Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus

English version

Autoři: Kathryn A. Coe ^aff001; Wonsik Lee ^aff001; Madeleine C. Stone ^aff001; Gloria Komazin-Meredith ^aff003; Timothy C. Meredith ^aff001; Yonatan H. Grad ^aff004; Suzanne Walker ^aff001
Působiště autorů: Department of Microbiology, Harvard Medical School, Boston, Massachusetts, United States of America ^aff001; School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea ^aff002; Department of Biochemistry and Molecular Biology, Pennsylvania State University, Pennsylvania, United States of America ^aff003; Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States of America ^aff004; Division of Infectious Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America ^aff005; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America ^aff006
Vyšlo v časopise: Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus. PLoS Pathog 15(11): e32767. doi:10.1371/journal.ppat.1007862
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1007862

Souhrn

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.

Klíčová slova:

Antibiotic resistance – Antibiotics – Genomic libraries – Genomic library construction – Methicillin-resistant Staphylococcus aureus – Polymerase chain reaction – Staphylococcus aureus – Transposable elements

Zdroje

1. Centers for Disease Control and Prevention (CDC). Antibiotic / antimicrobial resistance (AR / AMR) 2018 [updated September 10, 2018; cited 2018 December 21]. Available from: https://www.cdc.gov/drugresistance/about.html.

2. Centers for Disease Control and Prevention (CDC). Antibiotic resistance threats in the United States, 2013. 2013.

3. World Health Organization. Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. Geneva: 2017.

4. Monegro AF, Regunath H. Hospital acquired infections. StatPearls [Internet]: StatPearls Publishing; 2017.

5. Spagnolo AM, Orlando P, Panatto D, Amicizia D, Perdelli F, Cristina ML. Staphylococcus aureus with reduced susceptibility to vancomycin in healthcare settings. Journal of Preventive Medicine and Hygiene. 2014;55(4):137. 26137787

6. Stefani S, Campanile F, Santagati M, Mezzatesta ML, Cafiso V, Pacini G. Insights and clinical perspectives of daptomycin resistance in Staphylococcus aureus: a review of the available evidence. International Journal of Antimicrobial Agents. 2015;46(3):278–89. doi: 10.1016/j.ijantimicag.2015.05.008 26143590

7. Decousser J-W, Desroches M, Bourgeois-Nicolaos N, Potier J, Jehl F, Lina G, et al. Susceptibility trends including emergence of linezolid resistance among coagulase-negative staphylococci and meticillin-resistant Staphylococcus aureus from invasive infections. International Journal of Antimicrobial Agents. 2015;46(6):622–30. doi: 10.1016/j.ijantimicag.2015.07.022 26453147

8. Planet PJ, Narechania A, Chen L, Mathema B, Boundy S, Archer G, et al. Architecture of a species: phylogenomics of Staphylococcus aureus. Trends in Microbiology. 2017;25(2):153–66. doi: 10.1016/j.tim.2016.09.009 27751626

9. Bosi E, Monk JM, Aziz RK, Fondi M, Nizet V, Palsson BO. Comparative genome-scale modelling of Staphylococcus aureus strains identifies strain-specific metabolic capabilities linked to pathogenicity. Proceedings of the National Academy of Sciences. 2016;113(26):E3801–9.

10. van Opijnen T, Bodi KL, Camilli A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nature Methods. 2009;6(10):767. doi: 10.1038/nmeth.1377 19767758

11. Goodman AL, McNulty NP, Zhao Y, Leip D, Mitra RD, Lozupone CA, et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host & Microbe. 2009;6(3):279–89.

12. Chaudhuri RR, Allen AG, Owen PJ, Shalom G, Stone K, Harrison M, et al. Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH). BMC Genomics. 2009;10(1):291.

