Multidrug resistance and major facilitator superfamily antimicrobial efflux pumps of the ESKAPEE pathogen Staphylococcus aureus

Abstract

Multiple drug-resistantStaphylococcus aureus bacterial pathogens are causative agents of serious infectious disease and are responsible for significant morbidity and mortality rates. Of particular concern in the public health domain are strains of methicillin-resistant S. aureus (MRSA), a member of the Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Escherichia coli (ESKAPEE) group of bacterial pathogens, many of which are recalcitrant to effective chemotherapy in the clinical setting due to their resistance to multiple antimicrobial agents. An important mechanism that confers multi-drug resistance in MRSA involves the active efflux of structurally different antimicrobial agents by members of the major facilitator superfamily (MFS) of proteins. The multidrug efflux pumps of the MFS share similar amino acid sequences, protein structures, and a common evolutionary origin. As such, the multidrug efflux pumps of the MFS are thought to operate by a similar solute transport mechanism and, thus, represent suitable targets for modulating their transport activities. This review article addresses MRSA as a serious pathogen, the mechanisms of antimicrobial resistance, and the functional and structural roles of the multidrug efflux pumps of the MFS in conferring pathogenicity.

Keywords

Bacteria / pathogens / infection / antimicrobial agents / major facilitator superfamily / multidrug efflux pump / Staphylococcus aureus / antimicrobial resistance

References

[1] Ford CA, Hurford IM, Cassat JE. Antivirulence Strategies for the Treatment of Staphylococcus aureus Infections: A Mini Review. Front Microbiol. 2021; 11: 632706.

[2] Pollitt EJG, Szkuta PT, Burns N, Foster SJ. Staphylococcus aureus infection dynamics. PLoS Pathog. 2018; 14: e1007112.

[3] Zhu F, Zhuang H, Ji S, Xu E, Di L, Wang Z, et al. Household Transmission of Community—Associated Methicillin—Resistant Staphylococcus Aureus. Front Public Health. 2021; 9: 658638.

[4] Effelsberg N, Stegger M, Peitzmann L, Altinok O, Coombs GW, Pichon B, et al. Global Epidemiology and Evolutionary History of Staphylococcus aureus ST45. J Clin Microbiol. 2020; 59: e02198—20.

[5] Campanile F, Bongiorno D, Perez M, Mongelli G, Sessa L, Benvenuto S, et al. Epidemiology of Staphylococcus aureus in Italy: First nationwide survey, 2012. J Glob Antimicrob Resist. 2015; 3: 247—54.

[6] Rasigade JP, Dumitrescu O, Lina G. New epidemiology of Staphylococcus aureus infections. Clin Microbiol Infect. 2014; 20: 587—8.

[7] Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev. 2015; 28: 603-61.

[8] Gordon RJ, Lowy FD. Pathogenesis of methicillin—resistant Staphylococcus aureus infection. Clin Infect Dis. 2008; 46: S350-9.

[9] Peetermans M, Verhamme P, Vanassche T. Coagulase Activity by Staphylococcus aureus: A Potential Target for Therapy? Semin Thromb Hemost. 2015; 41: 433-44.

[10] Chambers HF. The changing epidemiology of Staphylococcus aureus? Emerg Infect Dis. 2001; 7: 178-82.

[11] Hindy JR, Quintero—Martinez JA, Lee AT, Scott CG, Gerberi DJ, Mahmood M, et al. Incidence Trends and Epidemiology of Staphylococcus aureus Bacteremia: A Systematic Review of Population—Based Studies. Cureus. 2022; 14: e25460.

[12] Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Global epidemiology of community—associated methicillin resistant Staphylococcus aureus (CA—MRSA) . Curr Opin Microbiol. 2012; 15: 588-95.

[13] Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H, et al. Meticillin—resistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods. Int J Antimicrob Agents. 2012; 39: 273-82.

[14] Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis. 2008; 197: 1079-81.

[15] Mahmood HY, Jamshidi S, Sutton JM, Rahman KM. Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps. Curr Med Chem. 2016; 23: 1062-81.

[16] GBD 2019 Antimicrobial Resistance Collaborators. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2022; 400: 2221—48.

[17] GBD 2019 Diseases and Injuries Collaborators. Global burden of 369 diseases and injuries in 204 countries and territories, 1990—2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2020; 396: 1204—22.

[18] Klein EY, Sun L, Smith DL, Laxminarayan R. The changing epidemiology of methicillin—resistant Staphylococcus aureus in the United States: a national observational study. Am J Epidemiol. 2013; 177: 666-74.

