The effects of NorA inhibition on the activities of levofloxacin, ciprofloxacin and norfloxacin against two genetically related strains of Staphylococcus aureus in an in-vitro infection model

Jeffrey R. Aeschlimanna,c, Glenn W. Kaatzb,c,d and Michael J. Rybaka,c,d,*

a The Anti-Infective Research Laboratory, Department of Pharmacy Services (1B), Detroit Receiving Hospital and University Health Center, 4201 St Antoine Blvd, Detroit, MI 48201; b Veterans' Administration Medical Center, Detroit; c College of Pharmacy and Allied Health Professions, and d Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, Wayne State University, Detroit, MI 48201, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
NorA is a membrane-associated multidrug efflux protein that can decrease susceptibility to fluoroquinolones in Staphylococcus aureus. We have previously determined that NorA inhibition can increase fluoroquinolone killing activity and post-antibiotic effect. In the current investigation, we studied the killing activity and development of resistance for levofloxacin, ciprofloxacin and norfloxacin with or without the H+/K+ ATPase inhibitor omeprazole, in a wild-type strain of S. aureus (SA-1199) and its NorA hyperproducing mutant (SA-1199-3) in an in-vitro pharmacodynamic model with infected fibrin-platetet matrices. Each drug was administered every 12–24 h for 72 h and human pharmacokinetics were simulated. Levofloxacin was the most potent fluoroquinolone against both strains and its activity was not significantly affected by combination with omeprazole. The addition of omeprazole to ciprofloxacin significantly lowered colony counts at all time-points against both strains and decreased the time to 99.9% kill from 72.2 h to 33.8 h against SA-1199. The addition of omeprazole minimally increased norfloxacin activity against both strains. Omeprazole decreased the frequency of ciprofloxacin resistance nearly 100-fold at the 24 h time-point, but the frequency of resistance was not significantly different for any of the fluoroquinolone regimens after this time-point. No resistance was detected during levofloxacin regimens. The hydrophobic fluoroquinolones such as levofloxacin appear to circumvent NorA efflux, which may contribute to their better activity and decreased resistance rates against staphylococci. More durable and potent NorA inhibitor compounds are needed that can improve killing activity and prevent resistance.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) rapidly become resistant to the first generation of fluoroquinolones (such as norfloxacin and ciprofloxacin), severely limiting their utility against these problematic pathogens.1,2 Fluoroquinolone resistance in S. aureususually occurs via three mechanisms: (i) mutations in the grl genes which alter the topoisomerase IV subunits,1,2,3,4,5 (ii) mutations in the gyr genes which alter the DNA gyrase subunits,4,5 and (iii) increased transcription of the norA gene leading to the presence of increased quantities of the membrane-associated NorA efflux protein.6,7,8,9,10,11,12,13 NorA is present in all wild-type S. aureus and appears to function in the same manner as other efflux proteins such as TetA and Bmr. It has greatest homology with Bmr and non-selectively removes naturally occurring compounds (such as chloramphenicol and puromycin) and synthetic compounds (such as fluoroquinolones and ethidium bromide) which are toxic to the bacterial cell.8,11,12,13

NorA appears to have higher affinity for more hydrophilic fluoroquinolones (ciprofloxacin, enoxacin, norfloxacin) than more hydrophobic compounds (levofloxacin, sparfloxacin, trovafloxacin).8,14 The pump's activity can be inhibited either by disruption of the proton gradient (by compounds like carbonylcyanide m-chlorophenyl-hydrazone (CCCP)) or by competitive inhibitors (such as reserpine and verapamil).6,7,8,9,10,11,12,13,15 Inhibition of NorA activity could possibly improve fluoroquinolone activity, as restoration of [3H]norfloxacin drug accumulation to wild-type levels has been reported in fluoroquinolone-resistant S. aureus with the addition of CCCP.8,9 Combinations with reserpine have produced similar effects on strains of S. aureus that constitutively and inducibly hyperproduce NorA.7 Although fluoroquinolone resistance in S. aureus occurs in a stepwise manner and constitutive hyperproduction of NorA is not typically observed initially,4 transient up-regulations might allow survival in the presence of these drugs and permit the emergence of grlAand gyrA mutations. If this hypothesis is true, inhibition of NorA might also delay or reduce fluoroquinolone resistance in susceptible strains of S. aureus.10,16

