Departments of Pathology, 1 Hershey Medical Center, 500 University Drive, Hershey, PA 17033; 2 Case Western Reserve University, Cleveland, OH, USA
Received 2 January 2002; returned 27 February 2002; revised 13 March 2002; accepted 5 April 2002
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Abstract |
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Introduction |
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There is an urgent need for compounds for in- and outpatient treatment of respiratory tract and other infections caused by pneumococci resistant to ß-lactams, macrolides, quinolones and other agents.5 The higher the penicillin MIC, the more likely the pneumococcus is to be macrolide resistant, with macrolide resistance rates of c. 65% in penicillin-resistant organisms.3
Quinolones such as ciprofloxacin and ofloxacin yield moderate in vitro activity against pneumococci, with MICs clustering around the breakpoints. Newer quinolones such as levofloxacin, gatifloxacin, moxifloxacin and gemifloxacin have lower MICs for pneumococci.2,8,9 Several recent reports from Hong Kong,10 Canada11 and Spain12 have documented a worrisome trend towards quinolone resistance in pneumococci isolated from adult patients, and this incidence may rise, especially if broad-spectrum quinolones are released into the paediatric market.
Linezolid, the first commercially available oxazolidinone, has excellent antipneumococcal activity, irrespective of the susceptibility of the strain to penicillin G, macrolides, quinolones or any other group of compounds.1317 This study examines the antipneumococcal activity of AZD2563 (Figure 1), a new oxazolidinone in development. AZD2563 differs from linezolid at positions 3 and 4 of the aryl ring and on the C-5 side chain. AZD2563 activity was compared with that of linezolid, erythromycin, azithromycin, clindamycin, penicillin G, amoxicillin, levofloxacin, quinupristin/dalfopristin and vancomycin by MIC testing of 300 penicillin-, macrolide- and quinolone-susceptible and -resistant isolates, and also by timekill analysis of 16 strains with differing susceptibilities to the latter three drug groups to all compounds except azithromycin and clindamycin.
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Materials and methods |
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Recent clinical isolates of pneumococci used in this study were from USA, France, Hungary, Czech Republic, Slovakia, Bulgaria, Romania, Poland, Greece, Israel, Japan, Korea and Hong Kong. They comprised 99 penicillin-susceptible (MICs 0.06 mg/L), 86 penicillin-intermediate (MICs 0.1251 mg/L) and 115 penicillin-resistant (MIC 216 mg/L) isolates. Of these, 185 were erythromycin resistant (MICs 1>64 mg/L) and 35 were levofloxacin resistant (MICs
8 mg/L). For timekill studies, a total of 16 penicillin-susceptible, penicillin-resistant, macrolide-resistant (ermB and mefE) and quinolone-resistant strains (with mutations in two or more sites in type II topoisomerase) were tested. Macrolide- and quinolone-resistant strains were characterized previously in our laboratory.18,19
Antimicrobials and MIC testing
AZD2563 was obtained from AstraZeneca, Cheshire, UK. Other antimicrobials were obtained from their respective manufacturers. Microbroth MICs were determined accord- ing to NCCLS recommendations20 using cation-adjusted MuellerHinton broth with 5% lysed defibrinated horse blood. MIC ranges tested (mg/L) were as follows: penicillin G, 0.0168; AZD2563, 0.068; linezolid, 0.12516; erythromycin, 0.01664; azithromycin and clindamycin, 0.0162; amoxicillin, 0.0168; levofloxacin, 0.2532; quinupristin/dalfopristin, 0.068; vancomycin, 0.034. Standard quality control strains, including Streptococcus pneumoniae ATCC 49619, were included in each run.
Timekill testing
For timekill studies, glass tubes containing 5 mL of cation-adjusted MuellerHinton broth (Difco Laboratories) + 5% lysed horse blood with doubling antibiotic concentrations were inoculated with 5 x 1055 x 106 cfu/mL and incubated at 35°C in a shaking water bath. Antibiotic concentrations were chosen to comprise three doubling dilutions above and three dilutions below the microdilution MIC. Growth controls with inoculum but no antibiotic were included with each experiment.21,22
Lysed horse blood was prepared as described previously.21,22 The bacterial inoculum was prepared by suspending growth from an overnight blood agar plate in MuellerHinton broth until turbidity matched a no. 1 McFarland standard. Dilutions required to obtain the correct inoculum (5 x 1055 x 106 cfu/mL) were determined by previous viability studies using each isolate.21,22
To inoculate each tube of serially diluted antibiotic, 50 µL of diluted inoculum was delivered by pipette beneath the surface of the broth. Tubes were then vortexed and plated for viability counts within 10 min (c. 0.2 h). The original inoculum was determined by using the untreated growth control. Only tubes containing an initial inoculum within the range 5 x 1055 x 106 cfu/mL were acceptable.21,22
Viability counts of antibiotic-containing suspensions were carried out by plating 10-fold dilutions of 0.1 mL aliquots from each tube in sterile MuellerHinton broth on to trypticase soy agar/5% sheep blood agar plates (BBL Microbiology Systems). Recovery plates were incubated for up to 72 h. Colony counts were carried out on plates yielding 30300 colonies. The lower limit of sensitivity of colony counts was 300 cfu/mL.21,22
Timekill assays were analysed by determining the number of isolates that yielded a log10 cfu/mL of 1, 2 and 3 at 3, 6, 12 and 24 h, compared with counts at time 0 h. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by
3 log10 cfu/mL (99.9%) at each of the time periods, and bacteriostatic if the inoculum was reduced by <3 log10 cfu/mL. With the sensitivity threshold and inocula used in these studies, no problems were encountered in delineating 99.9% killing, when present. The problem of drug carryover was addressed by dilution as described previously.21,22 For macrolide timekill testing, only isolates with macrolide MICs
8 mg/L were tested because of problems in solubilization at high concentrations and also because of lack of clinical significance.
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Results |
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Discussion |
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Timekill studies demonstrated that both AZD2563 and linezolid showed comparable kill kinetics, with slow bactericidal activity, but uniform bacteriostatic activity at the MIC after 24 h. Amoxicillin, levofloxacin and vancomycin displayed good bactericidal activity after 24 h, whereas quinupristin/dalfopristin gave excellent bactericidal activity even after 3 h. Erythromycin was bactericidal after 24 h, at 4 x MIC, with slower killing at earlier time periods. Kill kinetics for all compounds other than oxazolidinones were similar to those reported previously by our group.21,22,24
The results of this study show that both oxazolidinones were very active in vitro against pneumococci irrespective of their ß-lactam, macrolide or quinolone susceptibility, with AZD2563 having MICs usually one dilution lower than linezolid.
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Acknowledgements |
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Footnotes |
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References |
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