[19] Schaumburg F, Alabi AS, Peters G, Becker K. New epidemiology of Staphylococcus aureus infection in Africa. Clin Microbiol Infect. 2014; 20: 589-96.

[20] Chen CJ, Huang YC. New epidemiology of Staphylococcus aureus infection in Asia. Clin Microbiol Infect. 2014; 20: 605—23.

[21] Moellering RC Jr. MRSA: the first half century. J Antimicrob Chemother. 2012; 67: 4-11.

[22] Eriksen KR, Erichsen I. Resistance to methicillin, isoxazolyl penicillins, and cephalothin in Staphylococcus aureus. Acta Pathol Microbiol Scand. 1964; 62: 255-75.

[23] Harkins CP, Pichon B, Doumith M, Parkhill J, Westh H, Tomasz A, et al. Methicillin—resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice. Genome Biol. 2017; 18: 130.

[24] Wielders CL, Fluit AC, Brisse S, Verhoef J, Schmitz FJ. mecA gene is widely disseminated in Staphylococcus aureus population. J Clin Microbiol. 2002; 40: 3970—5.

[25] Kluytmans—Vandenbergh MF, Kluytmans JA. Community—acquired methicillin—resistant Staphylococcus aureus: current perspectives. Clin Microbiol Infect. 2006; 12: 9-15.

[26] Hassoun A, Linden PK, Friedman B. Incidence, prevalence, and management of MRSA bacteremia across patient populations—a review of recent developments in MRSA management and treatment. Crit Care. 2017; 21: 211.

[27] O’Neill J. Antimicrobial resistance: tackling a crisis for the health and wealth of nations. Rev Antimicrob Resist. 2014; 20: 1-6.

[28] Centers for Disease Control and Prevention (CDC). Four pediatric deaths from community—acquired methicillin—resistant Staphylococcus aureus—Minnesota and North Dakota, 1997—1999. MMWR Morb Mortal Wkly Rep. 1999; 48: 707-10.

[29] Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, Boyle—Vavra S, et al. Community—acquired methicillin—resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA. 1998; 279: 593—8.

[30] Hou Z, Liu L, Wei J, Xu B. Progress in the Prevalence, Classification and Drug Resistance Mechanisms of Methicillin—Resistant Staphylococcus aureus. Infect Drug Resist. 2023; 16: 3271—92.

[31] Maguire GP, Arthur AD, Boustead PJ, Dwyer B, Currie BJ. Emerging epidemic of community—acquired methicillin—resistant Staphylococcus aureus infection in the Northern Territory. Med J Aust. 1996; 164: 721—3.

[32] Mason E, Nsonwu O, Elmes J, Chudasama D, Pearson C, Hasan L, et al. Increased rates of hospital—onset Staphylococcus aureus bacteraemia in National Health Service acute trusts in England between June 2020 and March 2021: a national surveillance review. J Hosp Infect. 2024; 143: 33-7.

[33] Kavanagh KT. Control of MSSA and MRSA in the United States: protocols, policies, risk adjustment and excuses. Antimicrob Resist Infect Control. 2019; 8: 103.

[34] Kourtis AP, Hatfield K, Baggs J, Mu Y, See I, Epson E, et al. Vital Signs: Epidemiology and Recent Trends in Methicillin—Resistant and in Methicillin—Susceptible Staphylococcus aureus Bloodstream Infections — United States. MMWR Morb Mortal Wkly Rep. 2019; 68: 214—9.

[35] Lakhundi S, Zhang K. Methicillin—Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology. Clin Microbiol Rev. 2018; 31: e00020—18.

[36] Thiede SN, Snitkin ES, Trick W, Payne D, Aroutcheva A, Weinstein RA, et al. Genomic Epidemiology Suggests Community Origins of Healthcare—Associated USA300 Methicillin—Resistant Staphylococcus aureus. J Infect Dis. 2022; 226: 157-66.

[37] van Duin D, Paterson DL. Multidrug—Resistant Bacteria in the Community: An Update. Infect Dis Clin North Am. 2020; 34: 709—22.

[38] Preeja PP, Kumar SH, Shetty V. Prevalence and Characterization of Methicillin—Resistant Staphylococcus aureus from Community— and Hospital—Associated Infections: A Tertiary Care Center Study. Antibiotics (Basel). 2021; 10: 197.

