In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus

Gregory T Robertson; Eric J Bonventre; Timothy B Doyle; Qun Du; Leonard Duncan; Timothy W Morris; Eric D Roche; Dalai Yan; A Simon Lynch

doi:10.1128/AAC.01649-07

. 2008 Apr 28;52(7):2313–2323. doi: 10.1128/AAC.01649-07

In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus^▿

Gregory T Robertson ¹, Eric J Bonventre ^1,^†, Timothy B Doyle ^1,^‡, Qun Du ¹, Leonard Duncan ^1,^‡, Timothy W Morris ^1,^§, Eric D Roche ^1,^¶, Dalai Yan ^1,^‖, A Simon Lynch ^1,^*

PMCID: PMC2443886 PMID: 18443108

Abstract

Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe mode-of-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.

Antibiotics of the rifamycin class exhibit potent activity against an array of gram-positive bacteria, including the mycobacteria, and have been used globally for the treatment of tuberculosis. Rifamycins, however, exert their antibacterial activity as inhibitors of a single enzyme target, DNA-dependent RNA polymerase, and a variety of single point mutations in the rpoB gene (which encodes the β subunit of the enzyme) give rise to strains that exhibit highly elevated MICs (7, 12). This resistance development liability limits the approved use of the rifamycin class of agents to combination regimens.

Rifamycins, alone or in combination, exhibit activity against susceptible bacteria propagated in the biofilm state, as determined from data from in vitro biofilm assays (3, 28, 32, 37, 41) and animal models of biofilm-associated infections (6, 19, 20, 37). In addition, clinical studies of rifampin in combination with fluoroquinolones (24, 36, 42, 43), vancomycin (21, 38), fusidic acid (38), and amoxicillin (38) have led to the adoption of specific rifampin-containing regimens as standard therapies for the treatment of biofilm-associated infections of indwelling medical devices (5).

The efficacy of the rifamycins in the treatment of biofilm-associated infections and other persistent or latent infections that are often recalcitrant to other antibiotics is likely explained by two distinct features. First, at the mechanistic level, the transcription process is thought to be essential for the establishment and maintenance of bacteria in alternate survival modes, including biofilms and metabolically quiescent states typical of latent cell populations. Second, at the physicochemical level, rifamycin agents exhibit excellent tissue distribution (1), efficiently penetrate biofilms formed in vitro (41), and exhibit good activity against a number of obligate or facultative intracellular pathogens.

In a program directed toward the synthesis of rifamycin derivatives with improved resistance development properties, a series of compounds was prepared in which rifamycin and quinolone pharmacophores were covalently combined. The design of these rifamycin-quinolone hybrids was such that they act as stable, dual-pharmacophore agents and therein are not active as prodrugs. This strategy should ensure that the pharmacokinetics, pharmacodynamics, and tissue distribution of the composite pharmacophores are matched. In CBR-2092, the rifamycin SV pharmacophore is combined with a quinolone pharmacophore derived from the 4H-4-oxo-quinolizine (or 2-pyridone) subfamily of fluoroquinolones that exhibit equipotent (balanced) activity against both DNA gyrase and DNA topoisomerase IV and retain activity against ciprofloxacin-resistant strains (26). In an accompanying article (30), we describe the results of microbiology studies used to characterize the in vitro profile of activity of CBR-2092 against staphylococci and streptococci. Here, we describe the results of biochemical, cell biology, and genetic studies undertaken to characterize the mode of action of CBR-2092 against a primary target pathogen, Staphylococcus aureus.

(Portions of this work were previously presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2007 [27a].)

MATERIALS AND METHODS

Antimicrobial agents.

CBR-2092 and ABT-719 (A-86719.0) were synthesized at Cumbre Pharmaceuticals Inc. Rifampin, ethidium bromide, and reserpine were purchased from Sigma-Aldrich (St. Louis, MO). Ciprofloxacin, levofloxacin, gatifloxacin, and nadifloxacin were purchased from LKT Laboratories (St. Paul, MN).

Bacterial strains.

Table 1 shows the relevant details for a series of derivatives of S. aureus ATCC 29213 (CB190) and RN4220 (CB1244) that exhibit stable resistance to agents of the rifamycin and/or quinolone classes that were isolated and characterized as described below.

TABLE 1.

Bacterial strains used in this study

Strain	Relevant genotype	Relevant characteristics	Source or reference
CB190	Wild type	ATCC 29213 parent strain for isogenic strain panel	ATCC
CB370	rpoB^H481Y	Spontaneous rifampin-resistant variant of CB190	This work
CB808	parC^S80F	Spontaneous ciprofloxacin-resistant variant of CB190	This work
CB809	rpoB^H481YparC^S80F	Spontaneous ciprofloxacin-resistant variant of CB370	This work
CB811	gyrA^S84L	Spontaneous nadifloxacin-resistant variant of CB190	This work
CB812	rpoB^H481YgyrA^S84L	Spontaneous nadifloxacin-resistant variant of CB370	This work
CB814	parC^S80FgyrA^S84L	Spontaneous higher-level ciprofloxacin-resistant variant of CB808	This work
CB815	rpoB^H481YparC^S80FgyrA^S84L	Spontaneous higher-level ciprofloxacin-resistant variant of CB809	This work
CB1244	Wild type	RN4220 parent strain: rsbU agr, restriction negative, methylation plus laboratory strain	I. Chopra
CB1522	rpoB^H481Y	Spontaneous rifampin-resistant variant of CB1244	This work

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Isolation and characterization of resistant mutants.

Single-step selections of antibiotic-resistant mutants were undertaken by standard agar-based methods. In all cases, mutants were purified through drug-free passage, and the initial antibiotic resistance phenotype was then verified to ensure that stable, true-breeding mutants had been obtained. Stepwise passage for multistep resistance selection was undertaken in glass tubes with cation-adjusted Mueller-Hinton (MHII) medium with or without 0.002% (vol/vol) polysorbate 80 (P-80) and were inoculated with 10⁶ CFU/ml in the presence of sub-MICs of the test agents (see footnotes a of Tables 6 and 7 for details). Cultures were incubated with shaking at 37°C for 20 to 24 h. Thereafter, the cell inoculum was prepared from the highest consecutive drug concentration which had supported growth to an absorbance equivalent to or greater than ≈10⁸ CFU per ml. Daily passages were performed until compound solubility issues limited further elaboration of individual steps election studies or it was apparent that a terminal resistance endpoint had been attained in specific selections. Genotypic analysis of strains exhibiting stable resistance phenotypes was undertaken by standard methods with amplification and DNA sequencing of the target loci. Assessment of the contribution of mutations in efflux pathways toward altered antibiotic resistance phenotypes was undertaken by determining altered sensitivity to the unrelated efflux substrate ethidium bromide and/or whether quinolone susceptibility was affected by reserpine.

TABLE 6.

Characterization of mutants from step selections undertaken with wild-type strains

Strain	Selection agent^a	Day^b	Mutation(s) identified^g	MIC (μg/ml)					Doubling time (min)^c
				CBR-2092		Rifampin	Cipro^d	EtBr^e
				Broth	Agar	Rifampin	Cipro^d	EtBr^e
CB190	CBR-2092	NA^f	Wild type	0.015	0.008	0.008	0.25	4	38
CB1880	CBR-2092	2	rpoB^R484H	0.12	0.03	>250	0.25	4	43
CB1881	CBR-2092	5	rpoB^R484HgyrA^ΔL520	0.5	0.25	>250	0.5	2	41
CB1882	CBR-2092	10	rpoB^R484HgyrA^ΔL520parC^R236†	1	0.5	>250	1	2	45
CB1883	CBR-2092	15	rpoB^R484HgyrA^{ΔL520, S84L}parC^R236†	8	1	>250	1	2	47
CB1884	CBR-2092	26	rpoB^R484HgyrA^{ΔL520, S84L}parC^R236,†^H103Y	64	4	>250	8	2	65
CB1871	Rifampin	2	rpoB^H481Y	0.12	ND^h	>250	0.25	8	ND
CB1875	Ciprofloxacin	15	gyrA^S84LparC^R298K, ^LNVIKEE461*	0.008	ND	0.016	>128	64	ND
CB1891	ABT-719	15	gyrA^S84LparC^E84KparE^{P587S, F638V}	0.03	ND	0.008	>64	64	ND

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Step selections were undertaken in MHII broth medium without P-80 supplementation.

The day on which the indicated mutant strain was purified.

The time necessary for the optical density at 600 nm of a logarithmic culture propagated at 37°C in MHII broth medium to double.

Cipro, ciprofloxacin.

EtBr, ethidium bromide.

NA, not applicable.

†, duplication; *, insertion.

NA, not applicable.

TABLE 7.

