In Vitro Studies with DQ-113 and Comparison Fluoroquinolones To Determine Propensities To Select Resistance in Gram-Positive Cocci

Abstract

DQ-113 was compared in vitro to sitafloxacin, moxifloxacin, levofloxacin, and ciprofloxacin for potential to select mutational resistance in multiresistant staphylococci, pneumococci, and enterococci. Its ability to select less-susceptible mutants varied according to species, being lowest with staphylococci, intermediate with pneumococci, and greatest with enterococci.

DQ-113 is an investigational fluoroquinolone with enhanced anti- gram-positive activity (Fig. 1) (6, 8, 10). We report investigations into its potential to select less-susceptible mutants from clinical isolates of Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus spp.

FIG. 1.

Structure of DQ-113. (Reprinted from reference 10 with permission.)

Single-step mutational frequencies were determined after exposure of approximately 10⁷ to 10⁹ CFU of five clinical isolates each of methicillin-susceptible S. aureus (MSSA), methicillin- resistant S. aureus (MRSA), penicillin-resistant S. pneumoniae (PRSP), and vancomycin-resistant Enterococcus spp. (VRE) (one E. faecalis and four E. faecium strains) to DQ-113, sitafloxacin, moxifloxacin, ciprofloxacin, and levofloxacin (powders provided by their manufacturers) at the MIC, and two, four, and eight times the MIC in appropriate media for 48 to 72 h at 35°C in air (5% CO₂ for S. pneumoniae) as previously described (1). Mutants were also selected at subinhibitory fluoroquinolone concentrations by the agar gradient plate method of Bryson and Szybalski (2) after five passages on drug-containing media. The MRSA strains were relatively susceptible to fluoroquinolones and not representative of frequently encountered nosocomial strains. Antibiotic susceptibilities were determined by the Clinical and Laboratory Standards Institute (CLSI) agar dilution methodology using CLSI-recommended quality control strains (3). Both fluoroquinolones and the nonfluoroquinolone agents vancomycin, oxacillin, erythromycin, gentamicin, chloramphenicol, tetracycline, and penicillin were tested. Known quinolone resistance determining regions (QRDRs) for the DNA gyrase subunit genes gyrA and gyrB, the topoisomerase IV subunit genes grlA and grlB (S. aureus) and parC and parE (S. pneumoniae), and the promoter regions of the norA and pmrA efflux pump genes were amplified by PCR and sequenced directly. The primer sequences used for amplicon generation and sequencing are shown in Table 1.

TABLE 1.

S. aureus and S. pneumoniae primers for amplicon generation and sequencing

Strain	Primer	Sequence (5′ to 3′)	Type	GenBank accession no.
S. aureus	GyrAF	AATGAACAAGGTATGACACC	PCR/sequencing	D10489
	GyrAR	TACGCGCTTCAGTATAACGC	PCR/sequencing	D10489
	GyrBF	CAGCGTTAGATGTAGCAAGC	PCR/sequencing	D10489
	GyrBR	CCGATTCCTGTACCAAATGC	PCR/sequencing	D10489
	GrlAF	ACTTGAAGATGTTTTAGGTGA	PCR/sequencing	D67075
	GrlAR	TTAGGAAATCTTGATGGCAA	PCR/sequencing	D67075
	GrlBF	CGATTAAAGCACAACAAGCAAG	PCR/sequencing	D67075
	GrlBR	CATCAGTCATAATAATTACAC	PCR/sequencing	D67075
	NorAF	TCGTCAATTCCAGTGGCTCAG	PCR/sequencing	D90119
	NorAR	CCAGGCATWACCATACCAGCAC	PCR/sequencing	D90119
S. pneumoniae	GyrAF1	GTAGCGCGAGCTCTTCCTGATGTT	PCR	AF053121
	GyrAR1	CACTGCATCAATGGTTTCACCCAG	PCR	AF053121
	GyrAF2	CCGTCGCATTCTCTACGGAATG	Sequencing	AF053121
	GyrAR2	CAACCGCGATACCAGTTGCTC	Sequencing	AF053121
	GyrBF1	CGCCTCTTCAGTGAAGCCTTCTCC	PCR	Z67740
	GyrBR1	CTCGCTTCCAACCTTGACACC	PCR	Z67740
	GyrBF2	GCCAAACGTATCGTGGAAAAAGGG	Sequencing	Z67740
	GyrBR2	GGTGGTTGGGCAATATAAACATAACCA	Sequencing	Z67740
	ParCF1	GACCGGGCTTTGCCAGATATTCG	PCR/sequencing	Z67739
	ParCR1	GCCGAAATCCCAGTCGAACC	PCR/sequencing	Z67739
	ParEF1	GCTTCTAACCTCATCCGCAAGG	PCR/sequencing	Z67739
	ParER1	GAGCAAGGTCTGGATATGG	PCR/sequencing	Z67739
	PmrAF1	GGCCAGCCAGGTTCTTG	PCR	AJ007367
	PmrAR1	CAACACAAAGGCTTGTCGC	PCR	AJ007367
	PmrAR2	GCGACTTGCTGACTCCCTAC	Sequencing	AJ007367

