topoisomerase inhibitors: fluoroquinolone mechanisms of...

Topoisomerase Inhibitors: FluoroquinoloneMechanisms of Action and Resistance

David C. Hooper1 and George A. Jacoby2

1Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 021142Lahey Hospital and Medical Center, Burlington, Massachusetts 01805

Correspondence: [email protected]

Quinolone antimicrobials are widely used in clinical medicine and are the only current classof agents that directly inhibit bacterial DNA synthesis. Quinolones dually target DNA gyraseand topoisomerase IV binding to specific domains and conformations so as to block DNAstrand passage catalysis and stabilize DNA–enzyme complexes that block the DNA repli-cation apparatus and generate double breaks in DNA that underlie their bactericidal activity.Resistance has emerged with clinical use of these agents and is common in some bacterialpathogens. Mechanisms of resistance include mutational alterations in drug target affinityand efflux pump expression and acquisition of resistance-conferring genes. Resistance mu-tations in one or both of the two drug target enzymes are commonly in a localized domain ofthe GyrA and ParC subunits of gyrase and topoisomerase IV, respectively, and reduce drugbinding to the enzyme–DNA complex. Other resistance mutations occur in regulatory genesthat control the expression of native efflux pumps localized in the bacterial membrane(s).These pumps have broad substrate profiles that include other antimicrobials as well as quin-olones. Mutations of both types can accumulate with selection pressure and produce highlyresistant strains. Resistance genes acquired on plasmids confer low-level resistance thatpromotes the selection of mutational high-level resistance. Plasmid-encoded resistance isbecause of Qnr proteins that protect the target enzymes from quinolone action, a mutantaminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile effluxpumps. Plasmids with these mechanisms often encode additional antimicrobial resistancesand can transfer multidrug resistance that includes quinolones.

Quinolones have been used extensively for awide range of clinical applications (Owens

and Ambrose 2000; Kim and Hooper 2014).Nalidixic acid, a related naphthyridone struc-ture and the first member of the class used clin-ically, was discovered by George Lesher as abyproduct of chloroquine synthesis in 1962.Its use was limited to treatment of urinary tractinfections and resistance emerged quickly

(Lesher et al. 1962). Medicinal chemists froma number of companies, however, subsequentlymodified the core quinolone and related chem-ical scaffolds, generating compounds withgreater potency, broader spectra of activity, im-proved pharmacokinetics, and lower frequencyof development of resistance (Domagala andHagen 2003). An important addition of a fluo-rine substituent at position 6 added to po-

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Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a025320

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tency and became the common feature of thefluoroquinolone class with the introductionsof norfloxacin in 1986 and ciprofloxacin in1987 that showed substantially greater potencyagainst Gram-negative bacteria. Subsequently,other fluoroquinolones, such as levofloxacin,gatifloxacin, moxifloxacin, and gemifloxacinwere developed with increased activity againstGram-positive bacteria. Because of their po-tency, spectrum of activity, oral bioavailability,and generally good safety profile, fluoroquino-lones were used widely for a range of clinicalindications worldwide. Although still clinicallyvaluable, fluoroquinolone use has been com-promised by the emergence of bacterial resis-tance because of mutation and acquisition ofplasmid-encoded genes. In the sections that fol-low, we review the mechanisms of quinoloneaction and resistance, which are summarizedin Table 1.

QUINOLONE MECHANISM OF ACTION

Quinolones target two essential bacterial type IItopoisomerase enzymes, DNA gyrase and DNAtopoisomerase IV (Hooper 1997). Both en-zymes are heterotetramers with two subunits,gyrase being constituted as GyrA2GyrB2 andtopoisomerase IV as ParC2ParE2. GyrA is ho-mologous to ParC, and GyrB homologous toParE (Wang 1996). In Staphylococcus aureus,topoisomerase IV subunits historically havealso been referred to as GrlA and GrlB. Bothenzymes act by catalyzing a DNA double-strandbreak, passing another DNA strand through thebreak, and resealing the break (Aldred et al.2014). DNA strand passing occurs via domainsthat are localized in GyrA and ParC, whereasATPase activity, which is required for enzymecatalysis, occurs via domains localized in GyrBand ParE.

Quinolones bind reversibly to the complex-es of DNA with gyrase and topoisomerase IV atthe interface between protein and DNA near theactive site tyrosine (Tyr122 for GyrA, Tyr120 forParC in Escherichia coli numbering), which istransiently covalently linked to DNA duringDNA strand passage, with intercalation intothe cleaved DNA (Laponogov et al. 2009,

2013; Bax et al. 2010; Wohlkonig et al. 2010).In GyrA, a noncatalytic Mg2þ ion coordinatedwith four water molecules appears as a bridgefor hydrogen bonding between quinolone andSer83 and Asp87 (Wohlkonig et al. 2010), thetwo most commonly mutated amino acids inquinolone-resistant mutants. Although quino-lones can bind to mycobacterial gyrase in theabsence of DNA (Kumar et al. 2014), in E. coli,the gyrase–DNA complex shows increases inthe amount and specificity of quinolone bind-ing relative to gyrase alone (Shen et al. 1989;Willmott and Maxwell 1993).

Quinolones inhibit enzyme function byblocking the resealing of the DNA double-strand break, but, in addition, this process stabi-lizes a catalytic intermediate covalent complexof enzyme and DNA that serves as a barrier tomovement of the DNA replication fork (Went-zell and Maxwell 2000) or transcription com-plexes (Willmott et al. 1994) and can be con-verted to permanent double-strand DNA breaks(Drlica et al. 2008), thereby functioning astopoisomerase poisons (Kreuzer and Cozzarelli1979). Quinolone interactions with DNA gyraseappear to result in more rapid inhibition ofDNA replication than quinolone interactionswith topoisomerase IV (Khodursky et al. 1995;Fournier et al. 2000), possibly relating to theoverall proximity to the DNA replication com-plex of enzyme-binding sites on chromosomalDNA, with gyrase positioned ahead of the com-plex and topoisomerase IV behind it (Khodur-sky and Cozzarelli 1998). Quinolones can differin their potency for the two enzymes, with ageneral pattern among quinolones in clinicaluse that there is greater activity against DNAgyrase in Gram-negative bacteria and greateractivity against topoisomerase IV in Gram-pos-itive bacteria; but exceptions occur, and somequinolones have similar potency against bothenzymes (Blanche et al. 1996; Pan and Fisher1997; Strahilevitz and Hooper 2005).

