Eur J Clin Microbiol Infect Dis (2003) 22:203–221DOI 10.1007/s10096-003-0907-5

R E V I E W

A. Dalhoff · F.-J. Schmitz

In Vitro Antibacterial Activity and Pharmacodynamicsof New Quinolones

Published online: 1 April 2003� Springer-Verlag 2003

Abstract This synopsis of published literature summaris-es data on the in vitro antibacterial activity and pharma-codynamics of fluoroquinolones. Data were compiled forciprofloxacin, levofloxcin, moxifloxacin, gatifloxacin,grepafloxacin, gemifloxacin, trovafloxacin, sitafloxacinand garenoxacin. All of these quinolones are almostequipotent against gram-negative bacteria but demon-strate improved activity against gram-positive species.The new quinolones are uniformly active against gram-positive species except Streptococcus pneumoniae;against which gemifloxacin, sitafloxacin and garenoxacinare one to two dilution steps more active than moxi-floxacin. All of the new quinolones except gemifloxacindemonstrate enhanced activity against anaerobes. Sinceall the new quinolones show similar activity against themajor respiratory tract pathogens except Streptococcuspneumoniae and members of the family Enterobacteri-aceae, their pharmacokinetics and pharmacodynamicswill be clinically relevant differentiators and determinantsof their overall activity and efficacy. In vitro simulationsof serum concentrations revealed that (i) gemifloxacinand levofloxacin were significantly and gatifloxacinmoderately less active than moxifloxacin against Strep-tococcus pneumoniae and Staphylococcus aureus, and (ii)resistant subpopulations emerged following exposure tolevofloxacin and gatifloxacin (gemifloxacin not yetpublished) but not to moxifloxacin. The emergence ofresistance is a function of drug concentrations achievablein vivo and the susceptibility pattern of the targetorganisms. Therefore, the use of less potent fluoro-

quinolones with borderline or even suboptimal pharma-cokinetic/pharmacodynamic surrogate parameters willinadvertently foster the development of class resistance.Drugs with the most favourable pharmacokinetic/phar-macodynamic characteristics should be used as first-lineagents in order to preserve the potential of this drug classand, most importantly, to provide the patient with anoptimally effective regimen.

Introduction

Streptococcus pneumoniae, Haemophilus influenzae andMoraxella catarrhalis are the most important causes ofinfective bacterial exacerbation in patients with chronicobstructive pulmonary disease. The percentage of beta-lactam-resistant strains among all three species is contin-uously increasing in Europe and the USA. Production ofbeta-lactamases – usually of the TEM-1 type but some-times of the ROB-1 type – by Haemophilus influenzae isincreasing, approaching >40% in some countries [1, 2, 3,4]. Resistance to cephalosporins and tetracyclines isuncommon at present but is emerging in some patientgroups or geographical areas. Beta-lactamase-negativebut otherwise ampicillin-resistant (BLNAR) strains havebeen reported since the early 1980s [5, 6, 7, 8]. Theirincidence remains low in various countries, but in Japanmore than 25% of all beta-lactamase-negative strains areBLNAR, and the frequency seems to be increasing [1, 4,9, 10, 11, 12].

Before the 1970s, Moraxella catarrhalis was suscep-tible to penicillin G and aminopenicillins. In the 1970sand 1980s, the susceptibility patterns began to change,and today between 70 and 90% of Moraxella catarrhalisisolates are reported to be beta-lactamase producers [13,14]. Moraxella catarrhalis produces a constitutivelyexpressed beta-lactamase. The enzymes BRO-1 andBRO-2 are transposon mediated, which explains theirrapid spread [15].

Penicillin has long been the treatment of choice forrespiratory tract infections caused by Streptococcus

A. Dalhoff ())Institute for Medical Microbiology and Virology,Universit�tsklinikum Schleswig-Holstein,Brunswiker Strasse 4, 24105 Kiel, Germanye-mail: axel.dalhoff.ad@bayer-ag.deTel.: +49-431-5973300Fax: +49-431-5972216

F.-J. SchmitzInstitute for Medical Microbiology and Virology,Universit�tsklinikum D�sseldorf,Universit�tsstrasse 1, Geb. 22.21, 40225 D�sseldorf, Germany

pneumoniae. The first penicillin-resistant pneumococciwere isolated in Papua New Guinea in 1967 from healthycarriers [16, 17]. Since then, however, there have beenincreasing numbers of reports of penicillin-resistantisolates from many countries throughout the world [18].A multicentre, collaborative study in Europe and Asiaduring the winter of 1997–1998 revealed that penicillinresistance (MIC, �2 mg/l) amongst pneumococci rangedfrom 0.7% in Germany to 43.5% and 33.9% in Spain andFrance; penicillin resistance in Japan and China was10.1% and 3.2%, respectively. However, macrolideresistance amounted to 48% in Japan and 73–74% inChina; a 49% difference in macrolide resistance wasdetected between France and Germany (58% and 9%,respectively) [19]. A dramatic increase in macrolideresistance among invasive pneumococci isolated in Ger-many was noted (from 3% in 1992 to 13% in 1998 up to15.3% in 2000) [20]. The past 3 years in particular havewitnessed a dramatic increase worldwide in the incidenceof pneumococcal strains that are resistant not only topenicillin but also to macrolides and other antimicrobialagents [21, 22]. The problem is exacerbated by thetendency of penicillin-resistant clones to spread fromcountry to country and from continent to continent [18].This penicillin resistance has emerged as a majorproblem, creating a need for alternative non-beta-lactamtherapies.

In an effort to respond to these problems, the pastdecade has seen the search for and development of manynew quinolones. Monofluorinated quinolones, like cipro-floxacin, are predominantly active against gram-negativepathogens; di- and tri-halogenated quinolones, on theother hand, exhibit greatly enhanced antibacterial activityand pharmacokinetics in humans. However, the develop-ment of many of these promising di- and tri-halogenatedagents was discontinued due to adverse events, includingphototoxic and other unexpected adverse reactions. Oth-ers, like trovafloxacin, are still commercially available,but their use is limited to a very small number of specificindications only.

This review examines the in vitro activities of newfluoroquinolones and their propensities for selection ofresistance.

Problems Encountered in Comparing SusceptibilityData

During recent years it became increasingly difficult togenerate a representative synopsis of published data onthe antibacterial activity of new quinolones against thetarget pathogens because the scope of susceptibilitystudies varies significantly, thus impacting the data beinggenerated. The following points outline the nature of theproblem inherent in comparing the results of susceptibil-ity data.

1. The resistance problem in general is in the focus of thepublic and scientific interest; authors may therefore

have selected resistant strains from strain collections ormay have used laboratory-derived strains with specificresistance mechanisms. Such specific subpopulationsor genetically defined strains with specific resistancemechanisms do need to be studied to fully exploit thepotential of a new agent. However, such straincollections do not represent the clinically relevantpathogens found in the target population.

2. Similar to the quandary outlined above, drugs inclinical use for long periods of time or those consid-ered to be gold standards have been studied exten-sively; in particular, mechanisms of resistance againstthese drugs and the activities of these drugs againststrains with selected and specific resistance patternsare of special interest but do not mirror susceptibilitypatterns of common pathogens.

3. To meet the EMEA (European Agency for theEvaluation of Medical Products) requirements, resis-tant strains must be selected for susceptibility studies.In the “Note for Guidance” in the PharmacodynamicSection of the SPC (Summary of Product Character-istics) of Antibacterial Medicinal Products, the EMEArequests that “strains without known acquired resis-tance mechanisms and, when available, harbouringknown resistance mechanisms to other antibioticswithin the same or related classes of antibiotics shouldbe tested (e.g. a new quinolone should be tested againstnalidixic-sensitive Escherichia colibut also againstnalidixic acid-resistant and ciprofloxacin-resistant Es-cherichia coli, and a new macrolide should be testedagainst erythromycin-resistant organisms).”

4. The epidemiology of resistance varies locally, region-ally, nationally and internationally, and sometimesstrains included in studies may have been collectedfrom pockets where atypical resistance characteristicspredominate. Because resistance profiles differ dra-matically, it is important that the strain collectionsstudied include substantial numbers of recent clinicalisolates from diverse areas in order to reflect the “realworld scenario”.

5. Strains are most frequently collected during a definedstudy period; consequently, such collections mayinclude strains isolated during an outbreak situation,so that such collections probably include strains ofclonal origin. If resistant strains of clonal origin areoverrepresented within a strain collection studied, thedistribution of MICs determined for these strains willlikely not reflect the susceptibility pattern of commonpathogens.

6. Many studies originate from hospital-based laborato-ries, so that isolates studied may derive predominantlyfrom hospitalised patients. Although the newest andmost active antibacterial agents are used in hospi-talised patients, particularly those at risk, manyhospitalised patients acquire resistant and even mul-tiresistant bacteria. Therefore, neither the epidemiolo-gy of resistance nor the susceptibility pattern of suchstrains mirrors the bacterial etiology in the outpatientsetting.

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Similarly, strains obtained in the outpatient setting arealso likely to include resistant subpopulations. Mostbacterial infections in outpatients are treated adequate-ly and effectively by the general practitioner, withoutany bacteriological assessment. Most frequently, sam-ples are withdrawn and analysed only in thoseoutpatients in whom the initial therapy failed and inwhom resistant subpopulations may already have beenselected. Thus, such isolates do not reflect the “true”resistance epidemiology of community-acquired infec-tions.Consequently, new community-based surveillanceprogrammes are being pursued that differentiatebetween community- and hospital-acquired infections.However, at present the database originating fromthese programmes is too small to allow a comprehen-sive review of the in vitro activities of new quinolones.