DQ-113 was less effective than sitafloxacin, moxifloxacin, and levofloxacin in preventing the emergence of single step mutants from the PRSP strains. Sitafloxacin selected single-step mutants from only two of the five strains, moxifloxacin and levofloxacin selected single-step mutants from three strains, DQ-113 selected single-step mutants from four strains, and ciprofloxacin selected single-step mutants from all five strains. All mutants were selected at mutational frequencies of 10⁻⁵ to 10⁻⁷. DQ-113 was the most potent agent, with all mutants being susceptible to 0.007 μg/ml, compared to 0.06 μg of sitafloxacin/ml, 0.25 μg of moxifloxacin/ml, 2 μg of levofloxacin/ml, and 8 μg of ciprofloxacin/ml (data not shown). Mutants were selected from all strains after five exposures to subinhibitory drug concentrations and were more resistant. All were inhibited by 0.12 μg of DQ-113/ml, 4 μg of sitafloxacin/ml, 8 μg of moxifloxacin/ml, 64 μg of levofloxacin/ml, and 128 μg of ciprofloxacin/ml (Table 2).

TABLE 2.

Influence of exposure of S. pneumoniae to subinhibitory fluoroquinolone concentrations on activity of DQ-113 and comparator fluoroquinolones

Strain no. or mutant (M)	Selection agent, concn (μg/ml)a	MIC (μg/ml) of:
		DQ	SIT	MOX	LEV	CIP
335	-b	0.002	0.12	0.12	1	1
M	DQ, 0.06	0.015	0.5	1	2	4
M	SIT, 0.5	0.03	2	1	4	16
M	MOX, 1	0.03	0.5	2	4	4
M	LEV, 16	0.12	4	8	64	128
M	CIP, 16	0.007	0.5	0.5	4	16
334	-	0.004	0.06	0.12	1	1
M	DQ, 0.06	0.06	0.5	2	4	8
M	SIT, 0.5	0.06	0.5	4	16	64
M	MOX, 1	0.015	0.12	0.5	2	8
M	LEV, 4	0.03	0.5	2	8	32
M	CIP, 32	0.03	0.5	1	8	32
216	-	0.001	0.06	0.06	0.5	0.5
M	DQ, 0.06	0.06	0.5	2	16	16
M	SIT, 0.5	0.03	2	2	8	32
M	MOX, 2	0.12	4	8	64	128
M	LEV, 4	0.007	0.5	0.25	4	16
M	CIP, 32	0.015	1	0.5	8	32
217	-	0.004	0.12	0.25	1	1
M	DQ, 0.03	0.007	0.25	0.25	2	4
M	SIT, 0.5	0.03	1	2	16	32
M	MOX, 0.5	0.015	0.5	1	2	4
M	LEV, 4	0.002	0.12	0.25	2	4
M	CIP, 8	0.015	0.5	2	16	32
218	-	0.004	0.12	0.12	1	1
M	DQ, 0.06	0.03	1	1	4	8
M	SIT, 0.5	0.015	1	1	4	16
M	MOX, 2	0.03	1	4	32	128
M	LEV, 16	0.015	0.5	1	4	8
M	CIP, 16	0.06	2	4	32	128

^aSelecting agent and final (highest) concentration for mutant selections. This part of the study involved five passages on gradient plates containing DQ-113 (DQ), sitafloxacin (SIT), moxifloxacin (MOX), levofloxacin (LEV), or ciprofloxacin (CIP).

