AAC Accepted Manuscript Posted Online 7 December 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.02334-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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AAC02334-15 REVISED
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Ciprofloxacin Resistance in Klebsiella pneumoniae: Searching for the Optimal Predictor
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Using In Vitro Dynamic Models
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Elena N. Strukova,a Yury A. Portnoy,a Andrey V. Romanov,b Mikhail V. Edelstein,b
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Stephen H. Zinner,c Alexander A. Firsova
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Department of Pharmacokinetics & Pharmacodynamics, Gause Institute of New Antibiotics,
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Moscow,119021, Russiaa; Institute of Antimicrobial Chemotherapy, Smolensk State Medical
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University, Smolensk, Russiab; Department of Medicine, Mount Auburn Hospital, Harvard
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Medical School, Cambridge, Massachusetts, USAc
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Running title: Predicting Bacterial Resistance in In Vitro Models
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................................................................................................................
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Address correspondence to Alexander A. Firsov, [email protected].
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There is growing evidence of applicability of the hypothesis of the mutant selection window
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(MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within
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which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-
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based pharmacokinetic variables are preferable to the respective MIC-based variables as inter-
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strain predictors of resistance. To examine the predictive power of the ratios of the area under the
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curve (AUC24) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of
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three Klebsiella pneumoniae strains with different MPC/MIC ratios was studied. Each organism
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was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak
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antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC.
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Resistant K. pneumoniae mutants were intensively enriched at AUC24/MIC from 60 to 360 h
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(AUC24/MPC from 2.5 to 15 h) but not at the lower or higher AUC24/MIC and AUC24/MPC
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ratios, in accordance with the MSW hypothesis. AUC24/MPC and AUC24/MIC relationships with
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areas under the time courses of ciprofloxacin-resistant K. pneumoniae (AUBCM) were bell-
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shaped. These relationships predict highly variable “anti-mutant” AUC24/MPC ratios (20 to 290
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h) compared to AUC24/MIC ratios (1310 to 2610 h). These findings suggest that the potential of
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the AUC24/MPC ratio as an inter-strain predictor of K. pneumoniae resistance is lower than
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AUC24/MIC.
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An increasing number of reports on the isolation of resistant pathogens (1, 2) combined with a
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weak antibiotic pipeline suggests that optimization of antibiotic therapy should be aimed at the
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suppression of resistance (3, 4). In this regard, dynamic models that mimic antimicrobial
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pharmacokinetics in vitro have been proven to be a useful tool in predicting the amplification of
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resistant mutants at clinically achievable antibiotic concentrations (5). Using these models, bell-
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shaped relationships have been established between the emergence of resistance to
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fluoroquinolones and the ratios of 24-hour area under the concentration-time curve (AUC24) to
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the MIC (6-17). Such relationships have been reported with moxifloxacin, gatifloxacin,
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levofloxacin, ciprofloxacin, an investigational fluoroquinolone АВТ492, pazufloxacin,
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tosufloxacin, garenoxacin, levofloxacin against Staphylococcus aureus (6-8, 14-16),
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moxifloxacin against Streptococcus pneumoniae (12, 13), garenoxacin against Klebsiella
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pneumoniae (15), ciprofloxacin and moxifloxacin against Pseudomonas aeruginosa (11, 12), and
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with ciprofloxacin, marbofloxacin and enrofloxacin against Escherichia coli (9, 10, 17). In some
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of these studies (6-15, 17, 18), changes in susceptibility of antibiotic-exposed bacteria and/or
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their enrichment with resistant mutants were observed at fluoroquinolone concentrations above
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the MIC but below the mutant prevention concentration (MPC), i.e., inside the mutant selection
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window (MSW) but not outside the MSW, in accordance with the MSW hypothesis (19, 20).
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However, the potential of AUC24/MIC and AUC24/MPC as inter-strain predictors of bacterial
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resistance have been examined infrequently (8, 10, 16-18, 21).
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To compare the abilities of AUC24/MPC and AUC24/MIC to predict inter-strain
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thresholds that restrict mutant enrichment, ciprofloxacin-resistant mutants of three K.
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pneumoniae strains with various MPC/MIC ratios were studied over a wide range of
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AUC24/MICs simulated in an in vitro dynamic model.
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Materials and methods
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Antimicrobial agent, bacterial strains and susceptibility testing. Ciprofloxacin powder was
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purchased from AppliChem Biochemica Chemical Synthesis Services (Darmstadt, Germany).
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Three clinical isolates of K. pneumoniae, 1885, 1145, and 185, with different
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ciprofloxacin MICs (1, 0.5 and 0.125, respectively) and MPCs (8, 12 and 8, respectively) were
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selected for the study. The MICs were determined prior to, during and after three-day simulated
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treatments with ciprofloxacin. Susceptibility testing was performed at least in duplicate by broth
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microdilution techniques (22) at 24 h post-exposure with organisms grown in Ca2+- and Mg2+-
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supplemented Mueller-Hinton broth (MHB) at an inoculum size of 106 CFU/ml.
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The MPCs were determined as described elsewhere (8). Briefly, the tested
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microorganisms were cultured in MHB and incubated for 24 h. Then, the suspension was
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centrifuged (4000g for 10 min) and re-suspended in MHB to yield a concentration of ~1010
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CFU/ml. A series of agar plates containing known ciprofloxacin concentrations was then
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inoculated with ~1010 CFU of K. pneumoniae. The inoculated plates were incubated for 48 h at
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37°C and screened visually for growth. To estimate the MPC, logarithms of bacterial numbers
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were plotted against antibiotic concentrations. MPC was taken as the point where the plot
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intersected the lower limit of detection (log CFU/ml = 1).
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In vitro dynamic model and simulated pharmacokinetic profiles. A previously described dynamic
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model (23) was used in the study. The concordance between measured and designed
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concentrations of ciprofloxacin has been reported elsewhere (9). Briefly, the model consisted of
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two connected flasks, one containing fresh MHB and the other with a magnetic stirrer, the central
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unit, with the same broth containing a bacterial culture plus antibiotic. Peristaltic pumps
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circulated fresh nutrient medium to the flasks and from the central 100-ml unit at a flow rate of
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17.3 ml/h. The system was filled with sterile MHB and placed in an incubator at 37oC. The
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central unit was inoculated with an 18-hour culture of K. pneumoniae. After 2-hour incubation
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the resulting exponentially growing cultures reached approximately 108 CFU/ml and
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ciprofloxacin solutions were injected into the central unit at 12-h intervals. The duration of the
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experiments was at least 72 h.
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A series of monoexponential profiles that mimic twice-daily dosing of ciprofloxacin with a
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half-life (T1/2) of 4 h was simulated for 3 consecutive days. The simulated T1/2 represented weighted
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means of values reported for humans: 3.2 to 5.0 h (24). The simulated pharmacokinetic profiles
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were designed to provide 24-hour ratios of area under the curve (AUC24) to the MIC ratios from
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15 to 2880 h [corresponding to an AUC24 range from 15 to 2880 µg×h /ml (0.7-131 range of
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ciprofloxacin clinical doses against K. pneumoniae 1885)] and from 7.5 to 2880 h with K.
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pneumoniae 1145 and 185 [corresponding to an AUC24 range from 3.75 to 1440 and from 0.9 to
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360 µg×h /ml, respectively (0.2-66 and 0.04-16.4 range of the clinical dose , respectively)].
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Quantitation of antibiotic effect on susceptible and resistant subpopulations. In each
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experiment, bacteria-containing media from the central unit of the model was sampled every 24
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h to determine bacterial concentrations throughout the observation period. One hundred-
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microliter samples were serially diluted if necessary, in order to account for antibiotic carryover
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prior to plating, thereby reducing the antibiotic concentration below the MIC of the drug.
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Samples were spirally plated onto Mueller-Hinton agar plates without antibiotic (“0×MIC”) and
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those containing 4×, 8× and 16×MIC of ciprofloxacin using the Easy Spiral Pro system
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(Interscience, St. Nom, France). Colonies were counted by an automated colony counter
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Interscience Scan 1200 (Interscience, St. Nom, France).
