Detection of gyrA and parC mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by use of oligonucleotide biochip technology

Abstract

An oligonucleotide biochip that specifically detects point mutations in the gyrA and parC genes of Neisseria gonorrhoeae was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the N. gonorrhoeae strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in gyrA and parC and to confirm the biochip data. The study demonstrates that all of the point mutations in the gyrA and parC genes of N. gonorrhoeae were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in N. gonorrhoeae isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of N. gonorrhoeae.

FIG. 1.

Layout of probes on biochips and fluorescence images of clinical isolates. (A) Probe layout on the oligonucleotide biochip. Following hybridization of Cy5-labeled fragmented DNA, as described in Materials and Methods, the fluorescence signals of mutations in the gyrA and parC genes from four clinical isolates were detected, as follows: Ser91→Phe/Asp95→Gly/Ser87→Arg (B), Ser-91→Phe/Asp95→Gly/Ser87→Asn/Glu91→Gln (C), Ser91→Phe/Asp95→Ala/Asp86→Asn/Ser87→Ile (D), and Ser91→Phe/Asp95→Asn/Glu91→Lys (E). (F) Wild-type sequence in gyrA and parC (Ser91/Asp95/Asp86/Ser87/Glu91).