Most studies of NorA inhibition have evaluated drug uptake over periods of minutes and/or the effects on MICs. It is also important to determine whether the effects of NorA inhibitors can be sustained and whether the NorA inhibition can also improve fluoroquinolone pharmacodynamic parameters. Recently, we determined that reserpine and the H+/K+ ATPase inhibitors omeprazole and lansoprazole could significantly reduce MICs, increase killing activity (in static time–kill curves) and prolong the post-antibiotic effect of various fluoroquinolones.17 In the current study, we compared the activity of three fluoroquinolones alone or in combination with omeprazole against two genetically related strains of S. aureus in an in-vitro pharmacodynamic model with infected fibrin–platelet matrices. We also determined whether NorA inhibitors could affect the emergence of fluoroquinolone resistance during repeated simulated human dosing regimens.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

S. aureus 1199 (SA-1199, a wild-type clinical isolate) and S. aureus1199-3 (SA-1199-3, a laboratory-derived mutant with inducible NorA hyperproduction) were used in the infection models. These isolates have been described previously.6,7,8,9 Production of NorA was induced in SA-1199-3 prior to each experiment by overnight growth in Mueller–Hinton broth supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) (SMHB; Difco, Detroit, MI, USA) or on Mueller–Hinton agar that contained 0.25 x MIC concentrations of cetrimide (lot No. 36H04421; Sigma Chemical Co., St Louis, MO, USA).

Media and antibiotics

SMHB was used for all susceptibility testing and in the in-vitro infection models. Tryptic soy agar (TSA, Difco) plates were used to determine colony counts from experimental samples. Levofloxacin was provided by R. W. Johnson Pharmaceutical Research Institute (lots N8017 and N8018) and ciprofloxacin and norfloxacin were commercially purchased (Bayer, West Haven, CT, USA lot 7BF1 and Sigma, lot 83H0921, respectively). Omeprazole (lot E6828) was provided by Astra Merck (Södertälje, Sweden). All stock solutions of compounds were prepared in sterile water except for omeprazole. Initial stock solutions of omeprazole were prepared in dimethyl sulphoxide and then further diluted to desired concentrations using sterile water or SMHB.

In-vitro antibiotic susceptibility tests

Broth microdilution MICs and MBCs were determined for each fluoroquinolone and NorA inhibitor alone and in combination with omeprazole using NCCLS guidelines.18 Our previous work indicated that omeprazole concentrations of 100 mg/L caused maximal reductions in fluoroquinolone MIC but had no detectable antibacterial effects alone.17

In-vitro pharmacodynamic model with infected fibrin–platelet matrices

The in-vitro infection model used has been previously described.19,20 Infected fibrin–platelet matrices containing approximately 1 x 109 cfu/g were prepared by combining 0.1 mL of concentrated (approximately 1010 cfu/mL) organism suspension, 0.8 mL of human cryoprecipitate antihaemolytic factor from volunteer donors (American National Red Cross, Detroit, MI, USA), 0.05 mL of aprotinin solution (2000 kiu/mL, Sigma) and 0.05 mL of platelet suspension (1:100 dilution of platelet-rich plasma in 0.9% normal saline, providing approximately 250,000–300,000 platelets/g) in a sterile, siliconized 1.5 mL Eppendorf tube. A sterile monofilament line was inserted and 0.1 mL of bovine thrombin solution (5000 units/vial reconstituted with 5 mL of sterile 50 mM calcium chloride solution, GenTrac, WI, USA) was added. The resultant gelatinous mixtures were removed using a sterile 21-gauge needle and placed into the infection models. Models were placed in a water bath and maintained at 37°C for the duration of the experiment. Each experiment was conducted over 72 h and was performed in duplicate to ensure reproducibility.

