Multiple mutations conferring ciprofloxacin resistance in Staphylococcus aureus demonstrate long-term stability in an antibiotic-free environment

Mark E. Jonesa,*, Nienke M. Boeninka, Jan Verhoefa, Karl Köhrerb and Franz-Josef Schmitzb

a Eijkman-Winkler Institute for Clinical Microbiology, University Hospital Utrecht, Utrecht 3584CX, The Netherlands; b Institute for Medical Microbiology and Virology, Heinrich-Heine University, Düsseldorf 40225, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Two unrelated strains of Staphylococcus aureus, one with a single mutation in grlA, the other with multiple mutations in gyrA, gyrB, grlA, grlB, norA and the norA promoter region, encoding low-level and high-level ciprofloxacin resistance, respectively, were studied. The characterized mutations in these genes were conserved when both strains were passaged for at least 500 generations in an antibiotic-free environment. New, rapidly stabilized mutations and higher MICs were detected for strains passaged in sub-MIC ciprofloxacin concentrations. The seeming irreversibility of quinolone resistance may affect the long-term success of this drug class.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Several reports have documented a significant reduction in the incidence of certain bacterial antimicrobial resistance phenotypes through reduced use of specific antibiotics.1,2 In contrast, particularly in Gram-negative bacteria, in vitro studies have demonstrated the stability of specific resistance determinants in the absence of antibiotics.3,4

In Staphylococcus aureus, resistance to quinolones is primarily mediated through chromosomal point mutations in gyrA and gyrB (encoding subunits of DNA gyrase) and grlA and grlB (encoding subunits of DNA topoisomerase IV).5,6 Together these comprise the so-called quinolone-resistance-determining regions (QRDRs). Additionally, norA encodes an efflux pump contributing to reduced susceptibility. High levels of resistance can be achieved through the accumulation of mutations in QRDRs and the promoter region of norA.7 Earlier studies in Escherichia coli have demonstrated that the common mutations in gyrA have little or no effect on catalytic function of the gyrase.8 However, these studies in E. coli also detected rarer mutations in gyrA which did show a reduction in gyrase activity. By inference, it is therefore possible that some mutation types in the topoisomerase or gyrase of S. aureus may disadvantage the cell compared with wild-type organisms. For this study the hypothesis was made that strains of S. aureus with mutations in QRDRs giving rise to reduced susceptibility to ciprofloxacin would not be at a selective disadvantage relative to sensitive revertants spontaneously appearing in culture containing no antibiotic, and thus no detectable change in the culture composition would occur. Additional mutations may well arise in organisms grown with sub-MIC ciprofloxacin concentrations. Here we report the genotypic and phenotypic stability of quinolone resistance in S. aureus in an environment with or without a quinolone antibiotic selective pressure.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Two recent clinical isolates of S. aureus (NB1 and NB126) which were clonally distinct by PFGE type were studied. The two isolates were selected from a population of S. aureus clinical isolates that had been characterized with respect to nucleotide sequences of QRDRs and the NorA promoter and encoding region on the basis of possessing different mutations. S. aureus NB1 had a single point mutation in grlA conferring a ciprofloxacin MIC of 2 mg/L, close to the resistant breakpoint of 4 mg/L according to NCCLS breakpoint criteria.9 The second isolate, S. aureus NB126, had point mutations in grlA, grlB, gyrA, gyrB and the norA promoter and encoding region, which conferred high-level ciprofloxacin resistance (MIC 128 mg/L) (TableGo).


