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Antimicrobial Agents and Chemotherapy, November 1998, p. 3032-3034, Vol. 42, No. 11
Departments of Pathology (Clinical
Microbiology), Hershey Medical Center, Hershey, Pennsylvania
17033,1 and
Case Western Reserve
University, Cleveland, Ohio 441062
Received 6 July 1998/Returned for modification 17 August
1998/Accepted 25 August 1998
The microdilution MICs of HMR 3647, erythromycin A, azithromycin,
clarithromycin, roxithromycin, and pristinamycin against 50/90% of 249 Haemophilus influenzae and 50 Moraxella
catarrhalis isolates were 2/4, 0.06/0.125; 8/16, 0.25/0.25; 2/4,
0.06/0.125; 16/16, 0.25/0.25; 32/>32, 1/2; and 2/4, 0.5/0.5 µg/ml.
Azithromycin was bactericidal against all 10 H. influenzae
and 3 of 5 M. catarrhalis isolates and HMR 3647, erythromycin A, clarithromycin, roxithromycin, and
pristinamycin were bacteriostatic, against all 15 strains after 24 h at the MIC.
Haemophilus influenzae
and Moraxella catarrhalis remain important causes of
respiratory tract infections (2, 10). The in vitro
susceptibility of H. influenzae to macrolides is
variable, with azithromycin showing the lowest MICs, followed by
clarithromycin, erythromycin A, and roxithromycin. Pristinamycin and
other streptogramins are also active in vitro against H. influenzae (5, 7, 8, 11, 12, 18). M. catarrhalis has been reported to be more susceptible to all of the
above compounds than H. influenzae (2).
HMR 3647 is a new ketolide with expanded activity against many
multiresistant (especially erythromycin A-resistant)
gram-positive organisms (4, 7, 20); excellent
activity against M. catarrhalis and activity against
H. influenzae equivalent to that of azithromycin have
recently been documented (1, 3, 4, 6, 7, 20). The problem of
macrolide activity against H. influenzae is complicated by the difficulty of in vitro susceptibility testing (21).
Haemophilus test medium (HTM), the medium recommended by the
National Committee for Clinical Laboratory Standards (NCCLS), has a
shelf life of only a few weeks and is therefore difficult to obtain
commercially. Viability counts should also be performed on each
suspension used for susceptibility testing (10, 21).
The current study attempted to shed light on the above by using freshly
prepared HTM and Mueller-Hinton broth with added Fildes extract (MHF)
to test the activity of HMR 3647, erythromycin A, azithromycin,
clarithromycin, roxithromycin, and pristinamycin against 249 H. influenzae and 50 M. catarrhalis
isolates by microdilution. The activity of each drug against 10 H. influenzae and 5 M. catarrhalis isolates was also tested by time-kill assay.
The organisms used were all recent clinical isolates.
Frozen microdilution trays (MicroMedia Systems Inc., Cleveland,
Ohio) each contained all five antimicrobials prepared in freshly prepared HTM and MHF. HTM was prepared by adding 0.5% yeast extract, 15-µg/ml hematin, and 15-µg/ml NAD to cation-supplemented
Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), and MHF was
prepared by adding 1% yeast extract and 5% Fildes enrichment (Difco)
to cation-supplemented Mueller-Hinton broth (Difco). Wells were
inoculated with 100-µl suspensions and incubated in air at 35°C for
20 to 24 h (9). Standard quality control strains
(9) were included in each run.
Time-kill experiments were carried out with HTM as previously described
(13, 14). Dilutions required to obtain the correct inoculum
were determined by prior viability studies with each strain. Only tubes
containing an initial inoculum between 5 × 105 and
5 × 106 CFU/ml were acceptable. Viability counts of
antibiotic-containing suspensions were performed at 0, 3, 6, 12, and
24 h, respectively (13, 14), on chocolate agar plates
incubated for up to 48 h in CO2. Colony counts were
performed in duplicate, and means were taken.
Time-kill assays were analyzed by determining the number of strains
which showed viable count decreases of 1, 2, and 3 log10 CFU/ml compared to the counts at 0 h. Drugs were considered
bacteriostatic if they yielded a decrease in the count of <3
log10 CFU/ml compared to that at 0 h. With the
sensitivity threshold (250 CFU/ml) and inocula used, bactericidal
activity (99.9% killing and a decrease in the count of >3
log10 CFU/ml) could be determined when present. Bacterial
carryover was minimized by dilution (13, 14).
