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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of 200 recently clinically isolated pneumococci, 68 were penicillin susceptible (MIC 0.06 mg/L); 67 penicillin-intermediate (MIC 0.11.0 mg/L) and 65 penicillin- resistant (MIC
2.0 mg/L). The 200 strains included 39 with raised quinolone MICs (ciprofloxacin MICs
8 mg/L21 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 MuellerHinton agar (Difco) with 5% sheep blood. Inocula were prepared by suspending growth from overnight cultures in MuellerHinton 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 strainsStaphylococcus aureus ATCC 29213 and Streptococcus pneumoniae ATCC 49619were included in each run.
Microdilution MICs
These were determined by the NCCLS method,5 using cation-adjusted MuellerHinton 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 MuellerHinton broth, and further diluted 1:10 to obtain a final inoculum (10 µL) containing 5 x 105 cfu/mL. Trays were incubated for 2024 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 MuellerHinton 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 MuellerHinton 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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), 2122 (intermediate) and
20 (resistant) are suggested.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 120813.[Abstract]
3
.
Friedland, I. R. & McCracken, G. H. (1994). Management of infections caused by antibiotic-resistant Streptococcus pneumoniae. New England Journal of Medicine 331, 37782.
4 . Jacobs, M. R. (1992). Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clinical Infectious Diseases 15, 11927.[ISI][Medline]
5 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth 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, 357984.
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, 15648.[Abstract]
9 . Cormican, M. G. & Jones, R. N. (1997). Antimicrobial activity and spectrum of LB 20304, a novel fluoronaphthyridone. Antimicrobial Agents and Chemotherapy 41, 20411.[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