Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolone exposure

Richard A. Veneziaa,b,*, Beth E. Domarackia, Ann M. Evansa, Karen E. Prestona and Eileen M. Graffunderb

a Departments of Pathology and Laboratory Medicine, and b Epidemiology, Albany Medical Center, Albany, NY 12208, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To study the effect of fluoroquinolone exposure on the expression of mec(A)-encoded oxacillin resistance, population analysis profiling was performed on four strains of fluoroquinolone-susceptible, mec(A)-positive, heteroresistant Staphylococcus aureus. Growth in the presence of 0.5 x MIC of a fluoroquinolone resulted in >10-fold increase in the proportion of the population that grew on agar containing oxacillin 128 mg/L. Ciprofloxacin exhibited a greater effect than moxifloxacin, levofloxacin and gatifloxacin (average 3400-, 220-, 170- and 49-fold increase in oxacillin-resistant colonies versus the control, respectively). The increase was directly proportional to the fluoroquinolone concentration and could be detected as early as 8 h after exposure to the fluoroquinolone. At 8 h, the absolute number of colonies that grew on oxacillin 128 mg/L was similar whether or not the isolate was exposed to the fluoroquinolone, but the total cfu on non-selective media decreased. The resultant oxacillin-resistant colonies also showed a 1.5- to 3-fold increase in fluoroquinolone MIC. No oxacillin resistance was observed on two similarly treated fluoroquinolone-susceptible, mec(A)-negative strains. It appears that fluoroquinolones influence oxacillin resistance by selective inhibition or killing of the more susceptible subpopulations in heteroresistant S. aureus. The surviving populations are more resistant to both oxacillin and fluoroquinolone. The mechanisms of resistance to the two agents may be unrelated but tend to be associated. This could explain in part the observed increases in fluoroquinolone-resistant MRSA.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The increased incidence of methicillin-resistant Staphylococcus aureus (MRSA) has led to therapeutic problems for hospitalized patients.1,2 In addition there has been an increase in the prevalence of MRSA in individuals with infections acquired outside of hospitals.3 The reasons for the observed increase in MRSA are complex but can be attributed in part to antibiotic practices that promote the expression of methicillin resistance and lapses in infection control that allow patient-to-patient transmission.1,4

ß-Lactam antibiotics that can cause an alteration in peptidoglycan synthesis can influence the expression of methicillin resistance in Staphylococcus spp. Methicillin inhibits bacteria by binding to penicillin-binding proteins (PBPs) in the cell wall and resistance occurs through the substitution of PBP 2A for PBP 2.5,6 This change is enabled by the acquisition of the foreign mec locus, which encodes PBP 2A.7 PBP 2A can take over cell wall building functions when the other PBPs are inhibited by ß-lactams.8 The mec(A) gene and its product PBP 2A are not solely responsible for resistance, and the expression of methicillin resistance is often heterogeneous in that the subpopulations of one strain will express resistance at different levels. This heterogeneity, which differs depending on the strain, appears to be associated with the interaction of mec genes with other regulatory chromosomal genes that appear to affect the level of methicillin resistance.5,912 However, the level of methicillin resistance does not correlate to the amount of PBP 2A present as a cell wall component.6,13 Accordingly, the mechanisms for regulation and expression of methicillin heteroresistance in S. aureus are complex.

The association of fluoroquinolone use and fluoroquinolone-resistant S. aureus has been widely reported.14,15 Fluoroquinolone resistance in S. aureus results primarily from mutations in genes for DNA gyrase or DNA topoisomerase IV, or regulation of active efflux pumps.16,17 Although this is different from the mechanisms controlling methicillin resistance, some analyses have indicated an association between methicillin resistance in Staphylococcus spp. and the use of ciprofloxacin.14,15,18 Others have reported the appearance of heterotropic resistances or other metabolic changes following exposure to ciprofloxacin.19 Quinolones also have the ability to affect mutational rates and error-prone SOS response in bacteria20 and therefore may genetically alter bacterial responses to selective pressures.

Over the past several years, there has been an increase in the use of fluoroquinolones in both the community and hospitals. During this time, we have also observed an increase in the incidence of MRSA.18 These data indicate that an association between fluoroquinolone use and clinically significant methicillin resistance exists. This in vitro study investigates the effect of fluoroquinolones on the expression of methicillin resistance in S. aureus.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacteriology

S. aureus was isolated from clinical specimens submitted for routine culture to the Clinical Microbiology Laboratories at Albany Medical Center, Albany, NY, USA. S. aureus ATCC 29213 was used as an oxacillin- and fluoroquinolone-susceptible and mec(A)-negative strain.

