Activities of Garenoxacin against Quinolone-Resistant Streptococcus pneumoniae Strains In Vitro and in a Mouse Pneumonia Model

Abstract

Garenoxacin is a novel des-F(6) quinolone with enhanced in vitro activities against both gram-positive and gram-negative bacteria. We compared the activity of garenoxacin with that of trovafloxacin (TVA) against Streptococcus pneumoniae, together with their efficacies and their capacities to select for resistant mutants, in a mouse model of acute pneumonia. In vitro, garenoxacin was more potent than TVA against wild-type S. pneumoniae and against a mutant with a single mutation (parC), a mutant with double mutations (gyrA and parC), and a mutant with triple mutations (gyrA, parC, and parE). Swiss mice were infected with 10⁵ CFU of virulent, encapsulated S. pneumoniae strain P-4241 or its derived isogenic parC, gyrA, gyrA parC, and efflux mutants and 10⁷ CFU of poorly virulent clinical strains carrying a parE mutation or gyrA, parC, and parE mutations. The drugs were administered six times, every 12 h, beginning at either 3 or 18 h postinfection. The pulmonary pharmacokinetic parameters in mice infected with strain P-4241 and treated with garenoxacin or TVA (25 mg/kg of body weight) were as follows: maximum concentration of drug in serum (C_max; 17.3 and 21.2 μg/ml, respectively), C_max/MIC ratio (288 and 170, respectively), area under the concentration-time curve (AUC; 48.5 and 250 μg · h/ml, respectively), and AUC/MIC ratio (808 and 2,000, respectively). Garenoxacin at 25 and 50 mg/kg was highly effective (survival rates, 85 to 100%) against the wild-type strain and mutants harboring a single mutation. TVA was as effective as garenoxacin against these strains. TVA at 200 mg/kg and garenoxacin at 50 mg/kg were ineffective against the mutant with the parC and gyrA double mutations and the mutant with the gyrA, parC, and parE triple mutations. The efficacy of garenoxacin was reduced only when strains bore several mutations for quinolone resistance.

Streptococcus pneumoniae is the bacterium most frequently isolated from patients with community-acquired pneumonia and continues to be a significant cause of mortality (15, 16, 19). It colonizes the human nasopharynx and is a leading cause of upper and lower respiratory tract infections, which result in a secondary risk of bacteremia. The worldwide incidence of infections caused by pneumococci resistant to penicillin, macrolides, and other antimicrobials has increased at an alarming rate during the past two decades (1, 2). There is thus considerable interest in the use of alternative antimicrobials, such as fluoroquinolones. The available fluoroquinolones, such as ciprofloxacin (CIP), have limited effectiveness in this setting, but there has been interest in the use of recently developed compounds for the treatment of such infections (3, 4, 21).

Several studies have shown that low-level pneumococcal resistance can result from mutations in the parC gene, which codes for topoisomerase IV (17). Increased levels of resistance occur following acquisition of additional mutations in gyrA, which encodes the A subunit of the type II topoisomerase DNA gyrase (12). The impacts of these two resistance mechanisms depend on the class of antibacterial agent (21). Moreover, recent studies have identified an efflux mechanism as a further cause of low-level resistance in pneumococci (6, 25). Higher levels of resistance occur when this efflux mechanism is associated with mutations of the DNA gyrase (gyrA) or the topoisomerase IV (parC) gene.

Garenoxacin is a des-F(6) quinolone. This novel compound lacks the C-6 fluorine atom characteristic of existing fluoroquinolones and has a broad spectrum of activity in vitro against both gram-positive and gram-negative pathogens, including some quinolone-resistant strains (5, 9, 13). The aim of this study was to evaluate the efficacy of garenoxacin in a mouse model of acute S. pneumoniae pneumonia caused by a wild-type strain and its isogenic quinolone-resistant mutants, together with the capacity of garenoxacin to select for resistant strains in vitro and in vivo, in comparison with the efficacy and capacity for the selection of resistance of trovafloxacin (TVA), a trifluoronaphthyridone quinolone.

(This work was presented in part at the 11th European Congress of Clinical Microbiology and Infectious Diseases, Istanbul, Turkey, 1 to 4 April 2001 [E. Azoulay-Dupuis et al., 11th Eur. Congr. Clin. Microbiol. Infect. Dis. abstr. P832, 2001].)