Pharmacokinetics

Fresh stock solutions of each antibiotic were prepared on the day of the experiment and stored at 2–8°C between administration times. Levofloxacin, ciprofloxacin and norfloxacin were given as bolus injections into the central compartment to simulate regimens of 750 mg iv q24 h, 400 mg iv q12 h and 1600 mg iv q12 h, which produced target peak concentrations of 8 mg/L, 5 mg/L and 4 mg/L, respectively. A peristaltic pump was used to supply fresh SMHB and to remove antibiotic-containing SMHB from the models to simulate levofloxacin, ciprofloxacin and norfloxacin half-lives of 6 h, 3 h and 3 h. During the fluoroquinolone plus omeprazole combination regimens, drug elimination rates were set to the fluoroquinolone half-lives and omeprazole was added to the incoming broth at a constant 100 mg/L concentration. Samples were obtained from the central compartment at 5 min, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h post-infusion to determine antibiotic concentrations. Fluoroquinolone concentrations were determined via standard agar diffusion bioassay methods with Klebsiella pneumoniae ATCC 10031 as the indicator organism.19 This assay had low (0.1 mg/L) and high (3 mg/L) concentration intra- and inter-day coefficients of variation of <=5.5% and correlation coefficients of <=0.95. Fluoroquinolone half-lives, peak and trough concentrations, and area under the concentration–time curves (AUCs) were calculated using the RStrip software program (Micromath, Salt Lake City, UT, USA).

Pharmacodynamics

Two to three infected fibrin–platelet clots were removed from each model at 0, 8, 24, 32, 48 and 72 h. The samples were each weighed, placed in a 2 mL sterile capped vial that was prefilled with 3 mm glass beads and 1.0 mL of 1.25% trypsin solution (1:250 powder, Difco), and homogenized using a mini-bead beater grinder (Biospec Products, Bartlesville, OK, USA). Cold 0.9% normal saline was used to serially dilute the homogenized clots and 20 µL quantities were plated in triplicate on to TSA to determine bacterial densities. Plates were incubated for 24 h at 37°C and colonies counted. The limits of detection for this method were 2.0 log10 cfu/g. Average log10 cfu/g values for each time-point were plotted against time to produce killing curves for the 72 h period and the total reductions over 72 h were compared between regimens. The time to achieve a 99.9% reduction in the starting inoculum was determined by linear regression (if R <= 0.95) or by visual inspection. The area between the killing curves and the growth curves (AUKC) was determined for each regimen using the trapezoidal rule and was correlated with pharmacodynamic parameters such as the AUC24 h/MIC, peak:MIC ratio, trough:MIC ratio, time above the MIC and the MICalone.

Antibiotic resistance

Homogenized samples (0.1 mL) of fibrin–platelet clots were placed on to Mueller–Hinton agar containing levofloxacin, ciprofloxacin or norfloxacin at 2 x, 4 x and 8 x the baseline MIC. The average number of colonies was determined after 24–48 h of incubation at 37°C and was divided by the total number of viable bacteria in the samples at each time-point, to determine the frequency at which resistance at the multiple of MIC developed. MICs were determined for randomly selected colonies to verify decreased fluoroquinolone susceptibility.

Statistical analyses

Bacterial inocula at 72 h and the time required to achieve 99.9% killing were compared between regimens using one-way ANOVA followed by Tukey's test for multiple comparisons. Significant correlations between the AUKC and the fluoroquinolone MICs/MBCs, peak:MIC, time above the MIC or the AUC24 h/MIC were determined using linear regression. The high degree of colinearity of certain pharmacodynamic parameters (R > 0.98) precluded multivariate regression analysis. For all statistical tests, a P value of <0.05 was considered significant. All statistical analyses were performed using SPSS Statistical Software (Release 6.1.3; SPSS Inc., Chicago, IL, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility testing

MICs and MBCs are summarized in Table I. Omeprazole decreased ciprofloxacin and norfloxacin MICs/MBCs two- to four-fold for SA-1199 but had no effect on the levofloxacin susceptibility of this organism. Omeprazole decreased ciprofloxacin and norfloxacin MICs eight-fold and decreased levofloxacin MIC four-fold for SA-1199-3.


