtilis laboratory strains, which grow in long chains of fully septated cells during exponential growth. When the cultures approach stationary phase, they express high levels of autolysin, which breaks the chains apart and separates the individual cells to prepare for sporulation (Margot et al., 1999Margot P. Pagni M. Karamata D. Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD.Microbiology. 1999; 145: 57-65Crossref PubMed Scopus (101) Google Scholar). Streptococcus pneumoniae performs the reverse switch—diplococci to chains—when it encounters host-associated media, such as nasal lavage fluid (Dalia and Weiser, 2011Dalia A.B. Weiser J.N. Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody.Cell Host Microbe. 2011; 10: 486-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The long chains adhere better to surfaces, which aids S. pneumoniae in colonization of the host (Rodriguez et al., 2012Rodriguez J.L. Dalia A.B. Weiser J.N. Increased chain length promotes pneumococcal adherence and colonization.Infect. Immun. 2012; 80: 3454-3459Crossref PubMed Scopus (47) Google Scholar). Interestingly, chaining also increased the likelihood of cells being bound by complement and engulfed by macrophages, suggesting that morphotypes may confer context-specific advantages during host interactions (Dalia and Weiser, 2011Dalia A.B. Weiser J.N. Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody.Cell Host Microbe. 2011; 10: 486-496Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Any bacterium that colonizes the intestine encounters bile acids, which are rarely found elsewhere in nature (Hofmann et al., 2010Hofmann A.F. Hagey L.R. Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance.J. Lipid Res. 2010; 51: 226-246Crossref PubMed Scopus (274) Google Scholar). Bile acids are present throughout the intestine in concentrations ranging from low millimolar to low micromolar, and they affect gut microbes since whole bile is anti-microbial at high concentrations (Begley et al., 2005Begley M. Gahan C.G.M. Hill C. The interaction between bacteria and bile.FEMS Microbiol. Rev. 2005; 29: 625-651Crossref PubMed Scopus (1151) Google Scholar). Bile is produced in the liver and is chemically altered by the intestinal microbiota into so-called secondary bile acids (Ridlon et al., 2016Ridlon J.M. Harris S.C. Bhowmik S. Kang D.J. Hylemon P.B. Consequences of bile salt biotransformations by intestinal bacteria.Gut Microbes. 2016; 7: 22-39Crossref PubMed Scopus (500) Google Scholar). Dozens of chemically distinct bile acids have been identified and each one is likely to have a unique spectrum of chemical and biological activities. The universal distribution of enterococci among coelomates (Lebreton et al., 2014Lebreton F. Willems R.J.L. Gilmore M.S. Enterococcus diversity, origins in nature, and Gut Colonization.in: Gilmore M.S. Clewell D.B. Ike Y. Shankar N. Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Massachusetts Eye and Ear Infirmary, 2014Google Scholar) suggests that their association with vertebrate hosts is ancient. Thus, VRE may possess mechanisms to respond to bile acids and facilitate to host colonization. Enterococci mount a stress response to high levels of bile (Bøhle et al., 2010Bøhle L.A. Færgestad E.M. Veiseth-Kent E. Steinmoen H. Nes I.F. Eijsink V.G. Mathiesen G. Identification of proteins related to the stress response in Enterococcus faecalis V583 caused by bovine bile.Proteome Sci. 2010; 8: 37Crossref PubMed Scopus (32) Google Scholar, Choudhury et al., 2011Choudhury T. Singh K.V. Sillanpää J. Nallapareddy S.R. Murray B.E. Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence.J. Infect. Dis. 2011; 203: 1147-1154Crossref PubMed Scopus (26) Google Scholar, Michaux et al., 2011Michaux C. Martini C. Hanin A. Auffray Y. Hartke A. Giard J.C. SlyA regulator is involved in bile salts stress response of Enterococcus faecalis.FEMS Microbiol. Lett. 2011; 324: 142-146Crossref PubMed Scopus (14) Google Scholar, Saito et al., 2014Saito H.E. Harp J.R. Fozo E.M. Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents.Appl. Environ. Microbiol. 2014; 80: 6527-6538Crossref PubMed Scopus (49) Google Scholar, Solheim et al., 2007Solheim M. Aakra A. Vebø H. Snipen L. Nes I.F. Transcriptional responses of Enterococcus faecalis V583 to bovine bile and sodium dodecyl sulfate.Appl. Environ. Microbiol. 2007; 73: 5767-5774Crossref PubMed Scopus (62) Google Scholar, Zhang et al., 2013Zhang X. Bierschenk D. Top J. Anastasiou I. Bonten M.J.M. Willems R.J.L. van Schaik W. Functional genomic analysis of bile salt resistance in Enterococcus faecium.BMC Genomics. 2013; 14: 299Crossref PubMed Scopus (20) Google Scholar), but little else is known about the effects of individual bile acids. Here, we identified a reversible morphotype switch in VRE that is triggered by bile acids, is reversed by cations, and plays a key role in intestinal colonization. We used experimental evolution to select for loss of the morphotype switch and obtained mutants with reduced biofilm formation and deficient intestinal colonization in the presence of a competing gut microbiota. Unexpectedly, these strains were highly susceptible to the antibiotic daptomycin because of the accumulation of mutations in genes associated with resistance. This morphotype switch represents a potential target for decolonization therapies for VRE and illustrates the linkage between phenotypes selected for during the evolution of host-associated microbes and antibiotic resistance. To determine how individual bile acids may affect the growth of enterococci in the intestine, we grew a vancomycin-resistant strain Enterococcus faecium ATCC 700221 (VRE) in liquid media in the presence of physiologically relevant concentrations of single bile acids. Primary bile acids such as cholic acid (CA) had little effect on growth. The secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) caused a dose-dependent reduction of the growth rate of VRE (Figures S1A and S1B). The final optical density (OD) of the culture was significantly, but slightly, decreased at physiologically relevant concentrations of 125 μM of LCA (Devlin and Fischbach, 2015Devlin A.S. Fischbach M.A. A biosynthetic pathway for a prominent class of microbiota-derived bile acids.Nat. Chem. Biol. 2015; 11: 685-690Crossref PubMed Scopus (207) Google Scholar, Hamilton et al., 2007Hamilton J.P. Xie G. Raufman J.P. Hogan S. Griffin T.L. Packard C.A. Chatfield D.A. Hagey L.R. Steinbach J.H. Hofmann A.F. Human cecal bile acids: concentration and spectrum.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 293: G256-G263Crossref PubMed Scopus (172) Google Scholar), but was largely unaffected by a diverse panel of primary and secondary bile acids tested mostly at 250 μM (Figures S1C and S1D). After overnight culture, we noticed a visually striking precipitation of VRE treated with LCA. Light microscopy revealed that this was due to a switch from diplococci to long chains (Figure 1A). Growth in the presence of 250 μM LCA resulted in a 2-log reduction in VRE colony-forming units (CFUs) (Figure S1E). This reduction in CFUs was eliminated when cultures were grown in the presence of DNAse, which breaks cell chains of E. faecium (Paganelli et al., 2013Paganelli F.L. Willems R.J.L. Jansen P. Hendrickx A. Zhang X. Bonten M.J.M. Leavis H.L. Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA release.mBio. 2013; 4: e00154Crossref PubMed Scopus (42) Google Scholar), suggesting that LCA is not toxic at this concentration. Transmission electron microscopy of LCA-treated cells showed complete division septa; however, the daughter cells failed to separate into diplococci, which were the most common cell morphology in normal growth media (Figure 1A). LCA-treated cells also generated horn-like structures of cell-envelope material (Figure S2), which often appear to flank nascent mid-cell division sites. These structures resembled those reported in daptomycin-resistant isolates of Enterococcus faecalis (Arias et al., 2011Arias C.A. Panesso D. McGrath D.M. Qin