Characterization of mutants from step selections undertaken with strains exhibiting resistance to rifamycin and/or quinolone agents

Strain	Selection^a	Relevant genotype^d	MIC (μg/ml)
			CBR-2092		Rifampin	Cipro^b	EtBr^c
			Broth	Agar	Rifampin	Cipro^b	EtBr^c
CB1884	Parent	rpoB^R484HgyrA^{ΔL520, S84L}parC^R236,†^H103Y	64	4	>250	8	2
CB1887	CB1884 plus 20 days selection with ABT-719	rpoB^R484HgyrA^{ΔL520, S84L, E88K}parC^R236,†^{H103Y, S80Y, E84G}	>250	>8	>250	64	ND
CB370	Parent	rpoB^H481Y	0.12	0.03	>250	0.25	4
CB1947	CB370 plus 20 days selection with CBR-2092	rpoB^H481YgyrA^{S84L, ΔE697}parC^G167V	4	1	>250	2	4
CB814	Parent	rpoB^WTgyrA^S84LparC^S80F	0.015	0.06	0.008	16	4
CB1952	CB814 plus 21 days selection with CBR-2092	rpoB^S486LgyrA^{S84L, E88Q, G532S}parC^{S80F, E84L}	31	4	>250	64	4
CB815	Parent	rpoB^H481YgyrA^S84LparC^S80F	1	0.25	>125	16	4
CB1953	CB815 plus 7 days selection with CBR-2092	rpoB^H481YgyrA^{S84L, V598I}parC^{S80F, R570H}	16	2	>125	64	4
CB1954	CB815 plus 7 days selection with CBR-2092	rpoB^H481YgyrA^S84L, ^ΔK809parC^{S80F, A523D}	16	2	>125	64	4

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Step selections were undertaken in MHII broth medium with supplementation (CBR-2092) or without supplementation (ABT-719) with 0.002% (vol/vol) P-80.

Cipro, ciprofloxacin.

EtBr, ethidium bromide.

†, duplication.

Biochemical studies.

Recombinant forms of the S. aureus σ^A RNA polymerase holoenzyme and a rifampin-resistant RpoB (H481Y) variant were prepared as described previously (27). Inhibition of transcription of S. aureus σ^A RNA polymerase holoenzymes was measured in single-round G-less cassette assays that employed the pGL2B-T7A1-G-less template (27). Quantitative gel electrophoresis of a single 272-nucleotide G-less RNA product species was used to determine the minimal concentration necessary to inhibit 50% (IC₅₀) of the holoenzyme-specific RNA product formed in the absence of test agents. Derivatives of the pET28b vector (Novagen Inc.) were constructed to express recombinant forms of the S. aureus GyrA or GyrA^S84L and ParC or ParC^S80F subunits bearing amino-terminal six-histidine and T7 tag oligonucleotide epitopes and carboxyl-terminal FLAG epitope tags. The previously described vectors pTrcHisB-SA-GyrB and pTrcHisA-SA-GrlB (18) were used for expression of recombinant variants of the S. aureus GyrB and ParE (GrlB) proteins, respectively, bearing amino-terminal six-histidine oligonucleotide affinity tags. In all cases, the recombinant subunits were purified from Escherichia coli Rosetta cells (Novagen Inc.) by immobilized metal affinity chromatography (IMAC). The peak fractions that eluted from the IMAC resin via an imidazole gradient were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pooled; and further purified by combination of size-exclusion chromatography, affinity chromatography, and/or dialysis. The DNA topoisomerase holoenzymes were then reconstituted through combination of the purified subunits at ratios optimized for specific activity to yield either wild-type or quinolone-resistant forms of DNA gyrase and DNA topoisomerase IV. For the studies described here, DNA gyrase preparations exhibited specific activity in the range of 200 U per μg (for GyrA^S84L and GyrB) to 250 U per μg (for GyrA and GyrB), in which 1 U is equivalent to the minimal amount of enzyme necessary to effect the supercoiling of 150 ng of relaxed pBR322 DNA in 1 h at 37°C. Wild-type (ParC and ParE) and mutant (ParC^S80F and ParE) preparations of DNA topoisomerase IV enzymes exhibited specific activities of 25 to 30 U per μg (in which 1 U is equivalent to the minimal amount of enzyme necessary to effect the relaxation of 150 ng of supercoiled pUC19 DNA in 1 h at 37°C). Assays for measurement of the inhibition of the in vitro activity of type II topoisomerases employed the relaxed (DNA gyrase) or negatively supercoiled (DNA topoisomerase IV) forms of covalently closed DNAs as substrates. Assays of DNA gyrase were undertaken in 50 mM Tris-HCl (pH 7.5), 50 mM potassium glutamate, 5 mM MgCl₂, 5 mM dithiothreitol, 1 mg/ml acetylated bovine serum albumin, 1.5 mM ATP, 15 μg/ml relaxed pBR322 DNA, and 100 μg/ml E. coli tRNA for 1 h at 37°C. Assays of DNA topoisomerase IV were undertaken in 50 mM Tris-HCl (pH 7.5), 50 mM potassium glutamate, 5 mM MgCl₂, 5 mM dithiothreitol, 1 mg/ml acetylated bovine serum albumin, 1.5 mM ATP, 15 μg/ml supercoiled pUC19 DNA, and 100 μg/ml tRNA for 1 h at 37°C. In all cases, the reactions were terminated by the addition of sodium dodecyl sulfate and proteinase K. Quantitative gel electrophoresis of the linear DNA products was employed to determine the minimal concentration of inhibitor necessary to induce 50% cleavage over that for the background of a covalently closed DNA substrate (CC₅₀), with mean values calculated from two or more independent assays.

Macromolecular biosynthesis assays.

The effects of the test agents on macromolecular biosynthesis in S. aureus were determined by using assays in which the incorporation of specific radiolabeled precursors was measured in a time-course fashion. The radiolabels employed were [methyl-³H]thymidine (NET-027; Perkin-Elmer) for DNA synthesis, [5,6-³H]uridine (NET-367; Perkin-Elmer) for RNA synthesis, l-[3,4,5-³H]leucine (NET-460; Perkin-Elmer) for protein synthesis, and [2,3-³H]d-alanine (ART-179; American Radiolabeled Chemicals) for cell wall synthesis. At each time point, the ratio of the amount of radiolabel incorporated in the presence of the test agent relative to the amount of radiolabel incorporated by the dimethyl sulfoxide-treated control culture was used to determine the percent incorporation. The amount of radiolabel incorporated at time zero was set equal to 100%.

Antimicrobial susceptibility testing.

Determination of MICs was done in accordance with the Clinical and Laboratory Standards Institute (CLSI) methodology by either the broth microdilution or the agar dilution method with MHII as the base medium (10). Unless otherwise indicated, broth microdilution assays employed MHII medium supplemented with 0.002% (vol/vol) P-80.

RESULTS

Structures of CBR-2092 and related agents.

In a program directed toward the synthesis and evaluation of rifamycin-based hybrid antibiotics, a series of compounds was prepared in which rifamycin and a quinolone pharmacophore were covalently joined. In total, approximately 300 rifamycin-quinolone hybrids were synthesized in an effort that entailed the combination of different rifamycin backbone scaffolds with quinolones representative of various subseries, including experimental quinolone pharmacophores. Structure-activity relationships derived from the data generated in a range of assays described here revealed that the potency of the quinolone entity was a critical parameter in terms of the retention of antimicrobial activity of rifamycin-quinolone hybrid agents against strains exhibiting rifampin resistance. Of the quinolone pharmacophores tested, members of the experimental 4H-4-oxoquinolizine subfamily (25) proved one of the most promising. In CBR-2092 (Fig. 1), the rifamycin SV pharmacophore is combined via a chiral linking group with a quinolone entity from the 4H-4-oxoquinolizine subfamily (25); for comparison, Fig. 1 also shows the structures of rifampin, ciprofloxacin, and a representative 4H-4-oxoquinolizine (ABT-719) that were employed in this study as comparator agents. ABT-719 was studied as a comparator, as it is the most extensively characterized member of the 4H-4-oxoquinolizine series and exhibits equipotent (balanced) biochemical activity against the DNA gyrase and DNA topoisomerase IV enzymes and potent antimicrobial activity against both gram-positive and gram-negative pathogens, including ciprofloxacin-resistant isolates of S. aureus, and is efficacious in rodent infection models (26).

FIG. 1. — Chemical structures of CBR-2092 and related molecules.

Biochemical studies of CBR-2092 and comparator agents.

As shown in Table 2, CBR-2092 retained rifampin-like potency against wild-type S. aureus RNA polymerase with an IC₅₀ of 0.034 μM, which makes it approximately twofold less active than rifampin (IC₅₀, 0.015 μM). In contrast, neither rifampin nor CBR-2092 exhibited detectable activity (IC₅₀s, >25 μM) against a mutant form of the RNA polymerase enzyme bearing a high-level rifampin resistance-conferring RpoB^H481Y subunit. These combined data suggest that appendage of the 4H-4-oxoquinolizine pharmacophore via the 3′ position of the rifamycin SV scaffold does not significantly affect the ability of CBR-2092 to interact with RNA polymerase in a manner similar to that of rifampin.

TABLE 2.

Effects of CBR-2092 and comparator agents on the in vitro activity of target enzymes from S. aureus

Test agent	IC₅₀ (μM) forσ^A RNA polymerase		CC₅₀ (μM) for:				CC₅₀ ratio for type II DNA topoisomerases
	Rif^s (RpoB^WT)^a	Rif^r (RpoB^H481Y)	DNA topoisomerase IV		DNA gyrase		GyrA^WT/ParC^WT	ParC^S80F/ParC^WT
	Rif^s (RpoB^WT)^a	Rif^r (RpoB^H481Y)	ParC^WT	ParC^S80F	GyrA^WT	GyrA^S84LParC^WT	GyrA^WT/ParC^WT	ParC^S80F/ParC^WT
CBR-2092	0.034	>25	1.7	2.7	1.5	>150	0.9	1.6
Rifampin	0.015	>25	NR^b	NR	NR	NR	NR	NR
Ciprofloxacin	NR	NR	0.4	31	4.6	>150	11.5	77.5
Gatifloxacin	NR	NR	0.22	13	0.7	18	3.2	59.1
ABT-719	NR	NR	0.06	1.1	0.09	0.9	1.5	18.3

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WT, wild type.

