Selection of moxifloxacin-resistant Staphylococcus aureus compared with five other fluoroquinolones

Deborah J. Griggs, Herida Marona and Laura J. V. Piddock*

Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, UK

Received 22 January 2003; returned 5 February 2003; revised 7 March 2003; accepted 7 March 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Fluoroquinolone-resistant mutants were selected from Staphylococcus aureus NCTC 8532 (F77), and two GrlA mutants of F77 (F193 and F194) with moxifloxacin, sparfloxacin, ofloxacin, grepafloxacin, levofloxacin and trovafloxacin. For mutants selected from F77, moxifloxacin, grepafloxacin and sparfloxacin selected preferentially for mutations in gyrA (Glu-88->Lys). Ofloxacin and trovafloxacin selected most commonly for mutations in grlA, conferring substitutions for Ser-80. Three mutants of F77 were shown to have substitutions in both GrlA (Phe-80) and GyrA (Lys-88). Of the mutants selected from F193 (GrlA Phe-80), restriction fragment length polymorphism analysis of gyrA showed that 76/94 had a mutation at codon 84; those analysed in detail all had the substitution Ser->Leu. Two mutants selected with grepafloxacin contained the substitution Lys-88. One mutant selected with trovafloxacin contained a novel mutation in gyrA substituting Gly-82->Cys. Of the mutants selected from F194 (GrlA Tyr-80), 6/8 had a mutation in gyrA codon 84; of which three contained Leu. The MICs of most agents for mutants selected from F193 and F194 were similar, irrespective of the mutation selected. No mutants had any changes in grlB, and only one had a mutation in gyrB giving rise to the novel substitution Asp-437->His. The mutations arising in first-step mutants were influenced by the fluoroquinolone used for selection. The phenotypes and genotypes of second-step mutants, derived from mutants with existing mutations in grlA, were similar, regardless of the selecting antibiotic.

Keywords: fluoroquinolone, mutant selection, S. aureus


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Fluoroquinolone resistance in Staphylococcus aureus is associated with mutations in the genes encoding the target enzymes, topoisomerase IV (grlA and grlB) and DNA gyrase (gyrA and gyrB), and overexpression of norA, the gene encoding the multidrug efflux pump NorA.1 Mutations in topoisomerase genes associated with fluoroquinolone resistance occur in the highly conserved quinolone resistance-determining region (QRDR), although resistant S. aureus with mutations outside this region have recently been described.2

Topoisomerase IV was regarded as the primary target of fluoroquinolones in S. aureus and DNA gyrase was thought to be the secondary target.3 However, recent studies of Streptococcus pneumoniae suggest that which topoisomerase is the primary target for fluoroquinolones may be dependent on the structure and the relative affinity of each agent for the target enzyme.4 In S. aureus, recent studies have shown that whereas topoisomerase IV is the primary target of many fluoroquinolones, others preferentially target DNA gyrase.5 Some newer fluoroquinolones have equal capacity to act upon both enzyme targets.2

In the present study, fluoroquinolone-resistant mutants from a wild-type strain of S. aureus (NCTC 8532) and two mutants derived from this strain, each containing a single mutation in grlA, were selected with moxifloxacin and five other fluoroquinolones. The aim of the study was to compare the selection frequency, the resistance phenotypes and the location of mutations in S. aureus mutants selected with the six fluoroquinolones.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and media

S. aureus F77 (NCTC type strain 8532) was obtained from the National Collection of Type Cultures (Colindale, UK). S. aureus F193 (GrlA Phe-80) and F194 (GrlA Tyr-80) were previously selected from F77 after exposure to 0.18 mg/L moxifloxacin. Spontaneous mutants with decreased susceptibility to fluoroquinolones were selected as follows. The three parent strains were grown overnight in antibiotic-free broth, concentrated by centrifugation and resuspended in sterile broth to give a range of inocula (106–1010 cfu/mL). Agar plates containing moxifloxacin, grepafloxacin, levofloxacin, ofloxacin, sparfloxacin and trovafloxacin at concentrations 3 x, 5 x and 10 x MIC for the respective parent strain (see Table 2) and the break point concentration of 2 mg/L were inoculated with 100 µL (105–109 cfu) of each cell suspension and incubated at 37°C in air. Ten colonies with the typical size and morphology of the original strain were chosen randomly from each selecting plate and sub-cultured onto antibiotic-free media. These were examined for susceptibility to antibacterial agents, and one mutant selected with each selecting antibiotic at each concentration of each observed resistance phenotype was retained for further study of the mechanism of resistance. All strains were cultured on Iso-sensitest agar containing 5% defibrinated horse blood at 37°C in air. Bacteriological media were supplied by Unipath (Basingstoke, UK) and chemicals by BDH or Sigma (Poole, UK).