13. Fey PD, Endres JL, Yajjala VK, Widhelm TJ, Boissy RJ, Bose JL, et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio. 2013;4(1):e00537–12. doi: 10.1128/mBio.00537-12 23404398

14. Bae T, Banger AK, Wallace A, Glass EM, Aslund F, Schneewind O, et al. Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proceedings of the National Academy of Sciences. 2004;101(33):12312–7.

15. Christiansen MT, Kaas RS, Chaudhuri RR, Holmes MA, Hasman H, Aarestrup FM. Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus sequence type 398 survival. PLOS ONE. 2014;9(2):e89018. doi: 10.1371/journal.pone.0089018 24563689

16. Valentino MD, Foulston L, Sadaka A, Kos VN, Villet RA, Santa Maria J, et al. Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies. mBio. 2014;5(5):e01729–14. doi: 10.1128/mBio.01729-14 25182329

17. Santiago M, Matano LM, Moussa SH, Gilmore MS, Walker S, Meredith TC. A new platform for ultra-high density Staphylococcus aureus transposon libraries. BMC Genomics. 2015;16:252. doi: 10.1186/s12864-015-1361-3 25888466

18. Grosser MR, Paluscio E, Thurlow LR, Dillon MM, Cooper VS, Kawula TH, et al. Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence. PLOS Pathogens. 2018;14(3):e1006907. doi: 10.1371/journal.ppat.1006907 29554137

19. Carey AF, Rock JM, Krieger IV, Chase MR, Fernandez-Suarez M, Gagneux S, et al. TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities. PLOS Pathogens. 2018;14(3):e1006939. doi: 10.1371/journal.ppat.1006939 29505613

20. Poulsen BE, Yang R, Clatworthy AE, White T, Osmulski SJ, Li L, et al. Defining the core essential genome of Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences. 2019;116(20):10072–80.

21. Santiago M, Lee W, Fayad AA, Coe KA, Rajagopal M, Do T, et al. Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic. Nature Chemical Biology. 2018;14(6):601–8. doi: 10.1038/s41589-018-0041-4 29662210

22. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K-i, Oguchi A, et al. Genome and virulence determinants of high virulence community-acquired MRSA. The Lancet. 2002;359(9320):1819–27.

23. Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology. 2009;7(9):629. doi: 10.1038/nrmicro2200 19680247

24. Centers for Disease Control and Prevention (CDC). Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus—Minnesota and North Dakota, 1997–1999. Morbidity and Mortality Weekly Report. 1999;48(32):707. 21033181

25. Holden MT, Feil EJ, Lindsay JA, Peacock SJ, Day NP, Enright MC, et al. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proceedings of the National Academy of Sciences. 2004;101(26):9786–91.

26. Highlander SK, Hultén KG, Qin X, Jiang H, Yerrapragada S, Mason EO, et al. Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus. BMC Microbiology. 2007;7(1):1.

27. Kennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proceedings of the National Academy of Sciences. 2008;105(4):1327–32.

28. van Opijnen T, Dedrick S, Bento J. Strain dependent genetic networks for antibiotic-sensitivity in a bacterial pathogen with a large pan-genome. PLOS Pathogens. 2016;12(9):e1005869. doi: 10.1371/journal.ppat.1005869 27607357

29. Brown S, Santa Maria JP Jr., Walker S. Wall teichoic acids of gram-positive bacteria. Annual Review of Microbiology. 2013;67(1):313–36.

30. van Heijenoort J. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology. 2001;11(3):25R–36R. doi: 10.1093/glycob/11.3.25r 11320055

31. Connelly JC, De Leau ES, Leach DR. DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli. Nucleic Acids Research. 1999;27(4):1039–46. doi: 10.1093/nar/27.4.1039 9927737

32. Connelly JC, Kirkham LA, Leach DR. The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proceedings of the National Academy of Sciences. 1998;95(14):7969–74.