NR, not relevant.

As also shown in Table 2, biochemical studies indicated that CBR-2092 exhibits equipotent (balanced) activity against wild-type S. aureus DNA topoisomerase IV and DNA gyrase enzymes, with CC₅₀s of 1.7 and 1.5 μM, respectively, which are in the same potency range as ciprofloxacin and gatifloxacin and which correspond to a ratio of the CC₅₀ of DNA gyrase/CC₅₀ of DNA topoisomerase IV of 0.9. The apparent target balance of CBR-2092 for wild-type topoisomerase IV and DNA gyrase is improved over the target balances of ciprofloxacin (CC₅₀ ratio, 11.5) and gatifloxacin (CC₅₀ ratio, 3.2) and is similar to that of ABT-719, for which we observed a CC₅₀ ratio of 1.5 and for which a CC₅₀ ratio of 2.1 has been reported previously (33). However, in contrast to all of the other fluoroquinolone comparators tested, CBR-2092 maintains its activity against a prevalent fluoroquinolone-resistant variant of DNA topoisomerase IV, with CC₅₀s of 2.7 and 1.7 μM determined for the ParC^S80F and wild-type variants, respectively. These data yield a CC₅₀ ratio for CBR-2092 for DNA topoisomerase IV of ParC^S80F/DNA topoisomerase IV of the ParC wild type of 1.6 that is improved over the ratios for ciprofloxacin (CC₅₀ ratio, 77.5), gatifloxacin (CC₅₀ ratio, 59.1), and ABT-719 (CC₅₀ ratio, 18.3). Retention of the activity of CBR-2092 against the ParC^S80F variant of DNA topoisomerase IV was considered a key attribute of the quinolone-driven activity of the molecule, as this mutation most commonly underlies target-mediated resistance in ciprofloxacin-resistant isolates of S. aureus.

Activity of CBR-2092 against S. aureus strains exhibiting rifampin and/or quinolone resistance.

Table 3 shows the activities of CBR-2092 and comparator agents against an otherwise isogenic set of derivatives of S. aureus CB190 (ATCC 29213) that bear all possible combinations of a high-level rifampin resistance mutation rpoB^H481Y and the prevalent quinolone resistance mutations parC^S80F and gyrA^S84L. For CBR-2092, MIC endpoints are shown here and elsewhere from assays conducted in both the agar dilution and the broth microdilution formats, with the latter conducted with MHII medium supplemented with 0.002% (vol/vol) P-80. As observed with a number of other semisynthetic antibiotics, CBR-2092 exhibits an apparent high avidity for plastic and glass materials. In the broth microdilution format, P-80 minimizes the surface loss of CBR-2092 to the plastic surface of the assay plates (G. T. Robertson, Q. Du, and A. S. Lynch, unpublished data) and improves the concordance of MIC endpoints with those determined by the agar dilution method (in which it is assumed that such compound loss is minimized by slower diffusion through the agar matrix).

TABLE 3.

Antibacterial activities of CBR-2092 against isogenic Staphylococcus aureus strains bearing combinations of rifamycin or quinolone resistance mutations

Test agent (condition)^a	MIC (μg/ml)
Test agent (condition)^a	CB190 wild type	CB811 gyrA^S84L	CB808 parC^S80F	CB814 gyrA^S84LparC^S80F	CB370 rpoB^H481Y	CB812 rpoB^H481YgyrA^S84L	CB809 rpoB^H481YparC^S80F	CB815 rpoB^H481YgyrA^S84LparC^S80F
CBR-2092 (agar)	0.008	0.008	0.008	0.008	0.06	0.12	0.06	0.25
CBR-2092	0.015	0.015	0.015	0.015	0.12	0.50	0.25	2
Rifampin	0.008	0.008	0.008	0.008	>250	>250	>250	>250
Ciprofloxacin	0.25	0.25	2	16	0.25	0.25	2	16
Levofloxacin	0.25	0.25	1	8	0.25	0.25	1	8
Gatifloxacin	0.06	0.12	0.25	8	0.06	0.12	0.25	8
ABT-719	0.015	0.03	0.03	0.50	0.015	0.03	0.03	0.25

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MICs were determined by the broth microdilution method in the presence of 0.002% (vol/vol) P-80 unless otherwise indicated.

As anticipated from past studies, wild-type S. aureus strain CB190 (ATCC 29213) and its rifamycin-resistant derivative CB370 (rpoB^H481Y) were observed to exhibit marked differences in their susceptibilities to rifampin, with the MIC for CB370 (>250 μg/ml) being >31,250 times higher than that determined for CB190 (0.008 μg/ml). In contrast, the difference in MICs observed for these two strains with CBR-2092 was markedly smaller, with the broth microdilution MIC for CB370 (0.12 μg/ml) being only some eight times higher than that determined for CB190 (ATCC 29213) (0.015 μg/ml). Similarly, as anticipated from past studies, wild-type S. aureus strain CB190 (ATCC 29213) and its fluoroquinolone-resistant derivative CB814 (gyrA^S84L parC^S80F) were observed to exhibit marked differences in their susceptibilities to fluoroquinolone agents. For instance, the ciprofloxacin MIC for CB814 (16 μg/ml) was observed to be ∼67 times higher than that determined for CB190 (ATCC 29213) (0.25 μg/ml). In contrast, no difference in the potency of CBR-2092 against these two strains was observed, with broth microdilution MICs of 0.015 μg/ml observed for both CB814 and CB190. These combined data indicate that the primary antimicrobial activity of CBR-2092 is conferred by its rifamycin pharmacophore.

These data are also informative about the nature of the fluoroquinolone activities of the agents tested and, in particular, in delineating the relative contributions of cellular inhibition of each of the DNA topoisomerase type II targets. Ciprofloxacin showed the anticipated pattern of activity against this genetically defined strain panel, with eightfold differences in MICs observed between otherwise isogenic strains bearing either the gyrA^S84L or the parC^S80F resistance determinant. Analysis of the MICs determined for CBR-2092 against the four rifamycin-resistant strains in the panel suggest that CBR-2092 is a relatively well balanced inhibitor of both DNA topoisomerase type II targets in S. aureus, with a possible slight preference for DNA gyrase. The mechanistic basis underlying the antimicrobial activity that CBR-2092 exhibits against strain CB815 (rpoB^H481Y gyrA^S84L parC^S80F) cannot be directly determined from these data. However, as CBR-2092 exhibits in vitro activity nearly equivalent to that of an inhibitor of both wild-type and fluoroquinolone-resistant forms of S. aureus DNA topoisomerase IV (Table 2), it seems reasonable to assume that the activity of CBR-2092 against strain CB815 is mediated via residual effects on DNA topoisomerase IV.

Effects of CBR-2092 and comparator agents on macromolecular biosynthesis.

The effects of rifampin, ciprofloxacin, and CBR-2092 on the four macromolecular biosynthesis pathways studied in CB190 (ATCC 29213) are shown in Fig. 2. As anticipated, rifampin had a primary effect on de novo RNA synthesis, a delayed, secondary effect on protein synthesis, and tertiary and less significant overall effects on cell wall and DNA synthesis. Similarly, consistent with past studies, ciprofloxacin had a primary effect on de novo DNA synthesis but minimal effects on the other pathways studied. In contrast, CBR-2092 exhibited intermediary effects that were somewhat distinct from those of either rifampin or ciprofloxacin. Like rifampin, CBR-2092 had a primary effect on de novo RNA synthesis and delayed, secondary effects on protein and cell wall synthesis. However, CBR-2092 also appeared to have a secondary effect on DNA synthesis that was more pronounced than the effect observed with rifampin and that likely reflects the secondary mode of action conferred by the quinolone pharmacophore.

FIG. 2. — Effects of CBR-2092 and comparator agents on de novo macromolecular biosynthesis in *S. aureus*. The radiolabels employed were [*methyl*-³H]thymidine (closed circles) for DNA synthesis, [5,6-³H]uridine (open circles) for RNA synthesis, l-[3,4,5-³H]leucine (closed triangles, dashed line) for protein synthesis, and [2,3-³H]d-alanine (open triangles, dashed line) for cell wall synthesis. At each time point, the ratio of radiolabel incorporated in the presence of the test agent relative to that incorporated in the presence of the dimethyl sulfoxide (DMSO)-treated control culture was used to determine the percent incorporation. The amount of radiolabel incorporated at time zero was set equal to 100%.

Figure 2 also shows data from equivalent studies undertaken with rifampin-resistant strain CB370 (rpoB^H481Y). As expected, rifampin had no significant effects at the concentration tested (equivalent to 4× MIC for CB190 [ATCC 29213]) on any of the four macromolecular biosynthesis pathways studied, while ciprofloxacin continued to exhibit a primary effect on de novo DNA synthesis. In contrast, CBR-2092 appeared to have a primary effect on de novo DNA synthesis but minimal effects on the other pathways studied. Also shown in Fig. 2 are the effects of the same compounds on macromolecular biosynthesis in quinolone-resistant strain CB814 (gyrA^S84L parC^S80F). As expected, ciprofloxacin had no apparent effects on de novo DNA synthesis or other pathways, while rifampin exhibited an activity profile that was essentially similar to that observed with strain CB190 (ATCC 29213). CBR-2092 also exhibited effects on the four macromolecular pathways in CB814 that were similar to those observed in strain CB190 (ATCC 29213), with a primary effect on RNA synthesis and a somewhat diminished effect on DNA synthesis that more closely resembled that of rifampin. Finally, at the concentrations tested here, neither rifampin, CBR-2092, nor ciprofloxacin had discernible effects on the macromolecular pathways studied when they were tested with strain CB815 (rpoB^H481Y gyrA^S84L parC^S80F) (data not shown).