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The lower limit of counting susceptible and resistant cells was 200 CFU/ml (equivalent to
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20 colonies per plate); the lower limit of quantification of resistant mutants was 10 CFU/ml
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(equivalent to at least one colony per plate). To reveal changes in susceptibility of ciprofloxacin-
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exposed bacterial cultures, MICs were re-assessed every 24 h during the experiment and after the
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simulated treatment. To confirm the enhanced ciprofloxacin resistance in isolates with
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established MIC elevations, repetitive MIC testing was performed after several passages on
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antibiotic-free medium.
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Based on time-kill data, the area between the cut-off level at 108 CFU/ml and the time-
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kill curve (ABBC; 25) was calculated. The ABBC-log AUC24/MIC curve was fitted by the
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sigmoid function:
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Y = Y0 + a/{1+exp [-(x – x0)/b]}
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where Y is ABBC, x is log (AUC24/MIC), Y0 and a are the minimal and maximal values of the
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antimicrobial effect, respectively, x0 is x corresponding to a/2, and b is a parameter reflecting
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sigmoidicity.
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(1)
To delineate AUC24/MIC and AUC24/MPC relationships with resistance, areas under the
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bacterial mutant concentration-time curves (AUBCMs; 8) were determined for subpopulations
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resistant to 4×, 8×, and 16×MIC of antibiotic from the beginning of treatment to 72 h, corrected
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for the areas under the lower limit of quantification over the same time interval. To reveal
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changes in susceptibility of ciprofloxacin-exposed bacterial cultures, MICs were re-assessed
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every 24 h during the experiment and after antibiotic exposures (MICfinal). Each sample from the
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central compartment was plated on MHA. After a 18-24 h incubation a suspension of isolated
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colonies was made in MHB to achieve 0.5 McFarland turbidity standard. Susceptibility was
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evaluated by microdilution technique (22).To account for the different susceptibilities of K.
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pneumoniae strains, the MICfinals were related to the MICs determined with the starting inoculum
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(MICinitials). A modified Gaussian type function was used to fit the AUBCM or MICfinal/MICinitial
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versus AUC24/MIC or AUC24/MPC data sets:
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Y = Y0 + a exp [0.5 (x – x0)c/b]
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where Y is AUBCM or MICfinal/MICinitial, x is log (AUC24/MIC) or log (AUC24/MPC), Y0 is the
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minimal value of Y, x0 is log (AUC24/MIC) or (AUC24/MPC) that corresponds to the maximal
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value of Y, and a and b are parameters.
(2)
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Determination of “anti-mutant” AUC24/MIC and AUC24/MPC. To determine the AUC24/MIC
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and AUC24/MPC ratios that protects against the enrichment of mutants (“anti-mutant” ratios),
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points belonging to the descending portion of the AUBCM–AUC24/MIC and AUBCM -
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AUC24/MPC curves were used. The AUBCMs were plotted against the respective AUC24/MICs
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and AUC24/MPCs. The “anti-mutant” ratio was taken as the point where the regression line
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reaches the level of AUBCM = 30 log (CFU/ml) × h.
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Y = Y0 + a x
(3)
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where Y is AUBCM, x is AUC24/MIC or AUC24/MPC, Y0 is the minimal value of Y, a is a
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parameter.
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Mechanisms of resistance. Nucleotide sequences of the quinolone-resistance determining regions
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(QRDRs) of gyrA and parC genes were determined for both parental strains and mutants with
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enhanced ciprofloxacin resistance as previously described (26, 27) with some modifications. The
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PCR and sequencing primers were re-designed based on alignment of all K. pneumoniae gyrA
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and parC gene sequences available from GeneBank. The original forward primer, gyrA6: 5’-
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CGACCTTGCGAGAGAAAT-3’, described by Weigel et al. (26) was used in combination with
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the modified primer, gyrAR2: 5’-CCTTCAATGCTGATGTCTTCA-3’; the parC forward and
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reverse primers (27) were, respectively, extended by 2 bases at 3’-end (parC-F2: 5’-
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TACGTGATCATGGACAGGGC-3’) and shortened by one base at 5’-end (parC-R2: 5’-
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CCACTTCCCGCAGGTTG-3’). Bacterial genomic DNA was isolated using the InstaGene
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matrix kit (Bio-Rad Laboratories Inc., Hercules, CA). Amplified fragments were purified by
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exonuclease I and shrimp alkaline phosphatase treatment (Thermo Scientific Fermentas, Vilnius,
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Lithuania) and were sequenced on both strands using the same primers as for PCR, the BigDye
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terminator v3.1 cycle sequencing kit and ABI Prism 310 genetic analyzer (Applied Biosystems,
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Foster City, CA). Sequences obtained were compared with those previously reported for gyrA
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and parC genes of K. pneumoniae ATCC® 43816 (GeneBank Acc. No. APWN00000000.1).
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Results
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Time courses of simulated ciprofloxacin concentrations, related to MIC, and ciprofloxacin-
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exposed K. pneumoniae grown without antibiotic (0×MIC) and on media containing, for
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example, 4×MIC of ciprofloxacin at three characteristic AUC24/MIC ratios are shown in Fig. 1.
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As seen in the left column, small and transient, if any, reductions in the number of susceptible K.
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pneumoniae cells were observed at the lowest AUC24/MIC ratio (15 h). Mutants resistant to
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4×MIC of ciprofloxacin were enriched starting from the second day of treatment with each of the
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studied organisms. At the AUC24/MIC ratio of 60 h (middle column in Fig. 1), both killing of
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susceptible cells and enrichment of resistant K. pneumoniae were more pronounced than at the
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lower AUC24/MIC ratio. At least with K. pneumoniae 1145, ciprofloxacin-susceptible cells were
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completely replaced by resistant mutants by the end of treatment. With an increase in the
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AUC24/MIC ratio to 720 h (right column in Fig. 1), the numbers of susceptible cells decreased
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significantly except K. pneumoniae 1145 (maximal reduction of 3 log CFU/ml). Although
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resistant mutants were enriched beginning from the third-fourth day of treatment, their final
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numbers were 1.1-2.4-fold order smaller than at the intermediate simulated AUC24/MIC ratio.
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Mutants resistant to 8× and 16×MIC were isolated with each ciprofloxacin-exposed
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organism at each AUC24/MIC ratio except for the lack of growth of K. pneumoniae 185 mutants
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resistant to 16×MIC at AUC24/MIC of 15 h. Time courses of the more resistant mutants were
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similar to those observed with mutant resistant to 4×MIC of ciprofloxacin (data not shown). As a
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whole, the higher the resistance level the less pronounced was the respective mutant's growth.
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The enrichment of resistant mutants at AUC24/MIC of 15 and 60 h was accompanied by
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concomitant MIC elevations. The enhanced ciprofloxacin resistance in these isolates was
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confirmed by repetitive MIC testing after several passages on antibiotic-free medium. With K.
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pneumoniae 1885 and 1145 the maximal ratio of MICfinal to MICinitial (14 and 8, respectively)
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was observed at AUC24/MIC of 60 h, with K. pneumoniae 185 – at AUC24/MIC of 720 h
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(MICfinal/MICinitial = 14). More detailed MICfinal/MICinitial data obtained over a wider range of
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simulated AUC24/MIC ratios are discussed below.
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To determine the mechanisms of decreased ciprofloxacin susceptibility in mutants, partial
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nucleotide sequences of gyrA (nucleotides from 21 to 605 with respect to the translation start
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site) and parC genes (nucleotides from 82 to 585 with respect to the translation start site)
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spanning the entire quinolone-resistance determining regions (QRDRs) were analyzed. The
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sequences of the parental strains and mutant strains were identical and contained no missense
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mutations as compared to the wild-type K. pneumoniae ATCC® 43816 (GeneBank Acc. No.
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APWN00000000.1).