33. Wheeler R, Turner RD, Bailey RG, Salamaga B, Mesnage S, Mohamad SA, et al. Bacterial cell enlargement requires control of cell wall stiffness mediated by peptidoglycan hydrolases. mBio. 2015;6(4):e00660–15. doi: 10.1128/mBio.00660-15 26220963

34. Holland LM, O'donnell ST, Ryjenkov DA, Gomelsky L, Slater SR, Fey PD, et al. A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP. Journal of Bacteriology. 2008;190(15):5178–89. doi: 10.1128/JB.00375-08 18502872

35. Pasquina LW, Santa Maria JP, Walker S. Teichoic acid biosynthesis as an antibiotic target. Current Opinion in Microbiology. 2013;16(5):531–7. doi: 10.1016/j.mib.2013.06.014 23916223

36. Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, et al. A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity. Molecular microbiology. 2007;65(4):1078–91. doi: 10.1111/j.1365-2958.2007.05854.x 17640274

37. Kho K, Meredith TC. Salt-induced stress stimulates a lipoteichoic acid-specific three-component glycosylation system in Staphylococcus aureus. Journal of Bacteriology. 2018;200(12):e00017–18. doi: 10.1128/JB.00017-18 29632092

38. Neuhaus FC, Baddiley J. A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in Gram-positive bacteria. Microbiology Molecular Biology Reviews. 2003;67(4):686–723. doi: 10.1128/MMBR.67.4.686-723.2003 14665680

39. Gründling A, Schneewind O. Genes required for glycolipid synthesis and lipoteichoic acid anchoring in Staphylococcus aureus. Journal of Bacteriology. 2007;189(6):2521–30. doi: 10.1128/JB.01683-06 17209021

40. Gründling A, Schneewind O. Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus. Proceedings of the National Academy of Sciences. 2007;104(20):8478–83.

41. Kiriukhin MY, Debabov DV, Shinabarger DL, Neuhaus FC. Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase. Journal of Bacteriology. 2001;183(11):3506–14. doi: 10.1128/JB.183.11.3506-3514.2001 11344159

42. Sheen TR, Ebrahimi CM, Hiemstra IH, Barlow SB, Peschel A, Doran KS. Penetration of the blood-brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid. J Mol Med (Berl). 2010;88(6):633–9. doi: 10.1007/s00109-010-0630-5 20419283

43. Oku Y, Kurokawa K, Matsuo M, Yamada S, Lee B-L, Sekimizu K. Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells. Journal of Bacteriology. 2009;191(1):141–51. doi: 10.1128/JB.01221-08 18952789

44. Corrigan RM, Abbott JC, Burhenne H, Kaever V, Gründling A. c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLOS Pathogens. 2011;7(9):e1002217. doi: 10.1371/journal.ppat.1002217 21909268

45. Bæk KT, Bowman L, Millership C, Søgaard MD, Kaever V, Siljamäki P, et al. The cell wall polymer lipoteichoic acid becomes nonessential in Staphylococcus aureus cells lacking the ClpX chaperone. MBio. 2016;7(4):e01228–16. doi: 10.1128/mBio.01228-16 27507828

46. Karinou E, Schuster CF, Pazos M, Vollmer W, Gründling A. Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid. Journal of Bacteriology. 2018;201(1):1–18.

47. Campbell J, Singh AK, Santa Maria JP Jr., Kim Y, Brown S, Swoboda JG, et al. Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus. ACS Chem Biol. 2011;6(1):106–16. doi: 10.1021/cb100269f 20961110

48. Lee SH, Wang H, Labroli M, Koseoglu S, Zuck P, Mayhood T, et al. TarO-specific inhibitors of wall teichoic acid biosynthesis restore beta-lactam efficacy against methicillin-resistant staphylococci. Sci Transl Med. 2016;8(329):329ra32. doi: 10.1126/scitranslmed.aad7364 26962156

