Activity of gemifloxacin, a new broad-spectrum quinolone, against 200 pneumococci by four different susceptibility testing methods

Lois M. Edniea, Michael R. Jacobsb and Peter C. Appelbauma,*

a Departments of Pathology (Clinical Microbiology), Hershey Medical Center, PO Box 850, Hershey, PA 17033; b Case Western Reserve University, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Agar and microdilution (in air), Etest (in air and CO2) and disc diffusion (in air and CO2) susceptibility testing methods were used to investigate the activity of gemifloxacin against 200 pneumococci. MIC50s were 0.016–0.03 mg/L and MIC90s 0.125–0.25 mg/L for all methods. With agar dilution as reference, 187/200 strains gave essential agreement with microdilution and 196 with Etest (air and CO2). Disc zones were a few millimetres narrower in CO2 than in air. With discs in CO2, all ciprofloxacin-susceptible strains yielded zone diameters 26 mm; values in air were 28 mm. Zones for ciprofloxacin-resistant strains in CO2 were mostly 21–26 mm; zones in air were a few millimetres wider, but mostly <31 mm.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of pneumococci resistant to penicillin G and other ß-lactams and non-ß-lactams has increased worldwide at an alarming rate, including in the USA.1 In the USA there has been an increase in resistance to penicillin from <5% before 1989 (including <0.02% of isolates with MICs >= 2.0 mg/L) to 6.6% in 1991–1992 (with 1.3% of isolates with MICs >= 2.0 mg/L) and to 23.6% of 1527 strains during 1994–1995.2

Oral compounds for the out-patient treatment of respiratory tract infections caused by penicillin-intermediate and -resistant pneumococci are urgently required.3,4 Older quinolones such as ciprofloxacin and ofloxacin exhibit moderate in vitro activity against pneumococci, with MICs clustering around breakpoints.4 Methods for routine susceptibility testing of pneumococci include broth microdilution and disc diffusion (recommended by the National Committee for Clinical Laboratory Standards) (NCCLS), agar dilution and Etest.57 NCCLS recommends incubation of microdilution MICs in air, but disc diffusion in CO2,5,6 while the manufacturer of the Etest recommends incubation in CO2.7 There are no standard recommendations for agar dilution pneumococcal MIC testing methodology, although agar dilution has been extensively used in this laboratory.4,7

If new compounds are to be tested for antipneumococcal activity in the clinical laboratory, methodology must be standardized. This study used microdilution and agar dilution (in air), Etest (in air and CO2) and disc diffusion (in air and CO2) to test the activity of gemifloxacin (SB 265805; LB 20304a), a new fluoronaphthyridone with a novel pyrrolidone substituent, with good Gram-positive and -negative activity,810 against 200 pneumococci.


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

Of 200 recently clinically isolated pneumococci, 68 were penicillin susceptible (MIC <= 0.06 mg/L); 67 penicillin-intermediate (MIC 0.1–1.0 mg/L) and 65 penicillin- resistant (MIC >= 2.0 mg/L). The 200 strains included 39 with raised quinolone MICs (ciprofloxacin MICs >= 8 mg/L—21 penicillin-susceptible, 12 intermediate, six penicillinresistant). Quinolone-resistant strains were obtained from Case Western Reserve University and D. Felmingham, GR Micro, London, UK. All strains had been subcultured several times before use and so therefore all grew well in air. Cultures were maintained at –70°C in double-strength skim milk (Difco Laboratories, Detroit, MI, USA). Gemifloxacin susceptibility powder, discs and Etests (AB Biodisk, Solna, Sweden) were obtained from SmithKline Beecham Laboratories, Collegeville, PA, USA.

Agar dilution MICs

These were performed4,5 on Mueller–Hinton agar (Difco) with 5% sheep blood. Inocula were prepared by suspending growth from overnight cultures in Mueller–Hinton broth (Difco) to a turbidity of a 0.5 McFarland standard. Final inocula contained 104 cfu/spot. Plates were inoculated with a Steers replicator (Melrose Machine Shop, Woodlyn, PA, USA), and incubated overnight at 35°C in ambient air. The lowest concentration of antibiotic showing no growth was read as the MIC. Quality control strains—Staphylococcus aureus ATCC 29213 and Streptococcus pneumoniae ATCC 49619—were included in each run.