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To delineate concentration-resistance relationships, the AUBCMs were plotted against
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AUC24/MIC and AUC24/MPC. Fig. 2 shows AUBCM-AUC24/MIC and AUBCM-AUC24/MPC
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relationships observed with K. pneumoniae mutants resistant to 4×, 8× and 16×MIC of
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ciprofloxacin. With each organism, AUBCM versus AUC24/MIC and AUC24/MPC curves were
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bell-shaped. The AUBCM increased with an increase in the AUC24/MIC or AUC24/MPC ratio,
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reaching a maximum followed by a subsequent decrease in numbers of resistant mutants. The
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maximal amplification of mutants resistant to 4×, 8× and 16×MIC of ciprofloxacin was bacterial
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strain-specific: much more pronounced for K. pneumoniae 185 and 1145 than for K. pneumoniae
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1885. At the lower levels of resistance (4× and 8×MIC), maximal AUBCMs for resistant mutants
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of the studied organisms differ by 1.3 – 1.8 order, whereas with the highest level of resistance
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(16×MIC) strain-specific difference in the maximal AUBCMs was 2.5 –fold. Regardless of
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bacterial strain, AUBCMs that reflect amplification of mutants resistant to 4×MIC of
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ciprofloxacin were more pronounced than with mutants resistant to 8× and especially 16×MIC.
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Unlike maximal AUBCMs, AUC24/MICs and AUC24/MPCs that correspond to maximums
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on the bell-shaped curves did not depend on the level of resistance, but they were strain-specific.
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Maximal amplification of resistant K. pneumoniae 1145 occurred at a lower AUC24/MIC (60 h)
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than for K. pneumoniae 1885 (60-120 h) and especially K. pneumoniae 185 (180-360 h). The
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respective AUC24/MPC ratios were 2.5, 7.6-15 and 2.8-5.6 h. Also, bacterial strain-specific
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differences were inherent in the “anti-mutant” AUC24/MIC and AUC24/MPC ratios determined
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for individual organisms (Table 1). As seen in the Table, with mutants of the same level of
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resistance, estimated thresholds of AUC24/MIC were less variable than those of AUC24/MPC
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among the studied strains. For example, with mutants resistant to 4×MIC of ciprofloxacin
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individual “anti-mutant” AUC24/MIC ratios varied over a two-fold range (from 1312 to 2612 h),
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whereas the respective AUC24/MPCs – over an almost ten-fold range (from 20 to 290 h). With K.
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pneumoniae 1885 and 1145 but not with 185, strain-associated differences in the “anti-mutant”
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AUC24/MIC or AUC24/MIC were less pronounced with mutants resistant to 8× and 16×MIC
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(1.2-1.7- versus 4.5-7.5-fold ranges, respectively). Overall, both AUC24/MIC and AUC24/MPC
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thresholds were lower with more resistant than less resistant mutants.
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Despite individual differences in the AUBCM versus AUC24/MIC or AUC24/MPC curves,
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a Gaussian-like function fits the AUBCM-AUC24/MIC or AUBCM-AUC24/MPC combined data
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on the three K. pneumoniae mutants resistant to 4×MIC of ciprofloxacin (Fig. 3, upper plot) with
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relatively high r2s (0.76 and 0.72, respectively). Less clear correlations were found for mutants
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resistant to 8×MIC (0.63 and 0.60, respectively) and 16×MIC (0.61 and 0.55, respectively) –
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data not shown. As seen in the figure, the greatest enrichment of mutants resistant to 4×MIC of
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ciprofloxacin was seen at 90 h (AUC24/MIC) and 5 h (AUC24/MPC), i.e., within the ranges
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established for individual organisms (60-360 h and 2.5-15 h, respectively).
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To determine the “anti-mutant” AUC24/MIC and AUC24/MPC more precisely, only points
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on the descending branch of the bell were considered (Fig. 3, lower plot). Although AUC24/MPC
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and AUC24/MIC relationships with the AUBCM were similar, a linear function (equation 3) fits
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AUC24/MIC data combined for the three K. pneumoniae strains better than the AUC24/MPC: r2
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of 0.74 versus 0.59. The respective “anti-mutant” AUC24/MIC of 1800 h and AUC24/MPC of 130
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h were within the ranges established for individual organisms (1310-2610 h and 20-290 h,
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respectively) too.
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The same equation 2 also fits MICfinal/MICinitial versus AUC24/MIC and MICfinal/MICinitial
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versus AUC24/MPC data (Fig. 4). These relationships were bell-shaped similar to the respective
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AUC24/MIC or AUC24/MPC relationships with the AUBCM described above. As seen in the left
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plot in Fig. 4, the maximal MICfinal elevations were observed at the intermediate AUC24/MIC
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ratios (180-360 h), whereas at the lower and higher AUC/MICs the MICfinals did not increase.
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MICfinal/MICinitial versus AUC24/MIC showed good correlation (r2 0.75), comparable to AUBCM-
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AUC24/MIC or AUBCM-AUC24/MPC relationships. However, the MICfinal/MICinitial -
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AUC24/MPC relationship, shown in the right plot in Fig. 4, was described with significantly
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lower r2 (0.43) because of pronounced scattering of the points. As seen in the figure, the greatest
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losses in susceptibility occurred at AUC24/MIC ratio of 300 h and AUC24/MPC – 8.5 h. The
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respective “anti-mutant” AUC24/MIC and AUC24/MPC, at which MICfinal/MICinitial = 1, were
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2560 and 300 h.
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Pronounced individual variability was inherent also in killing of susceptible
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subpopulations of K. pneumoniae exposed to ciprofloxacin (Fig. 5). Using combined data on the
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three organisms, a sigmoid function (equation 1) fits the ABBC versus AUC24/MIC with r2 of
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0.78.
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Discussion
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Aimed at examination of the AUC24/MPC and AUC24/MIC as inter-strain predictors of bacterial
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resistance, this study demonstrates applicability of the MSW hypothesis to fluoroquinolone-
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resistant K. pneumoniae. With each of three clinical isolates, the maximal amplification of
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mutants resistant to 4×, 8× and 16×MIC of ciprofloxacin was observed when antibiotic
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concentrations fell into the MSW for most of the dosing interval, and both AUC24/MIC and
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AUC24/MPC relationships with resistance expressed by the population analysis data (AUBCM) or
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susceptibility testing (ratio of the post-exposure to pre-exposure MIC) were bell-shaped. These
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observations are consistent with earlier findings reported in studies with fluoroquinolones (6-17).
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Findings obtained in the present study predict the “anti-mutant” AUC24/MIC ratios for K.
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pneumoniae strains 1885, 1145, and 185 (2320, 2610 and 1310 h, respectively) that are much
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higher than the respective clinically attainable AUC24/MICs (22, 44 and 176 h, respectively).
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The same trend also was seen in our recent study with ciprofloxacin against four P. aeruginosa
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strains (11): “anti-mutant” AUC24/MIC ratios (220-1100 h) were higher than the respective
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clinically attainable AUC24/MICs (44 and 176 h). In contrast, in our studies with E. coli (9),
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despite relatively high “anti-mutant” AUC24/MICs (720-1440 h), these ratios can be achieved at
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clinically attainable exposures (AUC/MICs 2750 and 1375 h).
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Despite pronounced stratification of AUBCM versus AUC24/MIC or AUC24/MPC curves
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seen with individual organisms, it appeared possible to establish bacterial strain-independent
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AUC24/MIC or AUC24/MPC relationships with resistance using combined data with all three K.
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pneumoniae strains. Similar relationships were established between MICfinal/MICinitial and
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AUC24/MIC and AUC24/MPC. However, AUC24/MIC and AUC24/MPC ratios that correspond to
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the greatest loss in susceptibility were shifted towards higher values compared to those that
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correspond to the greatest AUBCM. A similar shift was reported with two of four strains in our
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study with ciprofloxacin-exposed P. aeruginosa (11).
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As in our recent study with ciprofloxacin against E. coli (10), to test significant levels of
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K. pneumoniae resistance we chose mutants resistant to 4×, 8× and 16×MIC, ignoring more
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abundant nontarget (e.g. efflux) mutants resistant to 2×MIC of ciprofloxacin because exposure of
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bacteria to twice their MICs can almost be regarded as positive controls (28). In the present study
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we did not identify any changes in nucleotide sequences of gyrA and parC QRDRs of
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ciprofloxacin-resistant K. pneumoniae mutants as compared to those of parental or reference
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strains suggesting that the enhancement of resistance was likely due to altered drug
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accumulation, although mutations causing resistance to fluoroquinolones in Enterobacteriaceae
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most often occur in the QRDR region of the primary target gene, gyrA (9, 29-31). Additional
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mutations that further increase resistance to fluoroquinolones may occur in the secondary target
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gene, parC (29-31), and, in K. pneumoniae, in various regulatory genes that affect the expression
295
of efflux pumps, e.g. AcrAB (32-34), and major porins, OmpK35 and OmpK36 (35, 36), leading
296
to decreased accumulation of the drug in bacterial cells.