MATERIALS AND METHODS

Drugs

The antibiotics used in this study comprised the quinolones garenoxacin (Bristol Myers Squibb Laboratories, Wallingford, Conn.), TVA (Pfizer Laboratories, Groton, Conn.), and CIP and moxifloxacin (MXF) (Bayer Laboratories, Sens, France). Ethidium bromide (Sigma, Saint Quentin Fallavier, Cedex France) was also used.

Bacterial strains

S. pneumoniae P-4241 is a blood isolate (serotype 3). This encapsulated strain is virulent in a mouse model of acute pneumonia (100% lethal dose, 3.3 log₁₀ CFU/mouse). It is susceptible to both penicillin and quinolones (amoxicillin MIC, 0.03 μg/ml; CIP MIC, 1 μg/ml). Strains 2500 and 2759 are poorly virulent clinical isolates carrying a single mutation in parE and triple mutations in gyrA, parC, and parE, respectively (see Table 1).

TABLE 1.

MICs and mutations in strains used for animal studies^a

Genotype (mutation)	Strain	MIC (μg/ml)
		GRN	TVA	MXF	SPX	CIP	NOR	EB
WT (parC, S79; gyrA, S81)	P-4241	0.06	0.125	0.125	0.5	2	8	4
ParC (S79Y)	C42-R2	0.125	0.5	0.125	1	8	64	8
GyrA (S81Y)	Sp42-R1	0.25	0.125	0.25	2	2	8	4
parC (S79Y) gyrA (S81Y)	C42-Sp6	1	8	4	16	32	128	8
Efflux (ND)	N42-R1	0.125	0.18	ND	0.5	4	16	16
parE (D435N + I460V)	2500	0.125	0.5	ND	0.5	16	>64	32
parC (S79F) gyrA (E85K) parE (I460V)	2759	1	16.0	ND	ND	64	>64	32

^aAbbreviations: WT, wild type; GRN, garenoxacin; EB, ethidium bromide; ND, not determined.

Selection and analysis of gyrA and parC isogenic mutants

Isogenic mutants were derived from parent strain P-4241, which was grown overnight at 37°C on Columbia agar plates containing 5% sheep blood. Cells were then scraped from the plate and resuspended in Columbia medium. One hundred microliters of cell suspension (10¹⁰ to 10¹¹ CFU/ml) was plated on Columbia agar containing 5% sheep blood and a selective antibiotic at a final concentration of 0.5 to 3 times the MIC. CIP was used to select for mutations in the parC subunit of topoisomerase IV (strain C42-R2), while sparfloxacin (SPX) was used to select for mutations in the gyrA subunit of topoisomerase II (strain Sp42-R1). Norfloxacin (NOR) at or slightly above the MIC preferentially selects for efflux pump mutants (strain N42-R1), as shown by increased resistance to ethidium bromide. A mutant with double mutations in parC and gyrA (strain C42-Sp6) was obtained by exposing strain C42-R2 to SPX. The quinolone resistance-determining regions (QRDRs) of gyrA and parC were amplified by PCR with primer pair VGA3 (5′-CCGTCGCATTCTTTACG) and VGA4 (5′ AGTTGCTCCATTAACCA) for gyrA and primer pair M0363 (5′-TGGGTTGAAGCCGGTTCA) and M4271 (5′ TGCTGGCAAGACCGTG) for parC (20). PCR conditions were as follows: 1 cycle of 1 min at 95°C, followed by 25 cycles of 30 s at 95°C, 30 s at 50°C, and 90 s at 72°C, with a final 5-min extension step at 72°C. PCR fragments were purified through spin columns (Qiagen, Hilden, Germany) and directly sequenced with the primers described above at the sequencing facility of the University of Geneva by use of an ABI A377 automatic sequencer (Applied Biosystems, Foster City, Calif.).

Selection of S. pneumoniae strains resistant to garenoxacin

Inocula of 10⁹ to 10¹⁰ CFU of S. pneumoniae strain P-4241 were plated on Columbia agar plates supplemented with 5% sheep blood and increasing concentrations of garenoxacin. The plates were monitored for the emergence of resistant colonies for 48 h at 37°C. Five of 18 colonies on the plate containing garenoxacin at 0.1 μg/ml were analyzed for their resistance profiles and mutations.