[39] Gittens—St Hilaire MV, Chase E, Alleyne D. Prevalence, molecular characteristics and antimicrobial susceptibility patterns of MRSA in hospitalized and nonhospitalized patients in Barbados. New Microbes New Infect. 2020; 35: 100659.

[40] Silva V, Correia S, Rocha J, Manaia CM, Silva A, García—Díez J, et al. Antimicrobial Resistance and Clonal Lineages of Staphylococcus aureus from Cattle, Their Handlers, and Their Surroundings: A Cross—Sectional Study from the One Health Perspective. Microorganisms. 2022; 10: 941.

[41] Chen H, Yin Y, van Dorp L, Shaw LP, Gao H, Acman M, et al. Drivers of methicillin—resistant Staphylococcus aureus (MRSA) lineage replacement in China. Genome Med. 2021; 13: 171.

[42] Knox J, Uhlemann AC, Lowy FD. Staphylococcus aureus infections: transmission within households and the community. Trends Microbiol. 2015; 23: 437-44.

[43] Tracy LA, Furuno JP, Harris AD, Singer M, Langenberg P, Roghmann MC. Staphylococcus aureus infections in US veterans, Maryland, USA, 1999—2008. Emerg Infect Dis. 2011; 17: 441—8.

[44] Moran GJ, Krishnadasan A, Gorwitz RJ, Fosheim GE, McDougal LK, Carey RB, et al. ; EMERGEncy ID Net Study Group. Methicillin—resistant S. aureus infections among patients in the emergency department. N Engl J Med. 2006; 355: 666-74.

[45] Varela MF, Stephen J, Lekshmi M, Ojha M, Wenzel N, Sanford LM, et al. Bacterial Resistance to Antimicrobial Agents. Antibiotics (Basel). 2021; 10: 593.

[46] Pantosti A, Sanchini A, Monaco M. Mechanisms of antibiotic resistance in Staphylococcus aureus. Future Microbiol. 2007; 2: 323-34.

[47] Varela MF, Kumar S. Molecular mechanisms of bacterial resistance to antimicrobial agents. In: Méndez—Vilas A, editor. Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education. Badajoz, Spain: Formatex Research Center; 2013. pp. 522—34.

[48] Driebe EM, Sahl JW, Roe C, Bowers JR, Schupp JM, Gillece JD, et al. Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus. PLoS One. 2015; 10: e0130955.

[49] Cavalcante FS, Schuenck RP, Ferreira DC, da Costa CR, Nouér SA, dos Santos KR. Meticillin—resistantStaphylococcus aureus: spread of specific lineages among patients in different wards at a Brazilian teaching hospital. J Hosp Infect. 2014; 86: 151—4.

[50] Ruffing U, Alabi A, Kazimoto T, Vubil DC, Akulenko R, Abdulla S, et al. Community—Associated Staphylococcus aureus from Sub—Saharan Africa and Germany: A Cross—Sectional Geographic Correlation Study. Sci Rep. 2017; 7: 154.

[51] Ip M, Yung RW, Ng TK, Luk WK, Tse C, Hung P, et al. Contemporary methicillin—resistant Staphylococcus aureus clones in Hong Kong. J Clin Microbiol. 2005; 43: 5069-73.

[52] Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin—resistant Staphylococcus aureus. PLoS One. 2011; 6: e17936.

[53] Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399: 629—55.

[54] Diekema DJ, Pfaller MA, Schmitz FJ, Smayevsky J, Bell J, Jones RN, et al. ; SENTRY Participants Group. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997—1999. Clin Infect Dis. 2001; 32: S114-32.

[55] Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, et al. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin—resistant Staphylococcus aureus infections in adults and children. Clin Infect Dis. 2011; 52: e18-55.

[56] Dyar OJ, Huttner B, Schouten J, Pulcini C ; ESGAP (ESCMID Study Group for Antimicrobial stewardshiP). What is antimicrobial stewardship? Clin Microbiol Infect. 2017; 23: 793-8.

[57] Boyce JM, Pittet D ; Healthcare Infection Control Practices Advisory Committee. Guideline for Hand Hygiene in Health—Care Settings: recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epidemiol. 2002; 23: S3-40.

[58] Siegel JD, Rhinehart E, Jackson M, Chiarello L ; Healthcare Infection Control Practices Advisory Committee. Management of multidrug—resistant organisms in health care settings, 2006. Am J Infect Control. 2007; 35: S165-93.

[59] Lekshmi M, Ammini P, Kumar S, Varela MF. The Food Production Environment and the Development of Antimicrobial Resistance in Human Pathogens of Animal Origin. Microorganisms. 2017; 5: 11.