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In conclusion, findings obtained in the present study are in support of the MSW
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hypothesis that explains the bell shape of the AUBCM versus AUC24/MIC and AUC24/MPC
299
curves. However, highly variable AUC24/MPC thresholds call into question the reliability of
300
their inter-strain predictions of the enrichment of K. pneumoniae mutants. From this point of
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view AUC24/MIC should be preferred to AUC24/MPC for predictions of the emergence of K.
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pneumoniae resistance. Similar conclusions were drawn in studies with ciprofloxacin-exposed E.
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coli (9, 10) and P. aeruginosa (11). In fact, a priori expectations of better predictive force of the
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AUC24/MPC ratio were confirmed only with fluoroquinolone-exposed S. aureus (5, 8).
305
Further studies with other bacterial species are needed to provide a more complete
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understanding of AUC24/MIC and AUC24/MPC as potential predictors of the enrichment of
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resistant mutants.
308 309
Funding information
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Studies performed at the Department of Pharmacokinetics & Pharmacodynamics, Gause Institute
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of New Antibiotics, were supported by a grant from Russian Science Foundation (#14-15-
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00970).
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prevent selection of resistant Staphylococcus aureus in an in vitro dynamic model. J
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Agents 32:488-493.
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10. Firsov AA, Portnoy YA, Strukova EN, Shlykova DS, Zinner SH. 2014. Predicting
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limitations. Int J Antimicrob Agents 44:301-305.
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related changes in susceptibility, resistance frequency and bacterial killing. J Antimicrob
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14. Oonishi Y, Mitsuyama О, Yamaguchi K. 2007. Effect of GrlA mutation on the
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16. Liang B, Bai N, Cai Y, Wang R, Drlica K, Zhao X. 2011. Mutant prevention concentration-
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enrichment of levofloxacin-resistant subpopulations of Staphylococcus aureus. Antimicrob
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mutants of Escherichia coli of canine origin. J Med Microbiol 60:1512-1522.
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18. Croisier D, Etienne M, Piroth L, Bergoin E, Lequeu C, Portier H, Chavanet P. 2004. In
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the enrichment of resistant mutants. J Antimicrob Chemother 54:640-647.
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19. Zhao X, Drlica K. 2001. Restricting the selection of antibiotic-resistant mutants: a general 294 strategy derived from fluoroquinolone studies. Clin Infect Dis 33:147–156.
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20. Blondeau JM, Hansen G, Metzler K, Hedlin P. 2004. The role of PK/PD parameters to
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ciprofloxacin resistance of Escherichia coli in an in vitro kinetic model: relation between
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drug exposure and mutant prevention concentration. J Antimicrob Chemother 57:1116-1121.
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22. CLSI. 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow
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aerobically. Approved Standard. Eighth Edition. CLSI document M-07-08, Clinical and
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23. Firsov AA, Golikova MV, Strukova EN, Portnoy YA, Romanov AV, Edelstein MV,
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Zinner SH. 2015. In vitro resistance studies with bacteria that exhibit low mutation
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frequencies: prediction of "antimutant" linezolid concentrations using a mixed inoculum
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containing both susceptible and resistant Staphylococcus aureus. Antimicrob Agents Chemother
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24. Wilson APR, Grüneberg RN. Ciprofloxacin: 10 years of clinical experience. Maxim Medical, Oxford, UK. 1997.
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1996. Predictors of effect of ampicillin-sulbactam against TEM-1 beta-lactamase-producing
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Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC. Antimicrob
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Agents Chemother 40:734-738.
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26. Weigel LM, Steward SD, Tenover FC. 1998. gyrA mutations associated with
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fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob Agents
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Chemother 42: 2661-2667.
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27. Zimhony O, Vilchèze C, Arai M, Welch JT, Jacobs WR Jr. 2006. Pyrazinoic acid and its
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n-propyl ester inhibit fatty acid synthase type I in replicating tubercle bacilli. Antimicrob
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28. Schubert S, Dalhoff A. 2006. Low propensity for development of resistance to MCB3681,
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the active moiety of oxaquin (MCB3837), in gram-positive bacteria with vancomycin-,
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linezolid-, methicillin- and/or ciprofloxacin resistances. University-Hospital Schleswig-
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Holstein, 46th ICAAC, San Francisco, CA, USA, F1-1968.
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29. Hooper DC, 2003. Mechanisms of quinolone resistance. In: Hooper DC, Rubinstein E (ed), Quinolone antimicrobial agents, 3rd ed, ASM Press, Washington DC, pp 41–67.
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30. Sáenz Y, Zarazaga M, Briñas L, Ruiz-Larrea F, Torres C. 2003. Mutations in gyrA and
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parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans
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and animals. J Antimicrob Chemother 51:1001-1005.
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31. Dalhoff A. 2012. Global fluoroquinolone resistance epidemiology and implictions for clinical use. Interdiscip Perspect Infect Dis 2012:976273. 32. Bialek-Davenet S, Marcon E, Leflon-Guibout V, Lavigne JP, Bert F, Moreau R,
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Nicolas-Chanoine MH. 2011. In vitro selection of ramR and soxR mutants overexpressing
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efflux systems by fluoroquinolones as well as cefoxitin in Klebsiella pneumoniae.
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Antimicrob Agents Chemother 55:2795-2802.
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33. Martínez-Martínez L, Pascual A, Conejo Mdel C, García I, Joyanes P, Doménech-
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Sánchez A, Benedí VJ. 2002. Energy-dependent accumulation of norfloxacin and porin
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expression in clinical isolates of Klebsiella pneumoniae and relationship to extended-
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spectrum beta-lactamase production. Antimicrob Agents Chemother 46:3926-3932.
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34. Aathithan S, French GL. 2011. Prevalence and role of efflux pump activity in
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ciprofloxacin resistance in clinical isolates of Klebsiella pneumoniae. Eur J Clin Microbiol
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Infect Dis 30:745–752.
431
35. Chen FJ, Lauderdale TL, Ho M, Lo HJ. 2003. The roles of mutations in gyrA, parC, and
432
ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae. Microb Drug Resist 9:265-
433
271.
434
36. Škopková-Žarnayová M, Siebor E, Rovná D, Bujdáková H, Neuwirth C. 2005. Outer
435
membrane protein profiles of clonally related Klebsiella pneumoniae isolates that differ in
436
cefoxitin resistance. FEMS Microbiology Letters 243:197–203.
437
438
Figure legends
439 440
FIG 1. Simulated pharmacokinetic profiles of ciprofloxacin and time courses of susceptible
441
(0×MIC) and resistant (4×MIC) subpopulations of K. pneumoniae exposed to ciprofloxacin. The
442
simulated AUC24/MIC ratios (in hours) are indicated as boxed numbers.
443
FIG 2. AUC24/MIC and AUC24/MPC relationships with AUBCM (for mutants resistant to 4×, 8×
444
and 16×MIC of ciprofloxacin) observed with individual K. pneumoniae isolates.
445
FIG 3. Combined data on all three K. pneumoniae isolates (for mutants resistant to 4×MIC of
446
ciprofloxacin). Upper panel: AUC24/MIC and AUC24/MPC relationships with AUBCM.
447
Estimated coefficients of equation 2: Y0 = 1, x0 = 1.926, a = 350.8, b = 0.6633, c = 2.007
448
(AUBCM versus AUC24/MIC); Y0 = 1, x0 = 0.4464, a = 349, b = 0.6887, c = 2.01 (AUBCM versus
449
AUC24/MPC). Bottom panel: “anti-mutant” AUC24/MIC and AUC24/MPC ratio determination
450
using the respective descending portions of the AUBCM versus AUC24/MIC and AUC24/MPC
451
curves. Estimated coefficients of equation 3: Y0 = 953.0, a = -283.8 (AUBCM versus
452
AUC24/MIC), Y0 = 442.5, a = -194.8 (AUBCM versus AUC24/MPC).
