In Vitro Activities of Garenoxacin (BMS-284756) against Haemophilus influenzae Isolates with Different Fluoroquinolone Susceptibilities

The in vitro activity of garenoxacin (BMS-284756) against 62clinical Haemophilus influenzae isolates with different fluoroquinolonesusceptibilities was determined by the microdilution susceptibilitytesting method and compared with the activities of other oralquinolones and nonquinolone oral antimicrobial agents. Cefiximepresented the highest intrinsic activity (MIC at which 50% ofthe isolates tested were inhibited [MIC₅₀], 0.01 µg/ml),followed by garenoxacin, moxifloxacin, and ciprofloxacin (MIC₅₀,0.06 µg/ml), levofloxacin (MIC₅₀, 0.12 µg/ml), cefuroxime(MIC₅₀, 1.0 µg/ml), and amoxicillin-clavulanate (MIC₅₀,1.0/0.5 µg/ml), amoxicillin (MIC₅₀, 2 µg/ml), azithromycin(MIC₅₀, 4 µg/ml), and erythromycin (MIC₅₀, 8 µg/ml).In strains with ciprofloxacin MICs of $<=$ 0.06 µg/ml, ciprofloxacinand garenoxacin displayed similar MIC₅₀s and MIC₉₀s, one dilutionlower than those of moxifloxacin and levofloxacin. For strainsfor which ciprofloxacin MICs were $>=$ 0.12 µg/ml,MIC₅₀s were similar for the four quinolones tested, althoughgarenoxacin presented the widest activity range (0.03 to 32µg/ml) and the highest MIC at which 90% of the isolatestested were inhibited (16.0 µg/ml). For strains withoutamino acid changes in the quinolone resistance determining region(QRDR) of GyrA and ParC, garenoxacin MICs were $<=$ 0.03 µg/ml;with a single amino acid change in GyrA, garenoxacin MICs were0.06 to 0.12 µg/ml; with one amino acid change each inGyrA and ParC, garenoxacin MICs were 0.5 to 2.0 µg/ml;one amino acid change in ParC combined with two amino acid changesin GyrA increased the MICs to $>=$ 4 µg/ml forall assayed quinolones. We conclude that garenoxacin has excellentactivity against H. influenzae, although progressive acquiredresistance was observed by step-by-step mutation in the QRDRof gyrA and parC.

INTRODUCTION

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Introduction

Garenoxacin is a novel des-fluoro(6)-quinolone that differsfrom earlier quinolones in its lack of a fluorine atom at theC-6 position and an isoindolin-5-yl substitution at the 7 position(17). It has reportedly shown increased activity relative tothe other quinolones against gram-positive organisms (3, 15),including methicillin (oxacilin)-resistant staphylococci andsome Enterococcus spp. (5). Broad antianaerobic coverage andsuperior activity against fastidious organisms has also beendescribed (9). The difluoromethoxy substituent at position 8,instead of a methoxy group, has been shown to improve bacteriostaticand bactericidal activity and decrease the selection of resistantmutants (10). Garenoxacin has good oral bioavailability (11),and toxicological findings indicate low chondrotoxicity in juvenilerats, making it a potentially suitable therapy for childrenand adolescents (12).

Little is known about the activity of this compound againstHaemophilus influenzae strains whether or not they have reducedsusceptibility to other fluoroquinolones. Although fluoroquinolonesremain among the most powerful in vitro antimicrobial agentsagainst H. influenzae and are also highly effective as oraltreatments of respiratory tract infections (6), resistance tothem has been recognized (4, 7, 16). Therapeutic failure incommunity-acquired pneumonia associated with levofloxacin resistancehas recently been described for H. influenzae (2). We have previouslyshown that strains with ciprofloxacin MICs of $<=$ 0.06 µg/mlremain free of quinolone resistance determining region (QRDR)mutations in both gyrA and parC, while the first mutation ingyrA appears in strains with ciprofloxacin MICs of $>=$ 0.12µg/ml (M. Pérez-Vázquez et al., submittedfor publication).

On the other hand, it is of interest to study the activitiesof new compounds that are to be introduced onto the market againstwell-characterized collections of resistant organisms (8). Thesestudies are useful for defining the intrinsic activities ofnew drugs, even when resistance mechanisms may compromise thesecompounds.

Our study was designed to determine the comparative activityof garenoxacin (BMS-284756) and those of other orally administeredquinolones and of other nonquinolone antimicrobial agents testedagainst H. influenzae isolates displaying different fluoroquinolonesusceptibility values. It also aimed to ascertain the influenceof GyrA and/or ParC QRDR amino acid changes on the activityof garenoxacin.

