Antipneumococcal activity of AZD2563, a new oxazolidinone, compared with nine other agents

Mihaela Peric1, Gengrong Lin1, Catherine L. Clark1, Michael R. Jacobs2 and Peter C. Appelbaum1,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of AZD2563, a new oxazolidinone, was compared with that of linezolid, vancomycin, quinupristin/dalfopristin, amoxicillin, levofloxacin, penicillin, erythromycin, azithromycin and clindamycin against a range of pneumococci by microdilution and time–kill studies. Against 300 pneumococci (99 penicillin susceptible, 86 penicillin intermediate, 115 penicillin resistant, 185 erythromycin resistant, 35 quinolone resistant), both oxazolidinones remained active against isolates less susceptible to other agents, with MICs ranging between 0.125 and 2 mg/L; AZD2563 MICs were generally one dilution lower than those of linezolid. Both quinupristin/dalfopristin and vancomycin were active against all groups (MIC ranges 0.125–2 and 0.125–0.25 mg/L, respectively). Apart from 35 isolates with levofloxacin MICs >= 8 mg/L, levofloxacin MICs were <=0.25–4 mg/L. MICs of amoxicillin and erythromycin rose with penicillin G MICs; most macrolide-resistant isolates were either penicillin-intermediate or -resistant. Against 16 organisms with differing ß-lactam, macrolide and quinolone MICs, time–kill studies showed that AZD2563 was bactericidal (99.9% killing) at 4 x MIC against nine strains at 24 h, with 90% killing of all 16 strains at 2 x MIC after 12 h. Similar results were obtained with linezolid. Both oxazolidinones were bacteriostatic at the MIC against all 16 strains. Amoxicillin, levofloxacin and vancomycin, at 2 x MIC, were bactericidal against 15 of the 16 strains after 24 h. Quinupristin/dalfopristin yielded the most rapid killing, with bactericidal activity against 13 of 16 strains at the MIC after 3 h and against 15 strains at 2 x MIC after 24 h. Erythromycin was bactericidal against all 10 strains with MICs <= 8 mg/L at 4 x MIC after 24 h.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of pneumococci resistant to penicillin G and other ß-lactam and non-ß-lactam compounds has increased worldwide at an alarming rate. Major foci of resistance currently include South Africa, Spain, Central and Eastern Europe and parts of Asia.1,2 In the USA, a recent survey has shown that 50.4% of 1476 clinically significant pneumococcal isolates were not susceptible to penicillin; overall, macrolide resistance was 33%, with 5% of penicillin-susceptible, 37% of penicillin-intermediate and 66% of penicillin-resistant isolates being macrolide resistant. No quinolone-resistant organisms were isolated.3 It is also important to note the high rates of isolation of penicillin-intermediate and -resistant pneumococci (c. 30%) in middle ear fluids from patients with refractory otitis media, compared with other isolation sites.4 The therapeutic problem of drug-resistant pneumococci5 is compounded by the ability of resistant clones to spread from country to country and from continent to continent.6,7

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 time–kill analysis of 16 strains with differing susceptibilities to the latter three drug groups to all compounds except azithromycin and clindamycin.



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Figure 1. Structure of AZD2563.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

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 0.125–1 mg/L) and 115 penicillin-resistant (MIC 2–16 mg/L) isolates. Of these, 185 were erythromycin resistant (MICs 1–>64 mg/L) and 35 were levofloxacin resistant (MICs >= 8 mg/L). For time–kill 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 Mueller–Hinton broth with 5% lysed defibrinated horse blood. MIC ranges tested (mg/L) were as follows: penicillin G, 0.016–8; AZD2563, 0.06–8; linezolid, 0.125–16; erythromycin, 0.016–64; azithromycin and clindamycin, 0.016–2; amoxicillin, 0.016–8; levofloxacin, 0.25–32; quinupristin/dalfopristin, 0.06–8; vancomycin, 0.03–4. Standard quality control strains, including Streptococcus pneumoniae ATCC 49619, were included in each run.