[60] Reygaert WC. An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiol. 2018; 4: 482-501.

[61] Lowy FD. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest. 2003; 111: 1265-73.

[62] Lim D, Strynadka NC. Structural basis for the β lactam resistance of PBP2a from methicillin—resistant Staphylococcus aureus. Nat Struct Biol. 2002; 9: 870-6.

[63] Vestergaard M, Frees D, Ingmer H. Antibiotic Resistance and the MRSA Problem. Microbiol Spectr. 2019; 7: 10.1128/microbiolspec.gpp3—0057.

[64] Fisher JF, Mobashery S. β—Lactams against the Fortress of the Gram—Positive Staphylococcus aureus Bacterium. Chem Rev. 2021; 121: 3412-63.

[65] Gregory PD, Lewis RA, Curnock SP, Dyke KG. Studies of the repressor (BlaI) of β—lactamase synthesis in Staphylococcus aureus. Mol Microbiol. 1997; 24: 1025-37.

[66] Zhang HZ, Hackbarth CJ, Chansky KM, Chambers HF. A Proteolytic Transmembrane Signaling Pathway and Resistance to β—Lactams in Staphylococci. Science. 2001; 291: 1962—5.

[67] Page MG. Beta—lactam antibiotics. In: Dougherty TJ, Pucci MJ, editors. Antibiotic Discovery and Development. Berlin/Heidelberg: Springer; 2011. pp. 79-117.

[68] Noto MJ, Fox PM, Archer GL. Spontaneous deletion of the methicillin resistance determinant, mecA, partially compensates for the fitness cost associated with high—level vancomycin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 2008; 52: 1221-9.

[69] Bitrus AA, Zakaria Z, Goni MD, Mshelia IT. Dissemination of resistance and virulence determinants in methicillin—resistant Staphylococcus aureus during colonization and disease: A Review. Adv Anim Vet Sci. 2018; 6: 44-54.

[70] Griffith RS. Vancomycin use—an historical review. J Antimicrob Chemother. 1984; 14: 1-5.

[71] Unni S, Siddiqui TJ, Bidaisee S. Reduced Susceptibility and Resistance to Vancomycin of Staphylococcus aureus: A Review of Global Incidence Patterns and Related Genetic Mechanisms. Cureus. 2021; 13: e18925.

[72] Micek ST. Alternatives to vancomycin for the treatment of methicillin—resistant Staphylococcus aureus infections. Clin Infect Dis. 2007; 45: S184-90.

[73] Haseeb A, Ajit Singh V, Teh CSJ, Loke MF. Addition of ceftaroline fosamil or vancomycin to PMMA: An in vitro comparison of biomechanical properties and anti—MRSA efficacy. J Orthop Surg (Hong Kong). 2019; 27: 2309499019850324.

[74] Arthur M, Molinas C, Depardieu F, Courvalin P. Characterization of Tn1546, a Tn3—related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1993; 175: 117-27.

[75] Walsh CT, Fisher SL, Park IS, Prahalad M, Wu Z. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story. Chem Biol. 1996; 3: 21-8.

[76] McGuinness WA, Malachowa N, DeLeo FR. Vancomycin Resistance in Staphylococcus aureus. Yale J Biol Med. 2017; 90: 269-81.

[77] Arredondo—Alonso S, Top J, Corander J, Willems RJL, Schürch AC. Mode and dynamics of vanA—type vancomycin resistance dissemination in Dutch hospitals. Genome Med. 2021; 13: 9.

[78] Sun L, Xu J, Wang W, He F. Emergence of vanA—Type Vancomycin—Resistant Enterococcus faecium ST 78 Strain with a rep2—Type Plasmid Carrying a Tn1546—Like Element Isolated from a Urinary Tract Infection in China. Infect Drug Resist. 2020; 13: 949—55.

[79] Vimberg V, Zieglerová L, Buriánková K, Branny P, Balíková Novotná G. VanZ Reduces the Binding of Lipoglycopeptide Antibiotics to Staphylococcus aureus and Streptococcus pneumoniae Cells. Front Microbiol. 2020; 11: 566.

[80] Johnson AP, Uttley AH, Woodford N, George RC. Resistance to vancomycin and teicoplanin: an emerging clinical problem. Clin Microbiol Rev. 1990; 3: 280-91.

[81] Howden BP, Davies JK, Johnson PD, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin—intermediate and heterogeneous vancomycin—intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev. 2010; 23: 99-139.