Susceptibility tests

To determine antibiotic activity, standard disc diffusion testing was performed on all isolates.21 Quantitative activities of oxacillin and ciprofloxacin were determined either by Etest (AB Biodisk, North America, Inc., Piscataway, NJ, USA) according to the manufacturer's instructions, or by microdilution assay with 2% NaCl to enhance methicillin resistance.21 To screen for methicillin resistance, S. aureus at a concentration of c. 108 cfu/mL was incubated at 30°C for 24 h on mannitol-salt agar (BBL, Becton Dickinson Cockeysville, MD, USA) with a 1 µg oxacillin susceptibility disc. Four clinical isolates of S. aureus (A, B, C and D) were chosen for experimentation because they were susceptible to fluoroquinolones and exhibited a double zone of inhibition around the oxacillin disc, which indicates heteroresistance to oxacillin. Two fluoroquinolone- and methicillin-susceptible isolates (ATCC 29213 and clinical strain E) were not observed to exhibit a double zone of inhibition with oxacillin. These served as control strains. Ciprofloxacin (Bayer Inc., West Haven, CT, USA), levofloxacin (Ortho-McNeil, Inc., Raritan, NJ, USA), gatifloxacin (Bristol-Myers Squibb Co., Wallingford, CT, USA) and moxifloxacin (Bayer) were obtained from their respective manufacturers.

Population analysis profiling

The isolates were grown to 109 cfu/mL in tryptic soy (TS) broth (BBL) for 20 h at 37°C while shaking at 145 rpm. The TS broth contained either no antibiotic (control) or 0.5 x MIC of the fluoroquinolone for each isolate. Cultures were then plated onto TS agar with 2% NaCl, and TS agar with 2% NaCl and oxacillin 128 mg/L. Colonies were counted after 24 and 48 h at 37°C, respectively. Population analysis profiling was used to determine the resistance index, which was defined as the proportion of cfu per millilitre expressing resistance to oxacillin. This was expressed as the ratio of the number of colonies growing on agar with oxacillin to the number of colonies on antibiotic-free agar (control). For two heteroresistant strains (A and B) the assays were repeated and cultures plated onto TS agar with oxacillin ranging from 0.5 to 128 mg/L for strain A, and from 16 to 128 mg/L for strain B. Strain E served as the methicillin-susceptible control.

Genetic fingerprinting and detection of the mec(A) gene

Agarose plugs containing total cellular DNAs from isolates of S. aureus were prepared using the GenePath Group 1 Reagent kit (Bio-Rad, Hercules, CA, USA). DNAs were restricted with SmaI and subjected to pulsed-field gel electrophoresis (PFGE) in the GenePath System according to the manufacturer's instructions. S. aureus strain NCTC 8325 was included on the gel as a standard. The resulting patterns of restriction fragments (‘fingerprints’) were compared for evidence of genetic similarity using the Bio-Rad Molecular Analyst software. The fingerprints were then transferred to a nylon Southern blot by a standard protocol, modified by extending the depurination treatment to 40 min to enhance the transfer of the large DNA fragments.22 mec(A) sequences on the blot were detected with a digoxigenin-labelled 1.3 kb PstI probe fragment from pGO158.23 Labelling, hybridization and detection of the probe were carried out with the Non-Radioactive Labeling and Detection kit [Boehringer-Mannheim (Roche), Indianapolis, IN, USA].

Growth curve

The effect of 0.5 x MIC of ciprofloxacin on bacterial growth for strain B was determined by standard broth growth curve. Briefly, 105 cfu/mL were inoculated into 50 mL of TS broth with and without ciprofloxacin 0.19 mg/L (0.5 x MIC). The cultures were incubated at 37°C for 20 h while shaking at 145 rpm. Aliquots were removed at various time points for plating onto TS agar with 2% NaCl, and TS agar with 2% NaCl plus oxacillin 128 mg/L. The number of cfu/mL was determined after 24 and 48 h at 37°C, respectively. The resistance index was calculated for each time point.