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Table 2.  Mutations in the QRDR of grlA, grlB, gyrA and gyrB and resistance phenotypes of representative mutant strains of S. aureus
 
Antibiotic susceptibility testing

The MICs of 16 antibacterial agents (including 10 fluoroquinolones and nalidixic acid), acriflavine and ethidium bromide were determined by the agar doubling dilution method as described previously.6

Restriction fragment length polymorphism (RFLP) and single stranded conformational polymorphism (SSCP) analysis

Staphylococcal DNA was extracted from all strains with guanidium thiocyanate using the method of Pitcher et al.7 The QRDR of grlA and gyrA was amplified by PCR using oligonucleotide primers based on the published sequences of these genes (Table 1). The amplimers were digested with restriction endonuclease HinfI and RFLP analysis was carried out as described by Sreedharan et al.8 A mutation at the codon Ser-80 of grlA or Ser-84 of gyrA results in the loss of the HinfI recognition site at this position.


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Table 1.  Oligonucleotide primers used for PCR of the QRDRs of topoisomerase genes in S. aureus
 
The QRDR of grlA, gyrA, grlB and gyrB of selected mutants was amplified by PCR (Table 1) and the amplimers analysed by SSCP to detect mutations in these genes, as described previously.9 The DNA sequences of the QRDR from mutants having novel SSCP patterns were determined by MWG-Biotech (Ebersberg, Germany).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mutant selection

Fluoroquinolone-resistant mutants were selected from wild-type S. aureus F77, and from two strains with an existing mutation in grlA (F193 and F194) using six fluoroquinolones. First-step mutants were readily selected from F77, but were selected less frequently with grepafloxacin (5.1 x 10–10) than with moxifloxacin and the other fluoroquinolones (3.8 to 8.5 x 10–9). Mutants were selected readily from F193 (GrlA Phe-80) on agar containing all agents except ofloxacin, but were selected less frequently with levofloxacin (1.7 x 10–11) and moxifloxacin (6.1 x 10–10) than the other agents (2.1 x 10–6 to 2.95 x 10–9). Mutants were selected from F194 (GrlA Tyr-80) only on agar containing levofloxacin. Sibling mutants were excluded from further analysis by selecting one mutant of each resistance phenotype, from each selecting antibiotic. In total, 60 F77 mutants, 94 F193 mutants and eight F194 mutants were analysed for the mechanism of resistance.

Antibiotic susceptibility of mutants

All first-step mutants selected from wild-type S. aureus F77 were resistant to nalidixic acid and were less susceptible than the parent to four or more fluoroquinolones (Table 2). However, mutants of F77 remained susceptible to moxifloxacin, grepafloxacin and trovafloxacin (MICs <= 1 mg/L). For second-step mutants selected from grlA mutants F193 and F194, moxifloxacin and trovafloxacin had lower MIC values (MIC90 1–8 mg/L) than the older fluoroquinolones (MIC90 32–>64 mg/L) but mutants were no more resistant to nalidixic acid than the parent strain (MIC 256 mg/L). All mutants selected were resistant to fluoroquinolones alone. None had a multidrug-resistant phenotype suggestive of an efflux mutant.

Mechanisms of fluoroquinolone resistance in mutants selected from wild-type S. aureus (F77)

The phenotypes and genotypes of the first-step mutants were more diverse than those of the second-step mutants, and the agent used for selection of resistance influenced the mutations selected. RFLP analysis of grlA showed that 16/60 (27%) of F77 mutants had a mutation in grlA at codon Ser-80. The number of grlA mutants selected with a mutation at this locus varied according to the selecting antibiotic: none was selected on sparfloxacin- and grepafloxacin-containing agar, compared with 9/10 mutants selected with ofloxacin, 5/8 with trovafloxacin and 2/17 with moxifloxacin.

RFLP analysis of gyrA revealed no nucleotide changes at codon Ser-84. However, DNA sequencing of 23 representative mutants covering all phenotypes revealed that 15/23 (65%) contained the gyrA mutation GAA->AAA (Glu-88->Lys; Table 2). These 15 mutants had been selected with moxifloxacin (6/11), grepafloxacin (4/4), trovafloxacin (3/3) and sparfloxacin (2/2). The remaining eight had a wild-type gyrA QRDR, however three (selected with ofloxacin) were shown to have a Ser-80 mutation in grlA, and two (selected with moxifloxacin) were less susceptible to acriflavine than the parent F77, and the MIC of ciprofloxacin was reduced two- to four-fold in the presence of reserpine. This may be evidence for weak activity of an efflux pump in these two mutants. The mechanism of resistance in the other three mutants is not known but the presence of mutations in topoisomerase genes outside the QRDR2 has yet to be excluded.

