In Vitro Development of Resistance to Six and Four Fluoroquinolones in Mycoplasma pneumoniae and Mycoplasma hominis, Respectively

Abstract

Selection of resistant mutants in sequential subcultures with increasing concentrations of six and four different fluoroquinolones was studied for one reference strain each of Mycoplasma pneumoniae and Mycoplasma hominis, respectively. All fluoroquinolones tested selected for resistance, with alterations affecting the quinolone resistance-determining regions of the four target topoisomerase genes.

Human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma hominis, are etiological agents of respiratory and genitourinary tract infections, respectively, for which fluoroquinolones offer the potential for empirical treatment. Newer compounds of this class have activity against mycoplasmas that is improved over that of older fluoroquinolones (1). To date, acquired resistance to fluoroquinolones has been reported among human mycoplasma clinical isolates only for the genital species, M. hominis, M. genitalium, and Ureaplasma spp. (3-5). In vivo target alterations located in the quinolone resistance-determining regions (QRDRs) of both DNA gyrase and topoisomerase IV subunits were confirmed by in vitro quinolone resistance studies with M. hominis (2, 8).

The purpose of this study was to determine and compare the abilities of six older and newer fluoroquinolones to select for fluoroquinolone-resistant mutants of M. pneumoniae. Furthermore, our previous data on fluoroquinolone resistance in M. hominis were completed by selecting for mutants of this mycoplasma that were resistant to the newer compounds levofloxacin, moxifloxacin, gemifloxacin, and gatifloxacin.

Growth conditions and antibiotic susceptibility testing of the mycoplasma strains have been previously described (12). MICs of fluoroquinolones were determined in the presence and absence of reserpine (20 μg/ml) as described previously (11). Two selection methods, with either broth or agar medium, were used for M. pneumoniae FH (ATCC 15531), while only the agar-based selection was done for M. hominis PG21 (ATCC 23114). Broth-selected mutants were obtained by serial transfers of M. pneumoniae FH in Hayflick modified broth medium containing subinhibitory concentrations of each fluoroquinolone, as previously described (10). For the first passage, the reference strain M. pneumoniae FH was inoculated in Hayflick modified medium with increasing twofold dilutions of each antibiotic. The MIC was determined as the lowest concentration of antimicrobial agent that prevented a color change in the medium at the time when the drug-free growth control first showed a color change (after about 5 days of incubation at 37°C). The culture containing the highest antibiotic concentration with visible growth (subinhibitory concentration) was used to inoculate another antibiotic dilution panel for the following passage. Fifteen passages were performed for each selector antibiotic except for gatifloxacin, and two of the five clones subcultured from passages 5, 7, 10, and 15 were studied. With gatifloxacin, the characterization of passage 2 was added and the selection was conducted up to 10 passages. Subinhibitory concentrations ranged from 0.06 to 16 μg/ml, depending on the selector fluoroquinolone and the selected passage. For the stepwise selection on agar, M. pneumoniae FH cells and M. hominis PG21 cells were concentrated 100-fold by centrifugation and directly filtered through a 0.45-μm-pore size filter (Millipore) to reach an inoculum titer of approximately 10⁹ and 10¹⁰ color changing units/ml, respectively. Then, selection of fluoroquinolone-resistant mutants was performed as previously described by plating a 100-μl inoculum onto Hayflick modified agar medium containing increasing inhibitory concentrations of the selector antibiotic (2). For each selection experiment, three steps were performed with fluoroquinolone concentrations at 2, 4, 8, and 10 times the MIC for the respective parent strain. The mutation frequency was determined as the number of colonies appearing on the plate with antibiotic divided by the number of colonies in the inoculum.