Analysis of strains recovered from mice

Bacteria were recovered from homogenated lungs of treated and untreated control animals. Lung homogenates were spread on Columbia agar plates supplemented with 5% sheep blood. Individual colonies were selected after overnight culture at 37°C, and their resistance profiles were determined. Strains for which MICs were higher than the control value were further investigated. The QRDRs were amplified and sequenced.

MIC determinations

MICs were determined by the broth microdilution method in Columbia medium supplemented with 5% sheep blood (18).

Infection of mice with S. pneumoniae

Swiss mice (weight, 20 to 22 g; Iffa Credo, L’Arbresle, France) were infected by the intratracheal route with 40 μl of bacterial suspension at a dose of approximately 10⁵ CFU of S. pneumoniae per immunocompetent mouse and 10⁷ CFU of the poorly virulent strains per leukopenic mouse. The animals were rendered leukopenic by intraperitoneal administration of 150 mg of cyclophosphamide/kg of body weight/day for 3 days, starting 4 days before infection. This treatment reduces leukocyte counts from about 7,000 to 1,000/ml of blood on the day of bacterial challenge (3).

Antibiotic treatment

Therapy was initiated 18 h after challenge with the wild-type virulent penicillin-susceptible strain (P-4241) and with the quinolone-resistant mutants (mutants with single parC, gyrA, and efflux mutations and the mutant with double parC and gyrA mutations). Treatment was initiated 3 h after challenge with the parE and the parC gyrA parE clinical strains. Garenoxacin and TVA were administered as six subcutaneous (s.c.) injections at doses of 12.5, 25, and 50 mg/kg. TVA was given at doses of 50, 100, and 200 mg/kg to mice challenged with the mutant with the double mutations. Infected, untreated control mice received the same volume of isotonic saline. Each treatment group comprised 15 animals. The observation period was 10 days. Death rates were recorded daily, and the cumulative survival rates were compared.

Bactericidal activity in vivo

The protocol used to study bactericidal activity in vivo was the same as that used for the mouse survival studies. The total CFU counts recovered from whole-lung homogenates were determined 6 h after the first treatment, which was initiated 18 h after bacterial challenge, and 12 h after the second, fourth, and sixth treatments at doses of 12.5 and 25 mg of garenoxacin per kg. Three mice were used for each dose and time point. Mice were killed by intraperitoneal injection of sodium pentobarbital and were exsanguinated by cardiac puncture; blood was used for culture. The lungs were removed and homogenized in 1 ml of normal saline. Serial 10-fold dilutions of the homogenates were plated on Columbia agar. Blood was cultured in brain heart infusion broth. After overnight culture, colonies were counted on agar plates seeded with lung samples, and blood cultures were examined for turbidity. Results are expressed as the mean ± standard deviation log₁₀ CFU per lung and as the number of positive or negative blood cultures for groups of three mice each.

Determination of garenoxacin concentrations in serum and lungs and PK analysis

Antibiotics were administered as a single s.c. dose of 25 mg of garenoxacin or TVA per kg to both infected and uninfected mice. Infected mice were treated at 18 h postinfection. Serum and lung samples were collected from groups of six mice at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after drug administration. All samples were stored at −20°C and protected from light to avoid garenoxacin degradation during analysis. Lung samples were crushed in liquid nitrogen with a magnetic crusher (Spex; Fisher Bioblock, Illkirch, France). Serum samples (100 μl) and lung tissue samples (20 to 50 mg of lung powder, as measured precisely) were prepared by mixing an internal standard (T-3811-IS01; Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, N.J.) with methanolic acid (100 and 500 μl, respectively). After precipitation or diffusion, vortexing or ultrasonic mixing, and centrifugation, 50 μl of the upper phase was injected into a high-performance liquid chromatographic system. The total drug concentration was determined by use of an octadecyl silyl column (Novapak C₁₈; 4.6 by 150 mm; Waters, Milford, Mass.) coupled to a spectrofluorometric detector operating at excitation and emission wavelengths of 280 and 415 nm, respectively. The mobile phase was a mixture of acetonitrile, sodium citrate buffer solution (pH 3.5), and water (22/15/63; vol/vol) with 0.2% triethylamine, adjusted to pH 4. The flow rate was 1.0 ml/min. The limits of quantification were 0.02 μg/ml and 0.05 μg/g for serum and lung tissue samples, respectively, and measurements were linear over the ranges of 0.2 to 10.0 μg/ml and 0.5 to 50.0 μg/g for serum and lung tissue samples, respectively. The coefficients of variation for quality control were below 10% for both serum and lung tissue samples. The pharmacokinetic (PK) parameters for TVA were evaluated as described elsewhere (4).

