Department of Medical Microbiology, City Hospital NHS Trust, Dudley Road, Birmingham B18 7QH, UK
Received 18 April 2003; returned 18 July 2003; revised 4 February 2004; accepted 11 February 2004
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Abstract |
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Methods: MICs were compared with those of two ß-lactams, telithromycin, ciprofloxacin and four later generation fluoroquinolones. The effects of human serum and of inoculum concentration were also investigated.
Results: MIC data indicate that ABT-492 has potent activity against Gram-positive organisms with enhanced anti-staphylococcal activity compared with earlier fluoroquinolones, in addition to activity against ß-haemolytic streptococci, pneumococci including penicillin- and fluoroquinolone-resistant strains and vancomycin-susceptible and -resistant Enterococcus faecalis but not Enterococcus faecium. ABT-492 was the most active agent tested against Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, fluoroquinolone-susceptible Neisseria gonorrhoeae and anaerobes. Good activity was observed for ABT-492 amongst the Enterobacteriaceae and anaerobes tested, but ciprofloxacin showed superior activity for species of Proteus, Morganella and Providencia, as well as for Pseudomonas spp. In common with the other fluoroquinolones tested, organisms with reduced susceptibility to ciprofloxacin had raised MIC90s to ABT-492. The one isolate of H. influenzae tested with reduced fluoroquinolone susceptibility had an ABT-492 MIC close to that of the population lacking a mechanism of quinolone resistance. ABT-492 was more active than ciprofloxacin against Chlamydia spp. An inoculum effect was observed with a number of isolates of Staphylococcus aureus, Streptococcus pneumoniae, E. faecium, Klebsiella spp. and Escherichia coli, in addition to moderately raised MICs in the presence of 70% serum protein. The clinical significance of these findings is yet to be determined.
Conclusions: ABT-492 is a new fluoroquinolone with excellent activity against both Gram-positive and Gram-negative organisms, with many potential clinical uses.
Keywords: antibacterials, bacterial resistance, quinolones
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Introduction |
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ABT-492 is 1-(6-amino-3,5-difluoropyridine-2-yl)-8-chloro-6-fluoro-(3-hydroxyyazetide-1-yl)-4-oxo-1,4-dihydroquinolone-3-carboxy acid (A-319492), currently being developed by Abbott Laboratories. Preliminary data indicate that it has a broad spectrum of activity rivalling that of other newer fluoroquinolones, with increased potency against multidrug-resistant bacteria (Abbott data on file). In this study, the in vitro activity of ABT-492 was compared with two ß-lactams (including co-amoxiclav at a 2:1 ratio), a ketolide and five fluoroquinolones against isolates including those with a known mechanism of resistance. The effects of human serum and of raising the inoculum concentration were also investigated. The activity of ABT-492 was compared with that of ciprofloxacin and erythromycin against three isolates of Chlamydia trachomatis and one strain of Chlamydia pneumoniae.
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Materials and methods |
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The antibiotics used in this study were ABT-492 and erythromycin (Abbott Laboratories, IL, USA), telithromycin (Aventis Pharma, Romainville, France), ciprofloxacin and moxifloxacin (Bayer AG, Wuppertal, Germany), levofloxacin (Aventis Ltd, Uxbridge, UK), gatifloxacin (Grunenthal GmbH, Aachen, Germany), amoxicillin, clavulanic acid and gemifloxacin (GlaxoSmithKline, Worthing, UK). Storage and preparation of agents were in accordance with the manufacturers instructions.
