a Departments of Pathology, Hershey Medical Center, 500 University Drive, Hershey, PA 17033; b Case Western Reserve University, Cleveland, OH 44106, USA
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Abstract |
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Introduction |
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There is an urgent need of oral compounds for outpatient treatment of respiratory tract infections caused by penicillin-intermediate and -resistant pneumococci. 2 ,3 ,4 8 Available quinolones such as ciprofloxacin and ofloxacin yield moderate in-vitro activity against pneumococci, with MICs clustering around the breakpoints. 4 ,9 ,10 ,11 Gatifloxacin (AM-1155, CG 5501) is a new broad-spectrum 8-methoxyquinolone which shares with sparfloxacin and grepafloxacin a methyl piperazinyl side-chain at position 7 and a cyclopropyl substituent at position 1. 12 ,13 ,14 ,15 ,16 ,17 Preliminary MIC studies with small numbers of strains have shown that gatifloxacin has good activity against penicillin-susceptible and -resistant pneumococci. 12 ,13 ,14 ,15 ,16 ,17
This study expands studies of the anti-pneumococcal activity of gatifloxacin by comparing its activity with that of four other quinolones, three ß-lactams and clarithromycin against 207 penicillin-susceptible, intermediate resistant and -resistant pneumococci. Drugs were chosen to represent a range of oral quinolone and non-quinolone agents, and one parenteral agent, which might be indicated for empirical treatment of community-acquired respiratory tract infections. The activity of the above compounds against 12 penicillin-susceptible and -resistant pneumococci was also investigated by broth MIC and time- kill methodology.
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Materials and methods |
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For agar dilution MICs, the pneumococci comprised 71 penicillin-susceptible (MIC 0.06
mg/L), 81 penicillin-intermediate (MIC 0.125-1.0 mg/L) and 55 penicillin- resistant (MIC
2.0-16.0 mg/L) strains. All susceptible, and some intermediate and resistant strains, were recent
USA
isolates. The remainder of the intermediate and resistant strains were isolated in South Africa,
Spain, France, central and eastern Europe and Korea. For time-kill studies, four
penicillin-susceptible, four -intermediate and four -resistant strains were tested.
Antimicrobials and MIC testing
Gatifloxacin powder was obtained from Bristol-Myers Pharmaceutical Research Institute (Wallingford, CT, USA) and other antimicrobials were obtained from their respective manufacturers. Two hundred and seven strains were tested by agar dilution methodology as described previously, 3 ,4 on Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 5% sheep blood. Broth dilution MICs for 12 strains were determined according to NCCLS recommendations 18 with cation-adjusted Mueller-Hinton broth with 5% lysed defibrinated horse blood. Standard quality control strains, including Streptococcus pneumoniae ATCC 49619, were included in each run of agar and broth dilution MICs. All strains used in this study have been subcultured many times and therefore do not require CO 2 for growth. All incubations were therefore in ambient air.
Time- kill testing
For time-kill studies, glass tubes containing 5 mL cation-adjusted Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) plus 5% lysed horse blood with doubling antibiotic concentrations were inoculated with 5 x 105 to 5 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 agar dilution MIC. Growth controls with inoculum but no antibiotic were included with each experiment. 19 ,20
Lysed horse blood was prepared as described previously. 19 ,20 The bacterial inoculum was prepared by diluting a 16 h broth culture (medium as above) in the same medium. Dilutions required to obtain the correct inoculum (5 x 105 -5 x 106 cfu/mL) were determined by prior viability studies with each strain.
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 viable counts within 10 min. The original inoculum was determined by using the untreated growth control. Only tubes containing an initial inoculum within the range of 5 x 105-5 x 106 cfu/mL were acceptable. 19 ,20
Viable counts of antibiotic-containing suspensions were determined according to standard methodology, by plating ten-fold dilutions of 0.1 mL aliquots from each tube in sterile Mueller- Hinton broth on to plates of trypticase soy agar with 5% sheep blood (BBL Microbiology Systems). Recovery plates were incubated for up to 72 h. Colony counts were performed on plates yielding 30- 300 colonies. The lower limit of sensitivity of colony counts was 300 cfu/mL. All incubation was in ambient air without CO 2.
Time-kill assays were analysed by determining the number of strains which yielded a change in
log
10 cfu/mL of -1, -2 and -3 at 0, 4, 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 log
10 cfu/mL (99.9%) at each of the time periods, and bacteriostatic if the
inoculum was reduced by 0-3 log
10 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
bacterial carryover was addressed as described previously.
19
,20 For clarithromycin time- kill testing, only strains with
MICs
4.0 mg/L were tested. Time-kill experiments were performed in duplicate and
arithmetic means of results taken. In all cases, results of repeat testing were practically identical.
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Results |
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Discussion |
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Several workers have reported that gatifloxacin is very active against a small number of
pneumococcal strains tested, with MIC
50s of 0.125 - 0.39 mg/L and MIC
90s of 0.2 - 0.5 mg/L.
12
,13
,15
,16
,17
Our findings of MIC
50 and MIC
90 values of 0.25 and 0.5 mg/L respectively, with a larger number of strains confirm
the earlier reports. Time-kill studies, which have not been reported previously, showed that at
0.5 mg/L gatifloxacin was bactericidal after 12 h against all pneumococcal strains tested.
Lower MICs of amoxycillin and ceftriaxone against penicillin-intermediate and -resistant pneumococci, and raised clarithromycin MICs in the latter two groups, have been described before. 4 ,21 Kill kinetics of other quinolones tested, including slightly more rapid killing by levofloxacin compared with other quinolones, have been described previously. 22 ,23
Nakashima and co-workers 14 have reported gatifloxacin C max values (mg/L) in healthy human volunteers of 0.873, 1.71, 3.35 and 5.41 after single oral doses of 100, 200, 400 and 600 mg, respectively. AUC values (mg·h/L) after the four doses were 7.0, 14.5, 32.4 and 53.5, respectively. Serum concentrations reached a peak between 1 and 2 h. With the above pharmacokinetic data taken into consideration together with the MIC and time-kill results obtained in our study, gatifloxacin shows promise in treatment of infections caused by penicillin-susceptible and -resistant pneumococci. Clinical studies will be necessary to validate these in-vitro findings.
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Acknowledgments |
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Notes |
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References |
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19 . 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]
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22 . Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1996). MIC and time- kill study of activities 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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Received 29 May 1998; returned 22 September 1998; revised 26 October 1998; accepted 30 January 1999