49. Eliopoulos GM, Thauvin C, Gerson B, Moellering R. In vitro activity and mechanism of action of A21978C1, a novel cyclic lipopeptide antibiotic. Antimicrobial Agents and Chemotherapy. 1985;27(3):357–62. doi: 10.1128/aac.27.3.357 3994349

50. Pogliano J, Pogliano N, Silverman JA. Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins. Journal of Bacteriology. 2012;194(17):4494–504. doi: 10.1128/JB.00011-12 22661688

51. Alborn W, Allen N, Preston D. Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 1991;35(11):2282–7. doi: 10.1128/aac.35.11.2282 1666494

52. Lakey JH, Ptak M. Fluorescence indicates a calcium-dependent interaction between the lipopeptide antibiotic LY 146032 and phospholipid membranes. Biochemistry. 1988;27(13):4639–45. doi: 10.1021/bi00413a009 2844233

53. Silverman JA, Perlmutter NG, Shapiro HM. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2003;47(8):2538–44. doi: 10.1128/AAC.47.8.2538-2544.2003 12878516

54. Müller A, Wenzel M, Strahl H, Grein F, Saaki TN, Kohl B, et al. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proceedings of the National Academy of Sciences. 2016;113(45):E7077–E86.

55. Heidary M, Khosravi AD, Khoshnood S, Nasiri MJ, Soleimani S, Goudarzi M. Daptomycin. Journal of Antimicrobial Chemotherapy. 2018;73(1):1–11. doi: 10.1093/jac/dkx349 29059358

56. Falord M, Karimova G, Hiron A, Msadek T. GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2012;56(2):1047–58. doi: 10.1128/AAC.05054-11 22123691

57. Meehl M, Herbert S, Gotz F, Cheung A. Interaction of the GraRS two-component system with the VraFG ABC transporter to support vancomycin-intermediate resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2007;51(8):2679–89. doi: 10.1128/AAC.00209-07 17502406

58. Cui L, Lian J-Q, Neoh H-m, Reyes E, Hiramatsu K. DNA microarray-based identification of genes associated with glycopeptide resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2005;49(8):3404–13. doi: 10.1128/AAC.49.8.3404-3413.2005 16048954

59. Fournier B, Hooper DC. A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus. Journal of Bacteriology. 2000;182(14):3955–64. doi: 10.1128/jb.182.14.3955-3964.2000 10869073

60. Fournier B, Klier A, Rapoport G. The two‐component system ArlS–ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Molecular Microbiology. 2001;41(1):247–61. doi: 10.1046/j.1365-2958.2001.02515.x 11454217

61. Bugg T, Walsh C. Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Natural Product Reports. 1992;9(3):199–215. doi: 10.1039/np9920900199 1436736

62. Blake KL, O'Neill AJ, Mengin‐Lecreulx D, Henderson PJ, Bostock JM, Dunsmore CJ, et al. The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors. Molecular Microbiology. 2009;72(2):335–43. doi: 10.1111/j.1365-2958.2009.06648.x 19298367

63. Kullik I, Jenni R, Berger-Bächi B. Sequence of the putative alanine racemase operon in Staphylococcus aureus: insertional interruption of this operon reduces D-alanine substitution of lipoteichoic acid and autolysis. Gene. 1998;219(1–2):9–17. doi: 10.1016/s0378-1119(98)00404-1 9756984

64. Peschel A, Jack RW, Otto M, Collins LV, Staubitz P, Nicholson G, et al. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine. Journal of Experimental Medicine. 2001;193(9):1067–76. doi: 10.1084/jem.193.9.1067 11342591

65. Eswara PJ, Brzozowski RS, Viola MG, Graham G, Spanoudis C, Trebino C, et al. An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics. eLife. 2018;7:e38856. doi: 10.7554/eLife.38856 30277210

66. Steele VR, Bottomley AL, Garcia‐Lara J, Kasturiarachchi J, Foster SJ. Multiple essential roles for EzrA in cell division of Staphylococcus aureus. Molecular Microbiology. 2011;80(2):542–55. doi: 10.1111/j.1365-2958.2011.07591.x 21401734