453
FIG 4. AUC24/MIC and AUC24/MPC relationships with MICfinal/MICinitial - combined data on all
454
three K. pneumoniae isolates. Estimated coefficients of equation 2: Y0 = 1, x0 = 2.309, a = 20.74,
455
b = 0.5127, c = 2.175 (MICfinal/MICinitial versus AUC24/MIC); Y0 = 1, x0 = 0.8559, a = 15.63, b =
456
0.7454, c = 2.207 (MICfinal/MICinitial versus AUC24/MPC).
457
FIG 5. AUC24/MIC-dependent effects of ciprofloxacin on susceptible subpopulation of K.
458
pneumoniae fitted by equation 1: Y0 = 0, x0 = 2,389, a = 208.2, b = 0.4083.
Concentration/MIC
100
15
60
720
0×MIC
0×MIC
0×MIC
4×MIC
4×MIC
4×MIC
10
1
0,1 10
8
Viable count (log CFU/ml)
6
4
2
0
10
8
6
4
2
0 0
24
48
72
0
K. pneumoniae 1885;
24
□
48
72
0
24
Time (h) K. pneumoniae 1145; K. pneumoniae 185
48
72
500
4×MIC
4×MIC
8×MIC
8×MIC
16×MIC
16×MIC
400
300
200
100
0
AUBCM ((log CFU/ml) × h)
500
400
300
200
100
0
500
400
300
200
100
0 1
10
100
1000
10000
0,1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
100
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
1000
500
400
r2 0.72
r2 0.76
300
AUBCM ((log CFU/ml) × h)
200
100
0
500
400
r2 0.74
300
r2 0.59
200
130
100
1800 0 1
10
100
1000
10000
0.1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
100
1000
MICfinal/MICinitial
30
r2 0.75
20
r2 0.43
10
0
1
10
100
1000
10000
0.1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
100
1000
ABBC ((log CFU/ml) × h)
300
200
r2 0.78 100
0
10
100
1000
10000
AUC24/MIC (h) K. pneumoniae 1885; □ K. pneumoniae 1145; K. pneumoniae 185
Table 1 Predicted “anti-mutant” (AUC24/MIC)/(AUC24/MPC) ratios in hours. Mutants resistant to ciprofloxacin
K. pneumoniae strain 4×MIC
8×MIC
16×MIC
1885
2320/290
1200/150
720/90
1145
2610/110
1440/60
1000/40
185
1310/20
1270/20
1250/20
1
AAC02334-15 REVISED
2 3
Ciprofloxacin Resistance in Klebsiella pneumoniae: Searching for the Optimal Predictor
4
Using In Vitro Dynamic Models
5 6
Elena N. Strukova,a Yury A. Portnoy,a Andrey V. Romanov,b Mikhail V. Edelstein,b
7
Stephen H. Zinner,c Alexander A. Firsova
8 9
Department of Pharmacokinetics & Pharmacodynamics, Gause Institute of New Antibiotics,
10
Moscow,119021, Russiaa; Institute of Antimicrobial Chemotherapy, Smolensk State Medical
11
University, Smolensk, Russiab; Department of Medicine, Mount Auburn Hospital, Harvard
12
Medical School, Cambridge, Massachusetts, USAc
13 14
Running title: Predicting Bacterial Resistance in In Vitro Models
15
................................................................................................................
16
Address correspondence to Alexander A. Firsov, [email protected].
17
18
There is growing evidence of applicability of the hypothesis of the mutant selection window
19
(MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within
20
which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-
21
based pharmacokinetic variables are preferable to the respective MIC-based variables as inter-
22
strain predictors of resistance. To examine the predictive power of the ratios of the area under the
23
curve (AUC24) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of
24
three Klebsiella pneumoniae strains with different MPC/MIC ratios was studied. Each organism
25
was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak
26
antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC.
27
Resistant K. pneumoniae mutants were intensively enriched at AUC24/MIC from 60 to 360 h
28
(AUC24/MPC from 2.5 to 15 h) but not at the lower or higher AUC24/MIC and AUC24/MPC
29
ratios, in accordance with the MSW hypothesis. AUC24/MPC and AUC24/MIC relationships with
30
areas under the time courses of ciprofloxacin-resistant K. pneumoniae (AUBCM) were bell-
31
shaped. These relationships predict highly variable “anti-mutant” AUC24/MPC ratios (20 to 290
32
h) compared to AUC24/MIC ratios (1310 to 2610 h). These findings suggest that the potential of
33
the AUC24/MPC ratio as an inter-strain predictor of K. pneumoniae resistance is lower than
34
AUC24/MIC.
35
36
An increasing number of reports on the isolation of resistant pathogens (1, 2) combined with a
37
weak antibiotic pipeline suggests that optimization of antibiotic therapy should be aimed at the
38
suppression of resistance (3, 4). In this regard, dynamic models that mimic antimicrobial
39
pharmacokinetics in vitro have been proven to be a useful tool in predicting the amplification of
40
resistant mutants at clinically achievable antibiotic concentrations (5). Using these models, bell-
41
shaped relationships have been established between the emergence of resistance to
42
fluoroquinolones and the ratios of 24-hour area under the concentration-time curve (AUC24) to
43
the MIC (6-17). Such relationships have been reported with moxifloxacin, gatifloxacin,
44
levofloxacin, ciprofloxacin, an investigational fluoroquinolone АВТ492, pazufloxacin,
45
tosufloxacin, garenoxacin, levofloxacin against Staphylococcus aureus (6-8, 14-16),
46
moxifloxacin against Streptococcus pneumoniae (12, 13), garenoxacin against Klebsiella
47
pneumoniae (15), ciprofloxacin and moxifloxacin against Pseudomonas aeruginosa (11, 12), and
48
with ciprofloxacin, marbofloxacin and enrofloxacin against Escherichia coli (9, 10, 17). In some
49
of these studies (6-15, 17, 18), changes in susceptibility of antibiotic-exposed bacteria and/or
50
their enrichment with resistant mutants were observed at fluoroquinolone concentrations above
51
the MIC but below the mutant prevention concentration (MPC), i.e., inside the mutant selection
52
window (MSW) but not outside the MSW, in accordance with the MSW hypothesis (19, 20).
53
However, the potential of AUC24/MIC and AUC24/MPC as inter-strain predictors of bacterial
54
resistance have been examined infrequently (8, 10, 16-18, 21).
55
To compare the abilities of AUC24/MPC and AUC24/MIC to predict inter-strain
56
thresholds that restrict mutant enrichment, ciprofloxacin-resistant mutants of three K.
57
pneumoniae strains with various MPC/MIC ratios were studied over a wide range of
58
AUC24/MICs simulated in an in vitro dynamic model.
59
60
Materials and methods
61 62
Antimicrobial agent, bacterial strains and susceptibility testing. Ciprofloxacin powder was
63
purchased from AppliChem Biochemica Chemical Synthesis Services (Darmstadt, Germany).
64
Three clinical isolates of K. pneumoniae, 1885, 1145, and 185, with different
65
ciprofloxacin MICs (1, 0.5 and 0.125, respectively) and MPCs (8, 12 and 8, respectively) were
66
selected for the study. The MICs were determined prior to, during and after three-day simulated
67
treatments with ciprofloxacin. Susceptibility testing was performed at least in duplicate by broth
68
microdilution techniques (22) at 24 h post-exposure with organisms grown in Ca2+- and Mg2+-
69
supplemented Mueller-Hinton broth (MHB) at an inoculum size of 106 CFU/ml.
70
The MPCs were determined as described elsewhere (8). Briefly, the tested
71
microorganisms were cultured in MHB and incubated for 24 h. Then, the suspension was
72
centrifuged (4000g for 10 min) and re-suspended in MHB to yield a concentration of ~1010
73
CFU/ml. A series of agar plates containing known ciprofloxacin concentrations was then
74
inoculated with ~1010 CFU of K. pneumoniae. The inoculated plates were incubated for 48 h at
75
37°C and screened visually for growth. To estimate the MPC, logarithms of bacterial numbers
76
were plotted against antibiotic concentrations. MPC was taken as the point where the plot
77
intersected the lower limit of detection (log CFU/ml = 1).