Single-step resistance studies with CBR-2092.

To further characterize the mode of action of CBR-2092 in S. aureus, we undertook a series of single-step genetic selections in which spontaneous mutants that exhibit reduced susceptibility to CBR-2092 were isolated and characterized. As summarized in Table 4, a series of agar-based selections undertaken with rifampin-sensitive strain CB190 (ATCC 29213) and CBR-2092 over the concentration range of 0.08 to 0.12 μg/ml (five- to eightfold the MIC) gave rise to 28 independently isolated “first-step” mutants that exhibited elevated MICs for CBR-2092 and rifampin in the ranges of 0.12 to 0.5 μg/ml and 0.25 to > 64 μg/ml, respectively, but that exhibited no apparent changes in their susceptibilities to ciprofloxacin. The recovery of such first-step resistance mutations at the indicated concentrations, but not at higher drug concentrations, is wholly consistent with the anticipated low spontaneous resistance potential of CBR-2092 at drug concentrations above the mutant prevention concentration at which the secondary antimicrobial activity of CBR-2092 exerts its effects; see the accompanying article (30). Genotypic characterization of these strains revealed a series of mutations corresponding to amino acid substitution or insertion mutations that span residues Gln468 to His481 in the RpoB subunit of RNA polymerase and therein overlap with cluster I of the previously characterized rifampin resistance-determining region of S. aureus and other pathogens. These genetic data are again wholly consistent with the notion that the primary antimicrobial activity of CBR-2092 in wild-type S. aureus is mediated by its rifamycin pharmacophore.

TABLE 4.

Characterization of CBR-2092 single-step mutants derived from a rifampin-sensitive strain

Mutation identified in rpoB	No. of independent isolations	Broth microdilution MIC (μg/ml)
Mutation identified in rpoB	No. of independent isolations	Rifampin	CBR-2092	Ciprofloxacin
Wild-type parent	NR^a	0.008	0.015	0.25
H481Y	12	>64	0.12	0.25
Q468L	1	>64	0.25	0.25
Q468K	1	>64	0.50	0.25
H481D	1	>64	0.25	0.25
H472 (insertion)	1	32	0.12	0.25
D471Y	2	16	0.25	0.50
E473 (insertion)	1	8	0.25	0.25
H481N	1	2	0.25	0.25
N474K	2	2	0.25	0.25
A477V	2	1	0.12	0.25
D471G	4	0.25	0.25	0.25

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NR, not relevant.

As shown in Table 5, a series of agar-based selections undertaken with rifampin-resistant strains CB370 (rpoB^H481Y) or CB1522 (rpoB^H481Y) and CBR-2092 over a concentration range of from 0.1 to 0.25 μg/ml (one- to twofold MIC) gave rise to a series of nine independently isolated mutants that exhibited elevated MICs for CBR-2092 in the range of 0.5 to 2 μg/ml. Genotypic characterization of these strains revealed a series of mutations corresponding to amino acid substitutions in the gyrA gene that, with one exception (gyrA^S84L), lie distal to the canonical quinolone resistance-determining region (QRDR) of the S. aureus gyrA gene. Further characterization of these strains revealed minimal (one- to twofold) shifts in MICs for a variety of fluoroquinolone agents (data not shown). In contrast, selections undertaken with CB370 (rpoB^H481Y) and ABT-719 (0.05 μg/ml) or gatifloxacin (0.2 μg/ml) gave rise to a series of independently isolated mutants that exhibited four- to eightfold shifts in ciprofloxacin MICs, and all were found to possess previously described QRDR mutations in either the parC or the parE locus (parC and parE encode the subunits of DNA topoisomerase IV) (data not shown). These combined data are consistent with the notion that, in contrast to gatifloxacin and ABT-719, the preferred DNA topoisomerase target of CBR-2092 in S. aureus is DNA gyrase.

TABLE 5.

Characterization of CBR-2092 single-step mutants derived from rifampin-resistant strains

Strain	Parent strain	Parent strain genotype	Selection concn (μg/ml)	Ending strain genotype	CBR-2092 broth microdilution MIC (μg/ml)
CB1522	CB1244	rpoB^H481Y	NA^a	NA	0.25
CB1523	CB1522	rpoB^H481Y	0.25	rpoB^H481YgyrA^A26V	1
CB1524	CB1522	rpoB^H481Y	0.25	rpoB^H481YgyrA^G773V	2
CB1416	CB370	rpoB^H481Y	0.1	rpoB^H481YgyrA^L795S	0.5
CB1417	CB370	rpoB^H481Y	0.1	rpoB^H481YgyrA^G532V	0.5
CB1418	CB370	rpoB^H481Y	0.1	rpoB^H481YgyrA^D705N	0.5
CB1419	CB370	rpoB^H481Y	0.1	rpoB^H481YgyrA^G572D	1
CB1420	CB370	rpoB^H481Y	0.1	rpoB^H481YgyrA^G584V	0.5
CB1326	CB370	rpoB^H481Y	0.2	rpoB^H481YgyrA^S784F	0.5
CB1327	CB370	rpoB^H481Y	0.2	rpoB^H481Y gyrA^S84L	0.5
CB1421	CB809	rpoB^H481YparC^S80F	0.18	rpoB^H481YparC^S80FgyrA^S84L	1
CB1430	CB812	rpoB^H481YgyrA^S84L	0.18	rpoB^H481YparE^E474KgyrA^S84L	16
CB1333	CB815	rpoB^H481YparC^S80FgyrA^S84L	0.8	rpoB^H481YparC^S80FparE^L517FgyrA^S84L	16
CB1334	CB815	rpoB^H481YparC^S80FgyrA^S84L	0.8	rpoB^H481YparC^{S80F, Q27H}gyrA^S84L	8
CB1335	CB815	rpoB^H481YparC^S80FgyrA^S84L	0.8	rpoB^H481YparC^S80FparE^E474QgyrA^S84L	>16
CB1353	CB815	rpoB^H481YparC^S80FgyrA^S84L	0.53	rpoB^H481YparC^S80FparE^V458GgyrA^S84L	16
CB1354	CB815	rpoB^H481YparC^S80FgyrA^S84L	0.53	rpoB^H481YparC^S80FparE^R455HgyrA^S84L	16

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NA, not applicable.

As is also shown in Table 5, a series of selections undertaken with strains CB809 (rpoB^H481Y parC^S80F), CB812 (rpoB^H481Y gyrA^S84L), and CB815 (rpoB^H481Y gyrA^S84L parC^S80F) and CBR-2092 over the concentration range of 0.2 to 0.8 μg/ml (one- to eightfold the MIC) gave rise to a series of independently isolated mutants that exhibited elevated MICs for CBR-2092 in the range of 1 to >16 μg/ml. Genotypic characterization of these strains revealed a series of mutations corresponding to amino acid substitutions in parE, parC, and gyrA, including both QRDR mutations described above and a further series of novel mutations. Importantly, when strains that possess both the rpoB^H481Y and the gyrA^S84L mutations were employed, CBR-2092 selections resulted exclusively in mutations in the ParC or the ParE subunit of DNA topoisomerase IV, including mutations outside of the previously described QRDR for DNA topoisomerase IV in S. aureus. These data further substantiate DNA topoisomerase IV as the secondary DNA topoisomerase target of CBR-2092 in S. aureus.

Finally, to determine the potential contribution of the efflux class of mutations in CBR-2092-selected mutants, the MICs for the efflux substrate ciprofloxacin were determined in the presence and the absence of the efflux pump inhibitor reserpine. In no case was a significant (more than twofold) shift in the ciprofloxacin MIC observed in the presence of reserpine (data not shown). Likewise, no significant shifts in MICs were apparent for any CBR-2092-selected mutants when they were tested against minocycline, a structurally unrelated antibiotic that is subject to additional efflux pathways in S. aureus (data not shown). These data suggest that efflux mechanisms do not contribute to the mutational mechanisms associated with first- or second-step resistance to CBR-2092 in S. aureus.

Multistep passage selections undertaken with CBR-2092 and comparator agents with CB190 (ATCC 29213).

To delineate the genetic mechanism(s) by which a wild-type, drug-naïve strain of S. aureus (CB190 [ATCC 29213]) may develop higher-level resistance to CBR-2092, we also undertook studies involving the stepwise enrichment of mutants with reduced susceptibility through serial passage in MHII broth medium starting from a sub-MIC range. Parallel studies were also undertaken with rifampin, ciprofloxacin, and ABT-719. Table 6 shows the characterization of intermediary and/or terminal isolates from each study.

Consistent with past precedents described in the literature, step selections undertaken with the comparator agents over the course of 2 to 15 days resulted in the isolation of strains that exhibited resistance to benchmark agents of the parental classes, with MICs elevated >31,250-fold for rifampin (2 days), >500-fold for ciprofloxacin (15 days), and >250-fold for ABT-719 (15 days) (data not shown) relative to those for parental strain CB190 (ATCC 29213). Furthermore, in the selections with the fluoroquinolone agents, it is apparent that efflux mechanisms contributed to the development of stable resistance to the test agents, as ethidium bromide MICs were elevated 16-fold (Table 6).