7. Another scenario in which the selection of resistantstrains is favoured is that of chronic disease. In patientsinfected by species that may cause chronic disease(e.g. Pseudomonas aeruginosainfections in cysticfibrosis), samples will be withdrawn repeatedly overlong periods of time. Because these patients receivelong-term therapy, the causative pathogens will likelyhave acquired resistance. Such isolates have a signif-icant impact on the susceptibility pattern of the straincollections studied, as the resistant subpopulation willbe over-represented if isolates from patients withshort-term versus long-term exposure are not reportedseparately.

The factors mentioned above have a significant impact onstudies of the in vitro activity of antibacterial agents. Theevaluation of a drug’s activity against specifically select-ed resistant strains or against isolates from hospitalisedpatients will result in an activity profile different fromthat of isolates causing community-acquired infections.Higher minimal inhibitory concentrations (MICs) ofantimicrobial agents will be reported as compared tostudies in which the most recent unselected isolates havebeen used, particularly if the isolates originate fromoutpatients. For example, in one study the level ofmacrolide resistance in invasive pneumococci is 50%higher (15.3%) than the level of macrolide resistance incommunity-acquired respiratory tract pathogens [20].Consequently, the modal MICs, the MIC50s and MIC90sof all drug classes other than quinolones differ forunselected surveillance isolates and invasive pneumococ-ci [20, 23].

Other examples of broad differences in the datareported for a given drug can be cited. For example,MIC90 values of ciprofloxacin against Escherichia colivary from 0.03 mg/l for nalidixic-acid- and ciprofloxacin-susceptible isolates to 1 mg/l for nalidixic-acid-resistantbut ciprofloxacin-susceptible isolates; MICs reached up to128 mg/l for the nalidixic-acid-resistant as well as theciprofloxacin-resistant subgroup [24]. King et al. [24]have mentioned explicitly that the organisms they studiedwere collected over a 10-year period and were selected to

include those known to be resistant. They also reportedthe data for every subgroup per species separately, thusdemonstrating clearly that the data described do notreflect the clinical isolation rates of resistant or suscep-tible bacteria and do not reflect the activities of the agentsstudied against unselected bacteria. Usually, however, theinclusion of quinolone-resistant strains into the studypopulation is not mentioned specifically. Instead, only thevery broad range of individual MICs for the pathogensstudied indicates that test strains known to be quinoloneresistant may have been selected. Consequently, thecompilation of all published data on the in vitro activity ofquinolones against bacterial isolates will result in a veryheterogeneous data set, even for one specific agent, andwill not be informative.

In conclusion, many of the strain collections used toevaluate the antibacterial activity of a new agent do notrepresent the resistance epidemiology of those strains thatpredominate in the target population. Rather, resistantstrains are likely to be over-represented.

Susceptibility Data for New Quinolones

Data on the susceptibilities of gram-positive and gram-negative isolates reported below originate from a very fewpublications in which almost all new quinolones havebeen compared with each other on the basis of directdrug-to-drug comparisons. Milatovic et al. [25] comparedthe activities of sitafloxacin, ciprofloxacin, trovafloxacin,levofloxacin, clinafloxacin, gatifloxacin and moxifloxacinagainst a total of 8,796 bacterial strains that were isolatedbetween April 1997 and February 1999 from patients in24 university hospitals in 14 European countries, 3hospitals in South Africa and 1 hospital in Israel. In thisreport, the MIC90 values are not subgrouped according toquinolone susceptibility or resistance of the bacteriaisolated. For example, the MICs of ciprofloxacin forEscherichia coli range from �0.008 up to �16 mg/l, thusillustrating the points mentioned above.

King et al. [24] studied ciprofloxacin, moxifloxacin,gemifloxacin, trovafloxacin, grepafloxacin, clinafloxacinand ofloxacin against strains that were specificallyscreened for ciprofloxacin and/or nalidixic acid resis-tance. If necessary, data on the susceptibility ofquinolones to gram-positive or gram-negative aerobicbacteria were complemented by results from other studies[26, 27].

The data on the investigational des-fluoro(6) quinoloneBMS 284756 (garenoxacin) are derived from recentpublications [28, 29, 30, 31, 32, 33]. Fung-Tomc et al.[28] compared garenoxacin with trovafloxacin, moxi-floxacin, levofloxacin, ofloxacin and ciprofloxacin, andthe SENTRY group compared garenoxacin with cipro-floxacin, gatifloxacin, levofloxacin and trovafloxacin aswell as with 11 non-quinolone agents.

In general, the new quinolones demonstrate improvedactivity against gram-positive microorganisms and anaer-obes as compared to previous quinolones such as

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ciprofloxacin and levofloxacin, which are active predom-inantly against gram-negative pathogens. The improvedactivity of the new quinolones against gram-positiveaerobes is exemplified by the low MIC90 values againststreptococci, ranging from 0.25 mg/l (moxifloxacin) and0.5 mg/l (gatifloxacin, grepafloxacin) to 0.06 mg/l(sitafloxacin, gemifloxacin) (Table 1).

Although ciprofloxacin-resistant pneumococci are ex-tremely rare and, consequently, the numbers tested aresmall, the improved activity of the newer quinolones isreflected by the low MIC values, even against highlyciprofloxacin-resistant pneumococci, for which MIC90values of 64 mg/l have been reported [24]. The MIC90s ofmoxifloxacin and garenoxacin against 53 selected clinicalstrains of Streptococcus pneumoniae demonstrating ef-flux-mediated fluoroquinolone resistance were within onedilution step (i.e. 0.5 and 0.12 mg/l, respectively) of theMIC90s reported for wild-type strains, thus indicating thatboth quinolones are unaffected by PmrA-mediated efflux[31].

Highly ciprofloxacin-resistant isolates will be resistant(based on breakpoint definition) to all the new quino-lones. Borderline ciprofloxacin-susceptible pneumococciare almost as susceptible as wild-type strains to the newerquinolones [33, 34, 35]. Thus, new quinolones withenhanced activity against streptococci and other gram-positive organisms may offer a useful therapeutic alter-native for pneumococcal infections, provided (i) theyretain activity against gram-positive bacteria resistant tothe early fluoroquinolones, and (ii) the development offluoroquinolone resistance can be minimised and con-trolled.

Activity of New Quinolones Against Pneumococciwith Genetic Mutations

In order to assess the potential of new fluoroquinolones astreatment alternatives for pneumococci resistant to theearly fluoroquinolones, their in vitro activity against

genetically defined resistant isolates was examined. TheMIC ranges for first-step mutants, i.e. isolates with singlealterations in gyrase or topoisomerase IV, generallyoverlapped those for wild-type isolates. In contrast,mutations in both loci increased the MICs of almost allnew fluoroquinolones up to levels of intermediate or high-level resistance, except for gemifloxacin and cli-nafloxacin [36, 37, 38]. However, no difference in thefrequency of selection of mutations in wild-type versusfirst-step mutants was detected [36]. Thus, fluoro-quinolone resistance and cross-resistance are consistentonly among the subset of double mutants with mutationsin both gyrase and topoisomerase IV.

Although, on the one hand, mutations in gyrA andparC, alone or in combination, most frequently contributeto fluoroquinolone resistance, mutations are, on the otherhand, much more heterogeneous than previously thought.Numerous in vitro studies (for summary, see [39]) haveindicated that a single amino acid change in either of thetwo targets results in an almost negligible increase inMICs of the new fluoroquinolones. However, a recentgenetic analysis of mutations contributing to fluoro-quinolone resistance in clinical isolates of Streptococcuspneumoniae revealed that mutations in the quinoloneresistance determining region (QRDR) were much moreheterogeneous [38]. The positions of amino acid changeswithin the genes gyrA, gyrB, parC and parE affectedresistance more than did the total number of QRDRmutations. The effect of a specific mutation on the MIC ofthat specific isolate varied significantly, depending on theagent tested. It seems to be conceivable that the hetero-geneity of mutations conferring resistance to fluoro-quinolones may increase, since pneumococci will beexposed to an increasing number of different fluoro-quinolones. The heterogeneity of mutations will compli-cate the prediction of cross-resistance amongst thefluoroquinolones.