DNA strand breaks trigger bacterial SOSDNA repair responses (Piddock et al. 1990;Drlica et al. 2008) and, to the extent that repairis incomplete, generate quinolone bactericidalactivity (Hiasa et al. 1996; Drlica and Zhao1997; Drlica et al. 2008, 2009). Inhibition of

D.C. Hooper and G.A. Jacoby

2 Cite this article as Cold Spring Harb Perspect Med 2016;6:a025320

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Fluoroquinolone Mechanisms of Action and Resistance

Cite this article as Cold Spring Harb Perspect Med 2016;6:a025320 3

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Tabl

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protein synthesis does not interfere with quin-olone-mediated inhibition of DNA replicationbut can reduce bacterial killing, with variationin the magnitude of reduction seen with differ-ent quinolones (Lewin et al. 1991; Howard et al.1993). These phenomena suggest that events inaddition to DNA replication inhibition thatmay affect DNA or other cellular damage mayalso contribute to quinolone bactericidal activ-ity, but the molecular mechanisms are not yetunderstood.

QUINOLONE RESISTANCE BECAUSEOF MUTATIONAL ALTERATIONIN TARGET ENZYMES

Single amino acid changes in either gyrase ortopoisomerase IV that confer quinolone resis-tance have been most commonly localized tothe amino-terminal domains of GyrA (residues67 to 106 for E. coli numbering) or ParC (resi-dues 63 to 102). These domains are near theactive site tyrosines (Tyr122 for GyrA, Tyr120for ParC) of both enzymes (Morais Cabral et al.1997; Laponogov et al. 2009, 2013; Wohlkoniget al. 2010). These domains have been termedthe “quinolone resistance-determining region”(QRDR) of GyrA and ParC (Yoshida et al.1990). The most common site of mutation inGyrA of E. coli is at Ser83 followed by Asp87,which as noted above are key residues in thebinding of quinolones to GyrA or ParC. Thereis conservation of a Ser and another acidic res-idue separated by four amino acids in bothGyrA and ParC in other species, and mutationin these residues can be frequently selected withquinolones in laboratory mutants and are fre-quently found in resistant clinical isolates(Hooper 2003; Drlica et al. 2009; Aldred et al.2014). Ser83Trp and Ser83Leu mutations ofE. coli GyrA have been associated with reducedbinding of the quinolone norfloxacin and enox-acin to gyrase–DNA complexes (Willmott andMaxwell 1993; Yoshida et al. 1993; Willmottet al. 1994). A Ser81Phe resistance mutation inParC of Bacillus anthracis appears also to showdecreased quinolone binding to the enzyme–DNA complex based on competition experi-ments with quinazolinediones, another gyr-

ase-targeting class of compounds (Aldred etal. 2012). Mutations in the Ser and nearby acid-ic residues differ in their effects on catalytic ef-ficiency of gyrase and topoisomerase IV, withmutation of the Ser residue generally havinglittle effect and mutation in the acidic residueresulting in a five- to 10-fold decrease in cata-lytic efficiency (Hiasa 2002; Aldred et al. 2014).

Resistance mutations in GyrB and ParE aresubstantially less common than those in GyrAand ParC in resistant clinical isolates. In E. coli,mutations at Asp426 and Lys447 of GyrB andLeu445 of ParE, as well as mutations at similarpositions in other species, can cause resistance(Yoshida et al. 1991; Ito et al. 1994; Gensberget al. 1995; Breines et al. 1997; Weigel et al.2001). Binding of enoxacin to enzyme–DNAcomplexes constituted with resistant mutantGyrB is reduced (Yoshida et al. 1993). The crys-tal structures of some conformations of yeasttopoisomerase II show proximity of the regionshomologous to the QRDRs of GyrA and GyrB(Fass et al. 1999). In E. coli crystal structures,the basic substituents at position C7 of cipro-floxacin and moxifloxacin were shown to befacing the GyrB subunit, and they could becross-linked to GyrB Cys466 (Mustaev et al.2014). In addition, in another crystal structureArg418 of Acinetobacter baumannii topoisom-erase IV is in proximity to the moxifloxacin C7basic substituent (Wohlkonig et al. 2010). No-tably, resistance mutations in acidic residues inthis domain of GyrB in E. coli (Asp426Asn) aswell as in ParC, suggest that drug–enzyme con-tacts could be mediated by charge interactions(Wohlkonig et al. 2010). Thus, resistance muta-tions in the QRDRs of both GyrA/ParC andGyrB/ParE appear to act by reducing the affin-ity of quinolones for the enzyme–DNA com-plex. Although direct drug binding data are notyet available for mutant topoisomerase IV–DNA complexes, the similarity of structuresbetween gyrase and topoisomerase IV and theconservation of key residues predict that resis-tance is similarly mediated by reduced affinityfor both enzyme–DNA complexes.

The magnitude of resistance caused by asingle-target mutation in one of the subunitsof gyrase or topoisomerase IV varies by quino-



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lone and bacterial species (Pan and Fisher 1997;Fournier et al. 2000). Because quinolone in-teraction with either target enzyme–DNA com-plex is sufficient to block cell growth and triggercell death (Drlica and Zhao 1997), the level ofsusceptibility of a wild-type bacterium is deter-mined by the more sensitive of the two tar-get enzymes, as noted above, often gyrase inGram-negative bacteria and topoisomerase IVin Gram-positive bacteria (Blanche et al. 1996;Pan and Fisher 1997). Thus, under quinoloneselection pressure, resistance mutation in themore sensitive or primary target enzyme willgenerally occur first because mutation in thesecondary less sensitive target enzyme alonedoes not have a resistance phenotype becauseof the dominance of the quinolone–primarytarget interaction (Trucksis et al. 1991; Ng etal. 1996; Breines et al. 1997). The magnitudeof the increase in resistance from such a first-step mutation in the primary target is then de-termined by either the magnitude of the effectof the mutation on enzyme sensitivity or theintrinsic level of sensitivity of the secondary tar-get enzyme. Thus, the level of sensitivity of thesecondary target enzyme can set a cap on themagnitude of resistance conferred by mutationin the primary target enzyme. The dominanceof sensitivity over resistance in the two targetenzymes also implies that quinolones with sim-ilar potency against both gyrase and topoisom-erase IV in an organism may require mutationsin both enzymes before the mutant bacteriumshows a substantial resistance phenotype (Panand Fisher 1998, 1999; Strahilevitz and Hooper2005). Fluoroquinolones currently in clinicaluse generally have differences in potency be-tween the two target enzymes, and single targetmutations produce eight- to 16-fold increasesin resistance.