X. Mojica M.F. Miller C. Diaz L. Tran T.T. Rincon S. Barbu E.M. et al.Genetic basis for in vivo daptomycin resistance in enterococci.N. Engl. J. Med. 2011; 365: 892-900Crossref PubMed Scopus (260) Google Scholar). We quantified the chain length of VRE after 24 h in the presence of the primary bile acids CA and chenodeoxycholic acid (CDCA) and secondary bile acids DCA and LCA. LCA was the strongest inducer of cell chaining at physiologically relevant concentrations with a mean chain length of 5 and a wide range that exceeded 20 cells (Figure 1B). This is likely an underestimate of the true mean chain length of the population, as longer chains often fail to separate from each other and often do not adhere to the coverslip in a single plane of focus. These data suggested that LCA treatment impacts cell separation without affecting cell division and that LCA may interfere with peptidoglycan synthesis. To determine if peptidoglycan synthesis was properly localized, we performed short pulse labeling of peptidoglycan synthesis by adding the fluorescent-labeled D-alanine probe HADA (Kuru et al., 2012Kuru E. Hughes H.V. Brown P.J. Hall E. Tekkam S. Cava F. de Pedro M.A. Brun Y.V. VanNieuwenhze M.S. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids.Angew. Chem. Int. Ed. 2012; 51: 12519-12523Crossref PubMed Scopus (409) Google Scholar) in the presence and absence of LCA. In untreated cells, the HADA incorporated at mid-coccus (Figure 1C), suggesting this was the location of the wall peptidoglycan synthesis machinery, which is consistent with other ovococci such as S. pneumoniae (Boersma et al., 2015Boersma M.J. Kuru E. Rittichier J.T. VanNieuwenhze M.S. Brun Y.V. Winkler M.E. Minimal peptidoglycan (PG) turnover in wild-type and PG hydrolase and cell division mutants of Streptococcus pneumoniae D39 growing planktonically and in host-relevant biofilms.J. Bacteriol. 2015; 197: 3472-3485Crossref PubMed Scopus (43) Google Scholar, Kuru et al., 2012Kuru E. Hughes H.V. Brown P.J. Hall E. Tekkam S. Cava F. de Pedro M.A. Brun Y.V. VanNieuwenhze M.S. In situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids.Angew. Chem. Int. Ed. 2012; 51: 12519-12523Crossref PubMed Scopus (409) Google Scholar). The dominant pattern at mid-log phase was of connected diplococci with triplet bands of HADA perpendicular to the long axis of the cells. Peptidoglycan synthesis co-localized with the site of membrane fission during cell division, suggested by the presence of overlapping HADA and MitoTracker Red at mid-cell (Figure 1C). In untreated cells, the septal HADA band, which marked the sites of autolysin activity that leads to eventual cell separation, was often shorter than the growth bands of HADA incorporation at mid-coccus (Figure 1C). In LCA-treated cells, the HADA also incorporated into a triplet pattern; however, the middle septal band was longer (Figure 1C). These longer septa could be due to an imbalance in peptidoglycan synthesis and autolysin activity, which is required for successful cell separation. The presence of triplets of HADA bands, however, suggests that overall cell polarity and geometry were unaffected by LCA and that the synthesis machinery of peptidoglycan assembled normally at, or near, the mid-coccus. Our laboratory strain of E. faecium undergoes spontaneous autolysis in static cultures left over the course of a few days. The presence of LCA, DCA, and CDCA resulted in significant reductions in spontaneous autolysis (Figure S1F), suggesting that bile acids may at least partially inhibit autolysin activity. Biofilm formation is an important factor in multiple colonization models with enterococci (Paganelli et al., 2012Paganelli F.L. Willems R.J. Leavis H.L. Optimizing future treatment of enterococcal infections: attacking the biofilm?.Trends Microbiol. 