Table 1 Comparative antibacterial activity (MIC90, mg/l) of quinolones against gram-positive bacteria (modified according to [24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34])

Agent S. pneumoniae Alpha-haemolyticstreptococci

Beta-haemolyticstreptococci

S. agalactiae MSSA

cipS cipRa cipS cipR

CIP 1 64/2* 2 1 2 0.5 >128LEV 1 –/1 – 1 1 0.25 >32MFX 0.25 4/0.25 0.5 0.25 0.5 0.06 4GAT 0.5 –/0.5 – 0.5 – 0.12 4GRE 0.5 8/– 1 1 0.5 0.12 32GEM 0.06 1/– 0.12 0.06 0.06 0.03 8TRO 0.25 4/0.25 0.25 0.25 0.5 0.06 2SIT 0.06 –/0.06 – 0.03 – 0.03 0.25GAR 0.12 –/0.12 – 0.25 0.12 0.03 1.4

CIP, ciprofloxacin; LEV, levofloxacin; MFX, moxifloxacin; GAT, gatifloxacin; GRE, grepafloxacin; GEM, gemifloxacin; TRO,trovafloxacin; SIT, sitafloxacin; GAR, garenoxacin (BMS-284756); MSSA, methicillin-susceptible Staphylococcus aureus; cipS,ciprofloxacin suscepitible; cipR; ciprofloxacin resistanta Data quoted from references [24/25], respectively

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Activity of Newer Quinolones AgainstStaphylococcus aureus

The newer quinolones also exhibit improved activityagainst highly ciprofloxacin-resistant but methicillin-susceptible Staphylococcus aureus isolates [33, 36, 40,41]. Although the markedly improved activity of all thenew quinolones against ciprofloxacin- and levofloxacin-resistant gram-positive organisms will not translate intoclinical efficacy, these data demonstrate that (i) the newquinolones exhibit markedly improved activity againstgram-positive aerobes in general due to high targetaffinity to both targets, i.e. gyrase as well as topoiso-merase IV, and (ii) quinolone resistance mechanisms ingram-positive bacteria have a differential effect on boththe older and the newer quinolones (see below).

Methicillin-resistant Staphylococcus aureus (MRSA)sensu strictu are as susceptible to moxifloxacin and,probably, the other new quinolones as methicillin-sus-ceptible Staphylococcus aureus (MSSA) [42, 43, 44, 45].The decreased susceptibility of MRSA to fluoro-quinolones is not the result of increased mutation rates;MSSA have been shown to have the same mutation ratesas MRSA [46]. It seems likely that an association existsbetween the action of fluoroquinolones on mec(A)-positive Staphylococcus aureus on the one hand, andthe increase in the proportion of colony-forming units permillilitre expressing methicillin resistance (i.e. the resis-tance index) on the other hand. Growth in the presence ofvarious fluoroquinolones increased the proportion of themec(A)-positive heteroresistant Staphylococcus aureusthat developed methicillin resistance (oxacillin MIC,�128 mg/l). Ciprofloxacin exhibited a >1,000-foldgreater effect than newer quinolones. The resultantoxacillin-resistant strains also showed a 1.5- to 3-foldincrease in fluoroquinolone MICs. This phenomenon isnot caused by the fluoroquinolone acting as a mutagen butrather to the selection of more fluoroquinolone-resistantsubpopulations, which were also predominantly moreresistant to oxacillin [47].

Resistance to methicillin emerged as early as 8 h afterexposure to the fluoroquinolone. Similarly, the 8-chlo-rine-substituted fluoroquinolone BAY y 3118 selected forquinolone resistance in MRSA strains as early as 6 h aftercommencement of exposure to BAY y 3118. Moxi-floxacin did not select for quinolone resistance in theseMRSA strains at all [48]. In the latter two studies [47, 48],no resistance emerged in MSSA strains. These two studiesindicate that quinolones differ in their potential to selectfor quinolone as well as methicillin resistance. Themechanisms of resistance to the two drug classes areunrelated, but their selective emergence tends to beassociated.

Since most of the clinical MRSA isolates are almostalways multidrug resistant, they are resistant to quino-lones, too. Therefore, quinolones in general, includingsitafloxacin and garenoxacin, do not offer a therapeuticalternative for treatment of MRSA infections.

Activity of Newer Quinolones AgainstMajor Pathogens

All of the new quinolones are active against the keyrespiratory tract pathogens Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis(Table 2). Haemophilus influenzae and Moraxella ca-tarrhalis are inhibited by lower concentrations thanStreptococcus pneumoniae [24, 25, 26, 49]. In general,resistance of these three species to beta-lactam agentsand/or macrolides does not affect their susceptibility toquinolones [49, 50, 51, 52].

Ciprofloxacin and levofloxacin as well as the newquinolones were very active against enterobacterialspecies, provided the isolates tested were susceptible tonalidixic acid and ciprofloxacin (Table 3). Interestingly,gemifloxacin, trovafloxacin, grepafloxacin and moxi-floxacin were as potent as or slightly less potent thanciprofloxacin against nalidixic-acid- and/or ciprofloxacin-resistant Enterobacteriaceae [24]. Gram-negative isolatesresistant to ciprofloxacin were also resistant to the newerquinolones. Against nalidixic-acid-resistant but ciproflox-acin-susceptible Escherichia coli isolates, MIC90 valuesof all the quinolones tested increased up to 1–2 mg/l, and,for the very few nalidixic-acid-resistant but ciprofloxacin-susceptible Klebsiella spp. (n=5), MIC values rangedfrom 0.06 to 1 mg/l. However, MIC90 concentrations forisolates of nalidixic-acid-resistant and ciprofloxacin-re-sistant Escherichia coli and Klebsiella spp. increased toconcentrations up to �128 mg/l. These data indicate that,on the one hand, the new quinolones have retainedactivity against gram-negative bacteria (except Pseudo-monas aeruginosa), but on the other hand, quinoloneresistance mechanisms in gram-negative bacteria affectall the quinolones tested to the same extent.

Against isolates of Acinetobacter spp. and Stenotro-phomonas maltophilia, moxifloxacin, gemifloxacin, tro-vafloxacin, gatifloxacin and grepafloxacin were morepotent than ciprofloxacin, and many isolates resistant tociprofloxacin were susceptible to these quinolones, par-ticularly to sitafloxacin [24, 25] (Table 3). However,

Table 2 Comparative antibacterial activity (MIC90, mg/l) ofquinolones against respiratory tract pathogens (modified accordingto references [24, 25, 49])

Agent Streptococcuspneumoniae

Haemophilusinfluenzae

Moraxellacatarrhalis

CIP 1–2 0.015 0.03LEV 1 0.03 0.06MFX 0.25 0.06 0.12GAT 0.5 0.015 0.03GRE 0.5 0.03 0.03GEM 0.06 0.015 0.03TRO 0.25 0.03 0.03SIT 0.06 �0.008 �0.008GAR 0.12 0.03 0.03

CIP, ciprofloxacin; LEV, levofloxacin; MFX, moxifloxacin; GAT,gatifloxacin; GRE, grepafloxacin; GEM, gemifloxacin; TRO,trovafloxacin; SIT, sitafloxacin; GAR, garenoxacin (BMS-284756)

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against Pseudomonas aeruginosa, ciprofloxacin is still themost potent quinolone; sitafloxacin tended to be onetitration step more active. However, assuming the samebreakpoints for ciprofloxacin and sitafloxacin as proposedby Jones et al. [53], almost complete cross-resistancebetween ciprofloxacin and sitafloxacin was observed.

King et al. [24] subgrouped the Pseudomonas aerug-inosa strains studied according to their ciprofloxacinsusceptibility pattern: the MIC90 of ciprofloxacin was0.5 mg/l for the susceptible strains and 128 mg/l for theresistant strains. Milatovic et al. [25] did not subgroup thePseudomonas aeruginosa strains studied, so the MIC90values exceed 16 mg/l. On the basis of the outcome of thetwo detailed studies mentioned below, it seems to belikely that the isolates evaluated by Milatovic et al. [25]or the ciprofloxacin-resistant subgroup studied by King etal. [24] may have originated from chronically infectedpatients. These examples illustrate the impact of asampling bias on the characterisation of the antibacterialactivities of both new and established drugs.

Bauernfeind et al. [54] recently evaluated the cipro-floxacin resistance pattern of Pseudomonas aeruginosaisolates from cystic fibrosis patients. Overall, 66.4% ofthe strains were ciprofloxacin susceptible. However,subgrouping the strains according to the age of patientsfrom whom Pseudomonas aeruginosa was isolated

revealed that, in the age groups �14 years and 15–19years (i.e. patients who either have not been or wererarely treated with ciprofloxacin), the resistance rateswere 14.8% and 15.4%, respectively. In patients olderthan 30 years (who were frequently treated withciprofloxacin), the resistance rate was as high as 48.6%.On average, ciprofloxacin resistance amounted to 28.7%.Surprisingly, in patients less than 14 years old – who mayvery rarely have been treated with a quinolone, if at all –ciprofloxacin resistance amounted to 14.8%. Although itcould have been anticipated that the ciprofloxacin resis-tance rate should have been much lower, this phenom-enon may likely result from the transmission of strainsfrom patients of older age groups (in whom resistanceranged from 30–49%) to younger patients.

The strains studied by Bauernfeind et al. [54] were notgenotyped, so it cannot be ruled out that isolates of clonalorigin were evaluated. Worlitzsch et al. [55] used for theirsusceptibility studies genotyped isolates only from youngcystic fibrosis patients, thus excluding copy strains. Forcomparison, isolates from healthy individuals and envi-ronmental sources (neonatal intensive care unit) werestudied. The mean MIC of ciprofloxacin for Pseudomonasaeruginosa isolates was 0.6 mg/l, independent of theorigin of the isolates; no ciprofloxacin resistance could bedetected.