Time–kill testing

For time–kill studies, glass tubes containing 5 mL of cation-adjusted Mueller–Hinton 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 Mueller–Hinton 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 Mueller–Hinton 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 30–300 colonies. The lower limit of sensitivity of colony counts was 300 cfu/mL.21,22

Time–kill assays were analysed by determining the number of isolates that yielded a {Delta}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 time–kill 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of microdilution MIC testing of the 300 isolates by penicillin G and erythromycin susceptibility are presented in Table 1. Both oxazolidinones were highly active, irrespective of susceptibility to other agents, with MICs ranging between 0.125 and 2 mg/L of both agents. AZD2563 MICs were generally one dilution lower than those of linezolid. Both quinupristin/dalfopristin and vancomycin were active against all groups (MIC ranges 0.125–2 and 0.125–0.25 mg/L, respectively). Apart from the 35 isolates with levofloxacin MICs >= 8 mg/L, levofloxacin MICs ranged between <=0.25 and 4 mg/L. MICs of amoxicillin and erythromycin rose with penicillin G MICs; most macrolide- and azalide-resistant isolates were found in the penicillin-intermediate and -resistant groups.


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Table 1.  MICs (mg/L) for 300 pneumococcal strains by penicillin G and erythromycin susceptibility
 
MICs for the 16 strains tested by time–kill are presented in Table 2. As can be seen, seven were penicillin susceptible, six penicillin intermediate and three penicillin resistant. Six strains were erythromycin susceptible and 10 erythromycin resistant. Six strains were highly quinolone resistant. Time–kill experiments (Table 3) showed that AZD2563, at 4 x MIC, was bactericidal (99.9% killing) against nine of the 16 strains tested at 24 h, with 90% killing of all 16 strains at 2 x MIC after 12 h. Similar results were obtained with linezolid. Both oxazolidinones were bacteriostatic at the MIC against all 16 strains tested. Amoxicillin, levofloxacin and vancomycin, at 2 x MIC, were bactericidal against 15 of the 16 strains after 24 h. Quinupristin/dalfopristin yielded the most rapid killing of all drugs tested, with bactericidal activity against 13 of 16 strains at the MIC after 3 h and against 15 strains at 2 x MIC after 24 h. Erythromycin was bactericidal against all 10 strains with MICs <= 8 mg/L at 4 x MIC after 24 h.


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Table 2.  MICs (mg/L) of 16 strains tested by time–kill
 

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Table 3.  Time–kill results of 16 pneumococcal strains
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the current study, AZD2563 and linezolid were highly active against all groups of pneumococci, regardless of their susceptibility to ß-lactams, macrolides and quinolones. No pneumococci with oxazolidinone MICs > 2 mg/L were found, and MICs were similar whether isolates were grouped by penicillin G, macrolide or quinolone susceptibility. Of other compounds primarily active against Gram-positive organisms, quinupristin/dalfopristin MICs were all between 0.125 and 2 mg/L and those of vancomycin were between 0.125 and 0.25 mg/L. Of other drugs tested, amoxicillin and erythromycin MICs rose with those of penicillin G. Levofloxacin was active against all organisms except those with raised quinolone MICs, which were expressly chosen for the current study. These MICs, both of linezolid and of other non-oxazolidinone compounds, reflect previous findings.2,3,8,9,13,1517,23

Time–kill 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.


    Acknowledgements
 
This study was supported by a grant from AstraZeneca, Cheshire, UK.


    Footnotes
 
* Corresponding author. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psu.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Jacobs, M. R. (1992). Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clinical Infectious Diseases 15, 119–27.[ISI][Medline]

2 . Jacobs, M. R. & Appelbaum, P. C. (1995). Antibiotic-resistant pneumococci. Reviews in Medical Microbiology 6, 77–93.

3 . Jacobs, M. R., Bajaksouzian, S., Zilles, A., Lin, G., Pankuch, G. A. & Appelbaum, P. C. (1999). Susceptibilities of Streptococcus pneumoniae and Haemophilus influenzae to 10 oral antimicrobial agents based on pharmacodynamic parameters: 1997 US surveillance study. Antimicrobial Agents and Chemotherapy 43, 1901–8.[Abstract/Free Full Text]

4 . Block, S., Harrison, C. J., Hedrick, J. A., Tyler, R. D., Smith, R. A., Keegan, E. et al. (1995). Penicillin-resistant Streptococcus pneumoniae in acute otitis media: risk factors, susceptibility patterns and antimicrobial management. Pediatric Infectious Disease Journal 14, 751–9.[ISI][Medline]

5 . Friedland, I. R. & McCracken, G. H., Jr (1994). Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. New England Journal of Medicine 331, 377–82.[Free Full Text]

6 . McDougal, L. K., Facklam, R., Reeves, M., Hunter, S., Swenson, J. M., Hill, B. C. et al. (1992). Analysis of multiply antimicrobial-resistant isolates of Streptococcus_pneumoniae from the United States. Antimicrobial Agents and Chemotherapy 36, 2176–84.[Abstract]