^b-, No parental data.

The QRDR sequences of gyrA, gyrB, parC, and parE revealed mutations only in the QRDR of parC and only in 2 of the 20 single-step mutants. No mutations were detected in the promoter of the pmrA gene. Both mutants with QRDR changes had the mutation Ser(79)Tyr. One of these was a double mutant with Glu(120)Asp as its second mutation (data not shown).

DQ-113 was the most effective agent for preventing the emergence of single-step mutants of MSSA and MRSA, with only one mutant selected from the 10 strains compared to mutants being selected from 4 strains by moxifloxacin, 5 strains by sitafloxacin, 7 strains by levofloxacin, and 8 strains by ciprofloxacin. All single-step mutants were susceptible to 0.03 μg of DQ-113/ml, 0.25 μg of sitafloxacin/ml, 0.5 μg of moxifloxacin/ml, 2 μg of levofloxacin/ml, and 8 μg of ciprofloxacin/ml, with frequencies of 10⁻⁶ and 10⁻¹⁰, with no apparent differences between the fluoroquinolones (data not shown).

Mutants were selected by subinhibitory drug concentrations from all parents with all mutants inhibited by 2 μg of DQ-113/ml, 16 μg of sitafloxacin/ml, 32 μg of moxifloxacin/ml, 256 μg of levofloxacin/ml, and >128 μg of ciprofloxacin/ml. Reduced susceptibility to gentamicin (four mutants with MICs increasing from 0.12 to 1 μg/ml [one mutant], 0.25 to 1 μg/ml [two mutants], and 0.25 to 2 μg/ml [one mutant]) and chloramphenicol (MIC increased from 4 to 16 μg/ml [one mutant]) was detected, and increased susceptibility to erythromycin (0.5 to 0.12 μg/ml [two mutants]) and tetracycline (MIC decreased from 1 to 0.25 μg/ml [1 mutant]) was detected (data not shown).

Mutations in the single-step mutants occurred in the QRDR of grlA in 17 of the 28 mutants, with the most frequent mutation being Ser(80)Phe. Other mutations were Ser(80)Tyr, Glu(84)Lys, and Pro(144)Ser. A Pro(144)Ser mutation occurred outside the defined QRDR of grlA of a ciprofloxacin-selected mutant, which also had a Ser(80)Phe mutation and three point mutations in the 5′untranslated region of the norA gene (data not shown).

Single-step mutants of VRE were selected at frequencies of 10⁻⁵ and 10⁻⁸ from each strain with each fluoroquinolone except sitafloxacin, which selected mutants from four strains. All mutants were susceptible to 2 μg of DQ-113/ml, 4 μg of sitafloxacin/ml, 8 μg of moxifloxacin/ml, and 16 μg of levofloxacin and ciprofloxacin/ml. The mutants selected by subinhibitory concentrations were more resistant than the single-step mutants, being inhibited by 8 μg of DQ-113 and sitafloxacin/ml, 64 μg of moxifloxacin/ml, 128 μg of ciprofloxacin/ml, and 256 μg of levofloxacin/ml. Vancomycin resistance was lost from a ciprofloxacin-selected mutant of E. faecium (MIC decreased from >128 to 2 μg/ml; data not shown).

Overall, DQ-113 was approximately 10-fold or more potent than the comparator fluoroquinolones. Its capability to select less-susceptible mutants varied according to species, being lowest with staphylococci, intermediate with pneumococci, and greatest with enterococci. The clinical relevance of this will be determined by the concentrations that can be safely achieved in serum and tissues. Its propensity to select less-susceptible mutants of PRSP in vitro may possibly be offset clinically if its high potency inhibits and prevents mutants from emerging during therapy. The absence of target mutations in many pneumococcal mutants suggested the involvement of another resistance mechanism. Although no mutations were detected in the promoter of the PmrA efflux pump (4), other types of mutations may have been responsible for increased PmrA activity, or other resistance mechanisms may have been involved, e.g., other efflux pumps (5) or ABC transporters such PatA and PatB (7, 9).

None of the study fluoroquinolones was highly active against the mutants of vancomycin-resistant E. faecium, suggesting that they would be unsuitable for monotherapy of serious infections caused by this pathogen.