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Table. Analyses of mutations (either amino acid or silent nucleotide changes) in grlA, grlB, gyrA, gyrB and the norA promoter and protein-encoding regions, and the corresponding ciprofloxacin MICs for test organisms NB1 and NB126
 
To investigate the genetic stability of these mutations, parallel cultures of NB1 and NB126 were established in duplicate using an established serial transfer culture technique.10 On day 1 of the experiment, 50 µL of fresh overnight stationary-phase cell culture (generation zero) was inoculated into two 100 mL flasks of fresh pre-warmed Luria-Bertani broth medium (Difco, Detroit, MI, USA), the first containing no antibiotic and the second containing ciprofloxacin at a concentration of 0.5 x original (generation zero) MIC (1 and 64 mg/L, respectively, for strains NB1 and NB126). These concentrations of ciprofloxacin remained constant throughout the remainder of the experiment. At the same time daily, 50 µL of overnight culture was inoculated into an additional set of flasks containing 100 mL of pre-warmed LB broth culture medium containing the appropriate ciprofloxacin concentration. All cultures were grown at 37°C with aeration. Before the start of the experiment, growth curves were plotted in duplicate for each test strain, ensuring that cultures reached a stationary phase (thus undergoing maximal doubling within the culture volume) within the 24 h incubation period. Cells counts confirmed the increase in cell numbers per unit volume. One of the duplicate cultures was reserved for use only in case of contamination of the study culture, which was checked by purity plate sampling daily. This serial transfer enabled a 2000-fold daily increase in cell numbers within the culture medium, corresponding to 10.9 doublings (46 x log2 2000) or approximately 10.9 generations of binary fission. Five hundred generations of binary fission thus equate to approximately 46 days of serial transfer culturing.10

Organisms from cultures equating to 0, 20, 40, 80, 160, 200 and 500 generations of binary fission were studied. For each sample, dilutions of bacterial culture were made and approximately 75–100 colonies were replica plated on to Mueller–Hinton agar plates containing dilutions of ciprofloxacin (provided by Bayer AG, Wüppertal, Germany) extending below and above the generation-zero MIC of the study isolates (1 and 128 mg/L, respectively). These experiments were done in duplicate using different sample dilutions. This allowed the screening of approximately 150–200 colonies from each test culture for raised or lowered MICs compared with the generation-zero value. In the case of no detectable changes using this screening method, ten colonies from each of the four study strains were randomly selected from the replica plate containing the same antibiotic concentration as the study broth culture, and ciprofloxacin MICs were redetermined for each using an agar dilution method according to NCCLS recommended guidelines.9 Target DNA molecules were also sequenced with methodologies defined previously by our group.11


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Using the replica plate method, no changes in susceptibility to ciprofloxacin were found with either NB1 or NB126 grown in antibiotic-free medium. In addition, in each of the ten colonies selected for further study, target gene nucleotide sequences and MIC values remained identical to those of the parent strains over 500 generations (TableGo). No mutations appeared in QRDRs or in the norA promoter and encoding region in wild-type S. aureus control isolates without mutations, grown without antibiotic. In the presence of 0.5 x MIC of ciprofloxacin, NB1 acquired an additional mutation in gyrA (Ser-84->Ala), which is known to confer resistance to ciprofloxacin. This was detected at generation 80 in all replica plated colonies and in all ten test colonies and remained stable in all subsequent cultures. Concomitant with the appearance of this mutation, susceptibility to ciprofloxacin decreased to an MIC of 8 mg/L, remaining stable thereafter (TableGo). In the presence of 0.5 x MIC of ciprofloxacin, target sequences in NB126 remained unchanged except for a stable point mutation in the norA promoter region (bp-125 T->A) introduced at generation 80. This mutation may enhance norA expression enabling increased ciprofloxacin efflux, but this was not tested. Concomitant with this, the MIC of ciprofloxacin increased to 1024 mg/L, remaining stable thereafter (TableGo).