78 79
In vitro dynamic model and simulated pharmacokinetic profiles. A previously described dynamic
80
model (23) was used in the study. The concordance between measured and designed
81
concentrations of ciprofloxacin has been reported elsewhere (9). Briefly, the model consisted of
82
two connected flasks, one containing fresh MHB and the other with a magnetic stirrer, the central
83
unit, with the same broth containing a bacterial culture plus antibiotic. Peristaltic pumps
84
circulated fresh nutrient medium to the flasks and from the central 100-ml unit at a flow rate of
85
17.3 ml/h. The system was filled with sterile MHB and placed in an incubator at 37oC. The
86
central unit was inoculated with an 18-hour culture of K. pneumoniae. After 2-hour incubation
87
the resulting exponentially growing cultures reached approximately 108 CFU/ml and
88
ciprofloxacin solutions were injected into the central unit at 12-h intervals. The duration of the
89
experiments was at least 72 h.
90
A series of monoexponential profiles that mimic twice-daily dosing of ciprofloxacin with a
91
half-life (T1/2) of 4 h was simulated for 3 consecutive days. The simulated T1/2 represented weighted
92
means of values reported for humans: 3.2 to 5.0 h (24). The simulated pharmacokinetic profiles
93
were designed to provide 24-hour ratios of area under the curve (AUC24) to the MIC ratios from
94
15 to 2880 h [corresponding to an AUC24 range from 15 to 2880 µg×h /ml (0.7-131 range of
95
ciprofloxacin clinical doses against K. pneumoniae 1885)] and from 7.5 to 2880 h with K.
96
pneumoniae 1145 and 185 [corresponding to an AUC24 range from 3.75 to 1440 and from 0.9 to
97
360 µg×h /ml, respectively (0.2-66 and 0.04-16.4 range of the clinical dose , respectively)].
98 99
Quantitation of antibiotic effect on susceptible and resistant subpopulations. In each
100
experiment, bacteria-containing media from the central unit of the model was sampled every 24
101
h to determine bacterial concentrations throughout the observation period. One hundred-
102
microliter samples were serially diluted if necessary, in order to account for antibiotic carryover
103
prior to plating, thereby reducing the antibiotic concentration below the MIC of the drug.
104
Samples were spirally plated onto Mueller-Hinton agar plates without antibiotic (“0×MIC”) and
105
those containing 4×, 8× and 16×MIC of ciprofloxacin using the Easy Spiral Pro system
106
(Interscience, St. Nom, France). Colonies were counted by an automated colony counter
107
Interscience Scan 1200 (Interscience, St. Nom, France).
108
The lower limit of counting susceptible and resistant cells was 200 CFU/ml (equivalent to
109
20 colonies per plate); the lower limit of quantification of resistant mutants was 10 CFU/ml
110
(equivalent to at least one colony per plate). To reveal changes in susceptibility of ciprofloxacin-
111
exposed bacterial cultures, MICs were re-assessed every 24 h during the experiment and after the
112
simulated treatment. To confirm the enhanced ciprofloxacin resistance in isolates with
113
established MIC elevations, repetitive MIC testing was performed after several passages on
114
antibiotic-free medium.
115
Based on time-kill data, the area between the cut-off level at 108 CFU/ml and the time-
116
kill curve (ABBC; 25) was calculated. The ABBC-log AUC24/MIC curve was fitted by the
117
sigmoid function:
118
Y = Y0 + a/{1+exp [-(x – x0)/b]}
119
where Y is ABBC, x is log (AUC24/MIC), Y0 and a are the minimal and maximal values of the
120
antimicrobial effect, respectively, x0 is x corresponding to a/2, and b is a parameter reflecting
121
sigmoidicity.
122
(1)
To delineate AUC24/MIC and AUC24/MPC relationships with resistance, areas under the
123
bacterial mutant concentration-time curves (AUBCMs; 8) were determined for subpopulations
124
resistant to 4×, 8×, and 16×MIC of antibiotic from the beginning of treatment to 72 h, corrected
125
for the areas under the lower limit of quantification over the same time interval. To reveal
126
changes in susceptibility of ciprofloxacin-exposed bacterial cultures, MICs were re-assessed
127
every 24 h during the experiment and after antibiotic exposures (MICfinal). Each sample from the
128
central compartment was plated on MHA. After a 18-24 h incubation a suspension of isolated
129
colonies was made in MHB to achieve 0.5 McFarland turbidity standard. Susceptibility was
130
evaluated by microdilution technique (22).To account for the different susceptibilities of K.
131
pneumoniae strains, the MICfinals were related to the MICs determined with the starting inoculum
132
(MICinitials). A modified Gaussian type function was used to fit the AUBCM or MICfinal/MICinitial
133
versus AUC24/MIC or AUC24/MPC data sets:
134
Y = Y0 + a exp [0.5 (x – x0)c/b]
135
where Y is AUBCM or MICfinal/MICinitial, x is log (AUC24/MIC) or log (AUC24/MPC), Y0 is the
136
minimal value of Y, x0 is log (AUC24/MIC) or (AUC24/MPC) that corresponds to the maximal
137
value of Y, and a and b are parameters.
(2)
138 139
Determination of “anti-mutant” AUC24/MIC and AUC24/MPC. To determine the AUC24/MIC
140
and AUC24/MPC ratios that protects against the enrichment of mutants (“anti-mutant” ratios),
141
points belonging to the descending portion of the AUBCM–AUC24/MIC and AUBCM -
142
AUC24/MPC curves were used. The AUBCMs were plotted against the respective AUC24/MICs
143
and AUC24/MPCs. The “anti-mutant” ratio was taken as the point where the regression line
144
reaches the level of AUBCM = 30 log (CFU/ml) × h.
145
Y = Y0 + a x
(3)
146
where Y is AUBCM, x is AUC24/MIC or AUC24/MPC, Y0 is the minimal value of Y, a is a
147
parameter.
148
Mechanisms of resistance. Nucleotide sequences of the quinolone-resistance determining regions
149
(QRDRs) of gyrA and parC genes were determined for both parental strains and mutants with
150
enhanced ciprofloxacin resistance as previously described (26, 27) with some modifications. The
151
PCR and sequencing primers were re-designed based on alignment of all K. pneumoniae gyrA
152
and parC gene sequences available from GeneBank. The original forward primer, gyrA6: 5’-
153
CGACCTTGCGAGAGAAAT-3’, described by Weigel et al. (26) was used in combination with
154
the modified primer, gyrAR2: 5’-CCTTCAATGCTGATGTCTTCA-3’; the parC forward and
155
reverse primers (27) were, respectively, extended by 2 bases at 3’-end (parC-F2: 5’-
156
TACGTGATCATGGACAGGGC-3’) and shortened by one base at 5’-end (parC-R2: 5’-
157
CCACTTCCCGCAGGTTG-3’). Bacterial genomic DNA was isolated using the InstaGene
158
matrix kit (Bio-Rad Laboratories Inc., Hercules, CA). Amplified fragments were purified by
159
exonuclease I and shrimp alkaline phosphatase treatment (Thermo Scientific Fermentas, Vilnius,
160
Lithuania) and were sequenced on both strands using the same primers as for PCR, the BigDye
161
terminator v3.1 cycle sequencing kit and ABI Prism 310 genetic analyzer (Applied Biosystems,
162
Foster City, CA). Sequences obtained were compared with those previously reported for gyrA
163
and parC genes of K. pneumoniae ATCC® 43816 (GeneBank Acc. No. APWN00000000.1).
164 165
Results
166
Time courses of simulated ciprofloxacin concentrations, related to MIC, and ciprofloxacin-
167
exposed K. pneumoniae grown without antibiotic (0×MIC) and on media containing, for
168
example, 4×MIC of ciprofloxacin at three characteristic AUC24/MIC ratios are shown in Fig. 1.
169
As seen in the left column, small and transient, if any, reductions in the number of susceptible K.
170
pneumoniae cells were observed at the lowest AUC24/MIC ratio (15 h). Mutants resistant to
171
4×MIC of ciprofloxacin were enriched starting from the second day of treatment with each of the
172
studied organisms. At the AUC24/MIC ratio of 60 h (middle column in Fig. 1), both killing of
173
susceptible cells and enrichment of resistant K. pneumoniae were more pronounced than at the
174
lower AUC24/MIC ratio. At least with K. pneumoniae 1145, ciprofloxacin-susceptible cells were
175
completely replaced by resistant mutants by the end of treatment. With an increase in the
176
AUC24/MIC ratio to 720 h (right column in Fig. 1), the numbers of susceptible cells decreased
177
significantly except K. pneumoniae 1145 (maximal reduction of 3 log CFU/ml). Although
178
resistant mutants were enriched beginning from the third-fourth day of treatment, their final
179
numbers were 1.1-2.4-fold order smaller than at the intermediate simulated AUC24/MIC ratio.