PK analysis was based on a noncompartmental model (WinNonlin, version 1.1; Pharsight, Mountain View, Calif.). Maximum concentrations in serum (C_max) were measured experimentally, while the area under the time-versus-serum concentration curve from 0 to 24 h (AUC_0-24) and the terminal half-life (t_1/2) were calculated by the use of WinNonlin software.

RESULTS

Activity of garenoxacin against S. pneumoniae strains with defined target mutations or the efflux phenotype

The MICs for the wild-type strain P-4241 and its isogenic target mutants are shown in Table 1. parC mutant C42-R2 was found to harbor a TCC to TAC mutation in parC codon 79, resulting in a Ser-to-Tyr substitution. The MXF MIC for this mutant was not affected, while the MICs of TVA, SPX, CIP, and NOR, which all preferentially select for parC mutants, were increased two- to eightfold. The garenoxacin MIC for the parC mutant increased twofold. gyrA mutant Sp42-R1 was found to harbor a TCC to TAC mutation, resulting in a Ser-to-Tyr substitution in gyrA codon 81. The MXF MIC for this mutant increased twofold, while the SPX and garenoxacin MICs increased fourfold and the TVA, NOR, and CIP MICs were unaffected. Mutant C42-Sp6, which had mutations in parC and gyrA, showed stronger increases in resistance, with the MICs for the mutant being from 16- to 32-fold higher than those for wild-type strain P-4241. Garenoxacin was the quinolone with the lowest MIC for this mutant (1 μg/ml). With the exception of the SPX MICs, the MICs of all quinolones increased twofold for efflux mutant N42-R1, which was selected with NOR. The garenoxacin and TVA MICs for the clinical strain carrying a parE mutation (strain 2500) increased twofold, while the CIP MIC increased eightfold. The garenoxacin MIC for the clinical strain carrying triple mutations (strain 2759) increased 16-fold, while the CIP and TVA MICs increased 32- and 128-fold, respectively.

In vitro selection of S. pneumoniae strains resistant to garenoxacin

Strain P-4241 was grown on garenoxacin-containing plates at concentrations ranging from 0.5 to 2 times the MIC, as determined by the broth dilution method. Initial screening of the gyrA and parC QRDRs in five resistant colonies by HinfI digestion indicated that mutations had occurred in gyrA rather than parC. The gyrA QRDRs of these strains were thus sequenced. One strain, designated 175, had an E85K substitution, while the other four strains, exemplified by strain 176, harbored S81Y substitutions (Table 2). GyrA is therefore the primary target of garenoxacin. The garenoxacin MICs for the resistant strains increased two- to fourfold. Second-step mutants of strains 175 and 176 were then selected on garenoxacin-containing medium, as described above. Each second-step mutant that we analyzed had acquired a mutation at S79 in ParC, resulting in an eightfold increase in the garenoxacin MIC compared to the MIC for parent strain P-4241 (Table 2).

TABLE 2.

Analysis of mutants generated with garenoxacin in vitro

Selection step (parent strain)	Selection concn (μg/ml)	Strain	Mutation(s)		MIC (μg/ml)
			ParC	GyrA	GRNa	CIP	SPX
		P-4241	S79	S81 and E85	0.125	2	0.5
First step	0.1	175	S79	E85K	0.5	2	2
First step	0.1	176	S79	S81Y	0.5	2	2
Second step (175)	0.3	186	S79Y	E85K	1	16	16
Second step (175)	0.3	187	S79Y	E85K	1	16	16
Second step (176)	0.3	191	S79F	S81Y	1	32	16
Second step (176)	0.3	192	S79Y	S81Y	1	32	16

^aGRN, garenoxacin.