Step selections undertaken with CBR-2092 over the course of 26 days of serial, stepwise passage enrichment resulted in the isolation of a strain (CB1884) that exhibited resistance elevated ∼4,000-fold and 500-fold over that for the parent strain (CB190 [ATCC 29213]), as determined by the broth microdilution and agar dilution MIC assays, respectively. Table 6 includes a summary of the phenotypic and genotypic characteristics of stable, true-breeding mutants derived from days 2, 5, 10, 15, and 26 of the CBR-2092 step selection study.

On day 2 of the CBR-2092 step selection study, a mutant derivative (CB1880) that had highly elevated resistance to rifampin was isolated, and genotypic analysis revealed an rpoB^R484H mutation in the canonical rifampin resistance-determining region of rpoB. A secondary mutational event was apparent on day 5, as the MICs for CBR-2092 were further elevated four- to eightfold and genotypic analysis of a purified day 5 isolate (CB1881) revealed a deletion mutation in the gyrA gene (gyrA^Δ^L520) corresponding to the leucine residue at position 520 of GyrA. Interestingly, this residue lies outside of the known QRDR of gyrA and is not associated with a significant change in the MIC for ciprofloxacin (Table 6).

A tertiary mutational event was apparent on day 10 of the CBR-2092 step selection study, with a further twofold shift in the MICs determined for CBR-2092. Genotypic analysis of a purified day 10 isolate (CB1882) revealed a duplication mutation in the parC gene (parC^R236 duplication) corresponding to a tandem duplication of the arginine residue at position 236 of the ParC subunit of DNA topoisomerase IV. Similarly, this residue also lies outside of the canonical QRDR of parC and was not associated with a significant change in the MIC for ciprofloxacin. A further mutational event was apparent on day 15, with CBR-2092 MICs elevated a further two- to eightfold. Genotypic analysis of a purified day 15 isolate (CB1883) revealed a substitution mutation in the gyrA gene (gyrA^S84L) that corresponded to a serine-to-leucine substitution at residue 84 of the GyrA subunit and to a prevalent QRDR mutation in fluoroquinolone-resistant S. aureus isolates. A final mutational event was apparent on day 26, as the CBR-2092 MICs were elevated a further four- to eightfold, and genotypic analysis of a purified day 26 isolate (CB1884) revealed a substitution mutation in the parC gene (parC^H103Y) corresponding to a histidine-to-tyrosine substitution at residue 103 of the ParC subunit of DNA gyrase. While this mutation is apparently atypical in fluoroquinolone-resistant clinical isolates, it has previously been reported in laboratory studies of the development of resistance to nonfluorinated quinolones (31) and lies within the canonical QRDR of parC in S. aureus. Hence, as expected, an eightfold shift in the ciprofloxacin MIC was observed between the day 15 (CB1883) and the day 26 (CB1884) isolates.

During the course of the CBR-2092 step selection study, it was apparent that the mutant derivatives that were isolated and characterized exhibited slower growth in vitro than the parental strain. Table 6 also shows the doubling times for each isolate characterized when it was grown in MHII broth medium at 37°C with good aeration. Interestingly, the doubling time of the terminal day 26 isolate (CB1884), which bears five mutations (rpoB^R484H, gyrA^Δ^L520, gyrA^S84L, parC^R236 duplication, and parC^H103Y), is 65 min and is thus significantly longer than that of parent strain CB190 (ATCC 29213) (38 min). The significance of these findings with regard to the in vivo fitness or pathogenicity of strains exhibiting decreased susceptibility to CBR-2092 remains to be determined.

Finally, in contrast to the fluoroquinolone comparator agents employed, it does not appear that the mutational activation of efflux systems is a contributory factor in the stepwise development of CBR-2092 resistance. In the case of ciprofloxacin and ABT-719, the terminal day 15 isolates exhibited MICs for ethidium bromide of 64 μg/ml, which were 16-fold elevated over the MIC for the parent strain (CB190 [ATCC 29213]; MIC, 4 μg/ml). In contrast, no decrease in susceptibility to ethidium bromide was apparent for the CBR-2092 selectants through the day 26 terminal endpoint.

Multistep passage selections undertaken with strains with preexisting rifamycin and/or quinolone resistance mutations.

In a follow-up study, the day 26 terminal isolate from the CBR-2092 step selection study (isolate CB1884) was used as the starting strain for a step selection undertaken with ABT-719 in MHII medium. As shown in Table 7, after 20 days of stepwise passage in increasing concentrations of ABT-719, a strain (strain CB1887) that had broth microdilution MICs for CBR-2092 and ciprofloxacin of >250 and 64 μg/ml, respectively, was isolated. Genotypic analysis of strain CB1887 confirmed that each of the five mutations previously characterized in strain CB1884 had been stably maintained and also revealed two further substitution mutations in parC, parC^E84G and parC^S80Y, which correspond to glutamate-to-glycine and serine-to-tyrosine substitutions at residues 84 and 80 of the ParC subunit of DNA topoisomerase IV, respectively, and a further substitution mutation in gyrA, gyrA^E88K, which corresponds to a glutamate-to-lysine substitution at residue 88 of the GyrA subunit of DNA gyrase. These combined data are consistent with the notion that the mode of action of CBR-2092 in the concentration ranges employed here (up to 250 and 8 μg/ml under the broth and agar conditions, respectively) can be accounted for by combined effects on the cellular functions of RNA polymerase, DNA gyrase, and DNA topoisomerase IV.

A final series of step selections were undertaken with CBR-2092 in studies employing starting strains that bore preexisting rifampin and/or quinolone resistance mutations. In these studies, MHII medium was supplemented with 0.002% (vol/vol) P-80 to enable selections involving higher concentrations of CBR-2092. Table 7 also shows a summary of the phenotypic and genotypic characteristics of the stable, true-breeding mutants derived from the terminal isolates resulting from each of these step selections. In toto, the combined data from the last set of genetic studies further substantiate conclusions from previously described genetic selections, including the notion that CBR-2092 acting as a quinolone (in rifamycin-resistant strains) elicits resistance mutations in DNA gyrase and DNA topoisomerases IV that are atypical of those elicited by traditional quinolones. In addition, the selection of secondary mutations in both GyrA and ParC subunits in each of two independent selections undertaken with a rifampin-resistant strain (CB815) bearing preexisting canonical fluoroquinolone resistance alleles (gyrA^S84L and parC^S80F) supports the notion that CBR-2092 exhibits residual activity against the prevalent fluoroquinolone-resistant variants and is consistent with the aforementioned biochemical data. Finally, it is noted again that the terminal isolates resulting from all CBR-2092 step selections exhibited no change with regard to their susceptibility to ethidium bromide, suggesting that the mutational activation of efflux systems is not a contributory factor in genotypic adaptations associated with decreased CBR-2092 susceptibility in vitro.

DISCUSSION

Examination of the rifampin-RNA polymerase cocrystal structures suggest that the tight rifampin binding interaction is mediated by key hydrogen bonds formed by hydroxyl groups at C-1, C-8, C-21, and C-23 as well as the carbonyl oxygen of the C-25 acetoxy group (Fig. 1), while the C-3-appended piperazine functionality of rifampin is spatially oriented away from the RNA polymerase binding surface and appears to be solvent accessible (4, 9). As all of the chemical features of the rifampin pharmacophore identified as critical elements in the RNA polymerase interaction are preserved in CBR-2092 and the quinolone moiety is appended to the C-3 position via a secondary hydrazone functionality identical to that of rifampin, it is perhaps not surprising that CBR-2092 exhibits in vitro potency as an RNA polymerase inhibitor that is nearly equivalent to that of rifampin. Preliminary evidence for distinct features of the interaction of CBR-2092 with RNA polymerase is suggested by the retention of activity of CBR-2092 against derivatives of a high-level quinolone-resistant strain (strain CB1623, gyrA^S84L parC^S80F parE^D434V norA^up) bearing intermediate-level rifampin resistance alleles, including rpoB^H481N, rpoB^S464P, or rpoB^I527F (Q. Du, L. Duncan, G. T. Robertson, and A. S. Lynch, unpublished observations).

The activity of CBR-2092 against rifampin-resistant strains, combined with the range of gyrA, parE, and parC mutations resulting from genetic selections undertaken with CBR-2092 in strains with preexisting high-level rifampin resistance, suggests that CBR-2092 exhibits secondary activity against S. aureus via its quinolone pharmacophore and that DNA gyrase is the preferred topoisomerase target in vivo. In this regard, it is interesting to note that other hybrid quinolone antibiotics, including quinolone dimers (14, 22, 23, 40), sulfonamide-quinolone hybrids (2, 29), and oxazolidinone-quinolone hybrids (13, 15, 16), similarly appear to preferentially target DNA gyrase in gram-positive bacteria. These data could be interpreted as indicating that the quinolone binding pocket of DNA gyrase in ternary complexes may be more accommodating of compounds that in effect bear large, bulky substitutions extending from position 7 of the classical 4-quinolone core nucleus.

The DNA gyrase and DNA topoisomerase IV mutations characterized in strains resulting from genetic selections with CBR-2092 include an array of mutational changes that have not previously been reported in studies of traditional quinolone agents and, as such, lie outside of the classical QRDRs. The selection of such mutants could reflect distinct aspects of the binding interaction between CBR-2092 and the DNA topoisomerase target proteins. Alternately, these atypical mutations may simply attenuate the activity of the enzyme such as to alter the sensitivity of the mutant toposiomerase to inhibition by CBR-2092. In this regard, it is of interest to note that past studies of laboratory-isolated quinolone-resistant mutants of S. aureus have similarly revealed mutations outside of the classical QRDRs and have been found to either reduce the expression of DNA topoisomerase IV (17) or yield enzymes with apparent reduced catalytic function (X. Zhang and D. Hooper, unpublished observations cited elsewhere [17]).