[82] Amberpet R, Sistla S, Sugumar M, Nagasundaram N, Manoharan M, Parija SC. Detection of heterogeneous vancomycin—intermediate Staphylococcus aureus: A preliminary report from south India. Indian J Med Res. 2019; 150: 194—8.

[83] Severin A, Tabei K, Tenover F, Chung M, Clarke N, Tomasz A. High level oxacillin and vancomycin resistance and altered cell wall composition in Staphylococcus aureus carrying the staphylococcal mecA and the enterococcal vanA gene complex. J Biol Chem. 2004; 279: 3398-407.

[84] Hu Q, Peng H, Rao X. Molecular Events for Promotion of Vancomycin Resistance in Vancomycin Intermediate Staphylococcus aureus. Front Microbiol. 2016; 7: 1601.

[85] Hiramatsu K, Kayayama Y, Matsuo M, Aiba Y, Saito M, Hishinuma T, et al. Vancomycin—intermediate resistance in Staphylococcus aureus. J Glob Antimicrob Resist. 2014; 2: 213-24.

[86] Suresh MK, Biswas R, Biswas L. An update on recent developments in the prevention and treatment of Staphylococcus aureus biofilms. Int J Med Microbiol. 2019; 309: 1-12.

[87] Yan J, Bassler BL. Surviving as a Community: Antibiotic Tolerance and Persistence in Bacterial Biofilms. Cell Host Microbe. 2019; 26: 15-21.

[88] Tao Q, Wu Q, Zhang Z, Liu J, Tian C, Huang Z, et al. Meta—Analysis for the Global Prevalence of Foodborne Pathogens Exhibiting Antibiotic Resistance and Biofilm Formation. Front Microbiol. 2022; 13: 906490.

[89] Silva V, Correia E, Pereira JE, González—Machado C, Capita R, Alonso—Calleja C, et al. Biofilm Formation of Staphylococcus aureus from Pets, Livestock, and Wild Animals: Relationship with Clonal Lineages and Antimicrobial Resistance. Antibiotics (Basel). 2022; 11: 772.

[90] Otto M. Staphylococcal Biofilms. Microbiol Spectr. 2018; 6: 10.1128/microbiolspec.gpp3—0023.

[91] Costa SS, Viveiros M, Amaral L, Couto I. Multidrug Efflux Pumps in Staphylococcus aureus: an Update. Open Microbiol J. 2013; 7: 59-71.

[92] Hassanzadeh S, Ganjloo S, Pourmand MR, Mashhadi R, Ghazvini K. Epidemiology of efflux pumps genes mediating resistance among Staphylococcus aureus; A systematic review. Microb Pathog. 2020; 139: 103850.

[93] Andersen JL, He GX, Kakarla P, KC R, Kumar S, Lakra WS, et al. Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens. Int J Environ Res Public Health. 2015; 12: 1487-547.

[94] Lekshmi M, Ammini P, Adjei J, Sanford LM, Shrestha U, Kumar S, et al. Modulation of antimicrobial efflux pumps of the major facilitator superfamily in Staphylococcus aureus. AIMS Microbiol. 2018; 4: 1-18.

[95] Jang S. Multidrug efflux pumps in Staphylococcus aureus and their clinical implications. J Microbiol. 2016; 54: 1-8.

[96] Stephen J, Salam F, Lekshmi M, Kumar SH, Varela MF. The Major Facilitator Superfamily and Antimicrobial Resistance Efflux Pumps of the ESKAPEE Pathogen Staphylococcus aureus. Antibiotics (Basel). 2023; 12: 343.

[97] Kumar S, He G, Kakarla P, Shrestha U, Ranjana KC, Ranaweera I, et al. Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation. Infect Disord Drug Targets. 2016; 16: 28-43.

[98] Varela MF, Kumar S. Strategies for discovery of new molecular targets for anti—infective drugs. Curr Opin Pharmacol. 2019; 48: 57-68.

[99] Lekshmi M, Ortiz—Alegria A, Kumar S, Varela MF. Major facilitator superfamily efflux pumps in human pathogens: Role in multidrug resistance and beyond. Curr Res Microb Sci. 2024; 7: 100248.

[100] Mahey N, Tambat R, Verma DK, Chandal N, Thakur KG, Nandanwar H. Antifungal Azoles as Tetracycline Resistance Modifiers in Staphylococcus aureus. Appl Environ Microbiol. 2021; 87: e0015521.