180
Mutants resistant to 8× and 16×MIC were isolated with each ciprofloxacin-exposed
181
organism at each AUC24/MIC ratio except for the lack of growth of K. pneumoniae 185 mutants
182
resistant to 16×MIC at AUC24/MIC of 15 h. Time courses of the more resistant mutants were
183
similar to those observed with mutant resistant to 4×MIC of ciprofloxacin (data not shown). As a
184
whole, the higher the resistance level the less pronounced was the respective mutant's growth.
185
The enrichment of resistant mutants at AUC24/MIC of 15 and 60 h was accompanied by
186
concomitant MIC elevations. The enhanced ciprofloxacin resistance in these isolates was
187
confirmed by repetitive MIC testing after several passages on antibiotic-free medium. With K.
188
pneumoniae 1885 and 1145 the maximal ratio of MICfinal to MICinitial (14 and 8, respectively)
189
was observed at AUC24/MIC of 60 h, with K. pneumoniae 185 – at AUC24/MIC of 720 h
190
(MICfinal/MICinitial = 14). More detailed MICfinal/MICinitial data obtained over a wider range of
191
simulated AUC24/MIC ratios are discussed below.
192
To determine the mechanisms of decreased ciprofloxacin susceptibility in mutants, partial
193
nucleotide sequences of gyrA (nucleotides from 21 to 605 with respect to the translation start
194
site) and parC genes (nucleotides from 82 to 585 with respect to the translation start site)
195
spanning the entire quinolone-resistance determining regions (QRDRs) were analyzed. The
196
sequences of the parental strains and mutant strains were identical and contained no missense
197
mutations as compared to the wild-type K. pneumoniae ATCC® 43816 (GeneBank Acc. No.
198
APWN00000000.1).
199
To delineate concentration-resistance relationships, the AUBCMs were plotted against
200
AUC24/MIC and AUC24/MPC. Fig. 2 shows AUBCM-AUC24/MIC and AUBCM-AUC24/MPC
201
relationships observed with K. pneumoniae mutants resistant to 4×, 8× and 16×MIC of
202
ciprofloxacin. With each organism, AUBCM versus AUC24/MIC and AUC24/MPC curves were
203
bell-shaped. The AUBCM increased with an increase in the AUC24/MIC or AUC24/MPC ratio,
204
reaching a maximum followed by a subsequent decrease in numbers of resistant mutants. The
205
maximal amplification of mutants resistant to 4×, 8× and 16×MIC of ciprofloxacin was bacterial
206
strain-specific: much more pronounced for K. pneumoniae 185 and 1145 than for K. pneumoniae
207
1885. At the lower levels of resistance (4× and 8×MIC), maximal AUBCMs for resistant mutants
208
of the studied organisms differ by 1.3 – 1.8 order, whereas with the highest level of resistance
209
(16×MIC) strain-specific difference in the maximal AUBCMs was 2.5 –fold. Regardless of
210
bacterial strain, AUBCMs that reflect amplification of mutants resistant to 4×MIC of
211
ciprofloxacin were more pronounced than with mutants resistant to 8× and especially 16×MIC.
212
Unlike maximal AUBCMs, AUC24/MICs and AUC24/MPCs that correspond to maximums
213
on the bell-shaped curves did not depend on the level of resistance, but they were strain-specific.
214
Maximal amplification of resistant K. pneumoniae 1145 occurred at a lower AUC24/MIC (60 h)
215
than for K. pneumoniae 1885 (60-120 h) and especially K. pneumoniae 185 (180-360 h). The
216
respective AUC24/MPC ratios were 2.5, 7.6-15 and 2.8-5.6 h. Also, bacterial strain-specific
217
differences were inherent in the “anti-mutant” AUC24/MIC and AUC24/MPC ratios determined
218
for individual organisms (Table 1). As seen in the Table, with mutants of the same level of
219
resistance, estimated thresholds of AUC24/MIC were less variable than those of AUC24/MPC
220
among the studied strains. For example, with mutants resistant to 4×MIC of ciprofloxacin
221
individual “anti-mutant” AUC24/MIC ratios varied over a two-fold range (from 1312 to 2612 h),
222
whereas the respective AUC24/MPCs – over an almost ten-fold range (from 20 to 290 h). With K.
223
pneumoniae 1885 and 1145 but not with 185, strain-associated differences in the “anti-mutant”
224
AUC24/MIC or AUC24/MIC were less pronounced with mutants resistant to 8× and 16×MIC
225
(1.2-1.7- versus 4.5-7.5-fold ranges, respectively). Overall, both AUC24/MIC and AUC24/MPC
226
thresholds were lower with more resistant than less resistant mutants.
227
Despite individual differences in the AUBCM versus AUC24/MIC or AUC24/MPC curves,
228
a Gaussian-like function fits the AUBCM-AUC24/MIC or AUBCM-AUC24/MPC combined data
229
on the three K. pneumoniae mutants resistant to 4×MIC of ciprofloxacin (Fig. 3, upper plot) with
230
relatively high r2s (0.76 and 0.72, respectively). Less clear correlations were found for mutants
231
resistant to 8×MIC (0.63 and 0.60, respectively) and 16×MIC (0.61 and 0.55, respectively) –
232
data not shown. As seen in the figure, the greatest enrichment of mutants resistant to 4×MIC of
233
ciprofloxacin was seen at 90 h (AUC24/MIC) and 5 h (AUC24/MPC), i.e., within the ranges
234
established for individual organisms (60-360 h and 2.5-15 h, respectively).
235
To determine the “anti-mutant” AUC24/MIC and AUC24/MPC more precisely, only points
236
on the descending branch of the bell were considered (Fig. 3, lower plot). Although AUC24/MPC
237
and AUC24/MIC relationships with the AUBCM were similar, a linear function (equation 3) fits
238
AUC24/MIC data combined for the three K. pneumoniae strains better than the AUC24/MPC: r2
239
of 0.74 versus 0.59. The respective “anti-mutant” AUC24/MIC of 1800 h and AUC24/MPC of 130
240
h were within the ranges established for individual organisms (1310-2610 h and 20-290 h,
241
respectively) too.
242
The same equation 2 also fits MICfinal/MICinitial versus AUC24/MIC and MICfinal/MICinitial
243
versus AUC24/MPC data (Fig. 4). These relationships were bell-shaped similar to the respective
244
AUC24/MIC or AUC24/MPC relationships with the AUBCM described above. As seen in the left
245
plot in Fig. 4, the maximal MICfinal elevations were observed at the intermediate AUC24/MIC
246
ratios (180-360 h), whereas at the lower and higher AUC/MICs the MICfinals did not increase.
247
MICfinal/MICinitial versus AUC24/MIC showed good correlation (r2 0.75), comparable to AUBCM-
248
AUC24/MIC or AUBCM-AUC24/MPC relationships. However, the MICfinal/MICinitial -
249
AUC24/MPC relationship, shown in the right plot in Fig. 4, was described with significantly
250
lower r2 (0.43) because of pronounced scattering of the points. As seen in the figure, the greatest
251
losses in susceptibility occurred at AUC24/MIC ratio of 300 h and AUC24/MPC – 8.5 h. The
252
respective “anti-mutant” AUC24/MIC and AUC24/MPC, at which MICfinal/MICinitial = 1, were
253
2560 and 300 h.
254
Pronounced individual variability was inherent also in killing of susceptible
255
subpopulations of K. pneumoniae exposed to ciprofloxacin (Fig. 5). Using combined data on the
256
three organisms, a sigmoid function (equation 1) fits the ABBC versus AUC24/MIC with r2 of
257
0.78.
258 259
Discussion
260
Aimed at examination of the AUC24/MPC and AUC24/MIC as inter-strain predictors of bacterial
261
resistance, this study demonstrates applicability of the MSW hypothesis to fluoroquinolone-
262
resistant K. pneumoniae. With each of three clinical isolates, the maximal amplification of
263
mutants resistant to 4×, 8× and 16×MIC of ciprofloxacin was observed when antibiotic
264
concentrations fell into the MSW for most of the dosing interval, and both AUC24/MIC and
265
AUC24/MPC relationships with resistance expressed by the population analysis data (AUBCM) or
266
susceptibility testing (ratio of the post-exposure to pre-exposure MIC) were bell-shaped. These
267
observations are consistent with earlier findings reported in studies with fluoroquinolones (6-17).