Accumulating mutations in both target en-zymes have been shown to cause increasingquinolone resistance. In many species, high-lev-el quinolone resistance is generally associatedwith mutations in both gyrase and topoisomer-ase IV (Schmitz et al. 1998). In several species,Mycobacterium tuberculosis, Helicobacter pylori,and Treponema pallidum, there is no topoisom-erase IV, and gyrase provides the functions of

both enzymes and is the only quinolone target(Hooper 2003). Thus, selection of mutationswith substantial resistance phenotypes is pre-dicted to occur readily in these pathogens,a prediction consistent with the frequent occur-rence of resistance with clinical use of quino-lones without use of other active agents to treatinfections with M. tuberculosis and H. pylori(Tsukamura et al. 1985; Megraud 1998).

QUINOLONE RESISTANCE BECAUSEOF DECREASED DRUG ACCESS TOTARGET ENZYMES

Quinolones must cross the bacterial envelopeto interact with their cytoplasmic gyrase andtopoisomerase IV targets. Active quinoloneefflux, reductions in influx, or both can de-crease cytoplasmic quinolone concentrationsand confer resistance. In Gram-positive bacte-ria, reduced diffusion across the cytoplasmicmembrane has not been found to cause resis-tance, but active efflux transporters that includequinolones in their substrate profiles have beenshown to cause low-level resistance. In contrast,in Gram-negative bacteria, reduced diffusionthrough outer membrane porin diffusion chan-nels can contribute to resistance. Reduced in-flux often acts in concert with basal or increasedexpression of efflux transporters with both con-tributing additively to resistance (Lomovskayaet al. 1999; Li and Nikaido 2009). Quinolonesthemselves generally do not induce expressionof efflux pumps. With the exception of plasmid-mediated quinolone resistance discussed later,acquired quinolone resistance by altered drugpermeation occurs largely by mutations ingenes encoding regulatory proteins that controlthe transcription of efflux pump or porin genes(Grkovic et al. 2002). Uncommonly, mutationsin efflux pump structural genes have causedchanges in pump substrate profiles that addquinolones (Blair et al. 2015). The levels of quin-olone resistance because of regulatory mutationand pump overexpression are often limited toabout four- to eightfold increases in inhibitoryconcentrations, likely because of counterbal-ancing regulatory factors and cellular toxicitiesof high levels of pump overexpression.



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Altered Permeation in Gram-Positive Bacteria

In Gram-positive bacteria, the major facilitatorsuperfamily (MFS) of transporters containsthe largest number of efflux transporters thatinclude quinolones in their substrate profiles.These efflux pumps are transporters energizedby the proton gradient across the bacterial mem-brane and are generally antiporters with ex-change of substrate and protons in opposite di-rections. The Nor MFS pumps of S. aureus havebeen most extensively studied (Li and Nikaido2009; Schindler et al. 2015). NorA (Ubukata et al.1989; Yu et al. 2002), NorB (Truong-Bolduc et al.2005), and NorC (Truong-Bolduc et al. 2006)efflux pumps cause four- to eight-fold increasesin resistance to quinolones when overexpressed.NorA confers resistance to hydrophilic quino-lones, such as norfloxacin and ciprofloxacin,whereas NorB and NorC each confer resistanceto both hydrophilic quinolones and hydropho-bic quinolones, such as sparfloxacin and moxi-floxacin (Yu et al. 2002; Truong-Bolduc et al.2005, 2006); these pumps also have structurallyunrelated substrates in addition to quinolones,in keeping with broad substrate profiles of manyMFS transporters. Although there are naturalquinolone-like compounds (Heeb et al. 2011),it is unlikely that synthetic antibacterial quino-lones are themselves the natural pump sub-strates, which are as yet unknown for Gram-pos-itive quinolone resistance pumps.

Regulation of expression of these transport-ers is complex and involves several transcrip-tional regulators. MgrA, the most studied, actsas a positive regulator of norA expression anda negative regulator of norB and norC expres-sion (Ingavale et al. 2005; Truong-Bolduc et al.2005). Posttranslational phosphorylation ofMgrA by the PknB kinase results in the loss ofthe ability of MgrA dimers to bind the norApromoter and an increase in their bindingto the norB promoter (Truong-Bolduc et al.2008; Truong-Bolduc and Hooper 2010). Acidicconditions alter the proportions of phosphory-lated and unphosphorylated MgrA, and oxida-tive and aeration conditions also affect dimeri-zation and promoter binding (Chen et al. 2006;Truong-Bolduc et al. 2011a, 2012). Thus, rela-

tive levels of expression of NorA, NorB, andNorC are modified in response to a variety ofenvironmental conditions. Notably, norB ex-pression is selectively increased in an abscessenvironment in response to low-free iron con-ditions and contributes to fitness and bacterialsurvival in abscesses (Ding et al. 2008), a com-mon form of S. aureus infection. The naturalsubstrate of NorB, transport of which may con-tribute to improving fitness in an abscess envi-ronment, is not known. In addition, physiologicincreased expression of NorB at the site of in-fection would suggest that susceptibility testingunder clinical laboratory conditions may notfully reflect susceptibility at the site of infection.