Dose–response

To determine the effect of various concentrations of ciprofloxacin on the enhancement of oxacillin resistance, c. 5 x 105 cfu/mL was inoculated into 5 mL of TS broth in 17 x 100 mm tubes with doubling dilutions of ciprofloxacin, and one broth tube without the antibiotic. After incubation at 37°C for 20 h while shaking at 145 rpm, aliquots were plated on to TS agar with 2% NaCl, and TS agar with 2% NaCl plus oxacillin 128 mg/L. The number of cfu/mL were determined after 24 and 48 h at 37°C, respectively, and the resistance index was calculated.

Fluctuation assay

Approximately 105 cfu/mL of strain B from an overnight blood agar plate (BBL) was inoculated into fifty 17 x 100 mm tubes containing 5 mL TS broth each. Twenty-five tubes contained ciprofloxacin 0.19 mg/L (0.5 x MIC of ciprofloxacin for strain B), and 25 served as controls without added ciprofloxacin. The inoculated tubes were incubated for 20 h at 37°C while shaking at 145 rpm. Serial dilutions to determine cfu/mL were plated on TS agar with 2% NaCl and oxacillin 128 mg/L. Five tubes from each group were selected for plating onto TS agar without oxacillin to determine total cfu/mL following the incubation period. The mutation rate was calculated as described previously.19


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Four S. aureus strains (A, B, C and D) were shown to be positive for mec(A) by hybridization, and had distinct PFGE patterns. Each had a double zone of inhibition around the oxacillin diffusion discs on media containing 2% NaCl, which indicated that they were heteroresistant to oxacillin. They also produced double zones of inhibition around oxacillin Etest strips, which were read as two distinct oxacillin MICs (Table 1Go). The lower MIC represented the major population and the higher MIC defined the lesser population. ATCC 29213 and strain E did not display heteroresistance and were negative for mec(A). Multiple zones of inhibition were not observed around the fluoroquinolone discs or strips for any of the strains tested (Table 1Go).


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Table 1. MICs (mg/L) of oxacillin and fluorquinolones for the S. aureus strains tested
 
In a dose–response assay for strain B, the resistance index of cells that grew in the presence of oxacillin 128 mg/L increased with exposure to higher concentrations of ciprofloxacin (Figure 1Go). Similar results were obtained for the other fluoroquinolones and mec(A)-containing strains. Fluoroquinolone concentrations at approximately 0.5 x MIC were chosen for the remaining assays since it was approximately midway on the exponential portion of the dose–response curve.



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Figure 1. Dose–response curve of mec(A)-positive strain B (ciprofloxacin MIC = 0.38 mg/L) to increasing concentrations of ciprofloxacin in TS broth after 20 h at 37°C. Population analysis profiling was used to determine the resistance index, which was defined as the proportion of cfu/mL expressing resistance to oxacillin. This was expressed as the ratio of the number of colonies growing on agar with oxacillin to the number of colonies on antibiotic-free agar (control).

 
The resistance index was calculated for strains A and B plated on various oxacillin concentrations after 20 h of exposure to 0.5 x MIC of ciprofloxacin (0.25 and 0.19 mg/L, respectively). The 20 h time-point was chosen from growth curve assays because at 0.5 x MIC, the fluoroquinolone-exposed strains just achieved stationary phase. At all concentrations of oxacillin >8 mg/L, the resistance index was higher for strains A and B cultures pre-exposed to ciprofloxacin than for those not exposed to ciprofloxacin (Figure 2Go).



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Figure 2. Resistance indices for mec(A)-positive strains A and B plated on increasing concentrations of oxacillin after growth for 20 h at 37°C in TS broth. This was in the presence (•) or absence ({circ}) of 0.5 x MIC of ciprofloxacin (0.25 mg/L) for strain A, and in the presence ({blacktriangleup}) or absence ({triangleup}) of 0.5 x MIC of ciprofloxacin (0.19 mg/L) for strain B. Strain E, a mec(A)-negative strain, following growth as above with ({blacksquare}) and without ({square}) exposure to 0.5 x MIC of ciprofloxacin (0.38 mg/L).