Finally, the mutational activation of efflux systems has been shown to be a common mechanism that contributes to the development of fluoroquinolone resistance in a variety of human pathogens. Studies of S. aureus suggest that up to ∼50% of fluoroquinolone-resistant strains of clinical origin exhibit an enhanced quinolone efflux phenotype (11, 34, 35, 39) and that mutations affecting the expression of two specific efflux pumps—NorA, a member of the major facilitator superfamily (MFS) class, and MepA, a multidrug and toxic extrusion (MATE) family member—are most commonly found. However, in contrast to most fluoroquinolones, including the comparator agents studied here, the mutational activation of efflux systems does not appear to be a contributory factor in the development of resistance to CBR-2092. Furthermore, in an accompanying article (30), we show through the use of a panel of genetically defined mutant strains of S. aureus that the increased expression of either norA or mepA has no discernible impact on the in vitro activity of CBR-2092. It is of interest to note that quinolone dimer agents linked via the C-7 position also appear to exhibit a lower propensity for quinolone efflux in S. aureus (14, 22, 23). In addition, the improved antimicrobial activities of other quinolone hybrid agents against gram-positive cocci compared to those of the parent agents (or cocktails thereof) may also be explained by the circumvention of intrinsic or mutationally activated efflux systems (8, 13, 15, 16). One possible explanation for this apparent commonality among quinolone hybrid antibiotics (including CBR-2092) is that the MFS and MATE classes of pumps involved in quinolone efflux in gram-positive cocci exhibit relatively narrow substrate specificities compared to those of the resistance-nodulation-division efflux systems principally involved in quinolone efflux in gram-negative pathogens. In light of the increasing prevalence of efflux-mediated resistance traits in gram-positive cocci, this feature may confer a selective advantage of CBR-2092 over rifamycin-fluoroquinolone cocktail combinations with regard to its activity against fluoroquinolone-resistant clinical isolates.

Acknowledgments

We thank David Hooper for provision of the plasmids used for the inducible overexpression of S. aureus GyrB and ParE subunits in E. coli, Ian Chopra for providing strain RN4220, and Douglas Beeman and Katrina Chapo for experimental contributions in the early stages of the work. We also acknowledge past contributors to the rifamycin-quinolone program at Cumbre Pharmaceuticals, including Donghui Bao, Keith Combrink, Jing Li, Zhenkun Ma, and Paul Renick, and the contributions of current colleagues Charles Ding, Steve Madden, and William Weiss.

Footnotes

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Published ahead of print on 28 April 2008.

REFERENCES

1.Acocella, G. 1983. Pharmacokinetics and metabolism of rifampin in humans. Rev. Infect. Dis. 5:S428-S432. [DOI] [PubMed] [Google Scholar]
2.Alovero, F. L., X.-S. Pan, J. E. Morris, R. H. Manzo, and L. M. Fisher. 2000. Engineering the specificity of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrob. Agents Chemother. 44:320-325. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Amorena, B., E. Gracia, M. Monzon, J. Leiva, C. Oteiza, M. Perez, J. L. Alabart, and J. Hernandez-Yago. 1999. Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro. J. Antimicrob. Chemother. 44:43-55. [DOI] [PubMed] [Google Scholar]
4.Artsimovitch, I., M. N. Vassylyeva, D. Svetlov, V. Svetlov, A. Perederina, N. Igarashi, N. Matsugaki, S. Wakatsuki, T. H. Tahirov, and D. G. Vassylyev. 2005. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins. Cell 122:351-363. [DOI] [PubMed] [Google Scholar]
5.Baddour, L. M., W. R. Wilson, A. S. Bayer, V. G. Fowler, Jr., A. F. Bolger, M. E. Levison, P. Ferrieri, M. A. Gerber, L. Y. Tani, M. H. Gewitz, D. C. Tong, J. M. Steckelberg, R. S. Baltimore, S. T. Shulman, J. C. Burns, D. A. Falace, J. W. Newburger, T. J. Pallasch, M. Takahashi, and K. A. Taubert. 2005. Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: endorsed by the Infectious Diseases Society of America. Circulation 111:e394-e434. [DOI] [PubMed] [Google Scholar]
6.Blaser, J., P. Vergeres, A. Widmer, and W. Zimmerli. 1995. In vivo verification of in vitro model of antibiotic treatment of device-related infection. Antimicrob. Agents Chemother. 39:1134-1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Bryskier, A. 2005. Ansamycins. In Antimicrobial agents: antibacterials and antifungals. ASM Press, Washington, DC.
8.Butler, M. M., W. A. LaMarr, K. A. Foster, M. H. Barnes, D. J. Skow, P. T. Lyden, L. M. Kustigian, C. Zhi, N. C. Brown, G. E. Wright, and T. L. Bowlin. 2007. Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound. Antimicrob. Agents Chemother. 51:119-127. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Campbell, E. A., N. Korzheva, A. Mustaev, K. Murakami, S. Nair, A. Goldfarb, and S. A. Darst. 2001. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell. Microbiol. 104:901-912. [DOI] [PubMed] [Google Scholar]
10.Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard, 7th ed. CLSI document M7-A7, vol. 26, no. 2. Clinical and Laboratory Standards Institute, Wayne, PA.
11.DeMarco, C. E., L. A. Cushing, E. Frempong-Manso, S. M. Seo, T. A. A. Jaravaza, and G. W. Kaatz. 2007. Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 51:3235-3239. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Floss, H. G., and T.-W. Yu. 2005. Rifamycin—mode of action, resistance and biosynthesis. Chem. Rev. 105:621-632. [DOI] [PubMed] [Google Scholar]
13.Gordeev, M. F., C. Hackbarth, M. R. Barbachyn, L. S. Banitt, J. R. Gage, G. W. Luehr, M. Gomez, J. Trias, S. E. Morin, G. E. Zurenko, C. N. Parker, J. M. Evans, R. J. White, and D. V. Patel. 2003. Novel oxazolidinone-quinolone hybrid antimicrobials. Bioorg. Med. Chem. Lett. 13:4213-4216. [DOI] [PubMed] [Google Scholar]
14.Gould, K. A., X.-S. Pan, R. J. Kerns, and L. M. Fisher. 2004. Ciprofloxacin dimers target gyrase in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 48:2108-2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Hubschwerlen, C., J. L. Specklin, D. K. Baeschlin, Y. Borer, S. Haefeli, C. Sigwalt, S. Schroeder, and H. H. Locher. 2003. Structure-activity relationship in the oxazolidinone-quinolone hybrid series: influence of the central spacer on the antibacterial activity and the mode of action. Bioorg. Med. Chem. Lett. 13:4229-4233. [DOI] [PubMed] [Google Scholar]
16.Hubschwerlen, C., J. L. Specklin, C. Sigwalt, S. Schroeder, and H. H. Locher. 2003. Design, synthesis and biological evaluation of oxazolidinone-quinolone hybrids. Bioorg. Med. Chem. 11:2313-2319. [DOI] [PubMed] [Google Scholar]
17.Ince, D., and D. C. Hooper. 2003. Quinolone resistance due to reduced target enzyme expression. J. Bacteriol. 185:6883-6892. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Ince, D., X. Zhang, L. C. Silver, and D. C. Hooper. 2002. Dual targeting of DNA gyrase and topoisomerase iv: target interactions of garenoxacin (BMS-284756, T-3811ME), a new desfluoroquinolone. Antimicrob. Agents Chemother. 46:3370-3380. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, R. Kimura, T. Purchio, and P. R. Contag. 2003. Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection. Antimicrob. Agents Chemother. 47:3130-3137. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, T. F. Purchio, and P. R. Contag. 2004. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrob. Agents Chemother. 48:2283-2287. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Karchmer, A. W. 2000. Infections of prosthetic heart valves, p. 145-172. In F. A. Waldvogel and A. L. Bisno (ed.), Infections associated with indwelling devices, 3rd ed. ASM Press, Washington, DC.
22.Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, and V. U. Diwadkar. 2003. Structural features of piperazinyl-linked ciprofloxacin dimers required for activity against drug-resistant strains of Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13:2109-2112. [DOI] [PubMed] [Google Scholar]
23.Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, V. U. Diwadkar, and T. D. Ward. 2003. Piperazinyl-linked fluoroquinolone dimers possessing potent antibacterial activity against drug-resistant strains of Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13:1745-1749. [DOI] [PubMed] [Google Scholar]
24.Konig, D. P., J. M. Schierholz, U. Munnich, and J. Rutt. 2001. Treatment of staphylococcal implant infection with rifampicin-ciprofloxacin in stable implants. Arch. Orthop Trauma Surg. 121:297-299. [DOI] [PubMed] [Google Scholar]
25.Li, Q., D. T. Chu, A. Claiborne, C. S. Cooper, C. M. Lee, K. Raye, K. B. Berst, P. Donner, W. Wang, L. Hasvold, A. Fung, Z. Ma, M. Tufano, R. Flamm, L. L. Shen, J. Baranowski, A. Nilius, J. Alder, J. Meulbroek, K. Marsh, D. Crowell, Y. Hui, L. Seif, L. M. Melcher, R. Henry, S. Spanton, R. Faghih, L. L. Klein, S. K. Tanaka, and J. J. Plattner. 1996. Synthesis and structure-activity relationships of 2-pyridones: a novel series of potent DNA gyrase inhibitors as antibacterial agents. J. Med. Chem. 39:3070-3088. [DOI] [PubMed] [Google Scholar]
26.Li, Q., L. A. Mitscher, and L. L. Shen. 2000. The 2-pyridone antibacterial agents: bacterial topoisomerase inhibitors. Med. Res. Rev. 20:231-293. [DOI] [PubMed] [Google Scholar]
27.Lynch, A. S., and Q. Du. 2007. Methods to identify and characterize inhibitors of bacterial RNA polymerase, p. 37-52. In New antibiotic targets. Molecular medicine series, Humana Press Inc., Totowa, NJ. [DOI] [PubMed]
27a.Lynch, A. S., E. J. Bonventre, T. B. Doyle, Q. Du, L. Duncan, G. T. Robertson, and E. D. Roche. 2007. In vitro mode-of-action studies of CBR-2092: a novel rifamycin-quinolone hybrid antibiotic, abstr. F1-2101. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
28.Monzon, M., C. Oteiza, J. Leiva, and B. Amorena. 2001. Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis. J. Antimicrob. Chemother. 48:793-801. [DOI] [PubMed] [Google Scholar]
29.Pan, X.-S., P. J. Hamlyn, R. Talens-Visconti, F. L. Alovero, R. H. Manzo, and L. M. Fisher. 2002. Small-colony mutants of Staphylococcus aureus allow selection of gyrase-mediated resistance to dual-target fluoroquinolones. Antimicrob. Agents Chemother. 46:2498-2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Robertson, G. T., E. J. Bonventre, T. B. Doyle, Q. Du, L. Duncan, T. W. Morris, E. D. Roche, D. Yan, and A. S. Lynch. 2008. In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: microbiology profiling studies with staphylococci and streptococci. Antimicrob. Agents Chemother. 52:2324-2334. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Roychoudhury, S., T. L. Twinem, K. M. Makin, M. A. Nienaber, C. Li, T. W. Morris, B. Ledoussal, and C. E. Catrenich. 2001. Staphylococcus aureus mutants isolated via exposure to nonfluorinated quinolones: detection of known and unique mutations. Antimicrob. Agents Chemother. 45:3422-3426. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Saginur, R., M. Stdenis, W. Ferris, S. D. Aaron, F. Chan, C. Lee, and K. Ramotar. 2006. Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections. Antimicrob. Agents Chemother. 50:55-61. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Saiki, A. Y., L. L. Shen, C. M. Chen, J. Baranowski, and C. G. Lerner. 1999. DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones. Antimicrob. Agents Chemother. 43:1574-1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Schmitz, F., A. Fluit, M. Luckefahr, B. Engler, B. Hofmann, J. Verhoef, H. Heinz, U. Hadding, and M. Jones. 1998. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 42:807-810. [DOI] [PubMed] [Google Scholar]
35.Schmitz, F. J., A. C. Fluit, S. Brisse, J. Verhoef, K. Kohrer, and D. Milatovic. 1999. Molecular epidemiology of quinolone resistance and comparative in vitro activities of new quinolones against European Staphylococcus aureus isolates. FEMS Immunol. Med. Microbiol. 26:281-287. [DOI] [PubMed] [Google Scholar]
36.Schrenzel, J., S. Harbarth, G. Schockmel, D. Genne, T. Bregenzer, U. Flueckiger, C. Petignat, F. Jacobs, P. Francioli, W. Zimmerli, D. P. Lew, and Swiss Staphylococcal Study Group. 2004. A randomized clinical trial to compare fleroxacin-rifampicin with flucloxacillin or vancomycin for the treatment of staphylococcal infection. Clin. Infect. Dis. 39:1285-1292. [DOI] [PubMed] [Google Scholar]
37.Schwank, S., Z. Rajacic, W. Zimmerli, and J. Blaser. 1998. Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrob. Agents Chemother. 42:895-898. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Stein, A., M. Drancourt, and D. Raoult. 2000. Management of infected orthopedic implants. In F. A. Waldvogel and A. L. Bisno (ed.), Infections associated with indwelling devices, 3rd ed. ASM Press, Washington, DC.
39.Tanaka, M., T. Wang, Y. Onodera, Y. Uchida, and K. Sato. 2000. Mechanism of quinolone resistance in Staphylococcus aureus. J. Infect. Chemother. 6:131-139. [DOI] [PubMed] [Google Scholar]
40.Zhao, X., B. Quinn, R. Kerns, and K. Drlica. 2006. Bactericidal activity and target preference of a piperazinyl-cross-linked ciprofloxacin dimer with Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 58:1283-1286. [DOI] [PubMed] [Google Scholar]
41.Zheng, Z., and P. S. Stewart. 2002. Penetration of rifampin through Staphylococcus epidermidis biofilms. Antimicrob. Agents Chemother. 46:900-903. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Zimmerli, W., A. Trampuz, and P. E. Ochsner. 2004. Prosthetic-joint infections. N. Engl. J. Med. 351:1645-1654. [DOI] [PubMed] [Google Scholar]
43.Zimmerli, W., A. F. Widmer, M. Blatter, R. Frei, P. E. Ochsner, et al. 1998. Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. JAMA 279:1537-1541. [DOI] [PubMed] [Google Scholar]