NorG, a member of the GntR-like transcrip-tional regulators, can also modulate pump ex-pression and levels of quinolone resistance;it is a direct activator of norA and norB expres-sion but a direct repressor of norC expression(Truong-Bolduc and Hooper 2007; Truong-Bolduc et al. 2011b). ArlRS, a two-componentregulatory system, has been shown to affect ex-pression of norA as well (Fournier and Hooper2000; Fournier et al. 2001). There are often hi-erarchies in regulatory networks, and other reg-ulators can affect expression of MgrA andNorG. Such complex regulatory networks af-fecting pump expression imply the importanceof modulation of pump functions in cellularphysiology and may contribute to different bac-terial responses to quinolones in different envi-ronments that affect pump expression.

Other MFS efflux transporters that can con-tribute to quinolone resistance in S. aureus in-clude MdeA (norfloxacin and ciprofloxacin)(Huang et al. 2004), SdrM (norfloxacin) (Ya-mada et al. 2006), QacB(III) (norfloxacin andciprofloxacin) (Nakaminami et al. 2010), andLmrS (gatifloxacin) (Floyd et al. 2010). MFStransporters in other Gram-positive bacteriahave also been shown to include quinolonesin their substrate profiles. These transportersinclude Bmr, Bmr3, and Blt of B. subtilis(Klyachko et al. 1997; Ohki and Murata 1997);PmrA of Streptococcus pneumoniae (Gill et al.1999); LmrP of Lactococcus lactis (Bolhuiset al. 1995), and Lde of Listeria monocytogenes(Godreuil et al. 2003).



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In addition to the MFS transporters, less-er numbers of efflux pumps of the multipleantibiotic and toxin extrusion (MATE) andATP-binding cassette (ABC) families havebeen shown to confer quinolone resistance inGram-positive bacteria. MATE family pumps,like those of the MFS, are secondary transport-ers energized by the membrane electrochemicalgradient. MepA confers resistance to norfloxa-cin, ciprofloxacin, moxifloxacin, and sparflox-acin, as well as other antimicrobials and dyes(Kaatz et al. 2006). MepA is negatively regulatedby MepR, and pentamidine, a MepA substrate,reduces MepR binding to the mepA promoter,thereby increasing mepA expression (Kumara-swami et al. 2009; Schindler et al. 2013). InL. monocytogenes, the FepA MATE familypump is overexpressed in quinolone-resistantstrains and is regulated by the FepR transcrip-tional regulator, a member of the TetR family.Mutation in FepR causes FepA overexpressionand resistance to norfloxacin and ciprofloxacin(Guerin et al. 2014).

Members of the ABC family of transportersare, in contrast to the other pump families dis-cussed, energized by ATP hydrolysis. PatAB ofS. pneumoniae (norfloxacin and ciprofloxacin)(Boncoeur et al. 2012), SatAB of S. suis (nor-floxacin and ciprofloxacin) (Escudero et al.2011), and LmrA of L. lactis (ciprofloxacinand ofloxacin) (Poelarends et al. 2000; Putmanet al. 2000) all have been shown to confer resis-tance to some quinolones.

ALTERED PERMEATION IN GRAM-NEGATIVEBACTERIA

In Gram-negative bacteria, the majority of ef-flux pumps that can effect quinolone resistanceare members of the resistance–nodulation–division (RND) superfamily (Li et al. 2015).The RND pumps are secondary antiporterscomposed of a pump protein localized in thecytoplasmic membrane, an outer membranechannel protein, and a membrane fusion pro-tein that links the pump and the outer mem-brane protein (Du et al. 2014). Some outermembrane components may link to morethan one pump–fusion protein pair, enabling

export of substrates across both inner and outermembranes (Li and Nikaido 2009). The best-studied systems have been in E. coli and Pseudo-monas aeruginosa.

In E. coli, the AcrAB-TolC pump complexhas been extensively studied. Crystal structuresof the complex have revealed a trimer of AcrBpump monomers that rotate around a centralaxis perpendicular to the membrane, with eachmonomer as its rotation position changes as-suming a different conformation mediating dif-ferent steps in substrate binding and extrusionthrough the channel (Nikaido and Takatsuka2009). Substrates enter the vestibule of AcrBfrom the periplasmic space between the innerand outer membranes or the outer leaflet of theinner membrane. Binding sites for ciprofloxacinand other substrates of diverse chemical typeshave been identified in the central cavity ofthe periplasmic domain of AcrB (Yu et al.2003, 2005; Li and Nikaido 2004). Fluoroquin-olones, which are zwitterionic, are presumed tocross the outer membrane through OmpF andOmpC porin diffusion channels, down-regula-tion or mutation of which may amplify resis-tance. Mutations in the MarR regulator can re-sult in both an increase in acrB expression aswell as a decrease in ompF expression, duallycontributing to quinolone resistance (Alekshunand Levy 1999). Mutations in the E. coli SoxRS(Miller et al. 1994; Chou et al. 1998) and Rob(Jair et al. 1996) regulons can also effect resis-tance to fluoroquinolones in part related toreductions in OmpF and in a manner that isdependent on AcrAB-TolC. Expression ofAcrAB-TolC also confers resistance to bile saltsand is induced by bile salts, likely one of itsnatural substrates (Rosenberg et al. 2003).Thus, AcrAB supports the ability of E. coli tosurvive in its natural habitat, the lower gastro-intestinal tract, and perhaps only incidentallyaffects quinolone susceptibility.

In P. aeruginosa, the OprF porin channelhas permeability a 100-fold lower than that ofOmpF in E. coli (Nikaido et al. 1991), contrib-uting to its intrinsic resistance to quinolonesand other antimicrobial agents relative toE. coli and other enteric bacteria. The MexAB-OprM efflux pump, a RND pump similar in