Amplification of the gyrA, gyrB, parC, and parE QRDRs was carried out with 2 μl of a broth culture for the resistant mutants and 1 μM (each) primer, as described elsewhere (2). For M. pneumoniae, primer sets were chosen from the complete genome sequence (6) to amplify a 550-bp gyrA fragment (nucleotides [nt] 28 to 577), a 297-bp gyrB fragment (nt 1258 to 1554), a 588-bp parC fragment (nt 22 to 609), and two parE fragments of 269 bp (nt 5 to 273) and 300 bp (nt 1219 to 1518). For M. hominis, gyrA and parC fragments were obtained with primer set MHA1 (5′-ATGAGTGTCATAGTTTCTCG-3′) and MH4 (2) and primer set MHC1 (5′-GCCGATATAATGTCTGATAG-3′) and MHC2 (5′-TGTTGCATCAATAACTTCGC-3′), respectively. gyrB and parE QRDRs were amplified with primers MH6-7 and MH28-29, respectively (2). A 5′ parE fragment was also amplified with primers MHE1 (5′-AAATAATTACGAAGCTAGCG-3′) and MHE2 (5′-ACTCGTGTTTATTGACAGG-3′). PCR products were directly sequenced by using an ABI PRISM dRhodamine terminator cycle sequencing ready reaction kit (Applied Biosystems).

We were able to select quinolone-resistant M. pneumoniae mutants after serial passages in subinhibitory concentrations of all the six fluoroquinolones used in this study. Two fluoroquinolones, ciprofloxacin and gatifloxacin, selected for M. pneumoniae mutants only with the broth method, using subinhibitory antibiotic concentrations. In contrast, resistant M. hominis mutants were obtained in the presence of inhibitory concentrations of the four fluoroquinolones studied. For M. pneumoniae, mutation frequencies ranged from 1.3 × 10⁻⁶ to 2.9 × 10⁻⁷ with sparfloxacin, while they ranged from 3 × 10⁻⁸ to 7 × 10⁻⁹ with levofloxacin, moxifloxacin, and gemifloxacin. For M. hominis, overall, mutation rates were lower with moxifloxacin, gemifloxacin, and gatifloxacin (about 10⁻⁸) than with levofloxacin (about 10⁻⁷). The susceptibility profiles of the mutants according to their gyrA, gyrB, parC, and parE QRDR status are shown Table 1 for M. pneumoniae and in Table 2 for M. hominis. These mutants exhibited cross-resistance for fluoroquinolones, with the MICs depending on the number of mutations and on the altered positions in the QRDRs. Moxifloxacin, gemifloxacin, and gatifloxacin but not ciprofloxacin and levofloxacin stayed active against M. pneumoniae or M. hominis mutants with one mutation. No efflux mechanism was detected for M. pneumoniae and M. hominis mutants in the presence of reserpine.

TABLE 1.

Characteristics of selected fluoroquinolone-resistant mutants of M. pneumoniae

Reference strain, selection method, or selected mutantc	MIC (μg/ml) ofa:						Amino acid change(s) in QRDR ofb:
	CIP	SPX	LVX	MXF	GEM	GAT	GyrA	GyrB	ParC	ParE
FH	2	0.12	0.5	0.12	0.12	0.12	None	None	None	None
SPX selection
On agar
IIPSA	8	1	2	0.5	1	NDd	—e	—	G81C	—
IIPSC1	8	1	2	0.5	1	ND	—	—	D87Y	—
IIIPSD1	8	2	2	0.5	1	ND	—	—	D87N	—
IIIPSD3	8	1	4	0.5	1	ND	—	—	A83V	—
In broth
S5A	4	1	1	0.5	0.5	ND	—	—	D87N	—
S7A	16	2	2	1	4	ND	—	E483G	D87N	—
S10A	32	8	16	4	8	ND	—	D443N	D87N	—
LVX selection
On agar
IPLA	8	1	2	0.5	0.5	ND	D99N	—	—	—
In broth
L7A	8	1	4	2	1	ND	D99N	—	—	P449S
L15A	32	16	16	8	4	ND	D99N	—	D87N	P449S
MXF selection
On agar
IPMB	2	0.25	1	0.25	0.5	ND	—	E483G	—	—
IIPMB	8	1	2	1	2	ND	—	E483G	D87N	—
In broth
M5A	16	8	16	2	4	ND	—	D443N	D87N	—
M15A	16	16	16	8	4	ND	—	D443N,     R464K	D87N	—
GEM selection
On agar
IIIPGB	8	1	2	0.25	1	ND	—	—	D87G	—
In broth
G5A	8	1	2	1	1	ND	D99N	—	—	—
G7A	16	16	16	2	4	ND	D99N	—	D87G	P449S
CIP selection (in broth only)
C7A	16	2	2	2	4	ND	—	E483G	G81C	—
C7B	8	2	2	1	1	ND	—	—	G81C	—
GAT selection (in broth only)
Ga2A	16	2	4	1	8	2	—	E483G	G81C	—