These data show that at least for these isolates clinical of S. aureus and ciprofloxacin the withdrawal of antibiotic from the environment does not reduce the proportion of resistant bacteria or levels of resistance. In addition, the characterized mutations within the QRDRs, in particular a single first-step mutation conferring low-level ciprofloxacin resistance in S. aureus NB1 and multiple mutations in different gene loci conferring high-level resistance in S. aureus NB126, remained stable in an antibiotic-free environment. Without a positive selection pressure, spontaneous reverse mutations occurring at any of the characterized mutated sites (presumably at a rate of approximately 106–107 per generation) would not have been detected in this experiment. Furthermore, new mutations induced by a sub-MIC antibiotic selective pressure were detected, and rapidly stabilized within cultures. It is worth noting that there may be other as yet unknown factors operating in vivo which favour selection of susceptible revertants; investigation of this would require animal studies. Identical mutations are responsible for ciprofloxacin resistance in other unrelated strains of S. aureus. In addition, DNA gyrase and topoisomerase IV genetic and functional homologues have been characterized in a number of other Gram-positive and Gram-negative species, and it is likely that the conclusions of this study could be extrapolated to organisms other than S. aureus. Finally, stability of ciprofloxacin resistance and of the mutations in the QRDRs conferring it have been observed by our group in clonal lineages of S. aureus in the clinical environment over a period of years.12

Quinolones have undergone a renaissance in recent years and several newly developed compounds will soon be available for clinical use. Although these compounds may be more active, they all share the same topoisomerase and gyrase target sites, which are largely homologous among different microorganisms. It seems reasonable to assume that mutations conferring resistance to these new quinolones will behave in a similar manner, in terms of stability, to those characterized in this study. These studies strongly suggest that for quinolone antibiotics the immediate implementation of prudent prescribing practices may be the wisest strategy to limit emergence of resistance, since reversing a high incidence of quinolone resistance may not be achievable simply by withdrawing the antibiotics from clinical use.


    Notes
 
* Present address. MRL Pharmaceutical Services, Den Brielstraat 11, 3554XD Utrecht, The Netherlands. Tel: +31-30-265-1794; Fax: +31-30-265-1784; E-mail: mjones{at}thetsn.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Kristinsson, K. G. (1997). Effect of antimicrobial use and others risk factors on antimicrobial resistance in pneumococci. Microbial Drug Resistance 3, 117–23.[ISI][Medline]

2 . Seppälä, H., Klaukka, T., Vuopio-Varkila, J., Muotiala, A., Helenius, H., Lager, K. et al. (1997). The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance amongst group A streptococci in Finland. New England Journal of Medicine 337, 441–6.[Abstract/Free Full Text]

3 . Bouma, J. E. & Lenski, R. E. (1988). Evolution of a bacteria/ plasmid association. Nature 335, 351–2.[ISI][Medline]

4 . Schrag S. J. & Perrot, V. (1996). Reducing antibiotic resistance. Nature 381, 120–1.[ISI][Medline]

5 . Ito, H., Yoshida, H., Bogaki-Shonnai, M., Niga, T., Hattori, H. & Nakamura, S. (1994). Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 38, 2014–23.[Abstract]

6 . Yamagishi, J., Kojima, T., Oyamada, Y., Fujimoto, K., Hattori, H., Nakumara, S. et al. (1996). Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 40, 1157–63.[Abstract]

7 . Yoshida, H., Bogaki, M., Nakamura, S., Ubukata, K., & Konno, M. (1990). Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. Journal of Bacteriology 172, 6942–9.[ISI][Medline]

8 . Aleixandre, V., Urios, A., Herrera, G. & Blanco, M. (1989). New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling. Molecular and General Genetics 219, 306–12.[Medline]

9 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing—Eighth Informational Supplement: Approved Standard M100-S8. NCCLS, Wayne, PA.

10 . Lenski, R. E., Rose, M. R., Simpson, S. C. & Tadler, S. C. (1991). Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations. American Nature 138, 1315–41.

11 . Schmitz, F.-J., Jones, M. E., Hofmann, B., Hansen, B., Scheuring, S., Luckefahr, M. et al. (1998). Characterization of grlA, grlB, gyrA and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrobial Agents and Chemotherapy 42, 1249–52.[Abstract/Free Full Text]

12 . Schmitz, F.-J., Hofmann, B., Hansen B., Scheuring, S., Verhoef, J., Fluit, A. et al. (1999). Stability of grlA, grlB, gyrA and gyrB mutations and corresponding MIC-values of fluoroquinolones in different clonal populations of methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection 5, 287–90.[Medline]

Received 16 June 1999; returned 28 September 1999; revised 9 November 1999; accepted 22 November 1999