Antimicrobial Agents and Chemotherapy, September 2004, p . 3630-3635, Vol . 48, No . 9

Designing Fluoroquinolone Breakpoints for Streptococcus pneumoniae by Using Genetics instead of Pharmacokinetics-Pharmacodynamics

H . J . Smith,^1,2,* A . M . Noreddin,^1,2 C . G . Siemens,¹ K . N . Schurek,¹ J . Greisman,¹ C . J . Hoban,¹ D . J . Hoban,^1,2 and G . G . Zhanel^1,3

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba,¹ Departments of Clinical Microbiology,² Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada³

Received 8 January 2004/ Returned for modification 17 March 2004/ Accepted 28 April 2004

Fluoroquinolones inhibit DNA synthesis through interactions with the type II topoisomerases DNA gyrase and topoisomerase IV (5, 18) . DNA gyrase and topoisomerase IV are heterotetramers composed of two A and two B subunits, encoded by gyrA parC and gyrB parE, respectively (5, 9, 17, 18) . Fluoroquinolone resistance in S . pneumoniae is primarily mediated by spontaneous point mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and/or parC (5, 9, 17, 18) . Fluoroquinolone efflux-mediated resistance has also been documented, although the role of efflux in resistance remains unknown (9, 14, 18, 19) .

The NCCLS designs fluoroquinolone breakpoints utilizing various factors including frequency distributions, clinical data, and pharmacokinetic-pharmacodynamic properties, which incorporate the MIC, to determine the probability of bacteriological and clinical success, the detection of resistant populations, or both (9, 11, 12) . Breakpoints may be subdivided into clinical breakpoints and microbiological breakpoints . Currently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) defines clinical breakpoints and epidemiological cutoff values, whereas the NCCLS does not (7) . Rather, the NCCLS focuses on clinical evidence as well as frequency distributions for setting clinical breakpoints . Clinical breakpoints are dependent on antimicrobial activity (MIC) as well as antimicrobial pharmacokinetics (i.e., pharmacodynamics) . These breakpoints are derived in order to predict the probability of achieving bacteriological eradication from an infection site and ultimately achieving clinical success . Microbiologic breakpoints, on the other hand, are established to identify isolates that may be categorized as susceptible when applying clinical breakpoints but that harbor resistance mutations that have been associated with reduced susceptibility to that antimicrobial agent or antimicrobial class . Microbiologic breakpoints may thus be useful in monitoring the emergence of resistance, especially over time . Like the EUCAST epidemiology cutoff values, the microbiological breakpoints separate wild-type organisms, isolates with no acquired or mutational resistance mechanisms to the particular antimicrobial, and non-wild-type organisms, isolates with acquired or mutational resistance mechanism for the evaluated antimicrobial (7) .

The aim of this study was to evaluate the use of genetic parameters to determine fluoroquinolone (gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin) microbiological breakpoints for S . pneumoniae . These microbiological breakpoints may serve to minimize mutant generation with the fluoroquinolones and reduce bacteriologic failures . We compared these microbiological breakpoints with the current NCCLS clinical breakpoints for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin versus S . pneumoniae . The probability of bacterial eradication of gatifloxacin and levofloxacin was tested in parallel to minimizing mutant generation by the Monte Carlo simulation technique . At this time, Monte Carlo simulations cannot be conducted with gemifloxacin and moxifloxacin, as there are no established models and no population pharmacokinetic studies .

The S . pneumoniae clinical isolates investigated in this study were collected as part of an ongoing national respiratory organism surveillance program (the Canadian Respiratory Organism Susceptibility Study [CROSS]) (19) . The isolates were obtained from 24 medical centers in 9 of the 10 Canadian provinces between 1997 and 2003 (19) . Isolates were identified by using conventional methodology and were deemed to be significant respiratory pathogens by each laboratory's existing protocols .

MICs were determined by the NCCLS broth microdilution technique (13) after the isolates were subcultured twice from frozen stock, grown on blood agar, and incubated at 37°C in 5% CO₂ for 24 h (19) . The antibiotics tested included ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin . The susceptibility interpretive criteria for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin were as described in NCCLS document M100-S12 (13), and ciprofloxacin nonsusceptibility was defined as an MIC of 4 µg/ml . Strains S . pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were used as controls for all MIC determinations . All MICs were determined a minimum of three times on separate days to ensure precision . These MICs are included in Table 1 .

 
As part of CROSS, the QRDRs of gyrA and parC are sequenced for all ciprofloxacin-resistant S . pneumoniae . All ciprofloxacin-resistant isolates that are susceptible to the other fluoroquinolones studied were included in this study (n = 40) . Additionally, 116 fluoroquinolone-susceptible S . pneumoniae isolates were randomly selected to encompass all years of CROSS (1997 to 2003) and all Canadian geographic regions . More isolates were selected from 2003 than other years, as it was hypothesized that mutations have likely become increasingly prevalent in recent years .