 
The initial population profiling assays were performed on three mec(A)-containing isolates (A, B and C) and showed a >100-fold enhancement of the population of cells that grew on oxacillin 128 mg/L after growing in the presence of 0.5 x MIC of ciprofloxacin for 20 h (Table 2Go). The two mec(A)-negative strains did not show this enhancement (strain E; Figure 2Go and Table 2Go). Ciprofloxacin provided the greatest enhancement when the growth was allowed up to 20 h. The colonies that grew on agar with oxacillin 128 mg/L were passed onto media without oxacillin for five consecutive days without reducing their level of oxacillin resistance.


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Table 2. Resistance indexa on oxacillin 128 mg/L after 20 h exposure to quinoloneb
 
The effect of 0.5 x MIC of ciprofloxacin on total cell growth was an average reduction of viable cells between 46% and 53% for growth curves of each of the four mec(A)-positive strains after 20 h of incubation at 37°C compared with the control growth curves without the antibiotic (data not shown). From the growth curves, the earliest time to detection of increased levels of oxacillin-resistant S. aureus was between 8–12 h of exposure to the fluoroquinolone just before entering exponential growth. At 8 h, the absolute number of colony-forming units on agar with oxacillin 128 mg/L was similar whether the strain was exposed to a fluoroquinolone or not. However, the proportion of oxacillin-resistant cells in the total population (resistance index) was greater following fluoroquinolone exposure (Table 3Go).


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Table 3. Resistance index (RI) of S. aureus at oxacillin 128 mg/L following exposure to fluoroquinolones
 
The fluctuation assay resulted in a narrow range of cfu/tube that would grow on agar with oxacillin 128 mg/L for both the control and fluoroquinolone-treated cultures (mean: 5 x 102 ± 3.9 x 102 and 1 x 107 ± 6.9 x 106, respectively).

The ability of heteroresistant MRSA to express subpopulations with enhanced resistance to both oxacillin and fluoroquinolones was examined, selecting colonies that grew on agar at or above their respective MICs. Colonies of strain B that grew on agar with oxacillin 128 mg/L were transferred to media containing ciprofloxacin. Of these, 100, 63 and 6% grew when transferred to agar with 0.38, 0.76 and 1.52 mg/L of ciprofloxacin, respectively (1 x, 2 x and 4 x MIC, respectively). None was detected without first being selected with oxacillin 128 mg/L. Similarly, colonies of strain B that were selected for growth on agar media containing 0.38, 0.76 and 1.52 mg/L of ciprofloxacin could be transferred to agar with oxacillin 128 mg/L at a rate of 80, 50 and 55%, respectively. Again, no growth on the oxacillin medium was detected unless strain B was first exposed to at least the MIC of ciprofloxacin. Colonies were also selected from media with oxacillin 128 mg/L and their MICs to the four fluoroquinolones were 1.5- to 3-fold higher as determined by Etest. The increase in MIC for all the fluoroquinolones occurred following exposure to oxacillin regardless of previous exposure to the fluoroquinolone. The higher resistance was also stable after five consecutive days of passage onto media without oxacillin or the fluoroquinolone.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
These observations indicate that there is an association between the action of fluoroquinolones on mec(A)-positive S. aureus and the increase in the resistance index for methicillin resistance. The data do not indicate a fluoroquinolone-mediated mutation. If mutational variants that allowed increased oxacillin resistance occurred, the mutations would have had to occur at the same time in each culture in the fluctuation assay. The more likely explanation is that ciprofloxacin was not acting as a mutagen but was selecting the more fluoroquinolone-resistant cells in the population, which were also predominantly more resistant to oxacillin.

This is similar to other reports in that the higher resistance of MRSA to fluoroquinolones is not the result of increased mutation rates. S. aureus strains that were highly susceptible to methicillin had the same mutation frequencies as MRSA.24 Our study cannot rule out completely the possibility that the fluoroquinolone may select for other types of mutations that lead to oxacillin resistance without directly mutating the oxacillin resistance genes. However, a relationship is indicated by our study, since the proportion of heteroresistant strains expressing methicillin resistance increased following exposure to fluoroquinolones, as did fluoroquinolone resistance following exposure to oxacillin. The apparent association between methicillin and fluoroquinolone resistance points to the high level of complexity of antimicrobial resistance in MRSA strains. The mechanism of interaction of fluoroquinolone exposure with methicillin resistance is unknown; however, a number of possibilities exist. Regulatory genes such as mec(I), which functions as a regulatory repressor of the mec(A) gene, could be effected by fluoroquinolone. Basal levels of mec(A) gene transcription were elevated in highly resistant MRSA that lacked or had a point mutation in the mec(I) gene.25 Fluoroquinolones might be repressing the mec(I) gene and, therefore, upregulation of the mec(A) gene is possible. The results of the fluctuation assay indicated that mutations were not occurring or, if they were, it was at a high rate. The strains grown in the presence of fluoroquinolone had approximately the same number of oxacillin-resistant colonies as those not exposed to fluoroquinolones. The representative colonies selected on oxacillin were also more resistant to inhibition by the fluoroquinolones and the resistance was across the class of fluoroquinolones used in this study. These data indicate that mec(A)-positive, heteroresistant S. aureus contains subpopulations that are resistant to oxacillin and fluoroquinolones. Selection by either antibiotic class enriches the overall population for resistance to both.

