Journal of Antimicrobial Chemotherapy (1999) 44, 679-688
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
The in-vitro activity and tentative breakpoint of gemifloxacin, a new fluoroquinolone
Department of Medical Microbiology, City Hospital NHS Trust, Birmingham, UK
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Abstract |
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The in-vitro activity of gemifloxacin, a new fluoroquinolone, against a wide range (c. 700) of recent clinical isolates, was compared with that of three other fluoroquinolones and other relevant agents. Gemifloxacin inhibited 90% of the Enterobacteriaceae strains at 0.5 mg/L or less, exceptions being Serratia spp. (MIC90 1 mg/L) and strains possessing a putative mechanism of resistance to fluoroquinolones. Ninety per cent of Pseudomonas aeruginosa were inhibited by 4 mg/L. Gemifloxacin had good activity against respiratory pathogens, with 90% of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis being inhibited by 0.06 mg/L or less. Staphylococcus aureus (MSSA) were highly susceptible (MIC90 0.06 mg/L) but MRSA less susceptible (MIC90 8 mg/L) to gemifloxacin. Enterococcus spp. were markedly more susceptible to the study agent than to ciprofloxacin. Gemifloxacin showed good activity against Bacteroides fragilis (MIC90 0.5 mg/L) and anaerobic cocci. A tentative in-vitro breakpoint of 0.5 mg/L was studied using a 1 µg disc content for all genera except Pseudomonas where a 5 µg disc content was employed. The false sensitivity reporting rate was 0.5% and false resistance rate was 6.0%, which was considered acceptable. In conclusion, gemifloxacin is a highly active fluoroquinolone that should prove clinically useful in the treatment of a wide range of infections. Susceptibility testing criteria have been developed that should prove robust in a clinical laboratory.
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Introduction |
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Gemifloxacin (LB20304) is a new fluoronaphthyridone compound. Preliminary studies suggest that it has enhanced activity in comparison with currently available fluoroquinolones against Gram-positive cocci.1,2 The compound was first developed by LG Chemical Ltd in Korea and is currently under investigation by SmithKline Beecham in the treatment of a wide range of clinical conditions.
In this study the activity of gemifloxacin was compared with commonly used antimicrobials against a wide range of bacterial strains. In addition, the techniques of the British Society for Antimicrobial Chemotherapy (BSAC) Working Party on Susceptibility Testing3 were employed to establish a tentative in-vitro breakpoint.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Antimicrobial agents
The following agents were used: gemifloxacin and co-amoxiclav (SmithKline Beecham, Worthing, UK); ciprofloxacin (Bayer AG, Wuppertal, Germany); trovafloxacin (Pfizer Ltd, Sandwich, UK); levofloxacin (Roussel Uclaf, Romainville, France) and nalidixic acid (Sigma, Poole, UK). All agents were prepared and stored following the manufacturer's instructions.
Susceptibility testing
A total of c. 700 recent clinical isolates, 11 control strains and 12 well characterized ß-lactamase-producing strains were studied. The control strains were Escherichia coli NCTC 10418, Pseudomonas aeruginosa NCTC 10662 and ATCC 27853, Staphylococcus aureus NCTC 6571, ATCC 25923 and ATCC 29213, Streptococcus pneumoniae NCTC 7465 and ATCC 49619, Haemophilus influenzae NCTC 11931 and ATCC 49247, Enterococcus faecalis ATCC 29212 and Bacteroides fragilis NCTC 9343.
Susceptibilities were determined by a standard agar plate dilution method3. Briefly, Iso-Sensitest agar (pH 7.2, Unipath, Basingstoke, UK), supplemented with 1-(4-nitrophenyl)-glycerol (BDH, Poole, UK) 50 mg/L where necessary to prevent swarming, was used for aerobic bacteria. Horse blood 5% (Bradsure Biologicals, Loughborough, UK) and NAD (Sigma) 20 mg/L were added to support the growth of fastidious bacteria.
For anaerobic bacteria, Wilkins–Chalgren agar (Unipath) supplemented with 5% horse blood was used. All strains were tested at a final inoculum of 104 cfu and for a few selected strains at an increased inoculum of 106 cfu, using a multipoint inoculator (Denley Instruments, Billingshurst, UK). Plates were incubated at 35–37°C for 18–24 h in air; or for fastidious bacteria, in an atmosphere enriched with 4–6% CO2; or for anaerobic bacteria in an anaerobic cabinet (Don Whitely, Shipley, UK) in an atmosphere of 10% hydrogen 10% CO2 and 80% nitrogen.
The MIC was defined as the lowest antibiotic concentration at which no more than two colonies were observed. Amoxycillin and clavulanic acid were combined in a ratio of 2:1, and the results were recorded in terms of the amoxycillin MIC.