Primers previously described by Morrissey and George were used to generate PCR products of the QRDRs of gyrA and parC (10) . Sequencing of the QRDRs was carried out with primers described by Morrissey and George in the forward and reverse directions (10) . An ABI PRISM Big Dye Terminator kit and an ABI PRISM 310 genetic analyzer (PE Applied Biosystems, Mississauga, Ontario, Canada) were used to conduct the sequencing (20) . A total of 156 isolates were selected based on the presence of mutations in the QRDRs of GyrA and ParC . Three groups of isolates were chosen to determine microbiological breakpoints: group 1, with no mutations in ParC or GyrA; group 2, with ParC mutations alone; and group 3, with mutations in both ParC and GyrA .

Monte Carlo simulation (3, 6, 16) was employed to estimate the probability of the once daily (OD) 400-mg doses of gatifloxacin and 500- and 750-mg doses of levofloxacin achieving free-drug area under the concentration-time curve from 0 to 24 h (AUC_0-24)/MIC ratios with both clinical breakpoints and microbiological breakpoints for S . pneumoniae . Gatifloxacin and levofloxacin exposure (free-drug AUC_0-24/MIC) was derived from previously validated population pharmacokinetic models (1, 15) . Variables from hospitalized patients with community-acquired pneumonia and MICs from a previous CROSS study (19) as well as the full variability of encountered drug exposure were integrated via Monte Carlo simulation by using the Professional Crystal Ball 2000 program (Decisioneering UK, Ltd.) . A 10,000-patient Monte Carlo simulation was performed to determine the percentage of patients achieving free-drug AUC_0-24/MIC ratios of 30, 40, 60, and 100 for levofloxacin as well as gatifloxacin dosing schemes evaluated against Canadian respiratory isolates of S . pneumoniae from the CROSS study .

The percentages of isolates with QRDR mutations at MICs considered susceptible by current NCCLS standards are presented in Table 1 . For gatifloxacin-susceptible isolates (MIC 1 µg/ml), 25% of the isolates (n = 143) had ParC mutations, 2% had GyrA mutations, 1% had mutations in both ParC and GyrA, and 72% had no QRDR mutations . For gemifloxacin-susceptible isolates (MIC 0.12 µg/ml) (n = 142), 25% had ParC mutations, 2% had GyrA mutations, 1% had mutations in both ParC and GyrA, and 72% had no QRDR substitutions . According to the current NCCLS susceptibility category for levofloxacin (MIC 2 µg/ml), 24% of the isolates (n = 156) had ParC mutations, 2% had GyrA mutations, 2% had both ParC and GyrA mutations, and 72% of the isolates had no QRDR mutations . For moxifloxacin-susceptible isolates (MIC 1 µg/ml), 24% of the isolates (n = 155) had ParC mutations, 2% had GyrA mutations, 1% had mutations in both ParC and GyrA, and 73% had no QRDR mutations .

Interestingly, at a gatifloxacin MIC of 1 µg/ml (n = 105), a gemifloxacin MIC of 0.12 µg/ml (n = 105), a levofloxacin MIC of 2 µg/ml (n = 116), and a moxifloxacin MIC of 1 µg/ml (n = 116) (all susceptible by NCCLS breakpoints), as well as a ciprofloxacin MIC of 2µg/ml (susceptible), 9% of isolates had ParC QRDR mutations and 2% had QRDR mutations in GyrA . However, at a gatifloxacin MIC of 1 µg/ml (n = 38), a gemifloxacin MIC of 0.12 µg/ml (n = 37), a levofloxacin MIC of 2 µg/ml (n = 40), and a moxifloxacin MIC of 1 µg/ml (n = 39) (all susceptible by NCCLS breakpoints), as well as a ciprofloxacin MIC of 4 µg/ml (resistant), 71, 70, 68, and 70% of isolates, respectively, have mutations in the QRDR of ParC, 3, 3, 3, and 3%, respectively, have mutations in the QRDR of GyrA, and 3, 5, 8, and 5%, repectively, have mutations in both ParC and GyrA .

Based on the high prevalence of QRDR mutations in isolates considered susceptible by current clinical breakpoints, we evaluated isolates with lower MICs and separated them into categories of few QRDR mutations (<15% of isolates), likely QRDR mutations, and very likely QRDR mutations (>60% of isolates) in order to establish microbiological breakpoints . These categories are presented in Table 2 .

 
A total of 156 isolates were sequenced to evaluate the presence of QRDR mutations in the proposed microbiological breakpoint categories . The sequencing results are presented in Table 1 . Based on the proposed few QRDR mutations category, 90, 94, 86, and 91% of the isolates had no QRDR mutations; 9, 6, 14, and 8% had ParC mutations; and 1, 0, 0, and 1% had GyrA mutations for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively . In the proposed likely QRDR mutation category, 48, 61, 96, and 47% had no QRDR mutations; 48, 33, 3, and 49% had ParC mutations; 2, 4, 1, and 2% had GyrA mutations; and 2, 2, 0, and 2% had double mutations for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively . In the proposed very likely QRDR mutations category, 20, 27, 36, and 25% had no QRDR mutations; 50, 64, 55, and 38% had ParC mutations; 10, 5, 3, and 13% had GyrA mutations; and 20, 5, 5, and 25% had double mutations for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively .

The probabilities of 500- and 750-mg levofloxacin and 400-mg OD achieving free-drug AUC₂₄/MIC ratios by using current NCCLS and new microbiological breakpoint categories for S . pneumoniae are shown in Table 3 . Levofloxacin 500- and 750-mg-dose OD probabilities of achieving a free-drug AUC₂₄/MIC ratio of 30 when using current NCCLS breakpoint categories were as follows: for susceptible or few QRDR mutations, 93.0 and 97.9%; for intermediate or likely QRDR mutations, 1.9 and 11.0%; and for resistant or very likely QRDR mutations, 0.0 and 0.0%, respectively . The levofloxacin 500- and 750-mg-dose OD probabilities of achieving a free-drug AUC₂₄/MIC ratio of 30 with new microbiological breakpoint categories were as follows: susceptible or few QRDR mutations, 99.6 and 97.9%; intermediate or likely QRDR mutations, 92.5 and 97.5%; and resistant or very likely QRDR mutations, 40.0 and 71.5% . For gatifloxacin 400-mg-dose OD, the probabilities of achieving a free-drug AUC₂₄/MIC ratio of 30 using current NCCLS breakpoint categories were as follows: susceptible or few QRDR mutations, 98.8%; intermediate or likely QRDR mutations, 12.1%; and resistant or very likely QRDR mutations, 0.0% . The probabilities of achieving a free-drug AUC₂₄/MIC ratio of 30 with new microbiological breakpoint categories were as follows: susceptible or few QRDR mutations, 99.6%; intermediate or likely QRDR mutations, 98.9%; resistant or very likely QRDR mutations, 67.9% .

 
Monte Carlo simulations showed that OD 500- and 750-mg doses of levofloxacin and a 400-mg dose of gatifloxacin have high probabilities of eradicating S . pneumoniae isolates that are classified as few QRDR mutations or likely QRDR mutations by using our new microbiological breakpoints . Although the probability of gatifloxacin achieving a free-drug AUC₂₄/MIC ratio of 30 for isolates with a gatifloxacin MIC of 0.5 µg/ml was 98.9%, we chose to classify isolates with gatifloxacin MICs of 0.5 µg/ml as resistant because the frequency of QRDR mutations in these isolates was 52% (Table 2) . For isolates that are very likely to harbor QRDR mutations in ParC and/or are likely to have mutations in both ParC and GyrA, none of the OD 400-mg dose of gatifloxacin and 500- and 750-mg doses of levofloxacin demonstrated acceptable probability for bacterial eradication (71.5, 40.0, and 67.9%, respectively) .

The currently used NCCLS breakpoints for fluoroquinolones and S . pneumoniae define many isolates as susceptible even though they harbor QRDR mutations . Based on the likelihood of QRDR mutations and, for gatifloxacin and levofloxacin, the probability of bacteriological eradication as determined by Monte Carlo analysis, we propose a microbiological resistance breakpoint . Our proposed microbiological resistance breakpoint is the MIC at which >50% of the isolates carry QRDR mutations and/or, when data are available, when Monte Carlo simulations demonstrate a <90% chance of bacteriological eradication . The proposed microbiological resistance breakpoints are as follows (in micrograms per milliliter): gatifloxacin, >0.25; gemifloxacin, >0.03; levofloxacin, >1, and moxifloxacin, >0.12 .

The recent occurrence of treatment failures resulting from the use of levofloxacin in the treatment of community-acquired pneumonia caused by susceptible S . pneumoniae isolates that harbored QRDR mutations (4, 8) has led to the need to reevaluate current breakpoints . It has previously been demonstrated that secondary mutations are acquired much more rapidly than first-step mutations, resulting in highly resistant isolates (2) which have led to the observed treatment failures . As Lim et al . have recently suggested, emerging resistance patterns cannot be detected based on clinical breakpoints that are unable to identify first-step mutations (9) . Thus, it is clinically important that we develop rapid identification methods for QRDR mutations to avoid treating an S . pneumoniae isolate carrying a first-step mutation with a fluoroquinolone in order to limit the development and propagation of highly resistant isolates . The MICs of numerous quinolones should be considered prior to fluoroquinolone treatment, as we see a much larger percentage of gatifloxacin-, gemifloxacin-, levofloxacin-, and moxifloxacin-susceptible isolates harboring mutations when they are ciprofloxacin resistant . We do no expect or recommend that the microbiological resistance breakpoint be used in clinical practice . The intent of this research is to create awareness of the potential for fluoroquinolone resistance propagation in S . pneumoniae, as many reportedly susceptible isolates carry resistance mutations and high-level resistance results from the sequential acquisition of mutations .

This research was funded in part by the University of Manitoba and Abbott Laboratories Ltd., AstraZeneca Canada Inc., Aventis Pharma, Bayer Inc., Bristol-Myers Squibb Pharmaceutical Group, GlaxoSmithKline, Janssen-Ortho Inc., Merck Frosst Canada & Co., Pfizer/Pharmacia Canada Inc., and Wyeth-Ayerst Canada Inc .

Present address: Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, Duluth, Minn .

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