Three mutants were shown to have substitutions in both GrlA (Phe-80) and GyrA (Lys-88; Table 2). It was unexpected to find mutations in both grlA and gyrA in three mutants derived from the wild-type strain F77 (F340, F341 both selected with moxifloxacin, and F350 with trovafloxacin) although all three were selected at high antibiotic concentrations (10 x MIC). Quinolone-resistant S. aureus shown to contain a gyrAgrlA double mutation have also been selected in a single step with ciprofloxacin.10

SSCP analysis and DNA sequencing of grlB and gyrB for 12 selected mutants of F77, including those for which no mutation was detected in grlA or gyrA, revealed no changes in these genes.

Mechanisms of fluoroquinolone resistance in mutants derived from GrlA mutants F193 and F194

Of the mutants selected from F193 (GrlA Phe-80), 76/94 (81%) were shown by RFLP analysis to have a mutation in gyrA at codon Ser-84. Most second step mutants selected by moxifloxacin (8/11), grepafloxacin (23/30), sparfloxacin (32/37) and trovafloxacin (13/16) contained the substitution Leu-84 (TCA->TTA; Table 2). RFLP analysis showed that seven of the mutants selected with grepafloxacin had no mutation in gyrA at Ser-84; SSCP analysis and DNA sequencing revealed a gyrA substitution Glu-88->Lys (GAA->AAA; Table 2) in two representative mutants. DNA sequencing of one mutant selected with trovafloxacin revealed a novel mutation in gyrA Gly-82->Cys (GGT->TGT; Table 2).

Only eight mutants were selected from F194 (GrlA Tyr-80; Table 2); all were selected with levofloxacin. RFLP analysis demonstrated that six had a mutation in gyrA encoding Ser-84; the three representative mutants analysed by SSCP and DNA sequencing were shown to have the substitution Ser-84->Leu (TCA->TTA).

For mutants of F193 and F194, different agents selected the same mutations in gyrA and the MICs of most agents were identical to each other, regardless of which secondary mutation was selected in gyrA (Table 2). The substitution Ser-84->Leu in GyrA was selected more commonly than Glu-88->Lys; both substitutions have been described previously for second-step mutants.11

Selected mutants, including those for which no mutation was detected in grlA or gyrA, were analysed by SSCP for changes in gyrB and grlB. The majority of mutants examined for changes in gyrB (15/19) were wild-type, however, one mutant F265 (selected from F193 with grepafloxacin; GrlA Phe-80, wild-type gyrA) had a mutation in gyrB giving rise to the novel substitution Asp-437->His (GAC->CAC). This mutant was less resistant to fluoroquinolones than those F193 mutants with alterations in gyrA (Table 2). A laboratory mutant S. aureus with the GyrB substitution Asp-437->Asn (GAC->AAC) has been described previously.12 There were no mutations detected in any of the 14 mutants examined for changes in the grlB gene.

In summary, our findings show that fluoroquinolone-resistant mutants were readily selected from a wild-type S. aureus and strains with an existing mutation in grlA, and confirm the importance of gyrA and grlA mutations in fluoroquinolone resistance. Two novel substitutions were found, GyrA Cys-82 and GyrB His-437. The location of mutations arising in a wild-type strain was influenced by the fluoroquinolone used for selection. Moxifloxacin, grepafloxacin and sparfloxacin select preferentially for mutations in gyrA at codon Glu-88, and ofloxacin and trovafloxacin select more commonly for mutations in grlA at codon Ser-80 suggesting that DNA gyrase and topoisomerase IV, respectively, are the primary targets of these fluoroquinolones. However, the majority of second step mutants had similar genotypes and phenotypes, regardless of the selecting antibiotic. First step mutants, even those with a mutation in both gyrA and grlA, remained susceptible to moxifloxacin.


    Acknowledgements
 
We thank Yu Fang Jin for selecting the mutants described in this manuscript. Since her return to China we have been unable to contact her to obtain her agreement to be a co-author. This study was supported in part by Bayer AG. H.M. was funded by CAPES, Brazil.


    Footnotes
 
* Corresponding author. Tel: +44-121-414-6966; Fax: +44-121-414-3454; E-mail: l.j.v.piddock{at}bham.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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