2012; 20: 40-49Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and it is possible that gut microbes may respond to gut-associated molecules by increasing biofilm formation. We tested whether a panel of bile acids also increased VRE biofilm formation. All bile acids tested increased VRE biofilm formation compared to DMSO control, but LCA was the most potent, with a significant increase at 125 μM and 250 μM (Figure 1D). Biofilm formation in E. faecalis and E. faecium requires autolysin (Guiton et al., 2009Guiton P.S. Hung C.S. Kline K.A. Roth R. Kau A.L. Hayes E. Heuser J. Dodson K.W. Caparon M.G. Hultgren S.J. Contribution of autolysin and sortase A during Enterococcus faecalis DNA-dependent biofilm development.Infect. Immun. 2009; 77: 3626-3638Crossref PubMed Scopus (114) Google Scholar, Mohamed et al., 2004Mohamed J.A. Huang W. Nallapareddy S.R. Teng F. Murray B.E. Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.Infect. Immun. 2004; 72: 3658-3663Crossref PubMed Scopus (243) Google Scholar, Paganelli et al., 2013Paganelli F.L. Willems R.J.L. Jansen P. Hendrickx A. Zhang X. Bonten M.J.M. Leavis H.L. Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA release.mBio. 2013; 4: e00154Crossref PubMed Scopus (42) Google Scholar, Thomas et al., 2009Thomas V.C. Hiromasa Y. Harms N. Thurlow L. Tomich J. Hancock L.E. A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis.Mol. Microbiol. 2009; 72: 1022-1036Crossref PubMed Scopus (138) Google Scholar). To determine if LCA-induced biofilm formation depended on the major autolysin AtlA in E. faecium E1162, we assayed an atlA deletion mutant and a plasmid-complemented strain. LCA-induced biofilm formation was lost in atlA mutant cells and was rescued in the plasmid-complemented strain (Figure 1E). These data suggest that LCA mediates biofilm formation through the canonical AtlA-dependent pathway in VRE. Bile acids are amphipathic molecules with the potential to chelate cations. Cation concentration is critical to peptidoglycan biosynthesis and mediates cell separation of S. pneumoniae (Rochford and Mandle, 1953Rochford E.J. Mandle R.J. The production of chains by Diplococcus pneumoniae in magnesium deficient media.J. Bacteriol. 1953; 66: 554-560PubMed Google Scholar). To test if cations played a role in VRE chaining, we treated cultures with MgCl2 and asked whether this ameliorated the effects of LCA on VRE morphotype. Growth in media supplemented with MgCl2 inhibited LCA-induced sedimentation of VRE chains after overnight growth (Figure S3A). VRE chain length and width was smaller in the presence of LCA plus MgCl2 after 4 h of growth, suggesting that normal cell separation had been partially restored (Figures 2A and 2B ). Treatment with MgCl2 also reversed the effect of LCA on biofilm formation (Figures S3B and 2C). These data support a link between chaining, biofilm formation, and VRE cell surface charge. To test if cation chelators can affect cell separation, we grew VRE in the presence of the cation chelator EDTA and observed significant chaining, which was also reversed by adding MgCl2 to the media (Figure S3D). We did not, however, detect significant differences in biofilm formation upon treatment with EDTA (Figure S3E). To determine if the effects of LCA and MgCl2 on biofilm formation were conserved more widely among enterococci, we investigated vancomycin-sensitive E. faecium E1162, vancomycin-resistant E. faecalis V583, and vancomycin-sensitive E. faecalis JH2-2 and OG1RF. LCA increased biofilm formation in all strains, and this was inhibited by 100 mM MgCl2 (Figure 2C). These results confirmed that the effect of bile acids on biofilms is conserved across diverse enterococci. Next, we examined the effects of cations and bile acids on VRE during intestinal colonization. We treated mice with ampicillin and then gavaged the mice with either MgCl2 or a control for three successive days before and concomitant with VRE gavage. (Figure S3C) We did not find a statistically significant decrease in VRE burden. This could be due to many complexities of the environment in vivo, such as the presence of other cation sequestering molecules or the absorbance of MgCl2 by the host. Enterococci colonize antibiotic-naive mice when added to drinking water over a period of weeks (Kommineni et al., 2015Kommineni S. Bretl D.J. Lam V. Chakraborty R. Hayward M. Simpson P. Cao Y. Bousounis P. Kristich C.J. Salzman N.H. Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.Nature. 2015; 526: 719-722Crossref PubMed Scopus (253) Google Scholar); however, it likely does not colonize at a titer high enough to detect by microscopy against the background of a full gut microbiota. Antibiotic treatment breaks anti-VRE colonization resistance but also alters the bile acid pool and greatly reduces secondary bile acid levels (Buffie et al., 2015Buffie C.G. Bucci V. Stein R.R. McKenney P.T. Ling L. Gobourne A. Liu H. Kinnebrew M. Viale A. et al.Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile.Nature. 2015; 517: 205-208Crossref PubMed Scopus (1104) Google Scholar). To determine if the morphotype switch occurs in a context more directly relevant to gut colonization, we cultured VRE ex vivo in liquid brain heart infusion media mixed 1:1 with an extract of cecal content from naive mice. In these conditions, we observed a modest but significant increase in mean chain length of VRE (Figure 2D). To determine if LCA affects VRE morphology in the mouse intestine, we treated mice with antibiotics then fed mice LCA in the presence or absence of MgCl2. We observed VRE in the cecum using fluorescence in situ hybridization (Caballero et al., 2015Caballero S. Carter R. Ke X. Sušac B. Leiner I.M. Kim G.J. Miller L. Ling L. Manova K. Pamer E.G. Distinct but spatially overlapping intestinal niches for vancomycin-resistant Enterococcus faecium and carbapenem-resistant Klebsiella pneumoniae.PLoS Pathog. 2015; 11: e1005132Crossref PubMed Scopus (76) Google Scholar). We were not able to clearly distinguish chains of VRE in LCA-treated mice; however, we did observe a substantial difference in aggregation of VRE in mice treated with LCA (Figures 2E and 2F). These VRE aggregates were not present in mice treated with both LCA and MgCl2. Taken together, these data suggest that LCA and MgCl2 affect VRE morphotype and aggregation in vitro and during intestinal colonization. To determine the molecular mechanism underlying the bile-acid-induced chaining, we adapted experimental evolution (van Ditmarsch et al., 2013van Ditmarsch D. Boyle K.E. Sakhtah H. Oyler J.E. Nadell C.D. Déziel É. Dietrich L.E.P. Xavier J.B. Convergent evolution of hyperswarming leads to impaired biofilm formation in pathogenic bacteria.Cell Rep. 2013; 4: 697-708Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Kawecki et al., 2012Kawecki T.J. Lenski R.E. Ebert D. Hollis B. Olivieri I. Whitlock M.C. Experimental evolution.Trends Ecol. Evol. 2012; 27: 547-560Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) to take advantage of the relative density of chains versus diplococci. Chains fall to the bottom of standing cultures while diplococci are enriched at the top, resulting in low OD of the planktonic portion of an overnight culture treated with LCA (Figure 3A). We started serial passages from the top of cultures to fresh culture media in two lineages in parallel: DMSO and LCA treated (50 μM), daily for 5 weeks, comprising approximately 350 generations. Cultures were saved as freezer stocks periodically during the course of selection, and then, the stocks were assayed for sedimentation, chain length, and mouse intestinal colonization. After 14 days, the planktonic OD of the LCA-treated lineage increased to a level similar to the ancestral untreated strain (Figure 3B). The mean chain length of the LCA-treated population, however, did not decrease to that of the ancestral untreated strain until day 35 of passage (Figure 3C). These data suggest that selection for buoyancy preceded selection for smaller chains. We then tested whether the day 35 LCA-treated population (D35LCA) could colonize ampicillin treated mice. Mice gavaged with D35LCA harbored less CFUs in fecal pellets at 12 h post-infection compared with mice gavaged with the same amount of the