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structure to AcrAB-TolC and expressed in wild-type strains, acts in concert with the low per-meability of OprF to augment the intrinsic levelof resistance of P. aeruginosa to fluoroquino-lones (Li et al. 2000b). Mutations in mexAand oprM cause increased uptake of norfloxacinand increased susceptibility to fluoroquino-lones (Poole et al. 1996b). Increased expressionof MexAB-OprM because of mutations in theMexR negative regulator causes increased re-sistance to ciprofloxacin and nalidixic acid,and mexR mutants can be selected with expo-sure to fluoroquinolones (Poole et al. 1993).P. aeruginosa also has three other efflux pumpsystems that include quinolones in their sub-strate profiles, MexCD-OprJ, MexEF-OprN,and MexXY-OprM (Masuda et al. 2000). Thesepumps vary in expression levels in wild-typestrains (Li et al. 2000a), but resistant mutantsoverexpressing these pumps can be selectedwith fluoroquinolones and other antimicrobialsubstrates (Kohler et al. 1997b). Mutation in theNfxB repressor, which is encoded upstream ofthe mexCD-oprJ operon, causes increased ex-pression of MexCD-OprJ and resistance tofluoroquinolones (Poole et al. 1996a). Mutationin nfxC results in overexpression of MexEF-OprN,but the exact regulatory mechanism is not yetknown (Kohler et al. 1997a). Mutations in theglobal regulator MvaT, which affects quorumsensing and virulence, also causes increased ex-pression of mexEF-oprM and resistance to nor-floxacin (Westfall et al. 2006). Expression ofboth MexEF-OprN and MexCD-OprJ vary in-versely with the level of expression of MexAB-OprM, but the mechanisms underlying thisproperty have not yet been elucidated (Li et al.2000a). Mutations in the MexZ repressor causeincreased expression of MexXY-OprM and re-sistance to fluoroquinolones, aminoglycosides,and other pump substrates (Matsuo et al. 2004;Hay et al. 2013). Specific quinolones differ inthe mutations they most commonly select(Kohler et al. 1997b). Most quinolones in clin-ical use have a fluorine at position 6 and a pos-itively charged substituent at position 7 (e.g.,norfloxacin, ciprofloxacin, levofloxacin, andmoxifloxacin) and tend to select nfxB-type mu-tants. In contrast, quinolones lacking a positive

charge at position 7 (e.g., nalidixic acid) oftenselect mexR and nfxC-type mutants, differencespresumably reflecting differences in the resis-tance profiles of the regulated pumps.

Additional RND pumps that cause quino-lone resistance have been found in a broad rangeof Gram-negative bacteria. Salmonella spp.(Baucheron et al. 2002) and Enterobacter aero-genes (Pradel and Pages 2002) have AcrABhomologs, and their increased expression hasbeen associated with quinolone resistance. TheCmeABC RND pump of Campylobacter jejunicontributes to the resistance of mutants selectedwith enrofloxacin, a veterinary quinolone sim-ilar to ciprofloxacin (Lin et al. 2002; Luo et al.2003). In Klebsiella pneumoniae, the OqxAB-TolC pump is encoded on the chromosome(Kim et al. 2009b) and was originally identifiedin E. coli isolates from pigs as a cause of plasmid-mediated resistance to olaquindox, a growthpromotant used in swine production; it alsoconfers resistance to quinolones.

Among nonenteric bacteria, in A. bauman-nii, the AdeIJK RND pump (Fernando et al.2014) is constitutively expressed and its broadresistance profile includes fluoroquinolones. Inaddition, overexpression of the AdeABC andAdeFGH RND pumps because of mutation intheir respective regulators, AdeRS, a two-com-ponent sensor-regulator system, and AdeL, aLysR family transcriptional regulator, also con-fer a similarly broad resistance profile contain-ing fluoroquinolones (Yoon et al. 2013, 2015).In Stenotrophomonas maltophilia, the SmeDEFpump (Alonso et al. 2000; Zhang et al. 2001)has been shown to contribute to quinoloneresistance based on pump knockout mutantswith increased susceptibility, resistant isolateswith increased pump gene expression, and itsability to confer resistance when overexpressedin E. coli.

Non-RND efflux pumps are much less com-mon in Gram-negative bacteria. A few casesMFS and MATE pumps associated with quino-lone resistance have been identified. AmongMFS pumps, in E. coli, EmrAB-TolC, an MFSpump that functions in tripartite structure likethe RND pumps, is negatively regulated byEmrR and confers resistance to nalidixic acid



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but not fluoroquinolones (Lomovskaya et al.1995). MdfA, originally termed CmlA, confersresistance to both chloramphenicol and fluoro-quinolones (Yang et al. 2003). MATE pumpsinclude the NorM pump, which can conferquinolone resistance in Vibrio parahaemolyticus(Morita et al. 2000), and in anaerobic Gram-negative bacteria the NorA pump of Bacteroidesfragilis (Miyamae et al. 1998) and the BexApump of B. thetaiotaomicron (Miyamae et al.2001), which have been shown to efflux fluoro-quinolones.

There are additional examples in bothGram-positive and Gram-negative bacteria inwhich there is evidence of efflux in quinolone-resistant isolates determined by either reductionin resistance with addition of a broad effluxpump inhibitor or reduced quinolone accumu-lation in resistant cells, but the contributingpump or its regulator have not been identified(Li and Nikaido 2009; Nikaido and Takatsuka2009). Information on efflux mechanisms andresistance in .50 bacterial species has recent-ly been extensively reviewed (Li and Nikaido2009; Li et al. 2015) and is beyond the scopeof this review. Thus, efflux-mediated resistanceto quinolones and many other antimicrobialsis widespread. The broad substrate profiles ofthese pumps link quinolone resistance to mul-tidrug resistance and constitute mechanismsby which use of non-quinolone antimicrobialscan also increase quinolone resistance. A simi-lar linkage to multidrug resistance occurs withplasmid-mediated quinolone resistance, whichis discussed in the next section.

PLASMID-MEDIATED QUINOLONERESISTANCE

Plasmid-mediatedquinoloneresistance(PMQR)was reported in 1998, 31 years after nalidixicacid began to be used clinically and 12 yearsafter modern fluoroquinolones were approvedfor use (Martınez-Martınez et al. 1998). Trans-ferable nalidixic acid resistance had been soughtunsuccessfully in the 1970s (Burman 1977), andplasmid-mediated resistance was thought un-likely to exist because quinolones are syntheticcompounds, and adequate resistance can arise

by chromosomal mutations (Courvalin 1990).The first PMQR was discovered in a multiresis-tant urinary isolate of K. pneumoniae from Ala-bama that could transfer low-level ciprofloxacinresistance to a variety of Gram-negative bacte-ria. When the responsible gene, named qnr andlater qnrA, was cloned and sequenced facilitat-ing its identification by PCR (Tran and Jacoby2002), qnr was soon found at low-frequency onplasmids in Gram-negative isolates around theworld. One qnrA plasmid from Shanghai con-ferred an unusually high-level of resistance andfurther study disclosed that it carried an addi-tional mechanism for PMQR, namely, modifi-cation of certain quinolones by a variant of thecommon aminoglycoside-modifying acetyl-transferase AAC(60)-Ib (Robicsek et al. 2006).A third mechanism for PMQR was the discov-ery of two plasmid-encoded quinolone effluxpumps: OqxAB and QepA (Sorensen et al.2003, Perichon et al. 2007; Yamane et al.2007). In the last decade, PMQR genes havebeen found in bacterial isolates worldwide.They reduce bacterial susceptibility to quino-lones, usually not to the level of clinical non-susceptibility, but facilitate the selection of mu-tants with higher level quinolone resistance andpromote treatment failure.