MATERIALS AND METHODS

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Materials and Methods

Test isolates. A total of 62 clinical H. influenzae isolates and two referencestrains from the American Type Culture Collection (ATCC 49247and ATCC 51907) were studied. This batch of strains was distributedinto two different groups: (i) group I included two Americanclinical strains and 30 Spanish clinical isolates from the collectionof the Haemophilus Reference Laboratory. They were selectedon the basis of their reduced susceptibility to ciprofloxacin(MIC, $>=$ 0.12 µg/ml). This group comprisedrespiratory specimens from patients with cystic fibrosis orchronic respiratory infections, obtained as part of antibioticresistance surveillance in Spanish clinical isolates. (ii) GroupII, which included a fully susceptible control group of 30 strainswith ciprofloxacin MICs of $<=$ 0.06 µg/ml, was selected andmatched according to the following criteria: similar date ofisolation, geographical area, clinical diagnosis, anatomicalsource, capsulation status, and biotype. The majority of strainswere collected between 1994 and 2002 from patients living inthe central area of Spain.

Susceptibility testing. Reference broth microdilution method was performed accordingto the NCCLS guidelines (13, 14). Haemophilus test medium wasprepared with Mueller-Hinton broth (Oxoid Ltd., Basingstoke,Hampshire, United Kingdom) supplemented with HTM supplement(Oxoid) and yeast extract (5 ${per thousand}$ ) (Difco, Detroit, Mich.). Microtiterplates were inoculated to produce a final inoculum density ofapproximately 5 x 10⁵ CFU/ml. This density was monitored atregular intervals by making colony counts. The inoculated plateswere incubated at 35°C for 20 to 24 h in ambient air beforeresults were interpreted. The MIC was defined as the lowestconcentration of antibiotic that inhibited growth.

Antimicrobial agents. Garenoxacin (BMS-284756) was supplied by Bristol-Myers Squibblaboratories (Madrid, Spain). The other antimicrobial agents,including a ß-lactamase inhibitor compound (amoxicillin,azithromycin, cefixime, cefuroxime, ciprofloxacin, erythromycin,levofloxacin, moxifloxacin, nalidixic acid, and clavulanate),were provided by their respective manufacturers or purchasedfrom Sigma (Madrid, Spain).

Amplification and sequence analysis of the QRDR regions of gyrA and parC genes. One isolate was randomly selected for QRDR analysis from eachgroup of strains with identical garenoxacin MICs (MIC range,0.007 to 32 µg/ml). Amplification was performed in a 50-µlfinal volume: 5 µl of DNA template, 10 mM Tris-HCl (pH8.3), 50 mM KCl, 2 mM MgCl₂, 1 µM (each) primer (Pharmacia),200 µM (each) deoxynucleoside triphosphate, and 2.5 Uof Taq polymerase (Roche). The PCR program applied was as follows:denaturation at 94°C for 5 min; 30 amplification cyclesof 94°C for 1 min, annealing at 54°C for 1 min, andpolymerization at 72°C for 1 min; and a final 10-min cycleat 72°C to extend amplicons fully.

A 400-bp fragment including the QRDR of gyrA and ParC was amplified.Specific primers used were the following: GYRA-F (5'-CCGCCGCGTACTATTCTCAAT-3'), GYRA-R (5'-GTTGCCATCCCCACCGCAATACCA-3'),PARC-F (5'-TCTGAACTTGGCTTAATTGCC-3'), and PARC-R (5'-GCCACGACCTTGCTCATAAAT-3').PCR products were purified with a PCR purification kit (Qiagen,Hilden, Germany). Sequencing of fragments was done on both DNAstrands with the Big DyeTM terminator cycle sequencing kit (Perkin-Elmer)according to the manufacturer's instructions. The products wereresolved and analyzed with an ABI PRISMR 377 DNA sequencer.Nucleotide sequences were analyzed using the DNAstar (Madison,Wis.) program.

RESULTS AND DISCUSSION

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Results and Discussion

When we compare in vitro activities of different antimicrobialagents against the 62 selected H. influenzae isolates in termsof the MICs at which 50% of the isolates tested were inhibited(MIC₅₀s), which normally represent modal MICs, cefixime hadthe highest intrinsic activity (0.01 µg/ml). This valuewas similar to that obtained with ciprofloxacin, moxifloxacin,and garenoxacin (0.06 µg/ml). Amoxicillin, amoxicillin-clavulanate,and cefuroxime presented similar MIC₅₀s of 1 to 2 µg/ml,and erythromycin and azitromycin were the less active agents,with MIC₅₀s of 8 and 4 µg/ml, respectively. However, asubstantial difference was observed in the MICs at which 90%of the isolates tested were inhibited (MIC₉₀s): 0.25 µg/mlfor cefixime and 2, 2, and 4 µg/ml for ciprofloxacin,moxifloxacin, and garenoxacin, respectively. For amoxicillin,the MIC₉₀ was 128 µg/ml; for amoxicillin-clavulanate,8 µg/ml; for cefuroxime, 16 µg/ml; for erythromycin,32 µg/ml; and for azithromycin, 8 µg/ml.

Table 1 shows the activity of the assayed antimicrobial agentsagainst H. influenzae isolates of groups I and II. Levofloxacin,moxifloxacin, and garenoxacin displayed similar MIC₅₀s and MIC₉₀sin group I of H. influenzae. For the group II strains, levofloxacin,moxifloxacin, and garenoxacin had markedly decreased activities.Nevertheless, for four isolates belonging to group II, MICsof garenoxacin were lower than those for ciprofloxacin; theMIC for one of them was 0.03 µg/ml for garenoxacin and0.12 µg/ml for ciprofloxacin, while the MICs for the threeadditional isolates were 0.06 µg/ml for garenoxacin and0.12 µg/ml (two strains) and 0.25 µg/ml (one strain)for ciprofloxacin.