Table 3 Comparative antibacterial activity (MIC90, mg/l) of quinolones (modified according to references [24, 25, 26, 27, 28, 29, 30, 31,32, 33, 49])

Agent Escherichia coli Klebsiella pneumoniea Acinetobacter spp. Stenotrophomonasmaltophilia

Pseudomonasaeruginosaa

CIP 0.03 6 0.06 0.25/0.5* 1/2 >16 2/8 2/0.25*LEV 0.03 8 0.13 0.25/0.5 –/1 16 1/2 4/2MFX 0.03 16 0.13 0.5/1 0.25/1 16 0.5/1 8/8GAT 0.03 8 0.13 –/1 –/– 8 1/– –/2GRE 0.03 – 0.13 –/– 0.12/– – 0.5/– –/1GEM 0.03 – 0.13 –/– 0.12/– – 0.5/– –/1TRO 0.03 >16 0.13 0.5/0.5 0.06/0.25 16 0.5/1 4/2SIT – 1 – –/0.12 – 2 0.12/– –/0.25GAR 0.06 – – 0.5/– –/0.25 – –/4 16/–

CIP, ciprofloxacin; LEV, levofloxacin; MFX, moxifloxacin; GAT, gatifloxacin; GRE, grepafloxacin; GEM, gemifloxacin; TRO,trovafloxacin; SIT, sitafloxacin; GAR, garenoxacin (BMS-284756)a Data quoted from references [24/25], respectively

Table 4 Comparative antibacterial activity (MIC90, mg/l) of quinolones against anaerobes (modified according to references [24, 25, 26,56, 59])

Agent Peptococcusspp.

Peptostreptococcusspp.

Fusobacteriaspp.

Bacteroidesfragilis

Porphyromonasspp.

Prevotellaspp.

CIP 2–4 2–4 2–8 8–32 1 2–>16LEV 4 4 8 2–8 0.5 4MFX 0.25–1 0.5–1 1–4 1–2 0.5 0.5–2GAT 2 2 8 1 0.12 2GRE 1 1 8 16 4 4GEM 0.12 0.06–4 0.25–>8 0.5–2 0.12–1 2–16TRO 0.5–1 0.5–1 0.5–4 0.25–1 0.25 1SIT 0.12 0.12 0.5 0.25 0.03 0.25GAR – 0.25 0.25–8 1 – 0.25

CIP, ciprofloxacin; LEV, levofloxacin; MFX, moxifloxacin; GAT, gatifloxacin; GRE, grepafloxacin; GEM, gemifloxacin; TRO,trovafloxacin; SIT, sitafloxacin; GAR, garenoxacin (BMS-284756)

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In conclusion, by excluding copy strains from thesusceptibility study, almost all isolates remained fluoro-quinolone susceptible. These patient isolates were assusceptible as the environmental strains collected on aneonatal intensive care unit, where quinolones are not beused for treatment. These detailed studies by Bauernfeindet al. [54], as well as those of Worlitzsch et al. [55],clearly indicate that the evaluation of drug activities isvery much dependent on the origin of the strains studied.

Activity of Quinolones Against Anaerobesand Atypical Bacteria

In general, data on the activity of quinolones againstanaerobes are rather less homogeneous than data availablefor aerobic bacteria, probably reflecting the differentmethods used by the various authors [24, 25, 26, 27, 56,57, 58, 59]. Despite this variability, published dataindicate that the newer quinolones are more active againstanaerobes than the older ones, with the exception ofgemifloxacin (Table 4). The activity of gemifloxacinagainst anaerobes appears to be more variable; anaerobicgram-positive bacteria are more susceptible than anaer-obic gram-negative bacteria. A significant intraspeciesvariability of gemifloxacin MICs for anaerobes wasnoted. Due to the variable activity of gemifloxacin,Goldstein [56] concluded that efficacy is unpredictable.Therefore, the susceptibilities of the isolates should betested before gemifloxacin is used clinically in thetreatment of anaerobic infections.

Against atypical bacteria in general and, in particular,Chlamydia spp. Mycoplasma spp. and Ureaplasma ure-alyticum, the newer quinolones are more active than theolder ones (Table 5) [27, 30, 60, 61, 62, 63, 64, 65, 66].

Summary of Activity of Newer Quinolones

In summary, the antibacterial activity of the newerquinolones is characterised by a retained activity againstgram-negative bacteria—with the exception of Pseudo-monas aeruginosa, against which ciprofloxacin is still the

most active quinolone – but a markedly enhanced activityagainst gram-positive bacteria, with sitafloxacin being themost active. Against most of the gram-positive species, allthe new quinolones are almost equipotent except againstStreptococcus pneumoniae, against which sitafloxacin,gemifloxacin and garenoxacin are more active thanmoxifloxacin by one to two dilution steps. Since all thenew quinolones show similar activity against the majorrespiratory tract pathogens (except Streptococcus pneu-moniae) and the Enterobacteriaceae, their pharmacody-namics, propensity for resistance selection, pharmaco-kinetics and toxicity/safety profile will be clinicallyrelevant differentiators and determinants of their overallactivity and efficacy. Thus, studies on the pharmacody-namics of quinolones will generate useful informationthat contributes to the differentiation of the various newquinolones.

Pharmacodynamics of New Quinolones

MICs of antibacterial agents provide a quantitativeparameter of the inhibition of bacterial growth at anendpoint of 18 h of incubation following exposure to aconstant antibiotic concentration. The endpoints are bynature discrete, so that MICs do not mirror an antibiotic’sbactericidal activity as a function of time and/or concen-tration. Antibacterial efficacy in vivo, however, is afunction of drug concentration at the focus of infectionversus time. Therefore, models that interpret both phar-macokinetics and pharmacodynamics provide more clin-ically meaningful information about the potential of anantibiotic than static endpoints. However, in vitro studiesassessing the antibacterial effects of fluctuating drugconcentrations that simulate serum- or “tissue” concen-tration versus time are cumbersome and therefore notroutinely performed.

Although not the optimal method for determiningefficacy, antibacterial effects in preclinical models ofinfection are most frequently correlated with one of threepharmacokinetic parameters: (i) time (T) of exposure of abacterium to serum concentrations exceeding the MIC(T>MIC); (ii) the ratio of peak concentration (Cmax) of an

Table 5 Comparative antibacterial activity (MIC90, mg/l) of quinolones (modified according to references [27, 30, 60, 66])

Agent Legionellapneumophila

Chlamydiapneumoniae

Chlamydiatrachomatis

Mycoplasmapneumoniae

Ureaplasmaurealyticum

CIP 0.03 1 2 1–4 4LEV 0.016 0.5–1 0.5 0.5–1 1MFX 0.016 0.06 0.06 0.12 1GAT 0.03 0.25 0.12 0.25 0.5GRE 0.016 0.5 NA 0.12 1GEM 0.016 0.25 NA 0.12 0.25TRO �0.004 1 NA 0.25 0.12SIT NA NA NA NA NAGAR 0.06 0.008 0.016 0.06 0.25

CIP, ciprofloxacin; LEV, levofloxacin; MFX, moxifloxacin; GAT, gatifloxacin; GRE, grepafloxacin; GEM, gemifloxacin; TRO,trovafloxacin; SIT, sitafloxacin; GAR, garenoxacin (BMS-284756); NA, information not available

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agent to its MIC for the bacterium (Cmax/MIC); and (iii)the ratio of the area under the concentration-versus-timecurve (AUC) to the MIC (AUC/MIC). Antimicrobialagents can be categorised by the best fit between thesecomposite parameters and their efficacy [67, 68]. Theparameters used for these calculations (Cmax, AUC andMIC90) are almost always the mean; usually, proteinbinding is not considered. Nevertheless, traditionalcalculations based on the means of pharmacodynamicparameters do not take into account their variabilities.Ideally, pharmacodynamic calculations should include allpossible drug exposures for standard dosage regimens andall MIC values likely to be found for the clinical isolates.Therefore, it would be more informative to publish theMIC distribution pattern for a large number of microor-ganisms. Similarly, maximal serum concentrations (Cmax)and AUC values demonstrate a Gaussian distribution ofthe individual values.

The integration of variability into both pharmacoki-netic parameters (Cmax and AUC) and of MIC distribu-tions into pharmacodynamic parameters can be achievedby various procedures. One approach could be to base thepharmacodynamic modelling on the Monte Carlo simu-lation. However, the information needed to integratevariability into pharmacokinetics as well as MIC distri-butions into pharmacodynamic modelling is, unfortunate-ly, usually not published. Another concept was developedby Schentag et al. [69], who, more than 15 years ago,suggested the concept of dual individualisation, whichintegrates patient-specific data on pharmacokinetics andsusceptibility of the causative pathogen into the dosageoptimisation specifically for that individual patient.Although this concept was a major advance, this approachwould have required real-time drug quantitation, MICtesting (qualitative disk diffusion would be inadequate)and computer modelling. As it was almost unrealistic toapply the dual individualisation concept in daily routinepatient care, the pharmacodynamics of intravenousciprofloxacin in seriously ill patients was analysedretrospectively in order to identify a drug exposuremeasure significantly associated with outcome. Based onthe retrospective study of Forrest et al. [70], in which thecorrelation between pharmacokinetics/pharmacodynam-ics outcomes and clinical efficacy was investigated, anAUC/MIC ratio of �125 is considered to be predictive foroptimal clinical and microbiological outcome. Thus, theAUC/MIC theory as defined by Schentag et al. [69]describes the probability of clinical and microbiologicalresponse to therapy.

Furthermore, it was demonstrated that reduced drugexposure and thus low AUC/MIC ratios correlateddirectly with the emergence of resistance [71]. With anMIC ratio below 100, there was a 50% probability thatresistance would emerge. Overall, 83% of bacteriadeveloped resistance if the initial AUC/MIC ratio was�100. However, if the initial AUC/MIC ratio was �101,resistant pathogens could be isolated from only 9% of thepatients [71].