^aCIP, ciprofloxacin; SPX, sparfloxacin; LVX, levofloxacin; MXF, moxifloxacin; GEM, gemifloxacin; GAT, gatifloxacin.

^bM. pneumoniae positions GyrA 99, GyrB 443, 464, and 483, ParC 81, 83, and 87, and ParE 449 correspond to E. coli coordinates GyrA 83, GyrB 426, 447, and 466, ParC 78, 80, and 84, and ParE 439.

^cM. pneumoniae FH is the parental strain. Agar-selected mutants are designated by a prefix corresponding to the selection step (I, II, or III) followed by the initial of the species (P) and of the selector fluoroquinolone (C, ciprofloxacin; S, sparfloxacin; L, levofloxacin; M, moxifloxacin; G, gemifloxacin; Ga, gatifloxacin). Broth-selected mutants are designated by the initial of the selector fluoroquinolone, followed by the passage number. Fifteen passages were performed for each antibiotic. For passages 5, 7, 10, and 15, two of the five clones subcultured were studied except with gatifloxacin, for which passage 2 was also studied. When both clones from one passage were identical, only one clone is represented in this table. For both agar and broth methods, only clones with significantly increased MICs and QRDR mutations are shown. See footnote a for three-letter drug abbreviations.

^dND, not determined.

^e—, identical to the reference strain.

TABLE 2.

Characteristics of selected fluoroquinolone-resistant mutants of M. hominis obtained from agar selection with various drugs

Reference strain, selection method, or selected mutantc	MIC (μg/ml) ofa:					Amino acid change in QRDR ofb:
	CIP	LVX	MXF	GEM	GAT	GyrA	GyrB	ParC	ParE
PG21	1	0.25	0.06	0.06	0.12	None	None	None	None
LVX selection
IHLA	4	4	0.12	0.12	0.5	—d	—	—	D426N
IHLB	4	1	0.12	0.12	0.25	—	—	R84H	—
IHLC	4	2	0.12	0.06	0.25	—	—	—	R447K
MXF selection
IHMA	>128	64	8	8	16	S153L	A453F	S91I	—
IHMI	>128	32	8	8	16	—	V450F	—	E466K
IIHMA	>128	256	32	32	64	S153L	A453F	S91I	D426N
GEM selection
IHGA	2	0.5	0.12	0.06	0.25	—	—	—	E466K
IHGC	2	0.25	0.12	0.25	0.25	D152N	—	—	—
IIHGB1	32	1	1	4	2	E157K	—	—	E466N
IIHGB9	32	0.5	1	4	2	S153L	—	—	E466K
IIIHB9A	32	0.5	1	8	4	S153L, A163T	—	—	E466K
IIIHB9B	>128	64	16	16	64	S153L	—	S91I	E466K
GAT selection
IHGaA	4	2	0.06	0.06	0.25	—	—	S91I	—
IHGaC	4	1	0.06	0.06	0.25	—	—	—	E466K
IIHGaB	>128	32	8	8	16	S153L	A453T	S91I	—
IIHGaC	32	1	1	4	2	S153L	—	—	E466K
IIHGaD	32	8	1	0.25	2	E157K	—	—	D426N
IIHGaA	>128	64	8	8	16	S153L	—	S91I	—
IIHGaC	>128	64	16	16	64	S153L	—	S91I	E466K

^aCIP, ciprofloxacin; SPX, sparfloxacin; LVX, levofloxacin; MXF, moxifloxacin; GEM, gemifloxacin; GAT, gatifloxacin.

