A novel real-time duplex PCR assay for detecting penA and ponA genotypes in Neisseria gonorrhoeae: Comparison with phenotypes determined by the E-test.
BACKGROUND
For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported.


METHODS
The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities.


RESULTS
The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results.


CONCLUSIONS
The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.
Vernel-Pauillac
Frédérique
F
Laboratoire de Recherche en Bactériologie, Institut Pasteur de Nouvelle-Calédonie, Nouvelle-Calédonie.
Merien
Fabrice
F
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D005838:Genotype; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D010406:Penicillins / Q000494:pharmacology; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins