Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing

Abstract

Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.

Fig. 1

Simulation sequence pattern display of expected mutations related to ciprofloxacin resistance in comparison with the wild-type sequence encoding amino acid positions 90–96 of the Neisseria gonorrhoeae gyrA gene: (a) wild-type sequence; (b–e) expected mutations.

Fig. 2

Pyrograms of the sequence encoding amino acid positions 90–96 of the gyrA gene in Neisseria gonorrhoeae isolates obtained by pre-programmed DNA sequencing: (a) wild-type sequence; (b–e) samples harbouring mutations. The arrows show the location of point mutations and alterations in the sequence signal peaks. This figure can be compared with Fig. 1.