Hartman-Neumann S, DenBleyker K, Pelosi L A, Lawrence L E, Barrett J F, Dougherty T J
Dept. of Microbiology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA.
Antimicrob Agents Chemother. 2001 Oct;45(10):2865-70. doi: 10.1128/AAC.45.10.2865-2870.2001.
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.
已知现有的喹诺酮类药物作用于细菌中的II型拓扑异构酶。为了确定在使用新型去氟（6）喹诺酮BMS-284756（T-3811ME）治疗的肺炎链球菌中，哪些靶点至关重要，通过将肺炎链球菌接种在一系列含有两倍递增浓度药物的血琼脂平板上，分几步筛选出耐药突变体，每一步的耐药性都有所增加。培养后，挑选出长出的菌落，并在选择水平的含抗生素培养基上传代两次。突变体的耐药性通常比先前的敏感水平提高了4至8倍。从上一轮耐药突变体中挑选出耐药性更高一级的突变体。随后，从亲本（R6）肺炎链球菌以及四个抗生素耐药性递增水平中每个水平的至少三个克隆中制备染色体DNA。使用PCR引物，从每个菌株中制备围绕gyrA、gyrB、parC和parE基因的喹诺酮耐药决定区（QRDR）的500至700碱基对扩增子。使用内部引物对以QRDR为中心的约400碱基对区域的两条DNA链进行测序。随着耐药性水平的增加而鉴定出的突变包括GyrA中Ser-81和Glu-85的变化以及ParC中Ser-79和Asp-83的变化。在本次筛选获得的耐药水平下，未观察到GyrB和ParE的变化。随着这些突变，对对照喹诺酮类药物（左氧氟沙星、环丙沙星和莫西沙星）的耐药性也以4至8倍的幅度增加。在本研究中，在所有耐药水平下观察到的BMS-284756固有的更高抗菌活性以及更低的最低抑菌浓度可能意味着在临床上能够覆盖这些耐药肺炎链球菌菌株。