In the study of Forrest et al. [70], elderly hospitalisedpatients suffering almost exclusively from gram-negativepneumonia were studied. Unfortunately, the target AUC/MIC ratio of �125 has been inappropriately applied toother patient groups and other causative pathogens. Thepharmacodynamic goal of an AUC/MIC ratio of �125 fortherapy of respiratory tract infections due to gram-negative bacteria in hospitalised patients appears to bedifferent from the target AUC/MIC ratio for gram-positive organisms, which cause community-acquiredrespiratory tract infections. Multiple data from in vitroand animal models of infection and recent human clinicaltrials provide strong evidence that the pharmacokinetics/pharmacodynamics parameter for quinolones that corre-lates to clinical outcome in the treatment of community-acquired pneumococcal infections is an AUC/MIC ratioof �30 [68, 72, 73, 74, 75, 76]. In addition, the Cmax wasfound to be linked to the propensity for selection ofresistant subpopulations and also antibacterial effects invitro and in animal models of infection [77, 78, 79, 80,81]; the pharmacodynamic goal of therapy should be apeak Cmax/MIC ratio of �10.

The most common approach to link pharmacokinetic/pharmacodynamic to the probability of response totherapy is to calculate the AUC/MIC and Cmax/MICratios by using the mean AUC and Cmax values and theMIC90 values of the relevant bacterial species. Thepharmacokinetic parameters used are summarised inTables 6 and 7. In addition, protein binding is taken intoaccount because only the unbound fraction is freelydiffusible and antibacterially active [82, 83]; Escherichiacoli and Streptococcus pneumoniae were used as repre-sentative indicator organisms. Since all fluoroquinolonesexhibit pronounced activity against ciprofloxacin-suscep-tible Enterobacteriaceae, they are all characterised byfavourable pharmacokinetic/pharmacodynamic surrogateparameters: for example, ciprofloxacin achieves the mostoptimal Cmax/MIC as well as AUC/MIC ratio (data notshown) for Enterobacteriaceae.

Lower Cmax and AUC values (as, for example, forgrepafloxacin, gemifloxacin or sitafloxacin) and highprotein binding (in the case of sitafloxacin, gemifloxacinand, in particular, garenoxacin) (Tables 6 and 7) have a

Table 6 Minimal inhibitory concentrations (MICs, mg/l) ofquinolones for wild type (WT) and single-step gyrase andtopoisomerase IV mutants of Streptococcus pneumoniae (MICsare derived from references [34, 84, 85]), used for the calculation ofpharmacokinetic/pharmacodynamic surrogate parameters

Agent WT GyrA ParC

Ciprofloxacin 1 8 4Levofloxacin 1 4 2Trovafloxacin 0.25 0.5 1Grepafloxacin 0.5 0.5 1Gatifloxacin 0.5 1 2Moxifloxacin 0.25 0.5 0.25Sitafloxacin 0.06 0.25 0.06Gemifloxacin 0.06 0.12 0.12Garenoxacin 0.06 0.25 0.12

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significant impact on the pharmacodynamics of the newquinolones against gram-positive bacteria. The improvedactivity of the new quinolones against gram-positivebacteria is only in part mirrored by their pharmacody-namics. Although garenoxacin, sitafloxacin and gemi-floxacin exhibit the highest in vitro activity against wildtype Streptococcus pneumoniae, their low MICs arecounterbalanced by either low serum concentrations and/or high protein binding. The ratios between the unboundand thus antibacterially active fraction of the AUC andthe corresponding MICs for wild type Streptococcuspneumoniae are highest for garenoxacin, followed inorder by those for sitafloxacin and moxifloxacin (almost

identical to those of sitafloxacin), but are lower forgatifloxacin and still lower for gemifloxacin. The un-bound Cmax/MIC ratios for the last two quinolones arealmost identical (Table 8, Table 9).

Although fluoroquinolone-resistant pneumococci arestill rare, isolates of Streptococcus pneumoniae withdecreased susceptibility to these agents can occasionallybe isolated. Thus, it is of interest to evaluate pharmaco-dynamic surrogate parameters for these strains, too. parCand gyrA mutations, alone and in combination, contributesignificantly to fluoroquinolone resistance [34]. Amongstthe gyrA mutations, Ser-81!Phe or Tyr and Ser-79!Phein parC were the most common and were not found in

Table 7 Fluoroquinolone phar-macokinetic constants used forthe calculation of pharmacoki-netic/pharmacodynamic surro-gate parameters

Agent Dose (mg) Cmax (mg/l) AUC 0–24 h(mg x h/l)

Protein binding(%)

Ref.

Ciprofloxacin 500 b.i.d. 2.9 28.0 35 [86]Ciprofloxacin 750 b.i.d. 4.1 44.14 35 [86]Levofloxacin 500 q.d. 5.2 47.7 26 [87]Trovafloxacin 200 q.d. 2.2 30.4 76 [88]Grepafloxacin 400 q.d. 0.9 11.4 50 [89]Grepafloxacin 600 q.d. 1.4 19.7 50 [89]Gatifloxacin 400 q.d. 3.4 32.4 20 [90]Moxifloxacin 400 q.d. 3.5 35.0 37 [91]Sitafloxacin 100 b.i.d. 1.0 11.1 50 [92]Gemifloxacin 320 q.d. 1.5 9.82 60 [93]Garenoxacin 400 q.d. 5.0 60 75, 87 [94, 95]

Table 8 Pharmacokinetic/phar-macodynamic surrogate param-eter AUC/MIC (=AUIC) forwild type (WT) and single-stepgyrase (GyrA) or topoisomeraseIV (ParC) mutants, differentiat-ed into total and protein un-bound fractions

Agent WT GyrA ParC

Total Unbound AUIC Total Unbound

Total Unbound

Ciprofloxacin 500 b.i.d. 28.0 18.2 3.5 2.3 7.0 4.5Ciprofloxacin 750 b.i.d. 44.2 28.6 5.5 3.6 11.0 7.2Levofloxacin 500 q.d. 47.6 35.2 11.9 8.8 23.8 17.6Trovafloxacin 200 q.d. 121.6 29.2 60.8 14.6 30.4 7.3Grepafloxacin 400 q.d. 22.8 11.4 22.8 11.4 11.4 5.8Grepafloxacin 600 q.d. 39.4 19.7 39.4 19.7 19.7 9.8Gatifloxacin 400 q.d. 64.8 51.8 32.4 25.9 16.2 12.9Moxifloxacin 400 q.d. 140.0 88.2 70.0 44.1 140.0 88.2Sitafloxacin 100 b.i.d. 185.0 92.5 44.4 22.2 185.0 92.5Gemifloxacin 320 q.d. 163.3 65.4 81.7 32.7 81.7 32.7Garenoxacin 400 q.d. 1,000.0 130.0 240.0 31.1 500.0 65.0

Table 9 Pharmacokinetic/phar-macodynamic surrogate param-eter Cmax/MIC for wild type(WT) and single-step gyrase(GyrA) or topoisomerase IV(ParC) mutants, differentiatedinto total and protein unboundfractions

Agent WT GyrA ParC

Total Unbound Cmax/MIC Total Unbound

Total Unbound

Ciprofloxacin 500 b.i.d. 2.8 1.8 0.3 0.2 0.7 0.4Ciprofloxacin 750 b.i.d. 4.0 2.6 0.5 0.3 1.0 0.6Levofloxacin 500 q.d. 5.2 3.8 1.3 0.9 2.6 1.9Trovafloxacin 200 q.d. 8.8 2.1 4.4 1.0 2.2 0.5Grepafloxacin 400 q.d. 1.8 0.9 1.8 0.9 0.9 0.4Grepafloxacin 600 q.d. 2.8 1.4 2.8 1.4 1.4 0.7Gatifloxacin 400 q.d. 6.8 5.5 1.7 1.4 3.4 2.7Moxifloxacin 400 q.d. 14.0 8.8 7.0 4.4 14.0 8.8Sitafloxacin 100 b.i.d. 16.8 8.4 4.0 2.0 16.7 8.4Gemifloxacin 320 q.d. 25.0 10.0 12.5 5.0 12.5 5.0Garenoxacin 400 q.d. 83.4 10.8 41.7 5.4 20.8 2.7

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quinolone-susceptible isolates. Therefore, the MIC90s ofthe new quinolones for gyrA and parC mutants, respec-tively, were used to calculate the pharmacokinetic/phar-macodynamic surrogate parameters in order to assesswhether the improved in vitro activity of the newfluoroquinolones against single-step, quinolone-resistant,gram-positive organisms may translate into improvedprobability of clinical efficacy.

The pharmacokinetic/pharmacodynamic surrogate pa-rameters (for the unbound fraction) calculated for single-step mutants are most favourable for moxifloxacin, sincefor both the gyrA as well as the parC mutant the AUC/MIC ratio exceeds the threshold value of 30 significantly.In contrast, the gyrA mutation reduces the AUC/MICratios below (sitafloxacin) or to borderline values,whereas the parC mutation affects the AUC/MIC ratiosless significantly (Table 8). The unbound Cmax/MIC ratiosare almost identical for sitafloxacin, moxifloxacin, gemi-floxacin and garenoxacin (Table 9). Least favourablesurrogate parameters were obtained for levofloxacin,trovafloxacin, grepafloxacin and gatifloxacin. Clearly,both parameters are suboptimal for levofloxacin. On thebasis of the above-mentioned threshold values of theCmax/MIC and AUC/MIC ratios, this finding indicates thatresistant subpopulations are likely to occur as a conse-quence of exposure to levofloxacin, trovafloxacin orgrepafloxacin (see below).