In vivo studies. (i) PK data

To establish the most appropriate antibiotic treatment schedule, we first determined the garenoxacin concentrations in sera and lung tissue. When immunocompetent mice were infected with virulent strain P-4241, the total drug concentrations in serum and lung tissue measured after administration of a single s.c. injection of 25 mg of garenoxacin per kg were similar to those in uninfected animals (Fig. 1). The AUC and t_1/2 values were also unaffected by infection status (Table 3). The TVA concentrations were higher in the sera and lung tissue of infected mice than in the controls, but the peak concentrations were similar. The t_1/2 and AUC values were two or three times higher than those in control animals (Fig. 2 and Table 3). The C_max and AUC values for both quinolones were much higher in lung tissue than in serum in both control and infected animals. Both quinolones were still detectable in infected serum and lung tissue from 12 to 24 h after injection. The PK and pharmacodynamic (PD) data for garenoxacin were similar to those for TVA, except that the AUC/MIC and t_1/2 values for TVA were higher than those for garenoxacin in infected lung tissues.

FIG. 1.

Concentration-time curves for serum and lungs of uninfected control and infected Swiss mice after a single s.c. injection of garenoxacin at 25 mg/kg.

TABLE 3.

PK-PD parameters in serum and lung tissues of immunocompetent control and infected mice after administration of a single s.c. dose of garenoxacin or TVA at 25 mg/kg

Group, drug, and sample	Cmax (μg/ml or μg/g)	Cmax/MIC ratio	δt MICa (h)	AUC0-24 (μg · h/ml)	AUC/MIC ratio	t1/2 (h)
Uninfected controls
Garenoxacin
Serum	8.0 ± 0.8b			25.5		7.5
Lung	17.7 ± 1.6			51.2		2.7
TVA
Serum	4.2c			19		2.8
Lung	24.2			155		4.3
Mice infected with strain P-4241d
Garenoxacin
Serum	7.5 ± 0.7	125	24	23.2	387	7.7
Lung	17.3 ± 2.0	288	12	48.5	808	3.1
TVA
Serum	5.7	45.6	>24	49	392	7.4
Lung	21.2	170	>24	250	2,000	11.6

^aδt MIC, time during which the total drug concentration exceeds the MIC for the test pathogen (P-4241).

^bValues for garenoxacin are means and standard deviations calculated for six serum and lung tissue samples taken 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdosing with garenoxacin.

^cValues for TVA are for six pooled samples of serum and lung tissue taken at the same times post dosing used for garenoxacin.

^dThe garenoxacin MIC for strain P-4241 was 0.06 μg/ml; the TVA MIC for the strain was 0.125 μg/ml.

FIG. 2.

Concentration-time curves for serum and lungs of uninfected control and infected Swiss mice after a single s.c. injection of TVA at 25 mg/kg.

(ii) Survival

All untreated control mice died within 4 to 6 days after challenge with isogenic strains P-4241, C42-R2, and Sp42-R1, whereas 92% of animals died when they were infected with efflux mutant N42-R1. Only 56% of untreated control animals treated with poorly virulent clinical strain 2759 (parC gyrA parE) died. Garenoxacin was then used to treat mice infected with virulent S. pneumoniae strain P-4241 and its fluoroquinolone-resistant derivatives generated in this study. Six injections of garenoxacin at 50 or 25 mg/kg yielded 100% survival of mice infected with wild-type strain P-4241, while 50% of mice were protected at a dose of 12.5 mg/kg (Fig. 3A). Garenoxacin at 50 and 25 mg/kg was also effective for mice infected with parC mutant C42-R2 (survival rates, 100 and 85%, respectively) and mice infected with gyrA mutant Sp42-R1 (survival rates, 93 and 55%, respectively) (Fig. 3B and C). All animals infected with efflux mutant N42-R1 (Fig. 3D) and parE mutant 2500 (Fig. 4A) survived when they were treated with garenoxacin at 25 and 50 mg/kg. Garenoxacin at 50 mg/kg was only slightly effective when animals were infected with mutant C42-Sp6 with double mutations or mutant 2759 with triple mutations (Fig. 4B and C). TVA was as efficacious as garenoxacin in mice infected with the wild-type strain and mutants with single mutations (gyrA, parC, and parE mutations and efflux mutations) (Fig. 5 and 6A). Garenoxacin and TVA were equally potent at doses of both 50 and 25 mg/kg. Animals infected with the mutant with gyrA and parC mutations died as quickly as the controls, despite the use of a high dose (200 mg/kg) of TVA (Fig. 6B).