Tentative MIC and zone diameter breakpoints
MICs (as described above) and zone size diameter were measured for 646 recent isolates following BSAC recommendations (BSAC Newsletter, Summer, 1998). Briefly 6 mm diameter discs prepared in-house containing 1, 2 and 5 µg gemifloxacin were placed on the appropriate medium seeded with an inoculum equivalent to semi-confluent growth and incubated (as above). The BSAC formula for breakpoint determination was applied3 assuming Cmax = 1.2 mg/L, elimination half life = c. 8–10 h and protein binding as 60%. This gives a breakpoint of 0.5–1 mg/L. Scattergrams of zone size plotted against MIC were performed and false sensitivity and resistance rates estimated.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Quality control
The MICs of all agents other than gemifloxacin for the reference strains were identical to, or within a two-fold dilution of, the reference values determined in this laboratory (data not shown).
Comparative in-vitro potency
The potency of gemifloxacin against the Enterobacteriaceae was similar to that of
ciprofloxacin, trovafloxacin and levofloxacin, all compounds having MICs within one doubling
dilution of each other (Table I). The E. coli strains tested
included 10
clinical isolates
with a ciprofloxacin MIC of 
1 mg/L. These strains showed cross-resistance (MIC 0.5
mg/L) to gemifloxacin. Against P. aeruginosa gemifloxacin displayed a similar degree
of potency to ciprofloxacin and when individual strains were compared both these compounds
were two-fold more potent than levofloxacin and trovafloxacin. There was a wide range of
susceptibility of Stenotrophomonas maltophilia to all the fluoroquinolones with
gemifloxacin and trovafloxacin being two-fold more potent than the other two fluoroquinolones.
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Against methicillin-susceptible S. aureus (MSSA), gemifloxacin and trovafloxacin were markedly more potent than ciprofloxacin (a 32-fold difference) or levofloxacin (an eight-fold difference) and co-amoxiclav (an eight-fold difference). Methicillin-resistant strains of S. aureus were all less susceptible to the fluoroquinolones than were the MSSA; the most susceptible strain had a gemifloxacin MIC of 1 mg/L and a ciprofloxacin MIC of 16 mg/L.
Similar differences between the fluoroquinolones were observed when the methicillin-susceptible S. epidermidis strains were tested: gemifloxacin and trovafloxacin were markedly more potent than the other agents studied. Generally, methicillin-resistant strains of S. epidermidis were susceptible to the fluoroquinolones but two strains that had a gemifloxacin MIC of 2 mg/L were susceptible to 4 and 8 mg/L of trovafloxacin and 64 and 16 mg/L ciprofloxacin, respectively.
Gemifloxacin was the most potent agent (other than co-amoxiclav) studied against E.
faecalis and E. faecium. Gemifloxacin was eight- to 16-fold more potent
than ciprofloxacin and four-fold more potent than trovafloxacin; however, strains with reduced
susceptibilities (gemifloxacin MIC 2 mg/L) were noted in both species.
Against streptococci (including S. pneumoniae) gemifloxacin was two- to four-fold
more potent than trovafloxacin and 16- to 32-fold more potent than ciprofloxacin. A total of 16
fluoroquinolone-resistant S. pneumoniae (ciprofloxacin MIC 1 mg/L) isolates
were studied and again gemifloxacin was the most potent agent of the class. There were 10
strains with ciprofloxacin MICs of 4–8 mg/L and these were susceptible to gemifloxacin
0.06–0.12 mg/L. Four strains had ciprofloxacin MICs of 128 mg/L and these were
susceptible to gemifloxacin 0.5–2 mg/L.
The H. influenzae, Neisseria spp. and Moraxella catarrhalis strains tested were highly susceptible to all the fluoroquinolones and those strains known to produce ß-lactamase were as susceptible as the ß-lactamase-negative strains.
Among the anaerobic organisms studied, Clostridium perfringens was highly susceptible to gemifloxacin, all strains being inhibited by 0.12 mg/L or less. Clostridium difficile was less susceptible with gemifloxacin MICs of 2 mg/L or less. The anaerobic cocci were equally susceptible to gemifloxacin and trovafloxacin. Against B. fragilis, trovafloxacin was marginally more potent or as potent as gemifloxacin, all strains being inhibited by 0.5 mg/L or less of the latter agent.
The susceptibility of 47 strains of both Gram-negative and Gram-positive organisms was studied at both the routine inoculum of 104 cfu per inoculum and at 106 cfu per inoculum (data not shown). Forty-two showed an MIC identical or within two-fold greater at the higher inoculum. The remainder showed a four-fold difference.