[101] Ubukata K, Itoh—Yamashita N, Konno M. Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1989; 33: 1535—9.

[102] Yoshida H, Bogaki M, Nakamura S, Ubukata K, Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990; 172: 6942—9.

[103] Truong—Bolduc QC, Dunman PM, Strahilevitz J, Projan SJ, Hooper DC. MgrA is a multiple regulator of two new efflux pumps in Staphylococcus aureus. J Bacteriol. 2005; 187: 2395-405.

[104] Maloney PC. Bacterial transporters. Curr Opin Cell Biol. 1994; 6: 571-82.

[105] Hirai T, Heymann JA, Maloney PC, Subramaniam S. Structural model for 12—helix transporters belonging to the major facilitator superfamily. J Bacteriol. 2003; 185: 1712—8.

[106] Law CJ, Maloney PC, Wang DN. Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol. 2008; 62: 289-305.

[107] Varela MF, Wilson TH. Molecular biology of the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1996; 1276: 21-34.

[108] Kumar S, Lekshmi M, Parvathi A, Ojha M, Wenzel N, Varela MF. Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps. Microorganisms. 2020; 8: 266.

[109] Saidijam M, Benedetti G, Ren Q, Xu Z, Hoyle CJ, Palmer SL, et al. Microbial drug efflux proteins of the major facilitator superfamily. Curr Drug Targets. 2006; 7: 793-811.

[110] Nikaido H, Pagès JM. Broad—specificity efflux pumps and their role in multidrug resistance of Gram—negative bacteria. FEMS Microbiol Rev. 2012; 36: 340-63.

[111] Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med. 2007; 39: 162-76.

[112] Li XZ, Plésiat P, Nikaido H. The challenge of efflux—mediated antibiotic resistance in Gram—negative bacteria. Clin Microbiol Rev. 2015; 28: 337-418.

[113] Ranaweera I, Shrestha U, Ranjana KC, Kakarla P, Willmon TM, Hernandez AJ, et al. Structural comparison of bacterial multidrug efflux pumps of the major facilitator superfamily. Trends Cell Mol Biol. 2015; 10: 131-40.

[114] Yaffe D, Radestock S, Shuster Y, Forrest LR, Schuldiner S. Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2. Proc Natl Acad Sci U S A. 2013; 110: E1332—41.

[115] Reddy VS, Shlykov MA, Castillo R, Sun EI, Saier MH Jr. The major facilitator superfamily (MFS) revisited. FEBS J. 2012; 279: 2022-35.

[116] Monteiro KLC, de Aquino TM, Mendonça Junior FJB. An Update on Staphylococcus aureus NorA Efflux Pump Inhibitors. Curr Top Med Chem. 2020; 20: 2168—85.

[117] Pasqua M, Bonaccorsi di Patti MC, Fanelli G, Utsumi R, Eguchi Y, Trirocco R, et al. Host — Bacterial Pathogen Communication: The Wily Role of the Multidrug Efflux Pumps of the MFS Family. Front Mol Biosci. 2021; 8: 723274.

[118] Dashtbani—Roozbehani A, Brown MH. Efflux Pump Mediated Antimicrobial Resistance by Staphylococci in Health—Related Environments: Challenges and the Quest for Inhibition. Antibiotics (Basel). 2021; 10: 1502.

[119] Majumder P, Khare S, Athreya A, Hussain N, Gulati A, Penmatsa A. Dissection of Protonation Sites for Antibacterial Recognition and Transport in QacA, a Multi—Drug Efflux Transporter. J Mol Biol. 2019; 431: 2163-79.

[120] Radestock S, Forrest LR. The alternating—access mechanism of MFS transporters arises from inverted—topology repeats. J Mol Biol. 2011; 407: 698-715.

[121] Kaback HR. Structure and mechanism of the lactose permease. C R Biol. 2005; 328: 557-67.

[122] Sigal N, Lewinson O, Wolf SG, Bibi E. E. coli multidrug transporter MdfA is a monomer. Biochemistry. 2007; 46: 5200—8.

[123] Heng J, Zhao Y, Liu M, Liu Y, Fan J, Wang X, et al. Substrate—bound structure of theE. coli multidrug resistance transporter MdfA. Cell Res. 2015; 25: 1060-73.

[124] Debruycker V, Hutchin A, Masureel M, Ficici E, Martens C, Legrand P, et al. An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity. Nat Struct Mol Biol. 2020; 27: 829—35.