Pharmacodynamic in vitro models simulating kineticprofiles provide more detailed information about dosingregimens of fluoroquinolones than the calculation ofAUC/MIC or Cmax/MIC ratios; these models can also beused to assess which parameter is associated withmaximising antibacterial effects or minimising the emer-gence of resistance. Data generated thus far by usingpharmacodynamic models of infection were summarisedrecently [96]. Only those in vitro pharmacodynamicstudies in which the emergence of quinolone resistanceduring drug exposure was evaluated are summarisedbelow. The synopsis provided in Table 10 clearlyindicates that (i) the antibacterial effect is dose and thusconcentration dependent, i.e. a once-daily regimen provedto be more efficacious than a twice-daily treatment of thesame total daily dose, (ii) resistance emerged as a functionof drug exposure, i.e. a Cmax/MIC ratio �10 and/or anAUC/MIC ratio �40–30 resulted in the emergence ofresistant bacteria. Therefore, if drug concentrations aretoo low as a consequence of inappropriate dosing and/or ifthe causative pathogen is borderline susceptible, resis-tance is likely to occur. These findings, derived from invitro pharmacodynamic models, were confirmed bystudies in experimental animals. The in vivo studies haveclearly demonstrated that emergence of resistance is afunction of drug concentrations at the focus of infection(for summary, see [76]).

On the basis of the current dosage regimens ofgatifloxacin and levofloxacin and the susceptibility ofthe predominant pathogen of community-acquired respi-ratory tract infections, i.e. Streptococcus pneumoniae, it islikely that these two quinolones will select for quinolone

resistance within this species as opposed to, e.g. moxi-floxacin or, probably, gemifloxacin or garenoxacin,whose propensity for resistance development, however,has not yet been studied in pharmacodynamic models.

Four studies are discussed in greater detail as pars prototo in order to support the conclusions drawn from thesynopsis presented in Table 9. Lister [103] compared thepharmacodynamics of moxifloxacin and levofloxacinagainst three strains each of Staphylococcus aureus andStaphylococcus epidermidis; two isolates from each groupwere methicillin and ciprofloxacin resistant, which re-sulted in moxifloxacin MICs ranging from 0.12 to 1 mg/land levofloxacin MICs ranging from 2 to 8 mg/l.Moxifloxacin was significantly bactericidal against allthe isolates, eradicating three strains and reducing viablecounts of three first-step mutants by 2–5 logs below theinoculum. Levofloxacin was initially as bactericidal asmoxifloxacin. However, resistant subpopulations wereselected from one wild type and three first-step mutantstrains that grew up to control viable counts. The selectionand outgrowth of resistant mutant subpopulations withlevofloxacin was observed only if Cmax/MIC ratios were4:1 or less.

No resistant subpopulations were observed followingexposure of wild type strains or first-step mutants tomoxifloxacin. It was of interest that no mutants wereselected with moxifloxacin, even if the peak Cmax/MICratios were 5:1. This may indicate that, in addition to thepharmacodynamic threshold values, structural elements ofthe drug molecule may contribute to either a lower or ahigher propensity for development of resistance (seebelow).

Bauernfeind et al. [104] evaluated the bactericidaleffect of gatifloxacin by exposing three different Strep-tococcus pneumoniae strains for which MICs ranged from0.25 to 1 mg/l to simulated serum concentrationsfollowing gatifloxacin doses of 100–800 mg p.o. oncedaily. Initially, all regimens reduced viable countssignificantly. However, after an incubation period ofapproximately 4 h, Streptococcus pneumoniae strainsbegan to regrow, provided both the Cmax/MIC and theAUC/MIC ratios were suboptimal (i.e. below 5 and 30,respectively). MICs of gatifloxacin for the regrownsubpopulations were 4–8 times higher than the initialMICs.

The pharmacodynamics and the impact of the C-8substitution of the fluoroquinolone core structure on theemergence of resistance were studied by Dalhoff [105].Moxifloxacin and the experimental fluoroquinolone BAYy 3118 differ structurally from each other only in the C-8substitution, moxifloxacin carrying a methoxy substituentand BAY y 3118 a chlorine substituent. Exposure of twociprofloxacin-susceptible MSSA strains to both agentsresulted in rapid elimination of the inoculum within 8 h,and regrowth did not occur. The two MRSA strains werekilled by moxifloxacin almost as effectively as the MSSAstrains, and, consequently, resistance did not emerge.

The two MRSA strains were, however, marginallyaffected by a single oral dose of BAY y 3118. Therefore,

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Table 10 Synopsis of published data on the use of in vitro pharmacodynamic models to evaluate the emergence of fluoroquinoloneresistance as a function of dosing regimens

Quinolone Dose Organism Effect Resistance Ref.

Ciprofloxacin 500 mgp.o., b.i.d.

Staphylococus aureus almost complete eradicationby ofloxacin and pefloxacin asopposed to a transitorybactericidal effect ofciprofloxacin and norfloxcacindue to 2–3 times lower Cmaxand ~2 times shorter t1/2

4- to 8-fold increase in MICsof ciprofloxacin and nor-floxacin in regrowing bacteria

[97]

Norfloxacin 400 mgp.o., b.i.d.

Pefloxacin 400 mgp.o., b.i.d.

Ofloxacin 400 mgp.o., b.i.d

Ciprofloxacin Cmax=1/2 xMIC

Pseudomonasaeruginosa(4 strains for whichMICs were different)

transient bactericidal effectfollowed by regrowth due tolow Cmax

up to 64-fold increase in MICs [98]

Enoxacin 500 mgp.o., b.i.d.

Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Staphylococcus aureus

q.d. regimen more potent andbactericidal if Cmax/MIC �8,otherwise regrowth

4- to 8-fold increase in MICsin regrowing bacteria

[78]

Levofloxacin 200 mgi.v., b.i.d.

MSSA, MRSA in general, q.d. regimen morepotent and more rapid kill thanb.i.d. treatment

16- and 64-fold increase inMIC after low-dose cipro-floxacin only; however,ciprofloxacin t1/2 was 2.7 h vs.ofloxacin/levoflxacin t1/2=6.5 h

[99]

Ofloxacin 400 mgi.v., b.i.d.

Ciprofloxacin 400 mgi.v., q.d.

Ciprofloxacin 200 mgi.v., b.i.d.

Klebsiella pneumoniae,Pseudomonas aeruginosa,Enterococcus faecalis,Staphylococcus aureus

dose-dependent kill 4-fold increase in MIC onlywith lowest dose or in border-line susceptible bacteria(MIC=0.25–1.0 mg/l)

[100]

400 mgi.v., b.i.d.600 mgi.v., b.i.d.400 mgi.v., t.i.d.

Ciprofloxacin 400 mgi.v., b.i.d.

Streptococcus pneumoniae(4 strains for which MICswere different)

effective AUC/MIC ratiosranged from 30 to 55

2- to 32-fold increase inciprofloxacin MICs, as allAUICs were below 28

[73]

Levofloxacin 500 mgi.v., b.i.d.

Ciprofloxacin 500 mgp.o., b.i.d.

Streptococcus pneumoniae ciprofloxacin was more rapidlybactericidal than sparfloxacindespite lower PK/PD surrogateparameters

4-fold increase in ciprofloxacinMICs only if Cmax/MIC �4, notcorrelated with AUIC; 2- to4-fold increase in sparfloxacinMICs despite a Cmax/MIC �12

[101]

Sparfloxacin 200 mgp.o., q.d.

Trovafloxacin 200 mgp.o., q.d.

Streptococcus pneumoniae(8 strains for which MICswere different)

�3 log kill was achieved onlyif the AUIC was �22

no resistant mutants weredetected

[75]

Ofloxacin 400 mgp.o., q.d.

Ciprofloxacin 750 mgp.o., q.d.

Moxifloxacin 400 mgp.o., q.d.

Streptococcus pneumoniae(10 strains for whichMICs were different)

a Cmax/MIC �10 and an AUIC�40 resulted in long times toor never-achieved eradication

no resistant mutants weredetected

[102]

Levofloxacin 500 mgp.o., q.d.

Sparfloxacin 200 mgp.o., q.d.

Moxifloxacin 400 mgp.o., q.d.

Staphylococcus aureus(n=3), Staphylococcusepidermidis (n=3)

regrowth and selection ofresistance if levofloxacinCmax/MIC �10 and AUIC�30; moxifloxacin wasbactericidal against all 6 strains

no moxifloxacin-resistantmutants; levofloxacin-resistantmutants in 4 strains if Cmax/MICwas 4 or less

[103]Levofloxacin

500 mgp.o., q.d.

Gatifloxacin 100 mgp.o., q.d.

Streptococcus pneumoniae,3 strains for which MICswere 0.25, 0.5 and1.0 mg/l

�3 log was achieved only ifAUIC was �30 and Cmax/MICwas >5

4- to 8-fold increase in MIC ifCmax/MIC was �5 and AUICwas <30

[104]

200 mgp.o., q.d.400 mgp.o., q.d.600 mgp.o., q.d.800 mgp.o., q.d.