Qnr Structure and Function

Cloning and sequencing qnrA revealed that itencoded a 218-residue protein with a tandemlyrepeating unit of five amino acids that indicatedmembership in the many thousand-memberpentapeptide repeat family of proteins (Tranand Jacoby 2002). Further searches led to thediscovery of related genes for plasmid-mediatedpentapeptide repeat proteins qnrS (Hata et al.2005), qnrB (Jacoby et al. 2006), qnrC (Wanget al.2009), qnrD (Cavacoet al.2009), andqnrVC(Fonseca and Vicente 2013), as well as chromo-somal qnr genes in bacteria from a varietyof clin-ical and environmental sources (Rodrıguez-Mar-tınez et al. 2008a; Sanchez et al. 2008). These newqnr genes generally differed by 35% or more insequence from qnrA and each other. Allelic vari-eties that differ by 10% or less have been de-scribed in almost all families: currently seven



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for QnrA, 78 for QnrB, one for QnrC, two forQnrD, nine for QnrS, and six for QnrVC (seelahey.org/qnrstudies) (Jacoby et al. 2008).

The first pentapeptide-repeat protein tohave its structure determined by X-ray crystal-lography was MfpA, which is encoded on thechromosome of M. smegmatus and other my-cobacteria (Hegde et al. 2005) and implicated inquinolone resistance (Montero et al. 2001).MfpA is a dimer, linked carboxyl terminus tocarboxyl terminus, and folded into a right-handed quadrilateral b helix with size, shape,and charge mimicking the B-form of DNA andjust the size to fit into the cationic G segmentDNA-binding saddle of DNA gyrase and topo-isomerase IV. In vitro, MfpA inhibits DNA su-percoiling by gyrase and, although it fails toblock gyrase inhibition by quinolone, it can stillconfer quinolone resistance to whole cells bycompeting with DNA to reduce the number oflethal double-strand breaks produced by quin-olone (Hegde et al. 2005).

As shown first with purified QnrA1 (Tranand Jacoby 2002), and subsequently with plas-mid-encoded QnrB1 (Jacoby et al. 2006) andQnrS1 (Tavio et al. 2014), and with chromo-somally encoded AhQnr from Aeromonas hy-drophila (Xiong et al. 2011) and EfsQnr fromE. faecalis (Hegde et al. 2011), Qnr proteins doprotect DNA gyrase from quinolone inhibitionand only inhibit the enzyme at high concentra-tion. Like MfpA, they form rod-like dimers buthave additional structural features. The struc-ture of QnrB1 is shown in Figure 1. The quad-rilateral b-helix is stabilized by interactionsbetween the middle, usually hydrophobic, ami-no acid (i) of the pentapeptide repeat and thefirst polar or hydrophobic residue (i-2), whichpoint inward, whereas the remaining aminoacids (i-1, iþ1, iþ2) are oriented outward,forming a generally anionic surface. Hydrogenbonding between backbone atoms of neighbor-ing coils stabilizes the helix.

The monomers of QnrB1 and AhQnr haveprojecting loops of eight and 12 amino acidsthat are important for their activity. Deletionof the small A loop reduces quinolone protec-tion, whereas deletion of the larger B loop orboth loops destroys protective activity (Vetting

et al. 2011; Xiong et al. 2011). Removal of even asingle amino acid in the larger loop compro-mises protection. Other essential residues inQnrB are found in pentapeptide repeat posi-tions i and i-2, in which alanine substitutionfor the native amino acid eliminates protectionas does deletion of .10 amino acids at the ami-no terminus or as few as three amino acids fromthe dimerization module at the carboxyl termi-nus (Jacoby et al. 2013). EfsQnr lack loops, butEfsQnr differs from MfpA in having a 25-ami-no-acid flexible extension required for full pro-tective activity (Hegde et al. 2011).

In vitro, more Qnr is required to protectDNA gyrase as the inhibiting concentration ofquinolone is increased (Tran and Jacoby 2002).In a gel-displacement assay (Tran et al. 2005) orbacterial two-hybrid system (Kim et al. 2015),Qnr binds to both gyrase holoenzyme and itsA and B subunits. Binding to GyrA is reducedby the same amino- and carboxy-terminal andloop B deletions in QnrB that destroy its pro-tective activity, whereas subinhibitory concen-trations of ciprofloxacin reduce binding toGyrA but not to GyrB, suggesting that Qnr pro-tects gyrase by blocking access of quinolone toGyrA sites essential for its lethal action.

Many naturally occurring antibiotics andsynthetic agents also target DNA gyrase. Qnrprotects against compounds with a somewhatquinolone-like structure (Jacoby et al. 2015), forexample, 2-pyridone (Flamm et al. 1995), quin-azoline-2,4-dione (Huband et al. 2007), or spi-ropyrimidinetrione (Kern et al. 2011), so it isnot strictly quinolone-specific. Qnr, however,does not block agents acting on the GyrB sub-unit, and it also does not block simocyclinoneD8, which, like quinolones, binds to the aminoterminus of GyrA and blocks DNA binding(Hearnshaw et al. 2014).