[125] Brawley DN, Sauer DB, Li J, Zheng X, Koide A, Jedhe GS, et al. Structural basis for inhibition of the drug efflux pump NorA fromStaphylococcus aureus. Nat Chem Biol. 2022; 18: 706—12.

[126] Kumar S, Athreya A, Gulati A, Nair RM, Mahendran I, Ranjan R, et al. Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single—domain camelid antibody. Commun Biol. 2021; 4: 836.

[127] Kumar S, Mahendran I, Athreya A, Ranjan R, Penmatsa A. Isolation and structural characterization of a Zn²⁺—bound single—domain antibody against NorC, a putative multidrug efflux transporter in bacteria. J Biol Chem. 2020; 295: 55-68.

[128] Yan N. Structural Biology of the Major Facilitator Superfamily Transporters. Annu Rev Biophys. 2015; 44: 257-83.

[129] Tamura N, Konishi S, Yamaguchi A. Mechanisms of drug/H⁺ antiport: complete cysteine—scanning mutagenesis and the protein engineering approach. Curr Opin Chem Biol. 2003; 7: 570—9.

[130] Varela MF, Ortiz—Alegria A, Lekshmi M, Stephen J, Kumar S. Functional Roles of the Conserved Amino Acid Sequence Motif C, the Antiporter Motif, in Membrane Transporters of the Major Facilitator Superfamily. Biology (Basel). 2023; 12: 1336.

[131] Kakarla P, KC R, Shrestha U, Ranaweera I, Mukherjee MM, Willmon M, et al. Functional Roles of Highly Conserved Amino Acid Sequence Motifs A and C in Solute Transporters of the Major Facilitator Superfamily. In: Arora G, Sajid A, Kalia VC, editors. Drug Resistance in Bacteria, Fungi, Malaria, and Cancer. Cham: Springer; 2017. pp. 111—40.

[132] Varela MF, Griffith JK. Nucleotide and deduced protein sequences of the class D tetracycline resistance determinant: relationship to other antimicrobial transport proteins. Antimicrob Agents Chemother. 1993; 37: 1253—8.

[133] Varela MF, Sansom CE, Griffith JK. Mutational analysis and molecular modelling of an amino acid sequence motif conserved in antiporters but not symporters in a transporter superfamily. Mol Membr Biol. 1995; 12: 313—9.

[134] Griffith JK, Baker ME, Rouch DA, Page MG, Skurray RA, Paulsen IT, et al. Membrane transport proteins: implication of sequence comparisons. Curr Opin Cell Biol. 1992; 4: 684—95.

[135] Tanford C. Translocation pathway in the catalysis of active transport. Proc Natl Acad Sci U S A. 1983; 80: 3701—5.

[136] Henderson PJ. Studies of translocation catalysis. Biosci Rep. 1991; 11: 477-538.

[137] Mitchell P. Vectorial chemiosmotic processes. Annu Rev Biochem. 1977; 46: 996-1005.

[138] Xiao Q, Sun B, Zhou Y, Wang C, Guo L, He J, et al. Visualizing the nonlinear changes of a drug—proton antiporter from inward—open to occluded state. Biochem Biophys Res Commun. 2021; 534: 272—8.

[139] Dang S, Sun L, Huang Y, Lu F, Liu Y, Gong H, et al. Structure of a fucose transporter in an outward—open conformation. Nature. 2010; 467: 734-8.

[140] Yin Y, He X, Szewczyk P, Nguyen T, Chang G. Structure of the multidrug transporter EmrD from Escherichia coli. Science. 2006; 312: 741—4.

[141] Marger MD, Saier MH Jr. A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993; 18: 13-20.

[142] Jiang D, Zhao Y, Wang X, Fan J, Heng J, Liu X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci U S A. 2013; 110: 14664—9.

[143] Luo J, Parsons SM. Conformational Propensities of Peptides Mimicking Transmembrane Helix 5 and Motif C in Wild—type and Mutant Vesicular Acetylcholine Transporters. ACS Chem Neurosci. 2010; 1: 381-90.

[144] Li Y, Ge X. Molecular Dynamics Investigation of MFS Efflux Pump MdfA Reveals an Intermediate State between Its Inward and Outward Conformations. Int J Mol Sci. 2022; 24: 356.

[145] Schaedler TA, van Veen HW. A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling. FASEB J. 2010; 24: 3653-61.

[146] Krämer R. Functional principles of solute transport systems: concepts and perspectives. Biochim Biophys Acta. 1994; 1185: 1-34.