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these two strains were exposed to fluctuating BAY y 3118concentrations, simulating a twice-daily dosing regimen(200 mg each, 12 h apart). Under these experimentalconditions, regrowth was not as rapid and marked asdescribed above, but regrowth still did occur. In parallelto a continuous increase in total viable counts, resistanceemerged rapidly.

Exposure of both Staphylococcus aureus strains tofluctuating BAY y 3118 concentrations simulating aregimen of 200 mg twice daily caused a rapid andextensive selection of resistance, independent from theMICs of the test strains. Resistance emerged within 6 hafter commencement of exposure to BAY y 3118 andcontinued to increase throughout the incubation period.

These data clearly demonstrate that the single substi-tution at the C-8 position significantly lowered thepropensity for mutant selection; that is, the methoxymoiety at the C-8 position, as in moxifloxacin, selectedmutants much less frequently than did the chlorine-substituted quinolone BAY y 3118. These in vitro datawere corroborated by studies in experimental animals[106, 107]. Analogue data were generated for gati-floxacin, another 8-methoxy quinolone.

The contribution of the 8-methoxy group to resistanceselectivity, target preference and antibacterial activityagainst Streptococcus pneumoniae was analysed byFukuda et al. [108]. Gatifloxacin selected mutant strainsat a low frequency, whereas the respective 8-H counter-part selected mutants at a high frequency (3.7�10–9 vs.>2.4�10–6). The 8-methoxy group contributed to en-hancement of antibacterial activity against target alteredmutant strains as well as the wild type strain. The 8-methoxy group also increased the level of target inhibi-tion, especially DNA gyrase, to the almost same extent asfor topoisomerase IV: thus, the 8-methoxy group leads topotent antibacterial activity and a low level of resistanceselectivity [108].

The emergence of multistep resistance to gatifloxacinwas compared with development of resistance to grepa-floxacin, which differs from gatifloxacin by a hydrogensubstitution at position C8 (and a methyl substitution at

position C5); ofloxacin and levofloxacin served ascontrols. Four strains each of Staphylococcus aureusand Streptococcus pneumoniae were exposed to subin-hibitory concentrations of fluoroquinolones for 6 days.The mean increase in MICs from baseline to day 6 forStaphylococcus aureus and Streptococcus pneumoniaewas 2 and 4 mg/l, respectively, for moxifloxacin, 32 and16 mg/l for BAY y 3118, 4 mg/l for gatifloxacin, 32 mg/lfor grepafloxacin, 16 and 32 mg/l for ofloxacin and 32and 64 mg/l for levofloxacin [109, 110, 111, 112, 113].As these numbers were based on small sample sizes,Schmitz et al. [114] compared the rates of fluoro-quinolone resistance development in 70 clinical isolatesof Streptococcus pneumoniae. The rate of resistancedevelopment was lowest for moxifloxacin and gati-floxacin, 50% higher for levofloxacin and approximatelytwice as high for gemifloxacin. These data indicate thatthe development of resistance amongst quinolones isdissociated. The C-8 methoxy group significantly de-creases the propensity for development of resistance toquinolones.

The possible contribution of efflux of fluoroquinolonesto the probability of resistance development was studiedby Madaras-Kelly et al. [115]. By exposing efflux andQRDR mutants to simulated moxifloxacin, sparfloxacinand levofloxacin dosing regimens, the ability of fluoro-quinolones to select out stepwise mutations was eval-uated. In general, moxifloxacin exhibited greaterantimicrobial effects against the wild type strain andagainst the mutants than the comparator agents. Effluxwas generally associated with partial loss of antimicrobialeffects, and sparfloxacin as well as levofloxacin retainedno effect against the parC-expressing mutant. Moxi-floxacin exhibited a relatively pronounced effect againstall isolates. Only levofloxacin selected out an effluxmutant from one of the two wild type strains. Amongstthe post-exposure isolates of the efflux mutant, one offour exposed to moxifloxacin, four of four exposed tosparfloxacin and two of five exposed to levofloxacindeveloped high-level fluoroquinolone resistance andcorresponding mutations in both parC and gyrA. Ana-

Table 10 (continued)

Quinolone Dose Organism Effect Resistance Ref.

Moxifloxacin 100–600 mgp.o., q.d.

Streptococcus pneumoniae;for which MICs were0.12–1 mg/l

as compared to gatifloxacin,moxifloxacin showed anenhanced rate of killing

no moxifloxain-resistantmutants; 4-fold increase ingatifloxacin MICs if Cmax/MIC�4 and AUIC �25

[105]

Gatifloxacin 100–800 mgp.o., q.d.

Moxifloxacin 100–600 mgp.o., q.d.

Staphylococcusaureus; 4 strains: 2 MSSA;1 MRSA ciprofloxacinsensitive; 1 MRSAciprofloxacin resistant

rapid initial bactericidal effectachieved with both drugs, butregrowth following exposureto 3118

no moxifloxain-resistantmutants; 4- to 8-fold increasein 3118 MICs for the 2 MRSAstrains, as Cmax/MIC �3.5

[106]

BAY y 3118 200 mgp.o., q.d.200 mgp.o., b.i.d.

PK/PD, pharmacokinetic/pharmacodynamic; AUIC, pharmacokinetic/pharmacodynamic surrogate parameter AUC/MIC

214

logue results were obtained with the parC-expressingefflux mutant. Post-exposure high-level fluoroquinolone-resistant mutants had mutations in both loci, the parC andgyrA.

The results of this study indicate that (i) differencesexist between the antimicrobial effects of the quinolonesagainst wild type strains, efflux and first-step mutants; (ii)overall genotypes of the selected mutants did not differ;and (iii) once organisms with a target mutation or thoseexhibiting phenotypic fluoroquinolone efflux appear, afurther selection of double mutants occurs. In conclusion,these results [115] support the notion that the probabilityof avoiding efflux reduces the potential for selection offurther resistance [116].

Efflux may permit short-term survival that then leadsto adaptive fluoroquinolone resistance via mutations inthe QRDR. Structural differences among fluoro-quinolones like overall molecular hydrophobicity and/orbulkiness of the C-7 substituent influence the efficiencyof efflux [116, 117]. Of the quinolones tested by Madras-Kelly et al. [115] and Beyer et al. [116], levofloxacin hasthe least bulky C-7 substituent, sparfloxacin a slightlylarger derivative and moxifloxacin the bulkiest C-7substituent. On the other hand, sparfloxacin is a ratherhydrophobic drug, while levofloxacin and moxifloxacinare much more hydrophilic. Moxifloxacin and sparflox-acin were relatively unaffected by pmrA- or norA-medicated efflux, whereas levofloxacin was highlyaffected. These data demonstrate that the bulk at C-7appears to be the key structural characteristic for avoid-ance of efflux. The ease of selection of fluoroquinolone-resistant mutants correlated with efflux susceptibility[116].

In summary, bulk at C-7 and the C-8 methoxy grouphas been shown to correlate with the prevention ofemergence of fluoroquinolone-resistant mutants. Previousfluoroquinolones such as ciprofloxacin and levofloxacinhave the least bulky C-7 substituent and a hydrogensubstitution at C-8, whereas, amongst the C-8 methoxy-quinolones, gatifloxacin has a slightly larger C-7 sub-stitution than levofloxacin; moxifloxacin has the bulkiestC-7 substitution, in addition to its C-8 methoxy group.These structural differences amongst the fluoroquinolones

provide a rationale for the dissociated propensity fordevelopment of resistance amongst gram-positive bacte-ria. On the basis of this theory, previous fluoroquinolonesshould have higher resistance selectivity in gram-positivebacteria than the “new” quinolones.

Apart from these structural specificities, the datasummarised in Table 8 convincingly support the theorythat (i) unfavourable pharmacokinetic/pharmacodynamicparameters, i.e. a Cmax/MIC ratio of �5–10 and/or anAUC/MIC ratio of �25–30 are linked to the emergence ofresistance in gram-positive bacteria, and (ii) activity in invitro models of infection is correlated with these param-eters, too. The clinical relevance of a potentially lowerpropensity to develop resistance should be analysed inanimal studies simulating human pharmacokinetics aswell as in surveillance studies to investigate increases influoroquinolone MICs for Streptococcus pneumoniae orMSSA as a consequence of the clinical use of differentfluoroquinolones.

Most recently, Boswell et al. [118] tried to link thepropensity for development of resistance in Streptococcuspneumoniae to focal drug concentrations of either mox-ifloxacin or levofloxacin. Four Streptococcus pneumoniaestrains (1 clinical isolate susceptible to fluoroquinolonesand 1 strain each of a ParC mutant, a GyrA mutant, and aParC/GyrA double mutant) were studied. These fourstrains were first exposed to drug concentrations quanti-tated in epithelial lining fluid (ELF) or lung mucosa 24 hafter oral administration of either 400 mg moxifloxacin or500 mg levofloxacin. First-step moxifloxacin-resistantmutants were elicited with a frequency ranging from1.3�10–10 to 1.4�10–8, while levofloxacin mutation ratesranged from 2.0�10–1 to 7.3�10–2, irrespective of whetherELF or mucosa concentrations were mimicked. Thesefirst-step mutants were transferred into fresh mediumcontaining maximal ELF or mucosal concentrations ofeither moxifloxacin or levofloxacin. Second-step mutantswere elicited with different frequencies that directlycorrelated to the absolute focal concentrations mimickedto which the first-step mutants were exposed (Fig. 1).Exposure to the highest concentrations studied, i.e.moxifloxacin ELF levels, gave rise to the lowest numbersof second-step mutants, which is in contrast to the high

Fig. 1 Propensity for resistancedevelopment in Streptococcuspneumoniae (mean of 3–4 ex-periments) as determined byusing focal drug concentrations(modified according to [118])

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numbers of mutation frequencies observed followingexposure to low focal concentrations of levofloxacin(Fig. 1).

