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Antimicrobial Agents and Chemotherapy, October 2001, p. 2865-2870, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2865-2870.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Selection and Genetic Characterization of Streptococcus
pneumoniae Mutants Resistant to the Des-F(6) Quinolone
BMS-284756
Sandra
Hartman-Neumann,
Kenneth
DenBleyker,
Lenore A.
Pelosi,
Laura E.
Lawrence,
John F.
Barrett,* and
Thomas
J.
Dougherty
Department of Microbiology, Bristol-Myers
Squibb Pharmaceutical Research Institute, Wallingford, Connecticut
06492
Received 26 February 2001/Returned for modification 17 May
2001/Accepted 9 July 2001
Existing quinolones are known to target the type II topoisomerases
in bacteria. In order to determine which of these targets are of key
importance in Streptococcus pneumoniae treated with BMS-284756 (T-3811ME), a novel des-F(6) quinolone, resistant mutants were selected in several steps of increasing resistance by plating pneumococci on a series of blood agar plates containing serial twofold-increasing concentrations of drug. After incubation, colonies that arose were selected and passaged twice on antibiotic-containing media at the selection level. Mutants generally showed increases in
resistance of four- to eightfold over the prior level of
susceptibility. Mutants in the next-higher level of resistance were
selected from the previous round of resistant mutants. Subsequently,
chromosomal DNA was prepared from parental (R6) pneumococci and from at
least three clones from each of four levels of increasing antibiotic resistance. Using PCR primers, 500- to 700-bp amplicons
surrounding the quinolone resistance determining regions (QRDR) of
gyrA, gyrB, parC, and
parE genes were prepared from each strain. Internal primers were used to sequence both DNA strands in the regions of
approximately 400 bp centered on the QRDR. Mutations identified with
increasing levels of resistance included changes in GyrA at Ser-81 and
Glu-85 and changes in ParC at Ser-79 and Asp-83. Changes in GyrB and
ParE were not observed at the levels of resistance obtained in this
selection. The resistance to comparator quinolones (levofloxacin,
ciprofloxacin, and moxifloxacin) also increased in four- to eightfold
steps with these mutations. The intrinsically greater level of
antibacterial activity and thus lower MICs of BMS-284756 observed at
all resistance levels in this study may translate to coverage of
these resistant pneumococcal strains in the clinic.
*
Corresponding author. Mailing address: Dept. of
Microbiology (Dept. 104), Bristol-Myers Squibb Co., 5 Research Parkway,
Wallingford, CT 06492. Phone: (203) 677-6449. Fax: (203) 677-6771. E-mail: John.Barrett{at}bms.com.

Present address: Pfizer Research Laboratories, Groton,
Conn.
Antimicrobial Agents and Chemotherapy, October 2001, p. 2865-2870, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2865-2870.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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