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Antimicrobial Agents and Chemotherapy, November 2000, p. 3101-3106, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Evernimicin (SCH27899) Inhibits a Novel Ribosome Target Site: Analysis of 23S Ribosomal DNA Mutants

Peter V. Adrian,1,* Cara Mendrick,2 David Loebenberg,2 Paul McNicholas,2 Karen J. Shaw,2 Keith P. Klugman,1 Roberta S. Hare,2 and Todd A. Black2

Pneumococcal Diseases Research Unit, South African Institute for Medical Research, University of the Witwatersrand, and the Medical Research Council, Johannesburg, South Africa,1 and Schering Plough Research Institute, Kenilworth, New Jersey2

Received 5 June 2000/Returned for modification 25 July 2000/Accepted 21 August 2000

Spontaneous mutants of susceptible clinical and laboratory isolates of Streptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH27899; MIC, 0.5 to 4.0 mg/liter) were selected on plates containing evernimicin. Four isolates that did not harbor mutations in rplP (which encodes ribosomal protein L16) were further analyzed. Whole chromosomal DNA or PCR products of the 23S ribosomal DNA (rDNA) operons from these mutants could be used to transform the susceptible S. pneumoniae strain R6 to resistance at frequencies of 10-5 and 10-4, respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeared slowly (48 to 72 h) on selective medium, and primary transformants passaged on nonselective medium produced single colonies that displayed heterogeneous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resistance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resistant mutants revealed single, unique mutations in each isolate at the equivalent Escherichia coli positions 2469 (A right-arrow C), 2480 (C right-arrow T), 2535 (G right-arrow A), and 2536 (G right-arrow C). The mutations map within two different stems of the peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, none of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of evernimicin may be unique.

* Corresponding author. Present address: Laboratory of Pediatrics, Erasmus University Rotterdam, Posbus 1738, 3000 DR Rotterdam, The Netherlands. Phone: 31-10-4087951. Fax: 31-10-4089486. E-mail: adrian{at}kgk.fgg.eur.nl.

Antimicrobial Agents and Chemotherapy, November 2000, p. 3101-3106, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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