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Antimicrobial Agents and Chemotherapy, January 1999, p. 166-168, Vol. 43, No. 1
Department of Pathology, Hershey Medical
Center, Hershey, Pennsylvania 17033,1 and
Department of Pathology, Case Western Reserve University,
Cleveland, Ohio 441062
Received 26 May 1998/Returned for modification 25 August
1998/Accepted 12 October 1998
Agar dilution was used to test the activities of HMR 3647, erythromycin A, azithromycin, clarithromycin, roxithromycin,
clindamycin, and quinupristin-dalfopristin against 235 strains of
Enterococcus faecalis. HMR 3647 was the most active
compound (MICs at which 50 and 90% of the isolates are inhibited
[MIC50 and MIC90, respectively] of 0.06 and
4.0 µg/ml, respectively). The MIC50 and MIC90
(with the MIC50 given first and the MIC90 given
second; both in micrograms per milliliter) for other compounds were as
follows: 4.0 and >32.0 for erythromycin A, 16.0 and >32.0 for
azithromycin, 2.0 and >32 for clarithromycin, 32.0 and >32.0 for
roxithromycin, 32.0 and >32.0 for clindamycin, and 8.0 and 16.0 for
quinupristin-dalfopristin. All compounds were only bacteriostatic.
Enterococci are increasing causes of
serious systemic infections, especially in debilitated hosts (6,
9, 20). The problem is complicated by the inherent drug
resistance of these species as well as recently developed resistance to
previously active drugs. Enterococcus faecalis has developed
resistance to ampicillin (chromosomal and plasmid mediated), high-level
resistance to aminoglycoside, and (rarely) resistance to
glycopeptide. Enterococcus faecium is inherently more
resistant than E. faecalis, with higher rates of
glycopeptide resistance (1, 4-6, 8, 9, 12, 14, 16, 18-20).
The ketolides are a new group of compounds characterized by a 3-keto
function replacing the cladinose moiety of other erythromycin A
derivatives (3). Previous studies have reported
improved activities of HMR 3004 and HMR 3647 against E. faecalis (especially vancomycin-susceptible strains) over those of
erythromycin A, azithromycin, clarithromycin, roxithromycin,
clindamycin, and quinupristin-dalfopristin (2, 3, 7, 10, 11, 13, 21-23). This study sheds further light on these
findings by (i) determination of the MICs of HMR 3647, erythromycin
A, azithromycin, clarithromycin, roxithromycin, clindamycin, and
quinupristin-dalfopristin against 235 E. faecalis
strains and (ii) examination of the activities of these compounds
against 10 strains by microdilution and time-kill studies.
All organisms were isolated from individual patients.
Agar dilution by standard methodology (15) was performed on
Mueller-Hinton agar. Time-kill studies were performed as described previously
(17), using cation-adjusted Mueller-Hinton broth (Difco).
Viability counts were performed at 0, 3, 6, 12, and 24 h. Data
were analyzed by determining the number of strains which yielded Results of agar dilution MICs for the 235 strains tested are presented
in Table 1. HMR 3647 was the most active,
with MICs at which 50 and 90% of the isolates are inhibited
(MIC50 and MIC90, respectively) of 0.06 and 4.0 µg/ml, respectively. The MIC50 and MIC90 (with the MIC50 given first and the
MIC90 given second; both in micrograms per milliliter) for
other compounds were as follows: 4.0 and >32.0 for erythromycin A,
16.0 and >32.0 for azithromycin, 2.0 and >32.0 for clarithromycin,
32.0 and >32.0 for roxithromycin, 32.0 and >32.0 for clindamycin, and
8.0 and 16.0 for quinupristin-dalfopristin. When enterococci
were divided into six groups (namely, (i) gentamicin
susceptible, (ii) high-level gentamicin resistant, (iii)
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Activity of HMR 3647 Compared to Those of Six
Compounds against 235 Strains of Enterococcus
faecalis
ABSTRACT
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-Lactamase-negative strains were recent clinical isolates from
Hershey Medical Center and University Hospital of Cleveland,
Ohio, and were not selected for specific antimicrobial
resistance. -Lactamase-producing and vancomycin-resistant
strains were obtained from sources cited in Acknowledgments.
-Lactamase testing was done with nitrocefin disks (Cefinase; BBL Microbiology Systems, Cockeysville, Md). For
-lactamase-producing strains, -lactam agar dilution MICs were repeated with inocula of 106 CFU/spot. Erythromycin A
breakpoints were 
0.5 µg/ml (susceptible), 1.0 to 4.0 µg/ml
(intermediate resistant), and 
8.0 µg/ml (resistant). High-level
gentamicin resistance was defined as MICs of >500 µg/ml. For the
purposes of this study, moderate susceptibility to clindamycin was
defined as MICs between 8.0 and 32.0 µg/ml (22).
Vancomycin resistance was defined as MICs of 16.0 µg/ml. For 10 selected strains examined by time-kill analysis, microbroth dilution
MICs were performed by using National Committee for Clinical Laboratory Standards methodology and cation-adjusted Mueller-Hinton broth (Difco
Laboratories, Detroit, Mich.) (15).
