Ciprofloxacin resistance caused by point mutations affects the treatment of N. gonorrhoeae infections, mainly owing to reduced binding of this antimicrobial drug to the target sites of DNA gyrase. Therefore, it is important to investigate the molecular mechanisms by which drug resistance is acquired in order to confront drug-resistant gonococcal infections.
Rapid and reliable methods are required for screening and surveillance of these mutations. Pyrosequencing technology has the unique and significant advantage of a pre-programmed nucleotide addition strategy for mutation detection. This is considered an important benefit, since nucleotides are dispensed into the reaction mixture in a programmed order and alterations are discovered easily. In contrast, de novo sequencing uses cyclic nucleotide dispensations, resulting in longer reaction times and, together with product accumulation due to more nucleotide dispensations in the system, causes lower sequence data quality when performing longer reads. The pre-programmed dispensation results in more efficient point mutation detection, as longer and faster reads and more time-effective screening can be achieved. The pre-programmed sequencing is not only suitable for quick screening of known mutations but also for identification of previously unknown mutations.
The sample preparation (single-strand preparation and primer annealing) method used in the present study was relatively quick, allowing for more rapid single-strand separation (ca. 15 min) prior to DNA sequencing. The pyrosequencer (PSQ HS 96A) used for this study has the advantage of using lower volumes of PCR product owing to a more sensitive CCD camera, significantly reducing the cost of performing PCR and sequencing reactions. The PSQ HS 96A system is primarily suitable for SNP analysis. By simple modifications in the SNP software, it is possible to incorporate both the cyclic and pre-programmed approaches in the system.
As the results in indicate, all isolates displaying a decreased susceptibility or resistance to ciprofloxacin comprised at least one mutation in codons 91 or 95 of the gyrA
gene. The minor discrepancies between the results of genotypic and phenotypic ciprofloxacin susceptibility testing may, in most cases, be due to other additional molecular mechanisms for ciprofloxacin resistance, for instance polymorphisms in the parC
We do not suggest that this method can replace cultivation and susceptibility testing for N. gonorrhoeae in patients with suspected gonorrhoea, but it may be useful for laboratories that use only molecular methods for detection of gonococci. Mutations in gyrA were also found in some susceptible strains, but these mutations are probably relevant, since they may represent the first step to complete resistance.
In conclusion, DNA sequencing is the most reliable tool to inspect and analyse the nucleotide sequences of point mutations in clinical samples. With the new approach described in the present study, clinical samples could be screened more efficiently, allowing for attainment of reliable DNA sequencing-based results conveniently and in a shorter period of time. The pre-programmed sequencing is a general approach and can be used for insertion, deletion and mutation detection in other microbial resistance surveillance studies.