[147] Poolman B, Konings WN. Secondary solute transport in bacteria. Biochim Biophys Acta. 1993; 1183: 5-39.

[148] Zomot E, Yardeni EH, Vargiu AV, Tam HK, Malloci G, Ramaswamy VK, et al. A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter. J Mol Biol. 2018; 430: 1368—85.

[149] Varela MF, Lekshmi M, Kumar S, Prakasan S, Stephen J. Molecular mechanisms of antimicrobial transport across the membrane by multidrug efflux pumps of the major facilitator superfamily in bacterial pathogens. Curr Top Biotechnol. 2022; 13: 1-15.

[150] Ranjana KC, Shrestha U, Kumar S, Ranaweera I, Kakarla P, Mukherjee MM, et al. Molecular Biology of Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily from Bacterial Food Pathogens. In: Singh OV, editor. Foodborne Pathogens and Antibiotic Resistance. John Wiley & Sons, Inc.; 2017. pp. 303—29.

[151] Lekshmi M, Stephen J, Ojha M, Kumar S, Varela M. Staphylococcus aureus antimicrobial efflux pumps and their inhibitors: recent developments. AIMS Med Sci. 2022; 9: 367-93.

[152] Chopra I. New developments in tetracycline antibiotics: glycylcyclines and tetracycline efflux pump inhibitors. Drug Resist Updat. 2002; 5: 119—25.

[153] Lewis K. In search of natural substrates and inhibitors of MDR pumps. J Mol Microbiol Biotechnol. 2001; 3: 247-54.

[154] Varela MF, Stephen J, Bharti D, Lekshmi M, Kumar S. Inhibition of Multidrug Efflux Pumps Belonging to the Major Facilitator Superfamily in Bacterial Pathogens. Biomedicines. 2023; 11: 1448.

[155] Kakarla P, Floyd J, Mukherjee M, Devireddy AR, Inupakutika MA, Ranweera I, et al. Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum. Arch Microbiol. 2017; 199: 465—74.

[156] Floyd JL, Smith KP, Kumar SH, Floyd JT, Varela MF. LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus. Antimicrob Agents Chemother. 2010; 54: 5406—12.

[157] Bruns MM, Kakarla P, Floyd JT, Mukherjee MM, Ponce RC, Garcia JA, et al. Modulation of the multidrug efflux pump EmrD—3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract. Arch Microbiol. 2017; 199: 1103-12.

[158] Nava AR, Mauricio N, Sanca AJ, Domínguez DC. Evidence of Calcium Signaling and Modulation of the LmrS Multidrug Resistant Efflux Pump Activity by Ca²⁺ Ions in S. aureus. Front Microbiol. 2020; 11: 573388.

[159] Meng F, Nie T, Lyu Y, Lyu F, Bie X, Lu Y, et al. Plantaricin A reverses resistance to ciprofloxacin of multidrug—resistant Staphylococcus aureus by inhibiting efflux pumps. Environ Microbiol. 2022; 24: 4818—33.

[160] Kumar G, Kiran Tudu A. Tackling multidrug—resistant Staphylococcus aureus by natural products and their analogues acting as NorA efflux pump inhibitors. Bioorg Med Chem. 2023; 80: 117187.

[161] GBD 2021 Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance 1990—2021: a systematic analysis with forecasts to 2050. Lancet. 2024; 404: 1199-226.

[162] World Health Organization. Antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, including tuberculosis [Internet]. Switzerland: Coherent Digital, LLC.: c2017 [cited 2025 Feb 15]. Available from: https://coilink.org/20.500.12592/pk2k9z

[163] Mahey N, Tambat R, Chandal N, Verma DK, Thakur KG, Nandanwar H. Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus. Microbiol Spectr. 2021; 9: e0095121.

[164] Zimmermann S, Klinger—Strobel M, Bohnert JA, Wendler S, Rödel J, Pletz MW, et al. Clinically Approved Drugs Inhibit the Staphylococcus aureus Multidrug NorA Efflux Pump and Reduce Biofilm Formation. Front Microbiol. 2019; 10: 2762.

[165] de Morais Oliveira—Tintino CD, Muniz DF, Dos Santos Barbosa CR, Silva Pereira RL, Begnini IM, Rebelo RA, et al. NorA, Tet(K), MepA, and MsrA Efflux Pumps in Staphylococcus aureus, their Inhibitors and 1,8—Naphthyridine Sulfonamides. Curr Pharm Des. 2023; 29: 323-55.