7 . Munoz, R., Musser, J. M., Crain, M., Briles, D. E., Marton, A., Parkinson, A. J. et al. (1992). Geographic distribution of penicillin-resistant clones of Streptococcus_pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis. Clinical Infectious Diseases 15, 112–8.[ISI][Medline]

8 . Appelbaum, P. C. (1992). Antimicrobial resistance in Streptococcus pneumoniae—an overview. Clinical Infectious Diseases 15, 77–83.[ISI][Medline]

9 . Bauernfeind, A. (1997). Comparison of the antibacterial activities of the quinolones Bay 12-8039, gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and ciprofloxacin. Journal of Antimicrobial Chemotherapy 40, 639–51.[Abstract]

10 . Ho, P.-L., Que, T.-L., Chang, D. N.-C., Ng, T.-K., Chow, K.-C. & Seto, W.-H. (1999). Emergence of fluoroquinolone resistance among multiply resistant strains of Streptococcus pneumoniae in Hong Kong. Antimicrobial Agents and Chemotherapy 43, 1310–3.[Abstract/Free Full Text]

11 . Chen, D. K., McGeer, A., de Azavedo, J. C. & Low, D. E. (1999). Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. New England Journal of Medicine 341, 233–9.[Abstract/Free Full Text]

12 . Liñares, J., de la Campa, A. G. & Pallares, R. (1999). Fluoroquinolone resistance in Streptococcus pneumoniae. New England Journal of Medicine 341, 1546–7.[Free Full Text]

13 . Cercenado, E., Garcia-Garrote, F. & Bouza, E. (2001). In-vitro activity of linezolid against multiply resistant Gram-positive clinical isolates. Journal of Antimicrobial Chemotherapy 47, 77–81.[Abstract/Free Full Text]

14 . Diekema, D. J. & Jones R. N. (2000). Oxazolidinones. A review. Drugs 59, 7–16.[ISI][Medline]

15 . Henwood, C. J., Livermore, D. M., Johnson, A. P., James, D., Warner, M., Gardiner, A. et al. (2000). Susceptibility of Gram-positive cocci from 25 UK hospitals to antimicrobial agents including linezolid. Journal of Antimicrobial Chemotherapy 46, 931–40.[Abstract/Free Full Text]

16 . Patel, R., Rouse, M. S., Piper, K. E. & Steckelberg, J. M. (1999). In-vitro activity of linezolid against vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. Diagnostic Microbiology and Infectious Disease 34, 119–22.[ISI][Medline]

17 . Wise, R., Andrews, J. M., Boswell, F. J. & Ashby, J. P. (1998). The in-vitro activity of linezolid (U-100766) and tentative breakpoints. Journal of Antimicrobial Chemotherapy 42, 721–8.[Abstract]

18 . Davies, T. A., Ednie, L. M., Hoellman, D. M., Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (2000). Antipneumococcal activity of ABT-773 compared to those of 10 other agents. Antimicrobial Agents and Chemotherapy 44, 1894–9.[Abstract/Free Full Text]

19 . Nagai, K., Davies, T. A., Pankuch, G. A., Dewasse, B., Jacobs, M. R. & Appelbaum, P. C. (2000). In vitro selection of resistance to clinafloxacin, ciprofloxacin, and trovafloxacin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 2740–6.[Abstract/Free Full Text]

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

21 . Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (1994). Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, ciprofloxacin and vancomycin by using time–kill methodology. Antimicrobial Agents and Chemotherapy 38, 2065–72.[Abstract]

22 . Pankuch, G. A., Lichtenberger, C., Jacobs, M. R. & Appelbaum, P. C. (1996). Antipneumococcal activities of RP 59500 (quinupristin/dalfopristin), penicillin G, erythromycin, and sparfloxacin determined by MIC and rapid time–kill methodologies. Antimicrobial Agents and Chemotherapy 40, 1653–6.[Abstract]

23 . Livermore, D. M. (2000). Quinupristin/dalfopristin and linezolid: where, when, which and whether to use? Journal of Antimicrobial Chemotherapy 46, 347–50.[Free Full Text]

24 . Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1996). MIC and time–kill study of DU-6859a, ciprofloxacin, levofloxacin, sparfloxacin, cefotaxime, imipenem, and vancomycin against nine penicillin-susceptible and -resistant pneumococci. Antimicrobial Agents and Chemotherapy 40, 362–6.[Abstract]





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