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Table I. MICs/MBCs (mg/L) of levofloxacin, ciprofloxacin and norfloxacin with or without omeprazole (100 mg/L) for SA-1199 and SA-1199-3
 
In-vitro infection models: fluoroquinolone monotherapy

The pharmacokinetic parameters obtained in the infection models are summarized in Table II. Levofloxacin caused significantly lower colony counts at 72 h compared with ciprofloxacin or norfloxacin against both SA-1199 and SA-1199-3 (Table III, Figure 1) and its activity was not substantially reduced by the NorA hyperproduction in SA-1199-3. In contrast, ciprofloxacin had significantly less activity against SA-1199-3. Norfloxacin had minimal activity against both strains.


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Table II. Pharmacokinetic parameters for levofloxacin, ciprofloxacin and norfloxacin in the in-vitro infection models
 

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Table III. Bacterial count at 72 h in the in-vitro infection models
 


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Figure 1. Killing curves obtained against (a) SA-1199 and (b) SA-1199-3 in the in-vitro infection models. Each time-point represents the mean log10 cfu/g ± S.D. from four to six samples. (•, growth control; {square}, levofloxacin; {blacksquare}, levofloxacin + omeprazole; {triangleup}, ciprofloxacin; {blacktriangleup}, ciprofloxacin + omeprazole; {lozenge}, norfloxacin; {blacklozenge}, norfloxacin + omeprazole.)

 
In-vitro infection models: fluoroquinolones + omeprazole

Omeprazole had no significant effect on levofloxacin killing activity against either strain. Its addition to ciprofloxacin resulted in significantly lower colony counts and AUKC values against both strains, and it significantly shortened time to 99.9% killing against SA-1199 (33.8 h compared with 72.2 h). The addition of omeprazole slightly reduced colony counts at 72 h and AUKCs for norfloxacin against both strains but activity was still marginal when compared with the other two fluoroquinolones.

Predictors of fluoroquinolone activity against SA-1199 and SA-1199-3

Table IV lists the pharmacodynamic parameters obtained in the infection model. A significant correlation existed between the AUKC and the Peak:MIC or logarithmic AUC:MIC for both the fluoroquinolone monotherapy (R = 0.91 or R = 0.90) and for the combinations with omeprazole regimens (R = 0.87 or R = 0.87). The combination of monotherapy with combination therapy data slightly weakened these correlations (R = 0.86 or R = 0.85).


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Table IV. Pharmacodymanics for levofloxacin, ciprofloxacin and norfloxacin in the in-vitro infection model
 
Frequency of resistance

The resistance frequencies (at 4 x MIC) in the infection models are summarized in Figure 2. Baseline resistance frequencies (at 2 x MIC) for SA-1199 and SA-1199-3 were lower for levofloxacin (<1 x 10-9.2 and 6.3 x 10-10) than for ciprofloxacin (1.4 x 10-8 and 8.0 x 10-9) and norfloxacin (1.0 x 10-8 and 1.8 x 10-–8). No resistance was detected over the 72 h test period for levofloxacin with or without omeprazole against SA-1199. Low-level resistance (at 2 x MIC) was detected for levofloxacin alone against SA-1199-3 at each time-point (average, less than five resistant colonies per sample), but no resistance occurred during combination with omeprazole. High frequencies of resistance (at 4 x MIC) were detected after 24 h for norfloxacin against both strains. Resistance frequencies after 24 h were lower for ciprofloxacin but became similar to norfloxacin frequencies at 48 and 72 h. The addition of omeprazole had no effect on the frequency of norfloxacin resistance for both strains. Omeprazole decreased the resistance frequency for ciprofloxacin by approximately 100-fold for SA-1199-3 at the 24 h time-point only. Resistance frequencies at 2 x and 8 x original MICs were typically one order of magnitude higher and lower than the 4 x MIC values (data not shown). Colonies of SA-1199 and SA-1199-3 that were recovered from norfloxacin-containing plates had MICs that increased to 32 mg/L and <=128 mg/L. The MICs for resistant colonies were similar for bacteria recovered from the omeprazole combination models. The levofloxacin MICs increased two-fold for SA-1199 and four-fold for SA-1199-3, while ciprofloxacin MICs increased four-fold for SA-1199 and 16-fold for SA-1199-3. For the resistant SA-1199 and SA-1199-3 recovered from ciprofloxacin infection models, MICs increased to 4–8 mg/L and 64–128 mg/L. Levofloxacin MICs were unchanged for the ciprofloxacin-resistant SA-1199. For the resistant SA-1199-3 bacteria from the ciprofloxacin and ciprofloxacin + omeprazole models, levofloxacin MICs for SA-1199-3 increased four-fold and 128-fold, respectively.



