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Antimicrobial Agents and Chemotherapy, February 2000, p. 414-417, Vol. 44, No. 2
Department of Pathology (Clinical
Microbiology), Hershey Medical Center, Hershey, Pennsylvania
17033,1 and Department of Pathology
(Clinical Microbiology), Case Western Reserve University, Cleveland,
Ohio 441062
Received 19 May 1999/Returned for modification 12 October
1999/Accepted 31 October 1999
The ability of 50 sequential subcultures in subinhibitory
concentrations of telithromycin (HMR 3647), azithromycin,
clarithromycin, erythromycin A, roxithromycin, clindamycin, and
pristinamycin to select for resistance was studied in five
macrolide-susceptible and six macrolide-resistant pneumococci
containing mefE or ermB. Telithromycin selected
for resistance less often than the other drugs.
Ketolides are semisynthetic
derivatives of erythromycin A characterized by a lack of
In a previous study (6) designed to determine if the recent
dramatic increase in incidence of drug-resistant pneumococci may be due
in part to abuse of oral drugs, such as macrolides and cephalosporins,
we found that sequential subcultures in subinhibitory concentrations of
azithromycin (used to represent the macrolide group), cefuroxime, and
cefaclor lead to increased pneumococcal MICs. Since repeated exposure
to azithromycin readily selected for pneumococci with increased
azithromycin MICs, we investigated how pneumococcal MICs were affected
by repeated exposure to a ketolide (an alternate choice to macrolides
in empiric therapy of pneumococcal infections), in comparison to other
antibiotics of the macrolide, lincosamide, and streptogramin class
(MLS). Specifically, we repeatedly exposed 11 strains of
Streptococcus pneumoniae to subinhibitory concentrations of
telithromycin, azithromycin, clarithromycin, erythromycin A,
roxithromycin, clindamycin, and pristinamycin to determine if
resistance developed.
Five strains were susceptible to erythromycin A (MICs MICs were determined by a standardized microdilution methodology in
Mueller-Hinton broth (Difco Laboratories) supplemented with 5% lysed
horse blood (4). Susceptibility breakpoints were those
approved by the National Committee for Clinical Laboratory Standards
(5): azithromycin, Resistant mutants were derived from parent strains as evidenced by (i)
serotyping using the standard Quellung method with sera from Statens
Seruminstitut (Copenhagen, Denmark) and (ii) pulsed-field gel
electrophoresis using a CHEF DR III apparatus (Bio-Rad, Hercules,
Calif.) as published previously (6). Strains were checked
for the presence of ermB and mefE by
amplification by PCR using primers and cycling conditions as described
by Sutcliffe et al. (9).
MIC results from subculturing in subinhibitory concentrations of
antibiotics are summarized in Table 1.
Among the five macrolide-susceptible pneumococci,
subculturing in telithromycin selected for two mutants, subculturing in
pristinamycin selected for three mutants, subculturing in azithromycin,
clarithromycin, and clindamycin selected for four mutants, and
subculturing in erythromycin A and roxithromycin selected for mutants
in all five strains. Among the three pneumococci containing
mefE, subculturing in all the drugs led to selection of
mutants with increased MICs in all three strains. The three pneumococci
containing ermB were highly resistant to all the macrolides and clindamycin and, therefore, were subcultured only in telithromycin and pristinamycin. Among these three strains, subculturing in telithromycin and pristinamycin led to selection of mutants with increased MICs in all three strains.
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In Vitro Development of Resistance to Telithromycin (HMR 3647),
Four Macrolides, Clindamycin, and Pristinamycin in
Streptococcus pneumoniae
ABSTRACT
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-L-cladinose on position 3 of erythronolide A, having a
three-keto function. Telithromycin (HMR 3647) is a ketolide that has
been shown to have good in vitro activity against penicillin- and
erythromycin-resistant pneumococci (1, 3, 7, 8).
0.25 µg/ml), and six were erythromycin A resistant: three strains had
mefE (erythromycin A MICs, 2 to 4 µg/ml) and three strains had ermB (erythromycin A MICs, >64 µg/ml). Antimicrobials
were obtained from their respective manufacturers.
