Relation between genetic markers of drug resistance and susceptibility profile of clinical Neisseria gonorrhoeae strains

Abstract

The main goal of this work is to clarify the predictive value of known genetic markers of Neisseria gonorrhoeae resistance to penicillin, tetracycline, and fluoroquinolones. The correlation between the presence of certain genetic markers and susceptibility of N. gonorrhoeae isolates to penicillin, tetracycline, and fluoroquinolones has been analyzed by means of statistical methods. Susceptibility testing with penicillin, tetracycline, and fluoroquinolones was performed by the agar dilution method. N. gonorrhoeae genomic DNA was isolated. The presence of bla(TEM-1) and tet(M) genes was analyzed by PCR. A novel method of polymorphism discovery based on a minisequencing reaction followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry was applied for the analysis of chromosomal N. gonorrhoeae genes involved in antimicrobial resistance development. Clinical N. gonorrhoeae isolates (n = 464) were collected. Susceptibility levels to penicillin, tetracycline, and fluoroquinolones were found to be 25.9%, 35.9%, and 54.1%, respectively. Among the 19 N. gonorrhoeae isolates with penicillin MICs of > or =4 microg/ml, the bla(TEM-1) gene was detected in 12. The Tet(M) determinant was found in 4 of 12 N. gonorrhoeae isolates with tetracycline MICs of > or =16 microg/ml. The chromosomal genetic markers of penicillin and tetracycline resistance were detected especially in isolates with penicillin MICs of 0.25 to 2.0 microg/ml and tetracycline MICs of 0.5 to 4 microg/ml. Mutations in GyrA and ParC were found in 208 of 211 quinolone-resistant N. gonorrhoeae isolates. This work is the first representative molecular research of the N. gonorrhoeae population in Russia. Information about the prevalence of antibiotic resistance mechanisms and the positive predictive value of certain genetic determinants is given. The positive predictive values of the analyzed genetic markers were found to be different for fluoroquinolones (90.3%), penicillin (91.1%), and tetracycline (81.9%).

FIG. 1.

Scheme of minisequencing reaction followed by MALDI-TOF mass spectrometry analysis. An amplified fragment of N. gonorrhoeae genomic DNA is used as a template. An internal oligonucleotide primer is selected close to the polymorphic site (the variable nucleotides are shown in bold). The annealing primer is extended by TermiPol DNA polymerase (Solis Biodyne, Estonia) at one or two nucleotides, in accordance with the nucleotide sequence of the polymorphic site. As a rule, a mixture of deoxynucleotides and dideoxynucleotides is used. The dideoxynucleotide which terminates primer elongation is indicated by an asterisk. The annealing primer and its extended products are shown in boxes. Subsequent MALDI-TOF mass spectrometry analysis of reaction products allows us to detect the diversity between the nonextended primer and products of the minisequencing reaction, which are different for the wild type (a) and the mutant (b).