These data corroborate the notion that emergence ofresistance is a function of drug concentrations achievablein vivo and the susceptibility pattern of the targetorganisms. The closer the MIC90 values or the modalMIC is to clinically relevant fluoroquinolone concentra-tions, the more likely resistant subpopulations willemerge. Once first-step mutants have been selected, theyare prone to mutate further.

These conclusions, drawn on the basis of in vitropharmacodynamic studies, suggest first that publishedclinical trials may lag behind the actual situation, as thesusceptibility of the causative pathogens may diminishbetween the time of the last patient/last visit and thepublication of study results. However, MICs of drugs forthe causative pathogens may increase in the meantime, sothat pharmacokinetic/pharmacodynamic surrogate param-eters become less favourable and resistance becomesmore likely to emerge. Therefore, drugs without any“comfort zone” at the time of the execution of the studiesmay, in the meantime, have converted into suboptimallyactive agents. Secondly, the use of weaker fluoro-quinolones with borderline or even suboptimal pharma-cokinetic/pharmacodynamic surrogate parameters willinadvertently foster the development of class resistance.

Consequently, it is tempting to speculate that fluoro-quinolone resistance should have emerged, particularly ingram-positive bacteria. The most frequently used fluoro-quinolones such as ofloxacin and ciprofloxacin wereintroduced in the mid 1980s, while levofloxacin wasintroduced in 1996 and 1998 in the USA and Europe,respectively. Although these agents are most frequentlyused to treat gastrointestinal disorders and urinary tractinfections, gram-positive bacteria in general and strepto-cocci in particular have been exposed to fluoroquinolonesunintentionally, as these microorganisms are commensalsof the oropharyngeal tract. In principle, oropharyngealstreptococci could have acquired and could be a reservoirof fluoroquinolone resistance. Thus, fluoroquinoloneresistance in Streptococcus pneumoniae could, in theory,have arisen from horizontal transfer between viridansgroup streptococci and Streptococcus pneumoniae. Nev-ertheless, the prevalence of fluoroquinolone-resistantStreptococcus pneumoniae is still very low [119, 120,121]. Hypothetically, various factors—either separatelyor in combination—may have contributed to the low ratesof resistance of streptococci to the fluoroquinolones, asdiscussed below.

Insufficient Exposure of Streptococci to Quinolones

Similar to the emergence of penicillin resistance inStreptococcus pneumoniae, the exposure of streptococcito fluoroquinolones may not have been sufficiently long.It took more than 25 years from the introduction ofpenicillin until the first moderately penicillin-resistant

pneumococci were isolated from healthy carriers [16, 17].A decade later the first multiresistant strains were isolatedfrom infected patients in South Africa [122, 123]. Sincethen, penicillin resistance has developed into a globalproblem.

Possible Existence of a Plasmid That ConfersResistance to Quinolones

Resistance to many antibacterial agents is plasmidmediated. Bacterial resistance of this type often precedesthe clinical use of a new antibiotic, the use of which inturn favors selection and, in particular, dissemination ofplasmid-mediated resistance. For several antibacterialagents, however, no plasmid-mediated resistance mech-anisms are known; chromosomal mutations confer resis-tance to, e.g., nitrofurans, novobiocin, polypeptides,rifampin and quinolones. Still, two reports [124, 125]indicated that quinolone resistance in Enterobacteriaceaemay originate from a transferable plasmid.

In the late 1980s there was a single report of plasmid-mediated resistance to nalidixic acid in Shigella dysen-teriae type 1. This strain caused an outbreak in southernBangladesh. In the first series of laboratory studies, thisstrain was found to harbour a 30-kilobase plasmidconferring resistance to nalidixic acid only [124]. Rein-spection of the data, however, revealed that the presumedtransconjugants were resistant mutants because they wereselected with nalidixic acid and exhibited no otherplasmid-mediated resistance [126]. The finding thatnalidixic-acid-resistant mutants rather than authentictransconjugants were obtained is attributable to the factthat the putative donor strain contained gyrA mutations[127] and that a plasmid was not directly involved [128].In the experiments performed by Munshi et al. [124], noserial transfers of the plasmid were attempted, and itselimination with acridine orange was unsuccessful. Allthese findings confirm that nalidixic acid resistance in theShigella dysenteriae isolate studied by Munshi et al. [124]was due to chromosomal mutation. The plasmid itselfdoes not code for nalidixic acid resistance but offers asurvival advantage to its host under nalidixic acid stress.

The second report [125] describes the transfer of amultiresistance plasmid (pMG252) from a clinical isolateof Klebsiella pneumoniae into (i) strains of Klebsiellapneumoniae deficient in outer membrane porins, and (ii)into Klebsiella pneumoniae or Escherichia coli withnormal porins. Ciprofloxacin resistance was high in thetwo membrane-deficient transconjugants (increase from 4to 32 mg/l and from 0.5 to 4 mg/l, respectively), whereasthe increase in MICs was much lower in the transconju-gants with normal porins (from 0.004 to 0.125 mg/l).Transconjugants were obtained by selection with a beta-lactam agent, so that there was no risk of selectingquinolone-resistant host mutations.

The findings described by Martinez-Martinez et al.[125], however, must be put into perspective. First,although this plasmid originated from a ciprofloxacin-

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resistant strain of Klebsiella pneumoniae, it mediatesbroad resistance to beta-lactam antibiotics and severalother classes of antibacterial agents, but not to quinolones.The plasmid conferred resistance to aztreonam, ceftazi-dime, cefotaxime, cefoxitin, cefotetan, chloramphenicol,kanamycin, gentamicin, streptomycin, tobramycin, sulfa-furazole, trimethoprim and mercuric chloride. Second, thepresence of the plasmid in the transconjugants exerts anindirect effect on susceptibility to quinolones. Themechanism of “plasmid-mediated quinolone resistance”is due to the protection of the gyrase from inhibition byciprofloxacin. The plasmid quinolone resistance gene qnrcodes for a protein belonging to the pentapeptide repeatfamily. This protein shares sequence homology with theimmunity protein Mcb G, which is thought to protectDNA gyrase from the action of microcin B17. Protectionof topoisomerase IV was not evident [129].

By itself, the qnr gene causes only a moderate increasein ciprofloxacin MICs, so that the transconjugants are stillclassified as susceptible. Its clinical importance lies in theaugmenting effect it produces between itself and otherresistance mutations. At present, the qnr determinant isvery uncommon among clinical isolates of ampC beta-lactamase-producing Klebsiella pneumoniae and wasundetectable in Escherichia coli [130]. So far, the closestrelatives to the qnr gene have been found in Caulobactercrescentus, Mycobacterium smegmatis, Bacillus subtilis,Enterococcus faecalis and Mycobacterium tuberculosis(nucleotide identity decreasing from 44.7 to 18.9%) butnot in other gram-positive bacteria [129].

Minimal Interspecies Horizontal Gene TransferBetween Streptococci

The horizontal transfer of genes between streptococcalspecies is well known for genes encoding penicillin-binding proteins and has contributed to the emergence ofbeta-lactam resistance [131]. The horizontal transfer ofparC and gyrA genes in fluoroquinolone-resistant clinicalisolates of Streptococcus pneumoniae has been suggested,assuming that viridans group streptococci could act as areservoir for fluoroquinolone resistance [132]. In princi-ple, the nucleotide sequences of the gyrase and topoiso-merase IV genes show high identity, and viridansstreptococci and Streptococcus pneumoniae share thesame quinolone resistance mechanisms [133, 134]. How-ever, the contribution of horizontal interspecies genetransfer to fluoroquinolone resistance has been disputed,as strains sharing such chimeric quinolone target genesare rarely found [135, 136]. Therefore, the horizontalinterspecies transfer of fluoroquinolone resistance genesdoes not appear to be a major mechanism by whichStreptococcus pneumoniae gains fluoroquinolone resis-tance.

Conclusions

Although it is an inevitable consequence of any antibac-terial therapy that resistance will emerge, it may be morelikely that fluoroquinolone resistance in streptococcioccurs sporadically through acquired point mutationsrather than by interspecies gene transfer that favours theemergence and rapid spread of resistance to other drugclasses. The above-mentioned factors—either separatelyor in combination—likely have contributed to the gener-ally low prevalence of fluoroquinolone resistanceamongst streptococcal species despite extremely highexposure rates. However, once first-step mutants havearisen, these strains are the progenitors of fully resistantstrains with dual mutations. Similar factors are probablyoperative for staphylococci [137]. Therefore, strategies toreduce resistance will become ever more important. Thenew fluoroquinolones offer demonstrable advantage overother agents. Those fluoroquinolones with the mostfavourable pharmacokinetic/pharmacodynamic character-istics should be used as first-line agents in order topreserve the potential of this drug class and, mostimportantly, to provide the patient with an optimallyeffective regimen and to reduce the likelihood of resis-tance selection.

Acknowledgement The assistance of Paul Higgins in the carefulpreparation of the manuscript is gratefully acknowledged.

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