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Figure 2. Frequencies of resistance (at 4 x MIC) in the in-vitro infection models for (a) ciprofloxacin and (b) norfloxacin, with or without omeprazole against SA-1199 and SA-1199-3. Resistance at 4 x MIC was undetectable for all levofloxacin regimens. {circ}, ciprofloxacin or norfloxacin against SA-1199; •, ciprofloxacin or norfloxacin + omeprazole against SA-1199; {square}, ciprofloxacin or norfloxacin against SA-1199-3; {blacksquare}, ciprofloxacin or norfloxacin + omeprazole against SA-1199-3. The y-axis values represent 1 raised to the appropriate base 10 value (i.e. 1e-2 is equivalent to 1x 10-2).)

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The normal function of the staphylococcal NorA efflux protein can be inhibited by direct competitors or protonophores causing increases in intracellular accumulation of fluoroquinolones, decreases MICs, improves killing activity, prolonged post-antibiotic effects, and possibly helping to prevent the emergence of resistant mutants.6,7,8,9,10,11,12,13,14,15,16,17 NorA preferentially affects the more hydrophilic fluoroquinolones and has its greatest impact on the antibacterial parameters of these drugs.6,7,8,9,17 Most of the studies that have generated this data have evaluated only the short-term effects of NorA inhibitors on drug uptake or have determined the effects on activity using constant drug concentrations. In the current study, we simulated typical concentration–time profiles obtained during fluoroquinolone dosing regimens in humans to determine whether improved activity might also be observed in the clinical setting. Although induction of NorA by cetrimide is not a phenomenon that would occur in vivo, we used this experimental technique with SA-1199-3 because it allowed for a clearer assessment of the effects of NorA inhibition on fluoroquinolone activity. Most constitutive NorA hyperproducing strains obtained clinically commonly have additional mutations that increase fluoroquinolone resistance, such as grl or gyr mutations.4,6

Levofloxacin had the lowest baseline MIC for SA-1199 and its susceptibility was not affected by omeprazole. This was expected as it is more hydrophobic than ciprofloxacin and norfloxacin and supports previous findings.6,7,8,9,14,17 The addition of omeprazole resulted in much more dramatic improvements in ciprofloxacin and norfloxacin susceptibility in both strains. Levofloxacin activity was decreased against SA-1199-3 but the presence of omeprazole reduced its MIC value four-fold. This observation could be related to the dramatically higher expression of NorA in SA-1199-3 as compared with the parent strain.7

Fluoroquinolone activity in the infection models was associated with the AUC:MIC or Peak:MIC ratios. These findings agree with previous studies of pharmacodynamic predictors of activity.21,22 The addition of omeprazole improved antibacterial activity for ciprofloxacin and norfloxacin against both strains and these improvements also correlated with typical pharmacodynamic factors. The improvements in activity in the infection models were not as dramatic as those previously observed in our test tube kill curve studies.17 Important factors such as a higher bacterial inocula, fluctuating antibiotic concentrations and a decreased penetration to the bacteria in the fibrin–platelet matrix probably helped cause the decreased activity.