Microdilution MICs

These were determined by the NCCLS method,5 using cation-adjusted Mueller–Hinton broth (Difco) with 5% lysed defibrinated horse blood. Trays were prepared in-house. Suspensions with a turbidity equivalent to that of a 0.5 McFarland standard were prepared by suspending growth from blood agar plates in 2 mL Mueller–Hinton broth, and further diluted 1:10 to obtain a final inoculum (10 µL) containing 5 x 105 cfu/mL. Trays were incubated for 20–24 h in ambient air at 35°C. Standard quality control strains (as above) were included in each run.

Etest MICs

Standard methodology was used.7 Mueller–Hinton plates supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, MD, USA) were inoculated with a 0.5 McFarland suspension harvested from plates, and Etest strips placed on each. After overnight incubation at 35°C, the MIC was read as the intersect where the ellipse of growth inhibition intersects the strip. Etest MICs were performed both in air and in CO2. Etest MICs were rounded up to the next highest doubling dilution.

Disc diffusion

This was by standard NCCLS methodology6 using 5 µg gemifloxacin discs (BBL) and Mueller–Hinton plates supplemented with 5% sheep blood (BBL), inoculated with a 0.5 McFarland suspension. After overnight incubation in both air and 5% CO2 at 35°C, zone diameters were measured with calipers.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
By agar dilution, ciprofloxacin MICs (mg/L) for all strains ranged between 0.5 and >=64, with an MIC50 of 2 mg/L and an MIC90 of 16 mg/L (Table IGo). In contrast, gemifloxacin MICs, which were practically identical with all methods, ranged between 0.004 and 1.0 mg/L, with MIC50s between 0.016 and 0.03 mg/L and MIC90s between 0.125 and 0.25 mg/L with agar dilution and microdilution in air, and Etest (both in air and CO2). Incubation of Etests in CO2 did not significantly influence MICs. When strains with ciprofloxacin MICs <= 4.0 mg/L were separated from strains with ciprofloxacin MICs >= 8 mg/L, gemifloxacin MIC50/90 values (mg/L) by agar dilution were 0.03/0.03 and 0.25/0.5, respectively. In contrast, ciprofloxacin MIC50/90 values (mg/L) for susceptible and resistant strains were 1/2 and 32/>=64, respectively (Table IGo). Results of agar dilution MICs for ciprofloxacin and gemifloxacin for all 200 strains tested showed a linear correlation.


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Table I. Comparison of agar dilution MIC (mg/L) results for ciprofloxacin-susceptible (161) and -resistant (39) strains
 
Agreements of microdilution and Etest (air and CO2) with agar dilution (used as the reference method) and Etests in air compared with CO2 are presented in Table IIGo. As can be seen, 187/200 strains (93.5%) gave essential agreement (± 1 log2 dilution) with microdilution, and 98.0% with the Etest (both in air and CO2). With a preliminary breakpoint of 0.5 mg/L, no major or very major discrepancies were found with microdilution in air or Etest in air or CO2. Etests incubated in air gave virtually identical results to those in CO2 (Table IIGo).


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Table II. Results of gemifloxacin pneumococcal susceptibility testing by four methods using agar dilution as the reference method
 