ancestral VRE strain (Figure 3D). After 12 h, mice remained stably colonized with no difference between ancestral VRE and D35LCA, indicating that the colonization defect was transient (Figure 3E). This selection could have been confounded by the simultaneous selection for buoyancy and other effects of prolonged serial passage. In order to identify the genes underlying LCA-induced chain formation and to minimize the number of accumulated mutations, we isolated clones from day 7 of the serial passage, a time point when the planktonic OD had begun to rise but had not yet reached the level of the ancestral strain. We screened 500 colonies from the LCA-treated passage for OD after overnight growth in 50 μM LCA. Cultures with a planktonic OD greater than 0.4 were observed by light microscopy. From this, we isolated 4 clones that appeared to form predominantly diplococci in the presence of LCA. We also isolated 4 clones from the LCA passage with a planktonic OD of greater than 0.4 that appeared to form chains when grown overnight in LCA. Finally, we isolated 4 clones with a planktonic OD of less than 0.1 to uncover SNPs associated with continuous LCA treatment, without an effect on chain formation. From the DMSO-treated passage, we isolated 4 clones with a planktonic OD of greater than 0.2 after growth in LCA and 4 clones with a planktonic OD less than 0.1. These clones comprised a collection of 5 distinct phenotypic classes of isolates, which we refer to as phenotypic classes I–V (Table 1). We sequenced the whole genomes of 19 clones using Illumina MiSeq, and we identified the nucleotide variants using breseq (Deatherage and Barrick, 2014Deatherage D.E. Barrick J.E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.Methods Mol. Biol. 2014; 1151: 165-188Crossref PubMed Scopus (646) Google Scholar). The isolates accumulated 12 variants (non-synonymous SNP, substitution, or deletion) shared among all members of a phenotypic class, suggesting that they were linked to the underlying phenotypes (Figure 4; Table S1). Surprisingly, two of the SNPs occurred in genes frequently mutated in daptomycin-resistant enterococci, the essential two-component kinase walK/yycG and the three-component response regulator liaR (Diaz et al., 2014Diaz L. Tran T.T. Munita J.M. Miller W.R. Rincon S. Carvajal L.P. Wollam A. Reyes J. Panesso D. Rojas N.L. et al.Whole-genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs.Antimicrob. Agents Chemother. 2014; 58: 4527-4534Crossref PubMed Scopus (95) Google Scholar, Miller et al., 2013Miller C. Kong J. Tran T.T. Arias C.A. Saxer G. Shamoo Y. Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance.Antimicrob. Agents Chemother. 2013; 57: 5373-5383Crossref PubMed Scopus (68) Google Scholar).Table 1Selection Strains and Phenotypic ClassesClassPassageChains?ODILCAnohighIILCAyeshighIIILCAyeslowIVDMSOyesmediumVDMSOyeslow Open table in a new tab To link phenotype to genotype, we screened the clone collection in a series of assays. We measured changes in LCA-induced chain length by flow cytometry for the 19 sequenced strains. Only the strains from class I had mean forward scatter values that were significantly lower than VRE treated with LCA (Figure 5A). As walK is the only gene to contain a SNP in all 4 clones sequenced from class I, it suggests that signaling through WalK modulates chain length in response to LCA. However, we cannot rule out the effects of pleiotropy from other SNPs in the strain background. Recent developments in the field suggest that attempts to genetically alter the function of walK, which is an essential gene, frequently results in confou" @default.
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• W2940808813 title "Intestinal Bile Acids Induce a Morphotype Switch in Vancomycin-Resistant Enterococcus that Facilitates Intestinal Colonization" @default.
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• W2940808813 doi "https://doi.org/10.1016/j.chom.2019.03.008" @default.
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