Chromosomal maps show the location of mec(A) between protein A and DNA gyrase genes.7 Since it is rare to have resistance to fluoroquinolones in methicillinsusceptible strains,17 the effect of fluoroquinolones on the selection of resistant gyrases could coincidentally influence mec(A)-mediated oxacillin resistance. This would be consistent with our observations that selection of resistant subpopulations by either fluoroquinolone or oxacillin enhances the detection of resistance for the other antimicrobial agent in mec(A)-bearing strains.

Other heterotropic associations have been reported in mec(A)-positive S. aureus. Expression of fibronectin-binding proteins was enhanced by fluoroquinolone-resistant S. aureus exposed to subinhibitory levels of ciprofloxacin.26 The susceptibilities of other structurally unrelated antibiotics were affected in MRSA by sub-MIC levels of ciprofloxacin.17 The isolation of multiply resistant variants for imipenem, fusidic acid and gentamicin increased >100-fold following exposure of MRSA to ciprofloxacin. Strain differences in the expression of resistant variants were also noted in their study. In our study, differences in the effect of the various fluoroquinolones were observed between the MRSA strains tested. Ciprofloxacin, which has the least activity against S. aureus, had the greatest effect. This effect diminished as the fluoroquinolones increased in Grampositive activity. We also noted that the resistance indices differed for the four mec(A) strains after fluoroquinolone exposure, although before exposure the resistance indices were not significantly different.

Fluoroquinolones cause an extended lag phase in MRSA that is due to the inhibition of DNA replication. As opposed to ß-lactams, which inhibit S. aureus growth for 1–3 h, the sub-MIC of fluoroquinolones can inhibit growth for up to 7 h and bactericidal activity can occur rapidly in 4–6 h.27,28 This timing is consistent with the extended 8 h lag phase in the growth of our strains in the presence of 0.5 x MIC of fluoroquinolones. During this period the more fluoroquinolone-susceptible populations would be inhibited or killed while the fluoroquinolone-resistant populations would continue to grow. The coincidence of death and growth would appear as the extended lag phase that we observed. If fluoroquinolone resistance is preferentially associated with the more oxacillin-resistant subpopulations, growth in the presence of subinhibitory levels of a fluoroquinolone would selectively enrich for the more oxacillin-resistant subpopulations. This explanation would be consistent with our observations. Therefore, the effect of the fluoroquinolone may be purely metabolic, as changes in pH, osmolarity and temperature that affect the growth rate of S. aureus enhance the detection of MRSA subpopulations. The control of the switch from heterologous to homologous expression of mec(A) is unknown29 but regulation of mec(A) transcription is correlated with expression of methicillin resistance.30 Fluoroquinolones, by delaying DNA replication, may be affecting transcription and therefore repressor protein production. The consequence of this may also cause an increase in higher level oxacillin resistance by decreased repressor levels.

An increase in MRSA carriage rate can be partly the result of the selective pressures of antimicrobial use.31,32 Other indirect factors such as antimicrobial agents that decrease bacterial competition, which allows increased colonization of MRSA in the host,33 and transmission of MRSA, may also be contributing to the increased incidence. It appears that S. aureus strains possessing mec(A) are capable of expressing heterotropic resistance to both methicillin and fluoroquinolones, which would be a selective advantage in an environment of widespread fluoroquinolone use. This study indicates that the use of fluoroquinolones with low potency for S. aureus may lead to an increased risk of colonization in individuals with high-level oxacillin-resistant strains. This is supported by the fact that countries which rarely use fluoroquinolones have a low incidence of MRSA.34 This study shows that the selection of high-level oxacillin resistance in heteroresistant S. aureus appears to be associated with fluoroquinolone exposure.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Benjamin Lomaestro for his comments and suggestions provided following his review of this manuscript.