FIG. 3.

Survival of garenoxacin-treated mice challenged with either a virulent penicillin-susceptible pneumococcal strain or mutant strains. (A) P-4241 (wild type); (B) C42-R2 (parC); (C) Sp42-R1 (gyrA); (D) N42-R1 (efflux mutant).

FIG. 4.

Survival of garenoxacin-treated mice challenged with strains harboring mutations. (A) 2500 (parE); (B) C42-Sp6 (gyrA parC); (C) 2759 (gyrA parC parE).

FIG. 5.

Survival of TVA-treated mice challenged with either a virulent penicillin-susceptible pneumococcal strain or mutant strains. (A) P-4241 (wild type); (B) C42-R2 (parC); (C) Sp42-R1 (gyrA); (D) N42-R1 (efflux mutant).

FIG. 6.

Survival of TVA-treated mice challenged with strains harboring mutations. (A) Strain 2500 (parE); (B) strain C42-Sp6 (gyrA parC).

(iii) Bacterial clearance

Bacterial growth in the lungs of mice infected with strain P-4241was observed from 16 h postinfection (4.4 log₁₀ CFU/ml) until death at 100 h postinfection (8 log₁₀ CFU/ml). Blood cultures were always positive for the untreated controls. The lungs and blood of mice infected with wild-type strain P-4241 were completely cleared after six injections of garenoxacin at 25 or 12.5 mg/kg (Table 4). In animals infected with the mutants with single isogenic mutations, the lungs and blood were completely cleared after six injections of garenoxacin at 25 mg/kg, while the lungs still contained 2.1 ± 1.1 log₁₀ CFU/ml 12 h after six treatments with a dose of 12.5 mg/kg. Bacterial clearance from the lungs and blood was also incomplete when animals infected with the mutants with double or triple mutations were treated with 50 mg of garenoxacin per kg: at 12 h after six treatments, the lungs contained 4.6 ± 0.6 log₁₀ CFU/ml and blood cultures were positive. Bacterial clearance correlated with survival in both garenoxacin- and TVA-treated animals.

TABLE 4.

Time course of bacterial clearance from lungs and blood of mice infected with strain P-4241 and treated with garenoxacin

Time postdosinga	Counts in animals treated with the following dosageb:
	Control	25 mg/kg	12.5 mg/kg
6 h after one Tt (24 h p.i.)	4.8 ± 0.8 (3/3)	3.4 ± 1.2 (3/3)	2.9 ± 1.6 (3/3)
12 h after two Tt (42 h p.i.)	4.5 ± 0.7 (3/3)	1.0 ± 0.2 (1/3)	1.9 ± 1.4 (3/3)
12 h after four Tt (66 h p.i.)	8.0c	LD (0/3)	1.3 ± 0.5 (1/3)
12 h after six Tt (90 h p.i.)		LD (0/3)	LD (0/2)

^aTt, treatment (the first treatment was given 18 h after bacterial challenge); p.i., postinfection.

^bValues are mean ± standard deviation (n = 3) log₁₀ CFU per milliliter of whole-lung homogenate. The values in parentheses are the number of animals with positive blood cultures/total number of animals. LD, less than the limit of detection (1 log₁₀ CFU/ml) in all three animals.

^cAll mice died.

(iv) In vivo emergence of resistance

Quinolone-resistant clones were isolated from 5 of 23 mice treated with garenoxacin (Table 5). Increased resistance was observed only when strains already carried a single target mutation in parC, gyrA, or parE. This concurred with our in vitro results, which showed that mutants of wild-type strain P-4241 with target mutations arose at a very low frequency (about 10⁻¹⁰). However, secondary mutations in strains initially carrying a single gyrA mutation were selected at frequencies of 4 × 10⁻⁹ to 1 × 10⁻⁷. Quinolone MICs for strain 182, which bore an initial parC mutation, were increased, although the gyrA, gyrB, and parE QRDRs were unchanged. Surprisingly, two strains (strains 184 and 185) lost their gyrA mutations, yet the MICs of all three quinolones tested for the two strains were increased relative to those for the parental strain and untreated strain Sp42-R1. The MICs for strains isolated from untreated animals showed no significant changes. Passage of quinolone-resistant strains in untreated control animals did not appear to select for the loss of quinolone resistance (encoded by the target or efflux phenotype), suggesting that single and even multiple target mutations do not affect the fitness of these strains.