Qnr ORIGIN

Qnr homologs can be found encoded on thechromosome of many Gram-positive as well asGram-negative bacteria, including species ofBacillus, Enterococcus, Listeria, and Mycobacteri-um, and anaerobes such as Clostridium difficileand Clostridium perfringens (Rodrıguez-Martı-



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http://lahey.org/qnrstudies

http://lahey.org/qnrstudies


nez et al. 2008b; Sanchez et al. 2008; Boulundet al. 2012; Jacoby and Hooper 2013). Aquaticbacteria are especially well represented, includ-ing species of Aeromonas, Photobacterium, She-wanella, and Vibrio (Poirel et al. 2005a,b).QnrA1 is 98% identical to the chromosomallydetermined Qnr of Shewanella algae (Poirel et al.2005a). QnrS1 is 97% identical to Qnr from Vib-rio parahemolyticus S022 (GenBank accessionnumber WP_029823919) or Vibrio mytili (Gen-Bank WP_041155100), and QnrC is 97%identical to Qnr in V. parahemolyticus S145(GenBank WP_025518018). QnrB homologs,on the other hand, are encoded on the chromo-some of members of the Citrobacter freundii

complex (Jacoby et al. 2011; Ribeiro et al.2015). The small, nonconjugative plasmidsthat carry qnrD are especially likely to be foundin Proteeae, such as Proteus mirabilis, P. vulgaris,and Providencia rettgeri and may have originat-ed there (Guillard et al. 2012, 2014; Zhang et al.2013).

The worldwide distribution of qnr sug-gests an origin well before quinolones werediscovered. Indeed, qnrB genes and pseudo-genes have been discovered on the chromo-some of C. freundii strains collected in the1930s (Saga et al. 2013). What the native func-tion of Qnr may have been is an as-yet unan-swered question.

N-term

Subunit A

Subunit B

α1

α1′

Loop A46–53

Loop A′46–53

Loop B′102–113

Loop B102–113

C-term

N-term′

N-term

C-term′

Face1 Face2 Face3 Face4

Coil0Coil1Coil2Coil3Coil4Coil5

173763

101133153173193214

83

Coil6Coil7Coil8Coil9

Loop A (*)

E K I

i–2

i+1

i+1

i+2

i+2

i–2i

–1i

–1i i

–2i

+1i

+2i

–1i i

–2i

+1i

+2i

–1ii

E NM A L A L V G E K I D R

C D F S GC N F S RA D F R NA D F R GA N F S KA Q V L GF D W R AL D I R G V D L Q G

A N F T HA T F S GV V L E K

S S A L GA M L K D

A D L S GN R F T G

A S F * *

S T F F N

C D L S MC R A Q GT N L S YN R W I GG E F S TS E L G DYQASL LMERL GIAVIG

C Q F *T E F I GA I F K SI E I R HA Y I T NC E L W ES D L S GC D L T NV K L D N

Y D R E S Q K G 53

M N M I T T R T W F C S 113Loop B (**)

46102

Figure 1. The rod-like structure of the QnrB1 dimer is shown (above) with the sequence of the monomer (below).The sequence is divided into four columns representing the four faces of the right-handed quadrilateral b-helix.Face names and color are shown at the top along with the naming convention for the five residues of thepentapeptide repeats. Loops A and B are indicated by one and two asterisks, respectively, with their sequencesindicated below and the loops shown as black traces on the diagram. The carboxy-terminal a-helix is coloredsalmon. The molecular twofold symmetry is indicated with a black diamond. Type II turn containing faces areshown as spheres and type IV-containing faces as strands. N-term, Amino terminal; C-term, carboxy terminal.(From Jacoby et al. 2014; reproduced, with permission, from the authors.)



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AAC(60)-Ib-cr

AAC(60)-Ib-cr is a bifunctional variant of acommon acetyltransferase, providing resistanceto such aminoglycosides as amikacin, kanamy-cin, and tobramycin, but also able to acetylatethose fluoroquinolones with an amino nitrogenon the piperazinyl ring such as ciprofloxacinand norfloxacin (Robicsek et al. 2006). Com-pared with other AAC(60)-Ib enzymes, the -crvariant has two unique amino acid substitu-tions—Trp102Arg and Asp179Tyr—both ofwhich are required for quinolone acetylatingactivity. Models of enzyme action suggest thatthe Asp179Tyr replacement is particularly im-portant in permitting p-stacking interactionswith the quinolone ring to facilitate quinolonebinding. The role of Trp102Arg is to positionthe Tyr face for optimal interaction (Vettinget al. 2008) or to hydrogen bond to keto orcarboxyl groups of the quinolone to fix it inplace (Maurice et al. 2008). The aac(60)-Ib-crgene is usually found in a cassette as part ofan integron in a multiresistance plasmid, whichmay contain other PMQR genes. Several alleleshave been described (Quiroga et al. 2015). Thegene has been found worldwide in a variety ofEnterobacteriaceae and even in P. aeruginosa(Ogbolu et al. 2011). Association with extendedspectrum b-lactamase (ESBL) CTX-M-15 isparticularly common (Oteo et al. 2009; Sabtch-eva et al. 2009).

OqxAB AND QepA

OqxAB was first identified as a plasmid-medi-ated efflux pump conferring resistance to theolaquindox, a food additive enhancing growthin pigs (Sorensen et al. 2003) and later shown toconfer resistance to ciprofloxacin and norflox-acin as well as other antimicrobials, includingchloramphenicol, nitrofurantoin, and trimeth-oprim (Hansen et al. 2007; Ho et al. 2016).Genes for oqxAB are commonly found on thechromosome of K. pneumoniae and Entero-bacter spp. but are expressed at a much higherlevel when captured on plasmids, often in asso-ciation with the IS26 mobilizing element (Rod-rıguez-Martınez et al. 2013; Wong et al. 2015).In a study of fecal samples from animals and

farmworkers in China, where olaquindox isused, 40% of E. coli isolated from animals and30% from humans carried plasmid-mediatedoqxAB, making it much more common thanother types of PMQR in this setting (Zhaoet al. 2010).

The QepA efflux pump was reported in2007 by investigators in Japan and France onplasmids in clinical isolates of E. coli often as-sociated with aminoglycoside resistance be-cause of ribosomal methylase rmtB (Perichonet al. 2007; Yamane et al. 2007). It has subse-quently been found worldwide (Habeeb et al.2014; Zhao et al. 2015).