Isolates
A total of 1146 isolates were studied, the majority of which were recent clinical isolates from City Hospital, Birmingham. Of the organisms with known mechanisms of resistance, 19 Streptococcus pneumoniae had reduced susceptibility to penicillin [high-level resistance (HLR) MIC 2 mg/L and intermediate-level resistance (LLR) MIC 0.121 mg/L] and eight with reduced susceptibility to the fluoroquinolones (including four isolates with a Ser-79
Tyr mutation in parC, two isolates with a Ser-79
Tyr mutation in parC and either a Glu-87
Lys or Glu-83
Tyr mutation in gyrA12,13 and two recent fluoroquinolone-resistant clinical isolates); 25 methicillin-resistant Staphylococcus aureus (MRSA); 33 vancomycin-resistant enterococci (VRE); eight imipenem-resistant Pseudomonas aeruginosa; 19 ciprofloxacin-resistant Escherichia coli; nine Neisseria gonorrhoeae with reduced susceptibility to quinolones (no zone of inhibition with a 30 µg nalidixic acid disc) and one isolate of Haemophilus influenzae with reduced susceptibility to the quinolones [quinolone resistance determining region (QRDR) mutation in gyrA Ser-84
Phe and no zone of inhibition with a 30 µg nalidixic acid disc].14 The following control strains were tested, E. coli ATCC 25922 and NCTC 10418, P. aeruginosa ATCC 27853 and NCTC 10662, Enterococcus faecalis ATCC 29212, H. influenzae NCTC 11931, S. pneumoniae ATCC 49619, N. gonorrhoeae NCTC 12700, Bacteroides fragilis NCTC 9343 and S. aureus NCTC 6571, ATCC 25923 and ATCC 29213.
Susceptibility testing
The MIC was determined by British Society for Antimicrobial Chemotherapy (BSAC) methodology.15 Briefly, Iso-Sensitest agar (Oxoid, Basingstoke, UK) supplemented where necessary with 5% defibrinated horse blood (TCS, Bodolph Clayton, UK) and 20 mg/L NAD (SigmaAldrich Co. Ltd, Poole, UK) for fastidious organisms or 50 mg/L 1-(4-nitro-phenyl)-glycerol (PNPG; BDH, Poole, UK) to prevent swarming. Plates were incubated for 1820 h at 3537°C in air or for fastidious organisms in an atmosphere of 46% CO2. In the case of anaerobes, Brucella agar (Oxoid) supplemented with 5% lysed horse blood was used and incubation was in an atmosphere of 10% CO2/10% H2/80% N2. A final inoculum of 104 cfu/spot was delivered using a multipoint inoculator (Mast, Bootle, UK). The MIC was defined as the lowest concentration of antimicrobial inhibiting bacterial growth, one or two colonies being ignored. To determine the effect of inoculum on the MIC, 163 isolates were each tested at an inoculum of 104 and 106 cfu/spot.
Effect of human serum
MICs and MBCs of ABT-492 were determined for two isolates each of E. coli, P. aeruginosa, S. pneumoniae, and Moraxella catarrhalis in the presence of human serum (TCS) as previously described.15 The basal medium, Iso-Sensitest broth (Oxoid), was supplemented with 5% laked horse blood (Oxoid) where necessary and either 0%, 20% or 70% human serum which had been screened for the presence of blood-borne viruses (HBsAg, HCV, HIV-1 and HIV-2). An inoculum of approximately 5 x 105 cfu/mL was used for testing. For broths with equivocal or no growth, 50 µL was subcultured onto blood agar and incubated under the appropriate conditions for 1820 h. The MBC was defined as the lowest concentration of antibiotic giving a growth of less than 25 colonies on subculture (99.9% kill).
Chlamydia susceptibility testing
MICs and minimum lethal concentrations (MLCs) of ABT-492, ciprofloxacin and erythromycin were determined for four chlamydia isolates (C. pneumoniae TW183, C. trachomatis L2, C. trachomatis CT 599, C. trachomatis CT 527), using a previously described technique.16 Briefly, McCoy cell coverslip cultures were inoculated to form a monolayer of approximately 1 x 103 inclusion forming units per coverslip and doubling dilutions of the antibiotics added. Eagles minimum essential medium with Earles salts (Invitrogen Ltd, UK) was used in place of antibiotic for the positive control. Plates were incubated at 3537°C for 48 h in 46% CO2 and stained with an immunofluorescent reagent (Dako Diagnostics, Ely, UK). The MIC was defined as the lowest concentration of antimicrobial to inhibit development of inclusions and the MLC as the lowest concentration at which no proper inclusions were formed after a further 48 h in antibiotic-free medium.