268
Findings obtained in the present study predict the “anti-mutant” AUC24/MIC ratios for K.
269
pneumoniae strains 1885, 1145, and 185 (2320, 2610 and 1310 h, respectively) that are much
270
higher than the respective clinically attainable AUC24/MICs (22, 44 and 176 h, respectively).
271
The same trend also was seen in our recent study with ciprofloxacin against four P. aeruginosa
272
strains (11): “anti-mutant” AUC24/MIC ratios (220-1100 h) were higher than the respective
273
clinically attainable AUC24/MICs (44 and 176 h). In contrast, in our studies with E. coli (9),
274
despite relatively high “anti-mutant” AUC24/MICs (720-1440 h), these ratios can be achieved at
275
clinically attainable exposures (AUC/MICs 2750 and 1375 h).
276
Despite pronounced stratification of AUBCM versus AUC24/MIC or AUC24/MPC curves
277
seen with individual organisms, it appeared possible to establish bacterial strain-independent
278
AUC24/MIC or AUC24/MPC relationships with resistance using combined data with all three K.
279
pneumoniae strains. Similar relationships were established between MICfinal/MICinitial and
280
AUC24/MIC and AUC24/MPC. However, AUC24/MIC and AUC24/MPC ratios that correspond to
281
the greatest loss in susceptibility were shifted towards higher values compared to those that
282
correspond to the greatest AUBCM. A similar shift was reported with two of four strains in our
283
study with ciprofloxacin-exposed P. aeruginosa (11).
284
As in our recent study with ciprofloxacin against E. coli (10), to test significant levels of
285
K. pneumoniae resistance we chose mutants resistant to 4×, 8× and 16×MIC, ignoring more
286
abundant nontarget (e.g. efflux) mutants resistant to 2×MIC of ciprofloxacin because exposure of
287
bacteria to twice their MICs can almost be regarded as positive controls (28). In the present study
288
we did not identify any changes in nucleotide sequences of gyrA and parC QRDRs of
289
ciprofloxacin-resistant K. pneumoniae mutants as compared to those of parental or reference
290
strains suggesting that the enhancement of resistance was likely due to altered drug
291
accumulation, although mutations causing resistance to fluoroquinolones in Enterobacteriaceae
292
most often occur in the QRDR region of the primary target gene, gyrA (9, 29-31). Additional
293
mutations that further increase resistance to fluoroquinolones may occur in the secondary target
294
gene, parC (29-31), and, in K. pneumoniae, in various regulatory genes that affect the expression
295
of efflux pumps, e.g. AcrAB (32-34), and major porins, OmpK35 and OmpK36 (35, 36), leading
296
to decreased accumulation of the drug in bacterial cells.
297
In conclusion, findings obtained in the present study are in support of the MSW
298
hypothesis that explains the bell shape of the AUBCM versus AUC24/MIC and AUC24/MPC
299
curves. However, highly variable AUC24/MPC thresholds call into question the reliability of
300
their inter-strain predictions of the enrichment of K. pneumoniae mutants. From this point of
301
view AUC24/MIC should be preferred to AUC24/MPC for predictions of the emergence of K.
302
pneumoniae resistance. Similar conclusions were drawn in studies with ciprofloxacin-exposed E.
303
coli (9, 10) and P. aeruginosa (11). In fact, a priori expectations of better predictive force of the
304
AUC24/MPC ratio were confirmed only with fluoroquinolone-exposed S. aureus (5, 8).
305
Further studies with other bacterial species are needed to provide a more complete
306
understanding of AUC24/MIC and AUC24/MPC as potential predictors of the enrichment of
307
resistant mutants.
308 309
Funding information
310
Studies performed at the Department of Pharmacokinetics & Pharmacodynamics, Gause Institute
311
of New Antibiotics, were supported by a grant from Russian Science Foundation (#14-15-
312
00970).
313
314
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435
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437
438
Figure legends
439 440
FIG 1. Simulated pharmacokinetic profiles of ciprofloxacin and time courses of susceptible
441
(0×MIC) and resistant (4×MIC) subpopulations of K. pneumoniae exposed to ciprofloxacin. The
442
simulated AUC24/MIC ratios (in hours) are indicated as boxed numbers.
443
FIG 2. AUC24/MIC and AUC24/MPC relationships with AUBCM (for mutants resistant to 4×, 8×
444
and 16×MIC of ciprofloxacin) observed with individual K. pneumoniae isolates.
445
FIG 3. Combined data on all three K. pneumoniae isolates (for mutants resistant to 4×MIC of
446
ciprofloxacin). Upper panel: AUC24/MIC and AUC24/MPC relationships with AUBCM.
447
Estimated coefficients of equation 2: Y0 = 1, x0 = 1.926, a = 350.8, b = 0.6633, c = 2.007
448
(AUBCM versus AUC24/MIC); Y0 = 1, x0 = 0.4464, a = 349, b = 0.6887, c = 2.01 (AUBCM versus
449
AUC24/MPC). Bottom panel: “anti-mutant” AUC24/MIC and AUC24/MPC ratio determination
450
using the respective descending portions of the AUBCM versus AUC24/MIC and AUC24/MPC
451
curves. Estimated coefficients of equation 3: Y0 = 953.0, a = -283.8 (AUBCM versus
452
AUC24/MIC), Y0 = 442.5, a = -194.8 (AUBCM versus AUC24/MPC).
453
FIG 4. AUC24/MIC and AUC24/MPC relationships with MICfinal/MICinitial - combined data on all
454
three K. pneumoniae isolates. Estimated coefficients of equation 2: Y0 = 1, x0 = 2.309, a = 20.74,
455
b = 0.5127, c = 2.175 (MICfinal/MICinitial versus AUC24/MIC); Y0 = 1, x0 = 0.8559, a = 15.63, b =
456
0.7454, c = 2.207 (MICfinal/MICinitial versus AUC24/MPC).
457
FIG 5. AUC24/MIC-dependent effects of ciprofloxacin on susceptible subpopulation of K.
458
pneumoniae fitted by equation 1: Y0 = 0, x0 = 2,389, a = 208.2, b = 0.4083.
Concentration/MIC
100
15
60
720
0×MIC
0×MIC
0×MIC
4×MIC
4×MIC
4×MIC
10
1
0,1 10
8
Viable count (log CFU/ml)
6
4
2
0
10
8
6
4
2
0 0
24
48
72
0
K. pneumoniae 1885;
24
□
48
72
0
24
Time (h) K. pneumoniae 1145; K. pneumoniae 185
48
72
500
4×MIC
4×MIC
8×MIC
8×MIC
16×MIC
16×MIC
400
300
200
100
0
AUBCM ((log CFU/ml) × h)
500
400
300
200
100
0
500
400
300
200
100
0 1
10
100
1000
10000
0,1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
100
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
1000
500
400
r2 0.72
r2 0.76
300
AUBCM ((log CFU/ml) × h)
200
100
0
500
400
r2 0.74
300
r2 0.59
200
130
100
1800 0 1
10
100
1000
10000
0.1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
100
1000
MICfinal/MICinitial
30
r2 0.75
20
r2 0.43
10
0
1
10
100
1000
10000
0.1
AUC24/MIC (h) K. pneumoniae 1885;
1
10
AUC24/MPC (h)
□ K. pneumoniae 1145;
K. pneumoniae 185
100
1000
ABBC ((log CFU/ml) × h)
300
200
r2 0.78 100
0
10
100
1000
10000
AUC24/MIC (h) K. pneumoniae 1885; □ K. pneumoniae 1145; K. pneumoniae 185
Table 1 Predicted “anti-mutant” (AUC24/MIC)/(AUC24/MPC) ratios in hours. Mutants resistant to ciprofloxacin
K. pneumoniae strain 4×MIC
8×MIC
16×MIC
1885
2320/290
1200/150
720/90
1145
2610/110
1440/60
1000/40
185
1310/20
1270/20
1250/20