[r1] 1.Acocella, G. 1983. Pharmacokinetics and metabolism of rifampin in humans. Rev. Infect. Dis. 5:S428-S432. [DOI] [PubMed] [Google Scholar]

[r2] 2.Alovero, F. L., X.-S. Pan, J. E. Morris, R. H. Manzo, and L. M. Fisher. 2000. Engineering the specificity of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrob. Agents Chemother. 44:320-325. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r3] 3.Amorena, B., E. Gracia, M. Monzon, J. Leiva, C. Oteiza, M. Perez, J. L. Alabart, and J. Hernandez-Yago. 1999. Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro. J. Antimicrob. Chemother. 44:43-55. [DOI] [PubMed] [Google Scholar]

[r4] 4.Artsimovitch, I., M. N. Vassylyeva, D. Svetlov, V. Svetlov, A. Perederina, N. Igarashi, N. Matsugaki, S. Wakatsuki, T. H. Tahirov, and D. G. Vassylyev. 2005. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins. Cell 122:351-363. [DOI] [PubMed] [Google Scholar]

[r5] 5.Baddour, L. M., W. R. Wilson, A. S. Bayer, V. G. Fowler, Jr., A. F. Bolger, M. E. Levison, P. Ferrieri, M. A. Gerber, L. Y. Tani, M. H. Gewitz, D. C. Tong, J. M. Steckelberg, R. S. Baltimore, S. T. Shulman, J. C. Burns, D. A. Falace, J. W. Newburger, T. J. Pallasch, M. Takahashi, and K. A. Taubert. 2005. Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: endorsed by the Infectious Diseases Society of America. Circulation 111:e394-e434. [DOI] [PubMed] [Google Scholar]

[r6] 6.Blaser, J., P. Vergeres, A. Widmer, and W. Zimmerli. 1995. In vivo verification of in vitro model of antibiotic treatment of device-related infection. Antimicrob. Agents Chemother. 39:1134-1139. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Bryskier, A. 2005. Ansamycins. In Antimicrobial agents: antibacterials and antifungals. ASM Press, Washington, DC.

[r8] 8.Butler, M. M., W. A. LaMarr, K. A. Foster, M. H. Barnes, D. J. Skow, P. T. Lyden, L. M. Kustigian, C. Zhi, N. C. Brown, G. E. Wright, and T. L. Bowlin. 2007. Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound. Antimicrob. Agents Chemother. 51:119-127. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r9] 9.Campbell, E. A., N. Korzheva, A. Mustaev, K. Murakami, S. Nair, A. Goldfarb, and S. A. Darst. 2001. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell. Microbiol. 104:901-912. [DOI] [PubMed] [Google Scholar]

[r10] 10.Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard, 7th ed. CLSI document M7-A7, vol. 26, no. 2. Clinical and Laboratory Standards Institute, Wayne, PA.

[r11] 11.DeMarco, C. E., L. A. Cushing, E. Frempong-Manso, S. M. Seo, T. A. A. Jaravaza, and G. W. Kaatz. 2007. Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 51:3235-3239. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Floss, H. G., and T.-W. Yu. 2005. Rifamycin—mode of action, resistance and biosynthesis. Chem. Rev. 105:621-632. [DOI] [PubMed] [Google Scholar]

[r13] 13.Gordeev, M. F., C. Hackbarth, M. R. Barbachyn, L. S. Banitt, J. R. Gage, G. W. Luehr, M. Gomez, J. Trias, S. E. Morin, G. E. Zurenko, C. N. Parker, J. M. Evans, R. J. White, and D. V. Patel. 2003. Novel oxazolidinone-quinolone hybrid antimicrobials. Bioorg. Med. Chem. Lett. 13:4213-4216. [DOI] [PubMed] [Google Scholar]

[r14] 14.Gould, K. A., X.-S. Pan, R. J. Kerns, and L. M. Fisher. 2004. Ciprofloxacin dimers target gyrase in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 48:2108-2115. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r15] 15.Hubschwerlen, C., J. L. Specklin, D. K. Baeschlin, Y. Borer, S. Haefeli, C. Sigwalt, S. Schroeder, and H. H. Locher. 2003. Structure-activity relationship in the oxazolidinone-quinolone hybrid series: influence of the central spacer on the antibacterial activity and the mode of action. Bioorg. Med. Chem. Lett. 13:4229-4233. [DOI] [PubMed] [Google Scholar]

[r16] 16.Hubschwerlen, C., J. L. Specklin, C. Sigwalt, S. Schroeder, and H. H. Locher. 2003. Design, synthesis and biological evaluation of oxazolidinone-quinolone hybrids. Bioorg. Med. Chem. 11:2313-2319. [DOI] [PubMed] [Google Scholar]

[r17] 17.Ince, D., and D. C. Hooper. 2003. Quinolone resistance due to reduced target enzyme expression. J. Bacteriol. 185:6883-6892. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r18] 18.Ince, D., X. Zhang, L. C. Silver, and D. C. Hooper. 2002. Dual targeting of DNA gyrase and topoisomerase iv: target interactions of garenoxacin (BMS-284756, T-3811ME), a new desfluoroquinolone. Antimicrob. Agents Chemother. 46:3370-3380. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, R. Kimura, T. Purchio, and P. R. Contag. 2003. Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection. Antimicrob. Agents Chemother. 47:3130-3137. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r20] 20.Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, T. F. Purchio, and P. R. Contag. 2004. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrob. Agents Chemother. 48:2283-2287. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Karchmer, A. W. 2000. Infections of prosthetic heart valves, p. 145-172. In F. A. Waldvogel and A. L. Bisno (ed.), Infections associated with indwelling devices, 3rd ed. ASM Press, Washington, DC.