Analysis of data
For the control strains, MICs were compared with expected values.15 Test MICs were evaluated with reference to existing BSAC breakpoints15 and where in sufficient numbers to allow comparison, with regard to the wild-type population as defined by the Swedish Reference Group for Antibiotics (SRGA).17 The population with reduced susceptibility to fluoroquinolones was identified by reference to the ciprofloxacin MICs.
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Results |
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P. aeruginosa MIC90 results were higher for ABT-492 than ciprofloxacin and gatifloxacin, but equal to levofloxacin, moxifloxacin and gemifloxacin. MIC90 results for ABT-492 were raised in the non wild-type compared with the wild-type populations, 0.5 and 64 mg/L, respectively.
ABT-492 was the most active agent against all staphylococci tested, but a higher MIC90 was observed for MRSA (0.5 mg/L) compared with methicillin-susceptible S. aureus (MSSA) (0.008 mg/L). ABT-492 was also the most active agent against the Streptococcus milleri group and the most active quinolone against group B streptococci. It shared equal highest quinolone activity with gemifloxacin for the group A streptococci tested. ABT-492 was equally active against penicillin-susceptible and -resistant pneumococci (data not shown). For the ciprofloxacin-resistant pneumococci (MIC90 128 mg/L), ABT-492 was superior in activity to the other quinolones tested (MICs 0.03 mg/L).
ABT-492 was 2- to 8-fold and 4- to 32-fold more active than the newer fluoroquinolones against Enterococcus faecium and E. faecalis, respectively, although this was offset by an eight-fold increase in MIC90 amongst the ciprofloxacin-resistant strains. It was 4- to 128-fold more active against vancomycin-resistant E. faecalis (MIC90 1 mg/L), but had higher MICs against vancomycin-resistant E. faecium (MIC90 8 mg/L) (data not shown).
For the nine isolates of N. gonorrhoeae with reduced fluoroquinolone susceptibility, ABT-492 MICs ranged from 0.001 to 0.015 mg/L and 0.03 to 0.25 mg/L, respectively for the organisms with low- and high-level resistance. ABT-492 had the lowest MICs for the anaerobes, N. meningitidis and the respiratory pathogens M. catarrhalis and H. influenzae. For the one isolate of H. influenzae with reduced susceptibility to the fluoroquinolones (ciprofloxacin, levofloxacin and gatifloxacin MICs 0.06 mg/L and moxifloxacin MIC 0.03 mg/L), the MIC of ABT-492 was only marginally higher than that of the susceptible population (MICs 0.004 and 0.001 mg/L, respectively).
Activity of ABT-492 was superior to ciprofloxacin and erythromycin against C. trachomatis (MIC and MLC 0.0150.06 mg/L) and C. pneumoniae (MIC and MLC 0.03 mg/L).
Increasing the inoculum from 104 to 106 cfu/spot raised the MIC 2-fold for most isolates, but greater differences were observed for three S. aureus (8- to 16-fold), one S. pneumoniae (8-fold), one E. faecium (8-fold), four Klebsiella spp. (16- to 64-fold) and three E. coli (32-fold) (Table 4).
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Discussion |
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Increasing levels of ciprofloxacin resistance amongst Pseudomonas spp. have been well-documented18 and greatly restrict clinical treatment options. Ciprofloxacin and gatifloxacin were the most active agents against P. aeruginosa and increased ABT-492 MICs were demonstrated in organisms where ciprofloxacin resistance already existed, suggesting that this new agent would not be a suitable alternative treatment option.