    Notes
 
* Corresponding author. Tel: +1-518-262-3506; Fax: +1-518-262-4337; E-mail: venezir{at}mail.amc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Ayliffe, G. A. J. (1997). The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clinical Infectious Diseases 24, Suppl. 1, S74–9.[ISI][Medline]

2 . Rubin, R. J., Harrington, C. A., Poon, A., Dietrich, K., Greene, J. A. & Moiduddin, A. (1999). The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerging Infectious Diseases 5, 9–17.[ISI][Medline]

3 . Anonymous. (2000). Four pediatric deaths from community-acquired methicillin-resistant Staphyococcus aureus – Minnesota and North Dakota 1997–1999. Morbidity and Mortality Weekly Report 48, 707–10.

4 . Villari, P., Farullo, C., Torre, I. & Nani, E. (1998). Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy. European Journal of Epidemiology 14, 807–16.[ISI][Medline]

5 . Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews 10, 781–91.[Abstract]

6 . de Lencastre, H., de Jonge, B. L., Matthews P. R. & Tomasz, A. (1994). Molecular aspects of methicillin resistance in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 33, 7–24.[Abstract]

7 . Archer, G. L., Niemeyer, D. M., Thanassi, J. A. & Pucci, M. J. (1994). Dissemination among staphylococci of DNA sequences associated with methicillin resistance. Antimicrobial Agents and Chemotherapy 38, 447–54.[Abstract]

8 . Roychoudhury, S., Dotzlaf, S., Ghag, S. & Yeh, W. K. (1994). Purification, properties and kinetics and enzymatic acylation with ß-lactams of soluble penicillin-binding protein. Journal of Biological Chemistry 269, 12067–73.[Abstract/Free Full Text]

9 . Hurlimann-Dalel, R. L., Ryffel, C., Kayser, F. H. & Berger-Bachi, B. (1992). Survey of the methicillin resistance-associated gene mecA, mecR1–mecI, and femA–femB in clinical isolates of methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 36, 2617–21.[Abstract]

10 . Kuwahara-Aral, K., Kondo, N., Hori, S., Tateda-Suzuki, E. & Hiramatsu, K. (1996). Suppression of methicillin resistance in a mecA-containing pre-methicillin-resistant Staphylococcus aureus strain is caused by the mecI-mediated repression of PBP 2' production. Antimicrobial Agents and Chemotherapy 40, 2680–5.[Abstract]

11 . Hartman, B. J. & Tomasz, A. (1986). Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 29, 85–92.[ISI][Medline]

12 . Ito, T., Katayama, Y. & Hiramatsu, K. (1999). Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrobial Agents and Chemotherapy 43, 1449–58.[Abstract/Free Full Text]

13 . Gustafson, J. E., Berger-Bachi, B., Strassle, A. & Wilkinson, B. J. (1992). Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 36, 566–72.[Abstract]

14 . Graham, K. K., Hufcut, R. M., Copeland, C. M., Stone, J. E., Lai, L. L., Villano, J. R. et al. (2000). Fluoroquinolone exposure and the development of nosocomial MRSA bacteremia. In Program and Abstracts of the Fourth Decennial International Conference on Nosocomial and Healthcare-Associated Infections, Atlanta, GA, 2000. Abstract S-M3-06, p. 52. Centers for Disease Control and Prevention, Atlanta, GA.

15 . Crowcroft, N. S., Ronveaux, O., Monnet, D. L. & Mertens, R. (1999). Methicillin-resistant Staphylococcus aureus and antimicrobial use in Belgian hospitals. Infection Control and Hospital Epidemiology 20, 31–6.[ISI][Medline]

16 . Nakamura, S. (1997). Mechanisms of quinolone resistance. Journal of Infection Chemotherapy 3, 128–38.

17 . Acar, J. F. & Goldstein, F. W. (1997). Trends in bacterial resistance to fluoroquinolones. Clinical Infectious Diseases 24, Suppl. 1, 567–73.

18 . Venezia, R. A., Domaracki, B. E., Evans, A. M., Preston, K. E. & Graffunder, E. M. (2000). Fluorquinolone (FQ) selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus strains (MRSA). In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 763, p. 84. American Society for Microbiology, Washington, DC.