1,
2, and 3 
log10 CFU/ml compared to the counts at time
0 h. Antimicrobial agents were considered bactericidal at the
lowest concentration which reduced the original inoculum by >3
log10 CFU/ml (99.9%) and bacteriostatic if the inoculum
was reduced by <3 log10 CFU/ml. Antibiotic carryover was
minimized by dilution (17). All strains were tested with
final inocula of 5 × 105 to 5 × 106
CFU/ml. For strains with macrolide MICs of >64.0 µg/ml, time-kill studies were performed with HMR 3647 and quinupristin-dalfopristin only.
-lactamase producing, (iv) erythromycin A susceptible
and moderately clindamycin susceptible, (v) erythromycin A and
clindamycin resistant, and (vi) vancomycin resistant), HMR 3647 yielded
the lowest MICs against strains susceptible to gentamicin (MIC50, 0.03 µg/ml; MIC90, 4.0 µg/ml) and
erythromycin A (MIC50, 0.03 µg/ml;
MIC90, 0.06 µg/ml). HMR 3647 MICs for the 102 strains with erythromycin A MICs in the intermediate range (1.0 to 4.0 µg/ml) were 0.008 to 0.125 µg/ml; clindamycin MICs for these strains ranged from 8.0 to 32.0 µg/ml. The highest MICs for all compounds were in -lactamase-producing strains. The clindamycin MICs
of all erythromycin A-susceptible strains (erythromycin A MICs of 0.5
µg/ml) were 8.0 to 32.0 µg/ml, while the clindamycin MICs of
erythromycin A-resistant strains (erythromycin A MICs of 8.0 µg/ml)
were 64.0 µg/ml. Vancomycin MICs for vancomycin-resistant strains
were 16.0 µg/ml for one strain and 256.0 µg/ml for the remaining
12 strains. Teicoplanin testing (5, 20) showed that nine
strains had phenotypes consistent with the VanA phenotype and four
strains had phenotypes consistent with the VanB phenotype. HMR 3647 MICs of VanA strains were 1.0 to 16.0 µg/ml, and those of VanB
strains were 0.06 to 8.0 µg/ml.
TABLE 1.
MICs of drug substances against 235 E. faecalis strains
The 10 strains tested by time-kill analysis had various
susceptibilities to erythromycin A, gentamicin, and vancomycin, with one -lactamase-producing organism. All compounds were bacteriostatic (0.1- to 1.9-log decrease compared to the counts at 0 h) only. No
99 or 99.9% killing was observed with any compound.
Our results indicate that, while overall activity of HMR 3647 was
greater than other agents, in vitro activity varied considerably, with
the MICs varying with susceptibilities to other macrolides, lincosamides, and nonmacrolides. Similar findings were reported for HMR
3004 (21), and HMR 3647 (22). However, lower
ketolide MICs were reported for -lactamase-producing strains
(MIC50 and MIC90 of 0.03 and 0.04 µg/ml,
respectively, compared to our values of 8.0 and 16.0 µg/ml,
respectively) (21, 22). Different strains and different
techniques may be responsible for this discrepancy. Time-kill studies
showed that compounds were uniformly bacteriostatic, with HMR 3647 yielding the lowest MICs. While strains of E. faecalis susceptible or intermediately resistant to erythromycin A were highly
susceptible to HMR 3647 (MICs of 0.125 µg/ml), strains with
high-level resistance to erythromycin A (8.0 µg/ml) and clindamycin
(64.0 µg/ml) were often but not always inhibited by 4.0 µg of
HMR 3647 per ml. Because of the intrinsic resistance of most
enterococci to lincosamides, it is possible that macrolide resistance
in these strains was inducible, rather than constitutive, despite a
phenotype which might suggest the latter pattern (21, 22).
Studies with E. faecalis strains with known mechanisms of resistance are necessary to resolve this issue.
Because approximately 50% of strains resistant to both erythromycin A
and clindamycin at 64.0 µg/ml were inhibited by 1.0 to 4.0 µg of
HMR 3647 per ml, concentrations of HMR 3647 which fall into the
intermediate category for erythromycin A, in vivo studies would be
useful to determine whether such isolates are truly susceptible to the
ketolide. This question is of special importance, considering the
current high rates of macrolide resistance among E. faecalis strains and the limited therapeutic options available for
treatment of infections caused by these organisms (21, 22).
Our results indicate that HMR 3647 yielded the lowest MICs against all
groups of E. faecalis strains tested, with
bacteriostatic activity comparable to those of other compounds.
Quinupristin-dalfopristin MIC50 and MIC90 were
8.0 µg/ml. Our results indicate a possible use for HMR 3647 in
treatment of E. faecalis infections, especially those
caused by erythromycin-susceptible strains. In vitro studies require
confirmation by pharmacokinetic studies and animal and human clinical studies.
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ACKNOWLEDGMENTS |
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This study was supported in part by a grant from Hoechst Marion Roussel Laboratories, Romainville, France.
We thank L. Rice (Cleveland, Ohio), G. Eliopoulos (Boston, Mass.), C. Stratton (Nashville, Tenn.), and R. Leclercq (Caen, France) for
providing -lactamase-producing and vancomycin-resistant strains.
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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, January 1999, p. 166-168, Vol. 43, No. 1
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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