ABT-492 showed superior activity to ciprofloxacin against C. trachomatis, with MICs lower than those quoted previously for levofloxacin, moxifloxacin, gatifloxacin and gemifloxacin (MIC90s 1, 0.12, 0.25 and 0.06 mg/L, respectively) against Chlamydia spp.19 It also showed superior activity against N. gonorrhoeae including those isolates with reduced susceptibility to fluoroquinolones. This indicates a role in the treatment of genitourinary infection, but future clinical application may be limited where resistance mechanisms already exist.
ABT-492 could have an important role as a single agent in the treatment of community-acquired pneumonia, particularly where ß-lactam and macrolide-resistant pathogens predominate. Activity against respiratory pathogens appeared superior to the ketolide, telithromycin, whose spectrum of activity includes pneumococci with reduced penicillin and erythromycin susceptibility. It was the most active agent against C. pneumoniae, H. influenzae (including the one isolate with reduced susceptibility to fluoroquinolones), M. catarrhalis and S. pneumoniae including LLR and HLR isolates. Indeed, pneumococci with existing mechanisms of fluoroquinolone resistance appeared susceptible to ABT-492. Although the chlamydia isolates were not tested against a range of fluoroquinolones, the ABT-492 MIC of the C. pneumoniae isolate (0.03 mg/L) was lower than the MIC ranges reported elsewhere for levofloxacin, moxifloxacin and gemifloxacin (0.5, 0.060.1 and 0.060.12 mg/L, respectively), supporting its use as an effective agent against this pathogen.20,21
Anti-staphylococcal activity of ABT-492 was superior for all species tested, with good activity displayed against MRSA strains (MIC90 0.25 mg/L). However, the relative increase in MIC90 (>64-fold) for MRSA compared with MSSA organisms remains a concern, and highlights potential limitations in its clinical use against these resistant pathogens. Amongst the fluoroquinolones, ABT-492 showed superior activity against enterococci and, in particular, vancomycin-resistant E. faecalis including vancomycin-resistant isolates.
An inoculum effect (defined as a 8-fold rise in MIC at a higher inoculum compared with a lower inoculum) was observed for 10 isolates including one each of S. aureus, E. faecium, Enterobacter aerogenes, three E. coli and four Klebsiella spp. (Table 4).
The MBC of ABT-492 was equal to or only two-fold higher than the MIC for five of six strains tested (two E. coli, two P. aeruginosa, one S. pneumoniae), and four-fold greater for one S. pneumoniae isolate. This implies that, at least in vitro, ABT-492 has a bactericidal action like existing fluoroquinolones. Although the addition of 70% human serum had little effect on the MICs, it had a marked effect on the MBCs causing a greater than or equal to four-fold increase in most cases. As the drug is 84% protein bound in humans (Abbott data), tissue distribution may be reduced in comparison with less highly bound fluoroquinolones22 and together with the inoculum effect this will need to be reflected in the setting of breakpoints.
This in vitro study indicates that ABT-492 could play an important role in the treatment of both Gram-positive and Gram-negative infections, including those due to multi-resistant bacteria, and atypical respiratory pathogens. However, the clinical significance of the evidence indicating the existence of resistance mechanisms, as well as an observed inoculum effect and of moderately high serum protein binding are unclear and the outcome of clinical studies, including those involving resistant pathogens like MRSA and VRE, are required to define the future clinical role of this new antimicrobial.
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Acknowledgements |
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Footnotes |
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References |
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2
.
Schito, G. C., Debbia, E. A. & Marchese, A. (2000). The evolving threat of antibiotic resistance in Europe: new data from the Alexander Project. Journal of Antimicrobial Chemotherapy 46, Suppl. T1, 39.
3
.
Granizo, J. J., Aguilar, L., Casal, J. et al. (2000). Streptococcus pneumoniae resistance to erythromycin and penicillin in relation to macrolide and ß-lactam consumption in Spain (19791997). Journal of Antimicrobial Chemotherapy 46, 76773.
4
.
American Thoracic Society. (2001). Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention. American Journal of Respiratory and Critical Care Medicine 163, 173054.