The development of levofloxacin resistance did not occur in SA-1199 and occurred in only a few colonies of SA-1199-3. In contrast, high levels of norfloxacin and ciprofloxacin resistance occurred in both strains of S. aureus. Omeprazole did not influence norfloxacin resistance but did appear to substantially decrease ciprofloxacin resistance rates at 24 h for SA-1199-3. These data differ from a report that described 100-fold decreases in resistant subpopulations when reserpine was added to agar containing 2 x MIC concentrations of norfloxacin, but support the results from recent investigations where deletion of the norA gene caused dramatic reductions in baseline ciprofloxacin resistance rates.10,16 Unlike the first investigation, we used a protonophore NorA inhibitor (as opposed to a direct NorA inhibitor) and also exposed the bacteria to both high and low fluoroquinolone concentrations for 72 h. These important differences might have helped to increase resistance development and suggest that the durability of NorA inhibition by protonophores may be limited. Interestingly, the addition of omeprazole to ciprofloxacin in the infection models caused higher-level ciprofloxacin resistance in SA-1199-3 and also caused high-level levofloxacin resistance. The mechanism(s) for these observations are currently unknown and warrant further investigation.

Peak:MIC ratios of >12 appear to decrease or prevent fluoroquinolone resistance in both Gram-positive and Gram-negative bacteria.21 In our experiments, peak:MIC ratios of <=12 were not adequate to prevent resistance, since SA-1199 developed resistance in the ciprofloxacin, ciprofloxacin + omeprazole and norfloxacin + omeprazole models where peak:MIC ratios were 24, 48 and 32. High initial bacterial inocula, the presence of small quantities of resistant mutants at baseline, repeated antimicrobial exposures and the extended duration of our infection models (72 h versus 24 h evaluation) may well account for the discrepancy with previous reports.

In conclusion, we determined that NorA inhibition by omeprazole modestly improves the activity of the hydrophilic fluoroquinolones norfloxacin and ciprofloxacin against a wild-type strain of S. aureus and a mutant strain with inducible NorA hyperproduction, in an in-vitro infection model. Levofloxacin, a more hydrophobic fluoroquinolone that is less affected by NorA, had the most potent activity against both of these strains. Inhibition of NorA by omeprazole did not substantially decrease the development of ciprofloxacin or norfloxacin resistance. Resistance to levofloxacin was minimal for both strains regardless of the presence of omeprazole. More potent and/or specific NorA inhibitors with prolonged effects on the efflux protein are needed, since the concentrations of omeprazole used in the current investigation cannot be attained in humans. The ability of levofloxacin (and the other newer generation fluoroquinolones) to avoid the effects of NorA appears to play a role in their improved activity against staphylococci and their lower resistance potential.


    Acknowledgments
 
This research was supported by a grant from the R. W. Johnson Pharmaceutical Research Institute.


    Notes
 
* Corresponding author. Tel: +1-313-745-4554; Fax: +1-313-993-2522; E-mail: mrybak{at}dmc.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Cambau, E. & Gutmann, L. (1993). Mechanisms of resistance to quinolones. Drugs 45, Suppl. 3, 15–23.[ISI][Medline]

2 . Nakanishi, N., Yoshida, S., Wakebe, H., Inoue, M., Yamaguchi, T. & Mitsuhashi, S. (1991). Mechanisms of clinical resistance to fluoroquinolones in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 35, 2562–7.[ISI][Medline]

3 . Wiedemann, B. & Heisig, P. (1994). Mechanisms of quinolone resistance. Infection 22, Suppl. 2, 73–9.