Tentative MIC and zone diameter breakpoints
For the purpose of initial analysis a conservative MIC breakpoint (BP) of 0.5 mg/L was chosen for all strains. Data obtained for 2 µg and 5 µg gemifloxacin disc contents revealed that zones of inhibition ranged from 30 to 45 mm for the sensitive population for all genera except Pseudomonas (data not shown). Zones of this magnitude have been shown to cause unacceptable zone merging when multidisc testing is performed on a 90 mm Petri dish and therefore analysis was only executed on data for a 1 µg disc content for all strains except Pseudomonas sp. (Figure 1). Using a zone diameter breakpoint (ZD BP) of 20 mm, three strains were falsely interpreted as sensitive and they comprised one E. coli (MIC of 1 mg/L, ZD 20 mm), one S. aureus (MIC 1 mg/L, ZD 32 mm) and one E. faecalis (MIC 2 mg/L, ZD 20 mm). With regard to false resistant reporting, 36 of the 603 strains tested (6%) were interpreted as falsely resistant; eight strains were enterococci with MICs close to the BP (MIC range 0.12–0.5 mg/L), 17 were strains of Enterobacteriaceae with reduced susceptibility to ciprofloxacin and higher gemifloxacin MICs than strains of the same genera with no demonstrated mechanism of resistance, and the remaining 11 organisms comprised Serratia, Acinetobacter, Providencia and Stenotrophomonas spp. with MICs close to the MIC BP. Figure 2 shows the results for Pseudomonas spp. tested using a 5 µg disc content. Using a ZD BP of 20 mm no false resistance was recorded and only two strains with MICs close to the MIC BP were reported as falsely sensitive. Tentative recommendations for gemifloxacin are summarized in Table II.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The data reported in this study are essentially similar (that is, within one doubling dilution) to that previously described1,2 with the following important exceptions. Oh and colleagues1 showed that 90% of MRSA were susceptible to 1 mg/L of gemifloxacin while we found the MIC90 to be 8 mg/L. This difference could well reflect the fact that fluoroquinolone resistance in S. aureus has increased in recent years and differs from one locality to another. Comican and Jones2 suggest that gemifloxacin is considerably less potent against B. fragilis (MIC90 8 mg/L) as against 0.5 mg/L found with our data. Methodological differences might explain these observations and require to be resolved.
The activity of gemifloxacin against S. pneumoniae is particularly noteworthy. Against penicillin-susceptible and -resistant strains our data confirm those of Kelly et al.,5 in that the fluoroquinolone maintains activity against both groups. In addition, our data show that gemifloxacin is also active against those strains that are intermediately resistant to ciprofloxacin (MIC 2–16 mg/L). Gemifloxacin MICs are 0.06–0.12 mg/L, below the tentative breakpoint. The most resistant stain we studied had a ciprofloxacin MIC of >128 mg/L and was susceptible to 1 mg/L of gemifloxacin. Recent studies on the mechanisms of resistance of pneumococci,6 particularly the substrate specificity of efflux pumps in this organism, may explain such differences in activity.
A tentative breakpoint of 0.5 mg/L was chosen which should readily distinguish between resistant and susceptible populations of common pathogens. It was interesting to note that strains of Enterobacteriaceae that have a presumed mechanism of resistance, such as a mutation in gyrA or parC (with an MIC of <0.5 mg/L) nevertheless were demonstrated as ‘falsely' resistant by our methodologies. It is possible therefore that the disc testing system is more sensitive than agar dilution procedures in identifying this population. It is possible that a breakpoint of 0.5 mg/L is too conservative and that clinical trials may suggest that 1 mg/L will be more appropriate, but we believe a cautious approach is justifiable.
Gemifloxacin is a significant advance on currently marketed fluoroquinolones, especially in the potential treatment of chest infections, and certainly merits clinical study.
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Acknowledgments |
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We thank SmithKline Beecham, Harlow, UK for financial support.
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Notes |
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* Corresponding author
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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1 . Oh, J. I., Paek, K.-S., Ahn, M.-J., Kim, M.-Y., Hong, C. Y., Kim, I.-C. et al. (1996). In vitro and in vivo evaluation of LB20304, a new fluoronaphthyridinone. Antimicrobial Agents and Chemotherapy 40, 1564–8.[Abstract]
2 . Comican, M. G. & Jones, R. N. (1997). Antimicrobial activity and spectrum of LB20304, a novel fluoronaphthyridone. Antimicrobial Agents and Chemotherapy 41,204 –11.[Abstract]
3 . Working Party of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D.
4
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Andrews, J. M. & Wise, R. (1989).
Disc
sensitivity testing with ciprofloxacin. Journal of Antimicrobial Chemotherapy 23, 156–8.
5 . Kelly, L. M., Jacobs, M. R. & Appelbaum, P. C. (1998). Anti-pneumococcal activity of gemifloxacin a new broad-spectrum quinolone, compared with nine compounds by MIC. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract F-087. p. 254. American Society for Microbiology, Washington, DC.
6 . Brenwald, N. P., Gill, M. J. & Wise, R. (1998). Cloning of a novel efflux pump gene associated with fluoroquinolone resistance in Streptococcus pneumoniae. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract LB-4. American Society for Microbiology, Washington, DC.
Received 22 February 1999; returned 30 April 1999; revised 20 May 1999; accepted 25 June 1999
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