0.5 µg/ml; clarithromycin, 0.25 µg/ml; erythromycin A, 0.25 µg/ml; and clindamycin, 0.25
µg/ml. The proposed susceptible breakpoint for telithromycin is 1
µg/ml (A. L. Barry, P. C. Fuchs, and S. D. Brown,
Programs Abstr. Fourth Int. Conf. Macrolides, Azalides, Streptogramins
Ketolides, abstr. 1.01, 1998). There are no roxithromycin and
pristinamycin National Committee for Clinical Laboratory Standards
breakpoints for S. pneumoniae. Daily passaging in
subinhibitory concentrations of antibiotic for a maximum of 50 days was
performed as published previously (6).
TABLE 1.
Resistance selection results
Resistance was stable (MIC of passaged strains remained within one doubling dilution of MIC after 10 passages on antibiotic-free media), in most cases, among mutants derived from macrolide-susceptible parent strains and ermB-containing parent strains. In contrast, resistance was not stable among mutants derived from parent strains containing mefE. In these mutants, telithromycin and macrolide MICs usually reverted back to baseline MICs (or close to) after 10 passages on antibiotic-free media.
There were 54 mutants with elevated MICs to at least one of the
antibiotics. Of these mutants, 3 were resistant to telithromycin (MICs
>1 µg/ml), 20 were resistant to clindamycin (MICs 
0.5 µg/ml), 36 were resistant to azithromycin (MICs 1 µg/ml), 37 were resistant to
clarithromycin (MICs 0.5 µg/ml), 39 were resistant to erythromycin A (MICs 0.5 µg/ml), 45 had roxithromycin MICs of 0.5 µg/ml, and
20 had pristinamycin MICs of 1.0 µg/ml (Table
2). Macrolide-resistant mutants usually
were still susceptible to telithromycin MICs (0.25 µg/ml). Mutants
with telithromycin MICs of 1 µg/ml were cross resistant to all
macrolides (MICs 16 µg/ml). In most cases, exposure of strains to
clindamycin or pristinamycin selected mutants with increased MICs to
the selecting drug while the MICs to telithromycin and the macrolides
remained unchanged or slightly higher.
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Pneumococcal mutants (derived from subculturing in azithromycin) from a previous study of ours (6) have recently been analyzed. Resistance was determined to be caused by (i) mutations in domain V of 23S rRNA leading to ML, MS, or MLSB phenotypes, depending on the specific mutation and (ii) mutations in ribosomal protein L4 leading to an MS phenotype (A. Tait-Kamradt, T. Davies, M. Jacobs, P. Appelbaum, and J. Sutcliffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-0842, 1999). At least two of the four copies of the 23S rRNA genes needed to be mutated to see elevated macrolide MICs (Tait-Kamradt et al., 39th ICAAC, abstr. C-0842). Similar studies to determine the mechanisms of resistance among the mutants generated in this study are in progress.
The clinical significance of these in vitro studies recently became evident, as we have documented that macrolide resistance among some clinical pneumococcal strains can be caused by the same or similar ribosomal mutations as those observed in the in vitro studies. We found mutations in ribosomal protein L4 among several clinical strains of S. pneumoniae from Central and Eastern Europe (some clonally related) and mutations in domain V of 23S rRNA among a few recent U.S. clinical strains (A. Tait-Kamradt, T. Davies, L. Brennan, P. Depardieu, P. Courvalin, J. Duigan, J. Petitpas, L. Wondrack, M. Jacobs, P. Appelbaum, and J. Sutcliffe, Final Program, Abstr. Exhibits Addendum 39th Intersci. Conf. Antimicrob. Agents Chemother. abstr. LB-8, 1999). The relationship between this L4 clone and macrolide use in Central and Eastern Europe is unknown, but clearly these drugs are used on a large scale in many of these countries (W. Hyrniewicz, personal communication). Thus far, macrolide-resistant pneumococci from clinical specimens that do not have mef or erm genes are extremely rare (8; Hyrniewicz, personal communication). This study suggests that overuse and misuse of MLS agents could lead to an increase in the incidence of macrolide resistance among pneumococci (by either mef or erm genes or ribosomal mutations). The need for cautious and judicious use of antimicrobials for treatment of pneumococcal infections is warranted.
In summary, telithromycin has been shown to have good in vitro activity against strains containing mefE and ermB and against in vitro-selected mutants resistant to other macrolides, clindamycin, and pristinamycin. While exposure to telithromycin did select for pneumococcal mutants with increased MICs (most of which were still susceptible), it did so in the least number of strains, compared to the other MLS agents.
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ACKNOWLEDGMENTS |
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This study was supported by a grant from Hoechst-Marion Roussel, Romainville, 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, February 2000, p. 414-417, Vol. 44, No. 2
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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