19 . Fung-Tomc, J., Kolek, B. & Bonner, D. P. (1993). Ciprofloxacin-induced, low-level resistance to structurally unrelated antibiotics in Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 1289–96.[Abstract]

20 . Mamber, S. W., Kolek, B., Brookshire, K. W., Bonner, D. P. & Fung-Tomc, J. (1993). Activity of quinolones in the Ames Salmonella TA102 mutagenicity test and other bacterial genotoxicity assays. Antimicrobial Agents and Chemotherapy 37, 213–7.[Abstract]

21 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Animicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M100-510. NCCLS, Villanova, PA.

22 . Sambrook, J., Fritch, E. F. & Maniatis, T. (1999). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

23 . Tomasz, A., Nachman, S. & Leaf, H. (1991). Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci. Antimicrobial Agents and Chemotherapy 35, 124–9.[ISI][Medline]

24 . Schmitz, F.-J., Fluit, A. C., Hafner, D., Beeck, A., Perdikouli, M., Boos, M. et al. (2000). Development of resistance to ciprofloxacin, rifampin, and mupirocin in methicillin-susceptible and -resistant Staphylococcus aureus isolates. Antimicrobial Agents and Chemotherapy 44, 3229–31.[Abstract/Free Full Text]

25 . Santo, T., Hiyama, E., Takesue, Y., Matsuura, Y. & Yokoyama, T. (1999). Analysis of mec regulator genes in clinical methicillin-resistant Staphylococcus aureus isolates according to the production of coagulase, types of enterotoxin, and toxic shock syndrome toxin-1. Hiroshima Journal of Medical Sciences 48, 49–56.[Medline]

26 . Bisognano, C., Vaudaux, P., Rohner, P., Lew, D. P. & Hooper, D. C. (2000). Induction of fibronectin-binding proteins and increased adhesion of quinolone-resistant Staphylococcus aureus by subinhibitory levels of ciprofloxacin. Antimicrobial Agents and Chemotherapy 44, 1428–37.[Abstract/Free Full Text]

27 . Fung-Tomc, J. C., Gradelski, E., Valera, L., Kolek, B. & Bonner, D. P. (2000). Comparative killing rates of fluroquinolones and cell wall-active agents. Antimicrobial Agents and Chemotherapy 44, 1377–80.[Abstract/Free Full Text]

28 . Guan, L., Blumenthal, R. M. & Burnham, J. C. (1992). Analysis of macromolecular biosynthesis to define the quinolone-induced postantibiotic effect in Escherichia coli. Antimicrobial Agents and Chemotherapy 36, 2118–24.[Abstract]

29 . Berger-Bachi, B., Strassle, A., Gustafson, J. E. & Kayser, F. H. (1992). Mapping and characterization of multiple chromosomal factors involved in methicillin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 36, 1367–73.[Abstract]

30 . Ryffel, C., Kayser, F. H. & Berger-Bachi, B. (1992). Correlation between regulation of mecA transcription and expression of methicillin resistance in Staphylococci. Antimicrobial Agents and Chemotherapy 36, 25–31.[Abstract]

31 . Pegues, A. A., Colby, C., Hibberd, P. L., Cohen L. G., Ausubel, F. M., Calderwood, S. B. et al. (1998). The epidemiology of resistance to ofloxacin and oxacillin among clinical coagulase-negative staphylococcal isolates: Analysis of risk factors and strain types. Clinical Infectious Diseases 26, 72–9.[ISI][Medline]

32 . L’Heriteau, F., Lucet, J.-C., Scanvic, A. & Bouvet, E. (1999). Community-acquired methicillin-resistant Staphylococcus aureus and familial transmission. Journal of the American Medical Association 282, 1038–9.[Free Full Text]

33 . Otto, M., Sussmuth, R., Vuong, C., Jung, G. & Gotz, F. (1999). Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives. FEBS Letters 450, 257–62.[ISI][Medline]

34 . Shopsin, B., Mathema, B., Martinez, J., Ha, E., Campo, M. L., Fierman, A. et al. (2000). Prevalence of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in the community. Journal of Infectious Diseases 182, 359–62.[ISI][Medline]

Received 15 December 2000; returned 28 March 2001; revised 25 April 2001; accepted 21 May 2001