QUINOLONE RESISTANCE PLASMIDS

Genes for quinolone resistance have been foundon plasmids varying in size and incompatibilityspecificity, indicating that the spread of multi-ple plasmids has been responsible for the dis-semination of this resistance around the worldand that plasmid acquisition of qnr and otherquinolone resistance determinants has occurredindependently multiple times. A mobile ortransposable element is almost invariably asso-ciated with qnr genes, especially ISCR1 andIS26. qnrD and qnrS2 are located within mobileinsertion cassettes, elements with bracketing in-verted repeats but lacking a transposase (Picaoet al. 2008; Guillard et al. 2014), whereas qnrVCis so far the only qnr gene located in a cassettewith a linked attC site (Fonseca et al. 2008; Be-lotti et al. 2015).

qnr genes are usually found in multiresis-tance plasmids linked to other resistance deter-minants. b-lactamase genes, including genesfor ESBLs, AmpC enzymes, and carbapene-mases, have been conspicuously common (Ja-coby et al. 2014).

PMQR genes have been found in a variety ofEnterobacteriaceae, especially E. coli and speciesof Enterobacter, Klebsiella, and Salmonella (Ja-coby et al. 2014). They have rarely been found innonfermenters but have occasionally been re-ported in P. aeruginosa and A. baumannii. qnrgenes are also found in a variety of Gram-pos-itive organisms but are chromosomal and notplasmid-mediated. The earliest known qnr out-



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side of Citrobacter spp. dates from 1988 (Jacobyet al. 2009). Studies in the last decade suggestthat the prevalence of PMQR is increasing (Kimet al. 2009a). In a recent study of .500 isolatesof E. coli from 30 county hospitals in China,37.3% carried at least one PMQR gene, includ-ing 19.7% with aac(6 0)-Ib-cr, 14.4% with qepA,3.8% with oqxAB, and 3.7% with qnr (Zhaoet al. 2015).

RESISTANCE BECAUSE OF PMQRDETERMINANTS

Table 2 shows the effect on susceptibility of acommon E. coli host of various PMQR genes.qnr alleles decrease ciprofloxacin or levofloxacinsusceptibility 30-fold to a level similar to that ofthe common Ser83Leu GyrA mutation but havemuch less effect on nalidixic acid resistance. Theother PMQR genes reduce susceptibility evenless but with more specificity. Levofloxacinand nalidixic acid are unaffected by AAC(60)-Ib-cr because they lack the C7 amino targetfor acetylation, and QepA affects ciprofloxacinsusceptibility more than that of the other quin-olones (Perichon et al. 2007). By themselvesnone reaches the CLSI breakpoint for loss ofsusceptibility. In combination, however, theymay do so, and all facilitate selection of high-er-level quinolone resistance (Martınez-Martı-nez et al. 1998; Robicsek et al. 2006; Rodrıguez-Martınez et al. 2007).

From a PMQR-free E. coli strain selection onciprofloxacin at �3 times minimum inhibitory

concentration (MIC) commonly yields mutantswith alterations in the QRDR of gyrA. Surpris-ingly, gyrase mutants are rarely selected fromthe same strain carrying qnr (Cesaro et al.2008). Rather, mutants have increased expres-sion of acrAB, mdtEF, or ydhE pumps, whichefflux quinolones, or alterations in genes of li-popolysaccharide core biosynthesis, which mayreduce quinolone entry via reduction in porinexpression (Vinue et al. 2015).

Despite the modest effect of PMQR geneson susceptibility, their presence makes in-fections in animal models harder to treat (Rod-rıguez-Martınez et al. 2008a; Jakobsen et al.2012), and there is some evidence for pooreroutcomes from human infections with qnr con-taining pathogens (Chong et al. 2010; Liao et al.2013).

SUMMARY

Resistance to quinolones has increased to sub-stantial levels, despite these agents being syn-thetic and having two essential bacterial targets.The microbial resistance has been affected by animpressive diversity of mechanisms that havelinked quinolone resistance to multidrug resis-tance, compounding the current public healthand medical challenges of broadly resistant bac-teria. Dual topoisomerase targets with differingsensitivities to many quinolones in clinical usecreate a pathway for additive mutational resis-tance to high levels. This core target-basedresistance has been further supplemented and

Table 2. Minimum inhibitory concentrations (MICs) produced in E. coli

MIC (mg/ml)

E. coli strain Ciprofloxacin Levofloxacin Nalidixic acid

J53 0.008 0.015 4J53 gyrA S83L 0.25 0.5 �512J53 pMG252 (qnrA1) 0.25 0.5 16J53 pMG299 (qnrB1) 0.25 0.5 16J53 pMG306 (qnrS1) 0.25 0.38 16J53 pMG320 (aac(60)-Ib-cr) 0.06 0.015 4J53 pAT851 (qepA) 0.064 0.032 4CLSI susceptibility breakpoint �1.0 �2.0 �16

From Jacoby et al. 2014.

MIC, Minimum inhibitory concentration.



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facilitated by more insidious low-level resis-tance. This low-level resistance has come fromboth overexpression of native multidrug effluxpumps as well as the unanticipated emergenceof multiple mechanisms of plasmid-mediatedquinolone resistance, which may not be readilydetected in clinical laboratories but can facili-tate selection of higher-level resistance and addsa plasmid linkage to multidrug resistance.

ACKNOWLEDGMENTS

This work is supported by Grants R01 AI057576(to D.C.H and G.A.J.), R37 AI023988 (toD.C.H.), and P01 AI083214 (to D.C.H.) fromthe U.S. Public Health Service, National Insti-tutes of Health.

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Aldred KJ, Kerns RJ, Osheroff N. 2014. Mechanism of quin-olone action and resistance. Biochemistry 53: 1565–1574.

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22, 20162016; doi: 10.1101/cshperspect.a025320 originally published online JulyCold Spring Harb Perspect Med

David C. Hooper and George A. Jacoby ResistanceTopoisomerase Inhibitors: Fluoroquinolone Mechanisms of Action and

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et al.Stefan Schwarz, Jianzhong Shen, Kristina Kadlec,

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Rifamycins, Alone and in CombinationDavid M. Rothstein

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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