Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

Abstract

A rapid method, using PCR-restriction fragment length and single-strand conformation polymorphism (SSCP), was applied to screen for mutations of the fluoroquinolone resistance determinants in Streptococcus pneumoniae. One hundred nonduplicate Streptococcus pneumoniae isolates with ciprofloxacin MICs of > or = 4.0 microg/ml from the Prince of Wales Hospital, Hong Kong, years 2000 to 2003, were examined. For each isolate, PCR amplicons of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were digested with AluI, HinfI, Sau3AI, and MspI, respectively, and analyzed by SSCP. Each SSCP pattern was given a number, and each isolate obtained a four-digit code, e.g., 1111, that represented the SSCP profile. The SSCP patterns were correlated to mutations characterized from sequence analyses of PCR amplicons. The most common SSCP profile obtained was no. 5232 (40%), which included strains with two amino acid substitutions in the ParC (Lys-137-Asn) and ParE (Ile-460-Val) genes, followed by the SSCP profile 5223 (17%), which included strains with amino acid substitutions in the ParE (Ile-460-Val) gene only. Ten isolates (10%) with amino acid substitutions at GyrA and ParE (+/-ParC) genes were resistant to levofloxacin with a MIC of > or = 16 microg/ml. Other SSCP profiles were unique in distinguishing the common amino acid substitutions in GyrA (Ser-81-Phe) and ParC (Lys-137-Asn, Ser-79-Phe plus Lys-137-Asn, Asp-83-Asn plus Lys-137-Asn, Ser-79-Phe, and Glu-96-Asp). SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.