67. Pang T, Wang X, Lim HC, Bernhardt TG, Rudner DZ. The nucleoid occlusion factor Noc controls DNA replication initiation in Staphylococcus aureus. PLOS Genetics. 2017;13(7):e1006908. doi: 10.1371/journal.pgen.1006908 28723932

68. Bayer AS, Mishra NN, Cheung AL, Rubio A, Yang S-J. Dysregulation of mprF and dltABCD expression among daptomycin-non-susceptible MRSA clinical isolates. Journal of Antimicrobial Chemotherapy. 2016;71(8):2100–4. doi: 10.1093/jac/dkw142 27121398

69. Yang S-J, Mishra NN, Rubio A, Bayer AS. Causal role of single nucleotide polymorphisms (SNPs) within the mprF gene of Staphylococcus aureus in daptomycin resistance. Antimicrobial Agents and Chemotherapy. 2013;57(11):5658–64. doi: 10.1128/AAC.01184-13 24002096

70. Neoh HM, Cui L, Yuzawa H, Takeuchi F, Matsuo M, Hiramatsu K. Mutated response regulator graR is responsible for phenotypic conversion of Staphylococcus aureus from heterogeneous vancomycin-intermediate resistance to vancomycin-intermediate resistance. Antimicrobial Agents and Chemotherapy. 2008;52(1):45–53. doi: 10.1128/AAC.00534-07 17954695

71. Blake KL, O'Neill AJ. Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents. Journal of Antimicrobial Chemotherapy. 2013;68(1):12–6. doi: 10.1093/jac/dks373 23045225

72. Humphries RM, Pollett S, Sakoulas G. A current perspective on daptomycin for the clinical microbiologist. Clinical Microbiology Reviews. 2013;26(4):759–80. doi: 10.1128/CMR.00030-13 24092854

73. Cafiso V, Bertuccio T, Purrello S, Campanile F, Mammina C, Sartor A, et al. dltA overexpression: A strain-independent keystone of daptomycin resistance in methicillin-resistant Staphylococcus aureus. International Journal of Antimicrobial Agents. 2014;43(1):26–31. doi: 10.1016/j.ijantimicag.2013.10.001 24183798

74. Mishra NN, Liu GY, Yeaman MR, Nast CC, Proctor RA, McKinnell J, et al. Carotenoid-related alteration of cell membrane fluidity impacts Staphylococcus aureus susceptibility to host defense peptides. Antimicrobial Agents and Chemotherapy. 2011;55(2):526–31. doi: 10.1128/AAC.00680-10 21115796

75. Friedman L, Alder JD, Silverman JA. Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2006;50(6):2137–45. doi: 10.1128/AAC.00039-06 16723576

76. Tran TT, Munita JM, Arias CA. Mechanisms of drug resistance: daptomycin resistance. Annals of the New York Academy of Sciences. 2015;1354(1):32–53.

77. Dhand A, Sakoulas G. Daptomycin in combination with other antibiotics for the treatment of complicated methicillin-resistant Staphylococcus aureus bacteremia. Clinical Therapeutics. 2014;36(10):1303–16. doi: 10.1016/j.clinthera.2014.09.005 25444563

78. Miró JM, Entenza JM, Del Río A, Velasco M, Castañeda X, Garcia de la Mària C, et al. High-dose daptomycin plus fosfomycin is safe and effective in treating methicillin-susceptible and methicillin-resistant Staphylococcus aureus endocarditis. Antimicrobial Agents and Chemotherapy. 2012;56(8):4511–5. doi: 10.1128/AAC.06449-11 22644033

79. Shaw Perujo E, Miró Meda JM, Puig-Asensio M, Pigrau C, Barcenilla F, Murillas J, et al. Daptomycin plus fosfomycin versus daptomycin monotherapy in treating MRSA: protocol of a multicentre, randomised, phase III trial. BMJ Open. 2015;5(3). doi: 10.1136/bmjopen-2014-006723

80. Choe D, Szubin R, Dahesh S, Cho S, Nizet V, Palsson B, et al. Genome-scale analysis of Methicillin-resistant Staphylococcus aureus USA300 reveals a tradeoff between pathogenesis and drug resistance. Science Reports. 2018;8(1):2215.