^bM. hominis positions GyrA 152, 153, 157, and 163, GyrB 450 and 453, ParC 84 and 91, and ParE 426, 447, and 466 correspond to E. coli coordinates GyrA 82, 83, 87, and 93, GyrB 450 and 453, ParC 73 and 80, and ParE 420, 441, and 460.

^cM. hominis PG21 is the parental strain. Agar-selected mutants are designated by a prefix corresponding to the selection step (I, II, or III) followed by initials of the species (H) and of the selector fluoroquinolone (L, levofloxacin; M, moxifloxacin; G, gemifloxacin; Ga, gatifloxacin). Only clones with significantly increased MICs and QRDR mutations are shown. See footnote a for three-letter drug abbreviations.

^d—, identical to the reference strain.

For M. pneumoniae, mutations were found at positions described as hot spots of fluoroquinolone resistance, such as GyrA 99 (83 for Escherichia coli), GyrB 443 and 464 (426 and 447 for E. coli), and ParC 83 and 87 (80 and 84 for E. coli) (7). Mutations ParC 81 (78 for E. coli) and GyrB 483 (466 for E. coli) were described previously for E. coli and Staphylococcus aureus (7) and for Proteus mirabilis and Pseudomonas aeruginosa (7, 13), respectively. Only position ParE 449 (439 for E. coli) was not previously associated with quinolone resistance in other bacteria. In M. hominis, besides the mutations previously found either in this mycoplasma or in other bacteria (7), several new mutations were found in this study, at positions GyrA 163 (93 for E. coli), GyrB 450 and 453 (same E. coli numbering), and ParE 447 (441 for E. coli). However, what real effect these new mutations have on quinolone susceptibility of both mycoplasmas has yet to be determined. In M. pneumoniae, gyrB mutations seem to be predominant over gyrA mutations, in contrast to the case for M. hominis and Ureaplasma spp. (1).

As in M. hominis, distinct fluoroquinolones seem to have different primary targets in M. pneumoniae. Thus, according to our genetic studies with M. pneumoniae, sparfloxacin and ciprofloxacin selected mutants with a single modification in parC alone, while levofloxacin and moxifloxacin selected single mutants with a mutation in gyrA or gyrB alone. Gemifloxacin selected single mutants with a modification in either gyrA or parC, in support of dual activity on both DNA gyrase and topoisomerase IV in M. pneumoniae, as in Streptococcus pneumoniae (9). It should be noted that the same fluoroquinolone did not have the same preferential target in both mycoplasmas species. For instance, sparfloxacin targeted primarily topoisomerase IV in M. pneumoniae (Table 1) but DNA gyrase in M. hominis (2, 8). For M. hominis, first-step mutants that were selected with newer fluoroquinolones, levofloxacin and gatifloxacin, had mutations in parC or parE. Here again, first-step mutants of M. hominis selected with gemifloxacin harbored mutations in either gyrA or parC. The preferential targets of moxifloxacin with M. hominis and gatifloxacin with M. pneumoniae were not elucidated in our study, with no single mutants selected with these compounds.

To our knowledge, this is the first description of fluoroquinolone resistance acquired in M. pneumoniae. Even though lower mutation rates were obtained with broader-spectrum quinolones, such as moxifloxacin, gemifloxacin, and gatifloxacin, than with ciprofloxacin for both mycoplasma species, all fluoroquinolones tested selected for resistance, confirming the necessity of a judicious use of these compounds in order to limit development of resistance.