[r22] 22.Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, and V. U. Diwadkar. 2003. Structural features of piperazinyl-linked ciprofloxacin dimers required for activity against drug-resistant strains of Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13:2109-2112. [DOI] [PubMed] [Google Scholar]

[r23] 23.Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, V. U. Diwadkar, and T. D. Ward. 2003. Piperazinyl-linked fluoroquinolone dimers possessing potent antibacterial activity against drug-resistant strains of Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13:1745-1749. [DOI] [PubMed] [Google Scholar]

[r24] 24.Konig, D. P., J. M. Schierholz, U. Munnich, and J. Rutt. 2001. Treatment of staphylococcal implant infection with rifampicin-ciprofloxacin in stable implants. Arch. Orthop Trauma Surg. 121:297-299. [DOI] [PubMed] [Google Scholar]

[r25] 25.Li, Q., D. T. Chu, A. Claiborne, C. S. Cooper, C. M. Lee, K. Raye, K. B. Berst, P. Donner, W. Wang, L. Hasvold, A. Fung, Z. Ma, M. Tufano, R. Flamm, L. L. Shen, J. Baranowski, A. Nilius, J. Alder, J. Meulbroek, K. Marsh, D. Crowell, Y. Hui, L. Seif, L. M. Melcher, R. Henry, S. Spanton, R. Faghih, L. L. Klein, S. K. Tanaka, and J. J. Plattner. 1996. Synthesis and structure-activity relationships of 2-pyridones: a novel series of potent DNA gyrase inhibitors as antibacterial agents. J. Med. Chem. 39:3070-3088. [DOI] [PubMed] [Google Scholar]

[r26] 26.Li, Q., L. A. Mitscher, and L. L. Shen. 2000. The 2-pyridone antibacterial agents: bacterial topoisomerase inhibitors. Med. Res. Rev. 20:231-293. [DOI] [PubMed] [Google Scholar]

[r27] 27.Lynch, A. S., and Q. Du. 2007. Methods to identify and characterize inhibitors of bacterial RNA polymerase, p. 37-52. In New antibiotic targets. Molecular medicine series, Humana Press Inc., Totowa, NJ. [DOI] [PubMed]

[r27a] 27a.Lynch, A. S., E. J. Bonventre, T. B. Doyle, Q. Du, L. Duncan, G. T. Robertson, and E. D. Roche. 2007. In vitro mode-of-action studies of CBR-2092: a novel rifamycin-quinolone hybrid antibiotic, abstr. F1-2101. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.

[r28] 28.Monzon, M., C. Oteiza, J. Leiva, and B. Amorena. 2001. Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis. J. Antimicrob. Chemother. 48:793-801. [DOI] [PubMed] [Google Scholar]

[r29] 29.Pan, X.-S., P. J. Hamlyn, R. Talens-Visconti, F. L. Alovero, R. H. Manzo, and L. M. Fisher. 2002. Small-colony mutants of Staphylococcus aureus allow selection of gyrase-mediated resistance to dual-target fluoroquinolones. Antimicrob. Agents Chemother. 46:2498-2506. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Robertson, G. T., E. J. Bonventre, T. B. Doyle, Q. Du, L. Duncan, T. W. Morris, E. D. Roche, D. Yan, and A. S. Lynch. 2008. In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: microbiology profiling studies with staphylococci and streptococci. Antimicrob. Agents Chemother. 52:2324-2334. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r31] 31.Roychoudhury, S., T. L. Twinem, K. M. Makin, M. A. Nienaber, C. Li, T. W. Morris, B. Ledoussal, and C. E. Catrenich. 2001. Staphylococcus aureus mutants isolated via exposure to nonfluorinated quinolones: detection of known and unique mutations. Antimicrob. Agents Chemother. 45:3422-3426. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r32] 32.Saginur, R., M. Stdenis, W. Ferris, S. D. Aaron, F. Chan, C. Lee, and K. Ramotar. 2006. Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections. Antimicrob. Agents Chemother. 50:55-61. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r33] 33.Saiki, A. Y., L. L. Shen, C. M. Chen, J. Baranowski, and C. G. Lerner. 1999. DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones. Antimicrob. Agents Chemother. 43:1574-1577. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Schmitz, F., A. Fluit, M. Luckefahr, B. Engler, B. Hofmann, J. Verhoef, H. Heinz, U. Hadding, and M. Jones. 1998. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 42:807-810. [DOI] [PubMed] [Google Scholar]

[r35] 35.Schmitz, F. J., A. C. Fluit, S. Brisse, J. Verhoef, K. Kohrer, and D. Milatovic. 1999. Molecular epidemiology of quinolone resistance and comparative in vitro activities of new quinolones against European Staphylococcus aureus isolates. FEMS Immunol. Med. Microbiol. 26:281-287. [DOI] [PubMed] [Google Scholar]

[r36] 36.Schrenzel, J., S. Harbarth, G. Schockmel, D. Genne, T. Bregenzer, U. Flueckiger, C. Petignat, F. Jacobs, P. Francioli, W. Zimmerli, D. P. Lew, and Swiss Staphylococcal Study Group. 2004. A randomized clinical trial to compare fleroxacin-rifampicin with flucloxacillin or vancomycin for the treatment of staphylococcal infection. Clin. Infect. Dis. 39:1285-1292. [DOI] [PubMed] [Google Scholar]

[r37] 37.Schwank, S., Z. Rajacic, W. Zimmerli, and J. Blaser. 1998. Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrob. Agents Chemother. 42:895-898. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r38] 38.Stein, A., M. Drancourt, and D. Raoult. 2000. Management of infected orthopedic implants. In F. A. Waldvogel and A. L. Bisno (ed.), Infections associated with indwelling devices, 3rd ed. ASM Press, Washington, DC.

[r39] 39.Tanaka, M., T. Wang, Y. Onodera, Y. Uchida, and K. Sato. 2000. Mechanism of quinolone resistance in Staphylococcus aureus. J. Infect. Chemother. 6:131-139. [DOI] [PubMed] [Google Scholar]

[r40] 40.Zhao, X., B. Quinn, R. Kerns, and K. Drlica. 2006. Bactericidal activity and target preference of a piperazinyl-cross-linked ciprofloxacin dimer with Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 58:1283-1286. [DOI] [PubMed] [Google Scholar]

[r41] 41.Zheng, Z., and P. S. Stewart. 2002. Penetration of rifampin through Staphylococcus epidermidis biofilms. Antimicrob. Agents Chemother. 46:900-903. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r42] 42.Zimmerli, W., A. Trampuz, and P. E. Ochsner. 2004. Prosthetic-joint infections. N. Engl. J. Med. 351:1645-1654. [DOI] [PubMed] [Google Scholar]

[r43] 43.Zimmerli, W., A. F. Widmer, M. Blatter, R. Frei, P. E. Ochsner, et al. 1998. Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. JAMA 279:1537-1541. [DOI] [PubMed] [Google Scholar]

PERMALINK

In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus▿

Gregory T Robertson

Eric J Bonventre

Timothy B Doyle

Qun Du

Leonard Duncan

Timothy W Morris

Eric D Roche

Dalai Yan

A Simon Lynch

Abstract

MATERIALS AND METHODS

Antimicrobial agents.

Bacterial strains.

TABLE 1.

Isolation and characterization of resistant mutants.

TABLE 6.

TABLE 7.

Biochemical studies.

Macromolecular biosynthesis assays.

Antimicrobial susceptibility testing.

RESULTS

Structures of CBR-2092 and related agents.

FIG. 1.

Biochemical studies of CBR-2092 and comparator agents.

TABLE 2.

Activity of CBR-2092 against S. aureus strains exhibiting rifampin and/or quinolone resistance.

TABLE 3.

Effects of CBR-2092 and comparator agents on macromolecular biosynthesis.

FIG. 2.

Single-step resistance studies with CBR-2092.

TABLE 4.

TABLE 5.

Multistep passage selections undertaken with CBR-2092 and comparator agents with CB190 (ATCC 29213).

Multistep passage selections undertaken with strains with preexisting rifamycin and/or quinolone resistance mutations.

DISCUSSION

Acknowledgments

Footnotes

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus^▿