5
.
British Thoracic Society. (2001). Guidelines for the management of community-acquired pneumonia in adults admitted to hospital. Thorax 56, Suppl. IV, 164.
6 . Bartlett, J. G., Dowell, S. F., Mandell, L. A. et al. (2000). Practice guidelines for the management of community-acquired pneumonia in adults. Clinical Infectious Diseases 31, 34782.[CrossRef][Medline]
7
.
ERS Task Force. (1998). Guidelines for management of adult community-acquired lower respiratory tract infections. European Respiratory Journal 11, 98691.
8
.
Chen, D. K., McGeer, A., De Azavedo, J. C. et al. (1999). Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. New England Journal of Medicine 341, 2339.
9
.
Jorgensen, J. H., Weigel, L. M., Ferraro, M. J. et al. (1999). Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci. Antimicrobial Agents and Chemotherapy 43, 32934.
10
.
Ho, P. L., Yung, R. W. H., Tsang, D. N. C. et al. (2001). Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 2000. Journal of Antimicrobial Chemotherapy 48, 65965.
11
.
Davidson, R., Cavalcanti, R., Brunton, J. L. et al. (2002). Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. New England Journal of Medicine 346, 74750.
12 . Brenwald, N. P., Gill, M. J. & Wise, R. (1999). Grepafloxacin vs pneumococci resistant to fluoroquinolones by a putative efflux mechanism. Drugs 58, Suppl. 2, 1178.[ISI]
13 . Pan, X.-S., Ambler, J., Mehtar, S. et al. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 23216.[Abstract]
14
.
Brenwald, N. P., Andrews, J. M., Jevons, G. et al. (2003). Detection of ciprofloxacin resistance in Haemophilus influenzae using nalidixic acid and BSAC methodology. Journal of Antimicrobial Chemotherapy 51, 13112.
15 . Andrews, J. M. (2003). BSAC disc diffusion method for antimicrobial susceptibility testing. Version 2.1.4. [Online.] http://www.bsac.org.uk/uploads/may%202003susceptibility1.pdf (30 January 2004, date last accessed).
16 . Webberley, J. M., Matthews, R. S., Andrews, J. M. et al. (1987). Commercially available fluorescent-conjugated monoclonal antibody for determining the in vitro activity of antimicrobial agents against Chlamydia trachomatis. European Journal of Clinical Microbiology and Infectious Diseases 6, 5879.[ISI]
17 . Kahlmeter, G. & Olsson-Liljequist, B. (1998). Swedish Reference Group for Antibiotics. Ciprofloxacin MIC values of isolates that lack resistance mechanisms. [Online.] http://www.srga.org/MICTAB/MIC/MICcip.html (30 January 2004, date last accessed).
18 . Thomson, C. J. (1999). The global epidemiology of resistance to ciprofloxacin and the changing nature of antibiotic resistance: a 10 year perspective. Journal of Antimicrobial Chemotherapy 43, Suppl. A, 3140.[Medline]
19
.
Andersson, M. L. & MacGowan, A. P. (2003). Development of the quinolones. Journal of Antimicrobial Chemotherapy 51, Suppl. S1, 111.
20 . Ball, P., Mandell, L., Niki, Y. et al. (1998). Therapeutic advances of new fluoroquinolones. Expert Opinion on Investigational Drugs 7, 76183.
21 . Felmingham, D., Robbins, M. J., Dencer, C. et al. (1999). In vitro activity of gemifloxacin against S. pneumoniae, H. influenzae, M. catarrhalis, L. pneumophila and Chlamydia spp. Abstract P408. Journal of Antimicrobial Chemotherapy 44, Suppl. A, 131.
22 . Craig, W. A. & Suh, B. (1986). Protein binding and the antimicrobial effects: methods for the determination of protein binding. In Antibiotics in Laboratory Medicine, 2nd edn (Lorian, V., Ed), pp. 477514. Williams & Wilkins, Baltimore, MD, USA.