TABLE 5.

Analysis of wild-type- and mutant-derived strains isolated from untreated controls and garenoxacin-treated mice

Strain	Treatment dose (mg/kg)	Code	MIC (μg/ml)			Mutationb
			GRNa	CIP	SPX
P-4241			0.06	2	0.5
	Control	C7	0.06	1	0.5
	12.5	B4	0.06	2	0.5
C42-R2			0.125	8	1	parC (S79Y)
	Control	F7	0.125	8	1	ND
	12.5	E10	0.125	4	1	ND
182c	12.5	E11	>1	16	>8	parC (S79Y)
	25	D5	0.125	8	1	ND
C42-Sp6			1	32	>8	gyrA (S81Y) + parC (S79Y)
	Control	K7	1	32	>8	ND
	50	J9	1	32	>8	ND
Sp42-R1			0.25	2	2	gyrA (S83Y)
	Control	18	0.25	2	2	ND
	25	H9	0.25	2	2	ND
183	25	H11	1	16	8	gyrA (S83Y), parC (wt)
184	50	G10	1	16	8	gyrA (wt), parC (S79Y)
185	50	G11	1	16	8	gyrA (wt), parC (S79Y)
N42-R1			0.125	4	0.5	Efflux
	Control	N4	0.125	4	0.5	Efflux
	12.5	M9	0.06	4	0.5	Efflux
	25	L8	0.06	4	0.5	Efflux
2500			0.125	16	0.5	parE (D435N + I460V)
	Control	G7	0.25	16	0.5	ND
	12.5	P6	1	32	8	ND
	25	O6	0.125	8	0.5	ND

^aGRN, garenoxacin.

^bND, not determined; wt, wild type.

^cBold face data are for quinolone-resistant clones isolated from mice treated with garenoxacin.

DISCUSSION

Garenoxacin displayed excellent in vitro activity against a virulent penicillin-susceptible wild-type strain of S. pneumoniae (P-4241). Its potency was twice those of TVA and MXF, 4 times that of SPX, and 32 times that of CIP. Our data are in agreement with those of Gales et al. (9), who obtained the following rank order of potency of the quinolones against 257 S. pneumoniae strains: garenoxacin > TVA > gatifloxacin (GAT) > levofloxacin (LVX) and CIP. Jones et al. (13) also found that garenoxacin was two and four times as potent as TVA and GAT, respectively, and showed (R. N. Jones et al., Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-58, p. 151, 2002) that the rank order of potency of the quinolones against 668 S. pneumoniae strains was as follows: gemifloxacin > garenoxacin > grepafloxacin, MXF, and TVA > LVX > CIP.

We found that garenoxacin was 8 times (gyrA mutant), 32 times (efflux mutant), and 64 times (parC mutant) more active than CIP against quinolone-resistant strains harboring a single mutation. It was also more active than TVA, MXF, SPX, and NOR. Reinert et al. (22) found that, among all the quinolones that they tested, garenoxacin and gemifloxacin had the best activities against parC or gyrA mutants. Lawrence et al. (14) found that garenoxacin had low MICs (0.03 to 0.125 μg/ml) for strains with parC or gyrA mutations and was more active than MXF, LVX, and CIP. Boswell et al. (5) showed that garenoxacin was the most active compound against strains with efflux-mediated fluoroquinolone resistance. We found that garenoxacin was also far more active than TVA (8- and 16-fold, respectively), CIP (32- and 64-fold, respectively), and NOR (>64-fold) against mutants with double and triple mutations. De Azavedo et al. (J. De Azavedo et al., Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-66, p. 153, 2002) also reported that garenoxacin was significantly more active than MXF, GAT, and LVX against S. pneumoniae isolates with double parC and double gyrA mutations (amino acid substitutions at S79 and D83 in ParC and at S81 and E85 in GyrA).