4 . Ferrero, L., Cameron, B. & Crouzet, J. (1995). Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 1554–8.[Abstract]

5 . Tanaka, M., Zhang, Y. X., Ishida, H., Akasaka, T., Sato, K. & Hayakawa, I. (1995). Mechanisms of 4-quinolone resistance in quinolone-resistant and methicillin-resistant Staphylococcus aureus isolates from Japan and China. Journal of Medical Microbiology 42, 214–9.[Abstract]

6 . Kaatz, G. W. & Seo, S. M. (1997). Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 41, 2733–7.[Abstract]

7 . Kaatz, G. W. & Seo, S. M. (1995). Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 2650–5.[Abstract]

8 . Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1993). Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 1086–94.[Abstract]

9 . Kaatz, G. W., Seo, S. M. & Ruble, C. A. (1991). Mechanisms of fluoroquinolone resistance in Staphylococcus aureus. Journal of Infectious Diseases 163, 1080–6.[ISI][Medline]

10 . Markham, P. N. & Neyfakh, A. A. (1996). Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 40, 2673–4.[Free Full Text]

11 . Neyfakh, A. A. (1992). The multidrug efflux transporter of Bacillus subtilis is a structural and functional homolog of the Staphylococcus NorA protein. Antimicrobial Agents and Chemotherapy 36, 484–5.[Abstract]

12 . Neyfakh, A. A., Borsch, C. M. & Kaatz, G. W. (1993). Fluoroquinolone resistance protein NorA of Staphylococcus aureusis a multidrug efflux transporter. Antimicrobial Agents and Chemotherapy 37, 128–9.[Abstract]

13 . Ng, E. Y., Trucksis, M. & Hooper, D. C. (1994). Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrobial Agents and Chemotherapy 38, 1345–55.[Abstract]

14 . Takenouchi, T., Tabata, F., Iwata, Y., Hanzawa, H., Sugawara, M. & Ohya, S. (1996). Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcusaureus. Antimicrobial Agents and Chemotherapy 40, 1835–42.[Abstract]

15 . Yoshida, S., Kojima, T., Inoue, M. & Mitsuhashi, S. (1991). Uptake of sparfloxacin and norfloxacin by clinical isolates of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 35, 368–70.[ISI][Medline]

16 . Roland, G. E., Sulavik, M. C., Barg, N. L., Li, J. & Miller, P. F. (1998). Inactivation of NorA by allelic replacement results in an antibiotic hypersensitive phenotype in Staphylococcus aureus. In Program and Abstracts of the Ninety-Eighth General Meeting of the American Society for Microbiology, Atlanta, GA. May 17–21, 1998. Abstract V-112, p. 532. American Society for Microbiology, Washington, DC.

17 . Aeschlimann, J. R., Dresser, L. D., Kaatz, G. W. & Rybak, M. J. (1999). Effects of NorA inhibitors on in-vitro antibacterial activities and postantibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 335–40.[Abstract/Free Full Text]

18 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

19 . Kang, S. L., Rybak, M. J., McGrath, B. J., Kaatz, G. W. & Seo, S. M. (1994). Pharmacodynamics of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with rifampin, against methicillin-susceptible and -resistant Staphylococcus aureus in an in-vitro infection model. Antimicrobial Agents and Chemotherapy 38, 2702–9.[Abstract]

20 . Palmer, S. M. & Rybak, M. J. (1996). Pharmacodynamics of once- or twice-daily levofloxacin versus vancomycin, with or without rifampin, against Staphylococcus aureus in an in-vitro model with infected platelet-fibrin clots. Antimicrobial Agents and Chemotherapy 40, 701–5.[Abstract]

21 . Blaser, J., Stone, B. B., Groner, M. C. & Zinner, S. H. (1987). Comparative study with enoxacin and netilmicin in a pharmacodynamic model to determine importance of ratio of antibiotic peak concentration to MIC for bactericidal activity and emergence of resistance. Antimicrobial Agents and Chemotherapy 31, 1054–60.[ISI][Medline]

22 . Dudley, M. N., Mandler, H. D., Gilbert, D., Ericson, J., Mayer, K. H. & Zinner, S. H. (1987). Pharmacokinetics and pharmacodynamics of intravenous ciprofloxacin. Studies in vivo and in an in-vitro model. American Journal of Medicine 82, Suppl. 4A, 363–8.[ISI][Medline]

Received 22 January 1999; returned 25 March 1999; revised 16 April 1999; accepted 11 May 1999