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TABLE 1. In vitro activities of four quinolones and different nonquinolone antimicrobials against H. influenzae isolates with different levels of susceptibility to ciprofloxacin

Table 2 shows the gyrA and parC QRDR mutations leading to aminoacid substitutions in selected H. influenzae isolates of thestudy collection. These isolates are representative of differentgarenoxacin MICs from group I and group II. For comparativepurposes, other quinolone MICs are also included in this table.

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TABLE 2. Quinolone susceptibility and amino acid changes in GyrA and ParC QRDR fragments from representative H. influenzae isolates^a

Garenoxacin displayed high intrinsic activity against isolateswithout gyrA or parC mutations, with MICs ranging from 0.007to 0.03 µg/ml, similar to ciprofloxacin, while the moxifloxacinand levofloxacin MIC range was 0.03 to 0.12 µg/ml (Table2). Garenoxacin displayed MICs of 0.06 to 0.12 µg/ml forisolates with a first step in the resistance mechanisms forquinolones (one amino acid change in GyrA). This value was slightlylower than that obtained with ciprofloxacin, moxifloxacin, andlevofloxacin (0.12 µg/ml). However, the existence of oneamino acid change in ParC combined with two amino acid changesin GyrA drastically diminished the in vitro activity of thefour assayed quinolones (Table 2).

Garenoxacin is a desfluoroquinolone with a broad spectrum ofactivity against both gram-positive and -negative pathogens,including fastidious strains that commonly cause community-acquiredrespiratory tract infections (5). The SENTRY antimicrobial surveillanceprogram (2000) demonstrated that the activity of garenoxacinagainst H. influenzae was similar to that of other quinolones,such as ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin(MIC₉₀, $<=$ 0.016 to 0.03 µg/ml) (1), consistent with dataobtained in the present study for strains without amino acidchanges in the QRDRs of gyrA and/or parC.

The H. influenzae population tested in our study includes bothstrains with and without resistance mechanisms for quinolones.The overall activity of garenoxacin was similar to that of ciprofloxacin(MIC₉₀, 4.0 µg/ml), but it was slightly lower than thoseof moxifloxacin and levofloxacin. To gain insight into garenoxacinactivity, we separated our H. influenzae collection accordingto its susceptibility to ciprofloxacin; for fully ciprofloxacin-susceptibleisolates (group I), ciprofloxacin, levofloxacin, and garenoxacinMIC₉₀s were similar (0.03 µg/ml), and for moxifloxacinthe MIC₉₀ was one log₂ dilution higher. In group II, garenoxacinwas the quinolone with the lowest activity, since its MIC₉₀was at least two log₂ dilutions higher than that of the othertested quinolones, although in this group, the garenoxacin MICrange included lower values (0.03 µg/ml for garenoxacin,0.06 µg/ml for moxifloxacin, and 0.12 µg/ml forciprofloxacin and levofloxacin). This result is concordant withthe fact that the older generation of quinolones (ciprofloxacinand ofloxacin) have relatively low gram-positive activity, andother new generation quinolones (including desquinolone) weremodified to enhance mainly gram-positive activity.

We have shown that the primary target of garenoxacin in H. influenzaeis DNA gyrase, the same as that of ciprofloxacin (7). A firstamino acid modification in the QRDR of GyrA increased the garenoxacinMIC to 0.06 to 0.12 µg/ml; a second amino acid changein the QRDR of ParC enhanced its MIC to 0.5 to 2 µg/ml;and a double amino acid change in GyrA yielded the highest MICsfor the strains in this collection (4.0 to 32 µg/ml).This increase in MICs was similar for ciprofloxacin, moxifloxacin,and levofloxacin, suggesting a step-by-step resistance mechanismcommon to the four quinolones tested.

In summary, we conclude that there was excellent garenoxacinactivity against H. influenzae strains lacking quinolone resistancemechanisms. However, some amino acid changes in the QRDR regionof GyrA and ParC clearly decreased the susceptibility of H.influenzae to garenoxacin, ciprofloxacin, levofloxacin, andmoxifloxacin, reaching a MIC of 32 µg/ml for strains withtwo modifications in the QRDR region of GyrA and one modificationin the QRDR of ParC.

ACKNOWLEDGMENTS

We thank Janet Hinder for providing us with two quinolone-resistantH. influenzae isolates.

M. Pérez-Vázquez is a recipient of grant 02/0056from the Instituto de Salud Carlos III, Majadahonda, Spain.This study was funded by a research grant from Bristol-MyersSquibb and a fellowship (99/0304) from the Spanish FIS agency.

FOOTNOTES

* Corresponding author. Mailing address: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda a Pozuelo, Km. 2, 28220 Majadahonda-Madrid, Spain. Phone: 34 91 5097901, ext. 3650 or 3643. Fax: 34 91 5097966. E-mail: jcampos{at}isciii.es.

REFERENCES

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