Our in vitro experiments show that garenoxacin can select for quinolone resistance, which, after the first exposure, exclusively involves the gyrA gene. SPX and MXF also preferentially select for gyrA mutations in S. pneumoniae. ParC mutants were selected only after the second exposure: each of the second-step mutants analyzed here acquired a mutation at S79 in parC. Our results are in keeping with those presented in a report from Hartman-Neumann et al. (10), who found that initial exposure to garenoxacin targeted the gyrA sequence, changing Ser81 to either Phe or Tyr. Second-level resistant mutants had an additional change in parC (Ser79 to Phe), followed during the next two steps by a second mutation in gyrA (Glu85 to Lys or Gly) or parC (Asp83 to Gly). The results of our in vitro studies show that target mutations occur at a very low frequency in the wild-type strain (1 × 10⁻¹⁰) and at higher frequencies (4 × 10⁻⁹ to 1 × 10⁻⁷) in strains already carrying a gyrA mutation. Clark et al. (7), using single- and multiple-step selections, found that garenoxacin tended to select for resistant clones at a lower rate than other quinolones (CIP, LVX, TVA, and MXF). Schmitz et al. (23) confirmed this low propensity of garenoxacin to induce resistance in vitro. Increased rates of resistance (0 to 20.5%) to the fluoroquinolones LVX and GAT has been reported among 3,328 CIP-resistant S. pneumoniae clones in Europe (M. Morosini et al., Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-107, p. 121, 2003), while the rate of MXF resistance reached 7.7% in 2002; conversely, all CIP-resistant strains remained susceptible to garenoxacin. Our in vivo results are also in keeping with the results of those in vitro studies, as resistant clones were observed in only 5 of 23 mice treated with garenoxacin and only among strains already carrying a single mutation.

In vivo, garenoxacin is as potent as TVA in terms of survival rates among mice infected with wild-type strains and resistant strains with single mutations and is slightly more effective than TVA against the mutants with double parC and gyrA mutations: 50 mg of garenoxacin per kg prolonged survival, whereas 200 mg of TVA per kg was ineffective. A comparison of the activity of garenoxacin with that of CIP, a well-characterized and widely distributed quinolone, showed that garenoxacin was far more effective. This was as expected, given the poor in vitro activity of CIP. The in vivo activity of garenoxacin is due to its better in vitro activity against wild-type and fluoroquinolone-resistant S. pneumoniae strains relative to that of CIP and its better activity against mutants with double and triple mutations compared to that of TVA. However, other factors, and particularly PK-PD parameters, are involved in the efficacies of quinolones in vivo. Forrest et al. (8) and Hyatt et al. (11) reported that the AUC/MIC ratio was the main parameter associated with bacterial eradication and clinical cure among patients with nosocomial pneumonia, with a minimal clinically effective ratio of 125. The favorable PK-PD parameters of garenoxacin thus contribute to its efficacy. Compared to CIP (3), garenoxacin has a longer half-life, larger AUCs, and superior in vitro activity, especially against S. pneumoniae; and garenoxacin yielded the highest AUC/MIC ratios in mouse serum and lung tissue samples. These PK and PD parameters are also very favorable for TVA, explaining why this quinolone is as effective as garenoxacin. Our pharmacokinetic data for garenoxacin closely match the mouse survival data, suggesting that serum protein binding has little influence on the therapeutic outcome, even though the level of serum protein binding reaches about 80% in mice (D. R. Andes and W. A. Craig, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-309, p. 10, 2003). This might be explained by the weak binding of garenoxacin to serum proteins. Moreover, inflammatory cells in lungs may serve as a reservoir, releasing garenoxacin in serum. TVA also shows high-level serum protein binding (24), while its efficacy is related to its good PK behavior. TVA was an interesting comparator in this mouse model of pneumococcal pneumonia, but it is clinically less relevant than garenoxacin because it has been withdrawn from the market.

In conclusion, garenoxacin is highly effective in a mouse model of pneumonia induced by both quinolone-susceptible and quinolone-resistant strains of S. pneumoniae. Garenoxacin could thus be a useful option for the empirical treatment of community-acquired respiratory tract infections.