81. Kumaraswamy M, Lin L, Olson J, Sun C-F, Nonejuie P, Corriden R, et al. Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia. Journal of Antimicrobial Chemotherapy. 2016;71(5):1264–9. doi: 10.1093/jac/dkv487 26832758

82. Goecks J, Nekrutenko A, Taylor J, Galaxy T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biology. 2010;11(8):R86. doi: 10.1186/gb-2010-11-8-r86 20738864

83. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Research. 2018;46(W1):W537–W44. doi: 10.1093/nar/gky379 29790989

84. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. doi: 10.1093/bioinformatics/btu153 24642063

85. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MT, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31(22):3691–3. doi: 10.1093/bioinformatics/btv421 26198102

86. Bernhardt J, Fuchs S, Mäder U, Mehlan H, Michalik S, Otto A, et al. AureoWiki 2018. Available from: http://aureowiki.med.uni-greifswald.de/Main_Page.

87. DeJesus MA, Ambadipudi C, Baker R, Sassetti C, Ioerger TR. TRANSIT—a software tool for Himar1 TnSeq analysis. PLOS Computational Biology. 2015;11(10):e1004401. doi: 10.1371/journal.pcbi.1004401 26447887

88. Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Research. 2016;45(D1):D183–D9. doi: 10.1093/nar/gkw1138 27899595

89. Gama-Castro S, Salgado H, Santos-Zavaleta A, Ledezma-Tejeida D, Muniz-Rascado L, Garcia-Sotelo JS, et al. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond. Nucleic Acids Research. 2016;44(D1):D133–43. doi: 10.1093/nar/gkv1156 26527724

90. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754–60. doi: 10.1093/bioinformatics/btp324 19451168

91. The Broad Institute. Picard Tools [cited 2018]. Available from: http://broadinstitute.github.io/picard.

92. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLOS ONE. 2014;9(11):e112963. doi: 10.1371/journal.pone.0112963 25409509

93. Chojnacki S, Cowley A, Lee J, Foix A, Lopez R. Programmatic access to bioinformatics tools from EMBL-EBI update: 2017. Nucleic Acids Research. 2017;45(W1):W550–W3. doi: 10.1093/nar/gkx273 28431173

94. Pasquina L, Santa Maria JP Jr., McKay Wood B, Moussa SH, Matano LM, Santiago M, et al. A synthetic lethal approach for compound and target identification in Staphylococcus aureus. Nature Chemical Biology. 2016;12(1):40–5. doi: 10.1038/nchembio.1967 26619249

95. Lee W, Do T, Zhang G, Kahne D, Meredith TC, Walker S. Antibiotic combinations that enable one-step, targeted mutagenesis of chromosomal genes. ACS Infectious Diseases. 2018;4(6):1007–18. doi: 10.1021/acsinfecdis.8b00017 29534563

96. Santa Maria JP Jr., Sadaka A, Moussa SH, Brown S, Zhang YJ, Rubin EJ, et al. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc Natl Acad Sci U S A. 2014;111(34):12510–5. doi: 10.1073/pnas.1404099111 25104751

97. Vickery CR, Wood BM, Morris HG, Losick R, Walker S. Reconstitution of Staphylococcus aureus lipoteichoic acid synthase activity identifies Congo red as a selective inhibitor. Journal of the American Chemical Society. 2018;140(3):876–9. doi: 10.1021/jacs.7b11704 29300473