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
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Abstract |
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Introduction |
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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 timekill 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 fibrinplatelet matrices. We also determined whether NorA inhibitors could affect the emergence of fluoroquinolone resistance during repeated simulated human dosing regimens.
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Materials and methods |
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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 MuellerHinton broth supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) (SMHB; Difco, Detroit, MI, USA) or on MuellerHinton 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 fibrinplatelet matrices
The in-vitro infection model used has been previously described.19,20 Infected fibrinplatelet 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 250,000300,000 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 28°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 concentrationtime curves (AUCs)
were calculated using the RStrip software program (Micromath, Salt Lake City, UT, USA).
Pharmacodynamics
Two to three infected fibrinplatelet 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 fibrinplatelet clots were placed on to MuellerHinton 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 2448 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).
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Results |
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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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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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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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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 48 mg/L and 64128 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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Discussion |
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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 fibrinplatelet 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.
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Acknowledgments |
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Notes |
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References |
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Received 22 January 1999; returned 25 March 1999; revised 16 April 1999; accepted 11 May 1999