Of 249 H. influenzae strains, 118 (47.4%) were
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Activity of HMR 3647 Compared to Those of Five Agents against
Haemophilus influenzae and Moraxella catarrhalis
by MIC Determination and Time-Kill Assay
ABSTRACT
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-Lactamase testing was by the nitrocefin disk method
(Cefinase; BBL Microbiology Systems, Cockeysville, Md.). Powders
were obtained from the respective manufacturers. Microdilutions
were performed on 249 H. influenzae and 50 M. catarrhalis strains by the NCCLS microdilution
method (9). Inocula were prepared from chocolate agar plates
(BBL) incubated for 24 h by the direct colony suspension method.
Final organism suspensions in trays yielded colony counts of 3 × 105 to 8 × 105 CFU/ml. Inoculum checks
were performed in every case; preparation of strains with inocula which
did not conform to the standard was repeated.
-lactamase positive. All 50 M. catarrhalis strains
were -lactamase producers. MICs did not differ for
-lactamase-positive and -negative H. influenzae
strains (data not shown). Microdilution MICs are presented in Table
1. HMR 3647, azithromycin, and
pristinamycin had the lowest MICs against H. influenzae, followed by erythromycin A, clarithromycin, and
roxithromycin. All compounds were active against M. catarrhalis, with HMR 3647 and azithromycin having the lowest MICs. Although MICs were sometimes higher in MHF than in HTM, the
values did not differ significantly (Table 1).
TABLE 1.
MICs for H. influenzae and M. catarrhalis strains in HTM and MHF
Azithromycin had the best kill kinetics against H. influenzae, with 99.9% killing of all strains after 24 h at the MIC. Pristinamycin was bactericidal against all strains after 24 h at twice the MIC. HMR 3647, erythromycin A, and clarithromycin had similar kill kinetics, with 99.9% killing of seven or eight strains at twice the MIC after 24 h and 90% killing of all strains after 24 h at two to four times the MIC. The kill kinetics of roxithromycin were slower. After 24 h, HMR 3647, erythromycin A, and clarithromycin had bacteriostatic activity at the MIC for all of the strains tested. All compounds were bactericidal at 24 h for three or four of the five M. catarrhalis strains at the MIC, and all were bacteriostatic at the MIC after 24 h.
Our results indicate that against both H. influenzae and M. catarrhalis, HMR 3647 and azithromycin had the greatest activity by MIC determination and azithromycin had the greatest activity by time-kill test followed by erythromycin A, clarithromycin, and roxithromycin. Pristinamycin gave MICs similar to those of HMR 3647 but showed more rapid kinetics of H. influenzae killing. Our HMR 3647 MICs were similar to those obtained by Felmingham et al. (4) and Wise and Andrews (20) but higher than those obtained by Agouridas and colleagues for HMR 3004, an older ketolide (1). However, ketolides consistently yield lower MICs than other macrolides, lincosamides, and streptogramins against these species (1, 3, 4, 6, 7, 20). Medium-related problems are probably responsible for published MIC differences.
Although HTM is recommended by the NCCLS as the method of choice for Haemophilus testing (9), the medium cannot be made reliably commercially and must be used within 2 to 3 weeks of in-house preparation for optimal growth. MICs in MHF did not differ significantly from those in HTM. Because most strains of Haemophilus and Moraxella have no specific macrolide-lincosamide-streptogramin mechanism such as erm or mef (16, 17), pharmacokinetic and pharmacodynamic factors (time above the MIC, area under the concentration-time curve/MIC, and concentration in specific body fluids) must also be taken into therapeutic consideration (10, 11, 19). There is an urgent need for a method of Haemophilus susceptibility testing which can readily be adapted for use in the clinical laboratory.
Despite the above technical problems, HMR 3647 and azithromycin had the lowest MICs of all of compounds tested. HMR 3647 was also bacteriostatic against all of the strains tested at the MIC after 24 h, with kill kinetics similar to those of erythromycin A and clarithromycin. HMR 3647 is very active against macrolide-susceptible and -resistant pneumococci (15), as well as other organisms responsible for community-acquired pneumonia (4, 7, 20). If results of pharmacokinetic, pharmacodynamic, and animal studies support its in vitro activity, clinical testing of HMR 3647 in respiratory infections is indicated.
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ACKNOWLEDGMENTS |
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This study was supported by a grant from Hoechst-Marion Roussel, Division of Clinical Anti-infectives, Paris, France.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, Hershey Medical Center, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum{at}psghs.edu.
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Antimicrobial Agents and Chemotherapy, November 1998, p. 3032-3034, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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