With discs incubated in CO2, all quinolone-susceptible strains yielded zone diameters >=26 mm; values in air were >=28 mm. Zone diameters for quinolone-resistant strains in CO2 varied between 18 and 31 mm but were mostly 21–26 mm; zone diameters in air were a few millimetres wider, but were also mostly <31 mm. Using a gemifloxacin breakpoint of 0.5 mg/L, >=20 mm for susceptible and <=19 mm (resistant) are proposed. All strains but one (with agar dilution) had MICs of <=0.5 mg/L and all but one strain yielded zones of >20 mm. With a breakpoint of 0.25 mg/L, zone diameters (mm) of >=23 (susceptible), 21–22 (intermediate) and <=20 (resistant) are suggested. Zone diameters in air were usually 1–3 mm wider than those in CO2.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs of gemifloxacin for pneumococci are similar to those described previously, including MICs (mg/L) of 0.004–0.06 if ciprofloxacin MICs are <=4810 and 0.03–1.0 if ciprofloxacin MICs are 8.0–64.0. MICs did not differ significantly whether determined on agar, by microdilution incubated in air or by Etests incubated in air and CO2. A previous study in this laboratory has demonstrated similar findings with levofloxacin.7 Disc diffusion showed zone sizes slightly smaller in CO2 than in air. Determination of breakpoints, both by disc diffusion and MIC, must await further studies, but ciprofloxacin-resistant strains with gemifloxacin MICs of 0.06–1.0 mg/L by agar dilution, gave zone diameters in CO2 between 21 and 31 mm (with the exception of one strain with a zone diameter of 18 mm), and most quinolone-susceptible strains yielded zone diameters > 25 mm.

Agar or microdilution MICs were not determined in CO2. NCCLS recommends incubation in air for microdilution and suggests (but does not recommend) incubation in air for agar dilution, in contrast to CO2 for disc diffusion, and Etest incubation in CO2 being recommended by the manufacturer. This is clearly not optimal, and there is a need for standardization of pneumococcal susceptibility testing, with all methods in CO2. However, a previous study has shown a negligible effect of CO2 on pneumococcal susceptibility testing with another quinolone, levofloxacin.7

Our results indicate an excellent correlation between agar dilution, microdilution and Etest, and all methods can confidently be recommended for pneumococcal susceptibility testing with gemifloxacin. Using a gemifloxacin breakpoint of 0.5 mg/L, >=20 mm for susceptible and <=19 mm are proposed (see Results). With a breakpoint of 0.25 mg/L, zone diameters (mm) of >=23 (susceptible), 21–22 (intermediate) and <=20 (resistant) are suggested.


    Acknowledgments
 
We thank D. Felmingham for provision of some quinolone-resistant strains.This study was supported by a grant from SmithKline Beecham Laboratories, Collegeville, PA, USA.


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


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Appelbaum, P. C. (1992). Antimicrobial resistance in Streptococcus pneumoniae—an overview. Clinical Infectious Diseases 15, 77–83.[ISI][Medline]

2 . Doern, G. V., Brueggemann, A., Holley, H. P. & Rauch, A. M. (1996). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-center national surveillance study. Antimicrobial Agents and Chemotherapy 40, 1208–13.[Abstract]

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

4 . Jacobs, M. R. (1992). Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clinical Infectious Diseases 15, 119–27.[ISI][Medline]

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

6 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility Tests —Sixth Edition: Approved Standard M2-A6. NCCLS, Villanova, PA.

7 . Clark, C. L., Jacobs, M. R. & Appelbaum, P. C. (1998). Antipneumococcal activities of levofloxacin and clarithromycin as determined by agar dilution, microdilution, E-test, and disk diffusion methodologies. Journal of Clinical Microbiology 36, 3579–84.[Abstract/Free Full Text]

8 . 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 evaluations of LB20304, a new fluoronaphthyridone. Antimicrobial Agents and Chemotherapy 40, 1564–8.[Abstract]

9 . Cormican, M. G. & Jones, R. N. (1997). Antimicrobial activity and spectrum of LB 20304, a novel fluoronaphthyridone. Antimicrobial Agents and Chemotherapy 41, 204–11.[Abstract]

10 . Kelly, L. M., Jacobs, M. R. & Appelbaum, P. C. (1998). Antipneumococcal activity of SB 265805 (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, 1996. Abstract F-87, p. 254. American Society for Microbiology, Washington, DC.

Received 15 June 1999; returned 3 September 1999; revised 13 September 1999; accepted 26 October 1999





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