Genotyping as a Tool for Antibiotic Resistance Surveillance of Neisseria gonorrhoeae in New Caledonia: Evidence of a Novel Genotype Associated with Reduced Penicillin Susceptibility

Abstract

Antibiotic resistance in Neisseria gonorrhoeae continues to be a major concern in public health. Resistance of N. gonorrhoeae bacteria to penicillin G is widespread in most developed countries, which has necessitated a change to newer drugs for treatment of gonococcal infections. Recent reports indicate that resistance to these newer drugs is increasing, highlighting the need for accurate therapeutic recommendations. In some countries or communities, however, N. gonorrhoeae isolates are still susceptible to penicillin, so the use of this antibiotic for single-dose treatments of medically under-resourced patients is beneficial. In order to evaluate the adequacy and sustainability of this treatment approach, we explored the presence and prevalence of chromosomally mediated resistance determinants in N. gonorrhoeae isolates collected from 2005 to 2007 in New Caledonia. We developed two new real-time PCR assays targeting the penB and mtrR determinants, to be used together with a previously described duplex assay targeting the penA and ponA determinants. The results of this study provided evidence that neither the most-common mtrR determinants nor the most-resistance-associated penB alleles are currently circulating in New Caledonia, suggesting that penicillin should still be considered a valuable treatment strategy. Additionally, using our genotyping assay, we observed an unexpected penB genotype at a relatively high frequency that was associated with a decreased susceptibility to penicillin (average MIC, 0.15 g/ml). Sequencing revealed that this genotype corresponded to an A102S mutation in the penB gene. The molecular tools developed in this study can be used successfully for prospective epidemiological monitoring and surveillance of penicillin susceptibility.Neisseria gonorrhoeae is an obligate gram-negative human pathogenic bacterium that continues to be a public health problem worldwide (31). It is well documented that N. gonorrhoeae bacteria have evolved resistance mechanisms to almost all antimicrobial compounds that have been used to treat infections, an ability that has largely been related to the capacity for natural recombination between strains of the same species within a human host and with other members of the natural human mucosal flora, notably saprophytic and other pathogenic Neisseria species (7,27,29,30).Antimicrobial resistance mechanisms include the production of antibiotic-degrading enzymes, modification of the target site, and decreased influx or increased efflux of the antibiotic (1, 13). Penicillin G was used to treat gonococcal infections up to the mid-1980s, until the emergence and spread of resistance rendered the antibiotic ineffective. These strains utilized either plasmid-mediated or chromosomally mediated mechanisms to become resistant to penicillin G (9, 36). The spread of strains harboring a plasmid-encoded TEM-like ␤-lactamase has been very rapid and has forced the withdrawal of penicillin for treating gonococcal infections in many areas. Chromosomally mediated resistance to penic...

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

“…This assay tested two adjacent SNPs (PorB 120/121), giving a WT result if there was a WT sequence in both positions or a SNP result if there was variation in at least one position. It is possible that only a specific amino acid change contributes to increased MICs to ESCs at this locus (22)(23)(24). In addition, previous studies have shown that porB mutations do not affect ESC susceptibility in the absence of the mtrR Ϫ35del SNP (11,25).…”

Quinolone Resistance in Neisseria gonorrhoeae : Rapid Genotyping of Quinolone Resistance-Determining Regions in gyrA and parC Genes by Melting Curve Analysis Predicts Susceptibility

“…Our assay clearly detects all mutated QRDRs and allows the identification of common genotypes, whether the QRDRs contain single or double mutations, providing valuable epidemiological tools. When this method is used in conjunction with similar assays and in vitro analyses, essential antibiotic resistance surveillance can be performed for public health purposes.Gonorrhea rates generally remain high in New Caledonia and Oceania, where penicillins are the recommended treatment (17,19,22). Increasing penicillin resistance means that fluoroquinolones are the next therapeutic option, but monitoring of relevant antibiotics for effectiveness for public health purposes (2,7,15,16,23) is required.…”

“…Gonorrhea rates generally remain high in New Caledonia and Oceania, where penicillins are the recommended treatment (17,19,22). Increasing penicillin resistance means that fluoroquinolones are the next therapeutic option, but monitoring of relevant antibiotics for effectiveness for public health purposes (2,7,15,16,23) is required.…”

Simple, Rapid, and Inexpensive Detection of Neisseria gonorrhoeae Resistance Mechanisms Using Heat-Denatured Isolates and SYBR Green-Based Real-Time PCR

“…Molecular tools are being used to provide an understanding of the genetic basis of resistance as well as to supplement phenotypic antimicrobial susceptibility testing (9,15,16,20,22,23,24). These tools have not yet advanced to a stage where they can be used in place of phenotypic testing, mainly because the genetic determinants of resistance to most antibiotics are not yet fully known.…”

“…These tools have not yet advanced to a stage where they can be used in place of phenotypic testing, mainly because the genetic determinants of resistance to most antibiotics are not yet fully known. Nevertheless, for well-characterized resistance mechanisms, molecular tools offer an accurate and objective means of detection and systems for the detection of pivotal resistance determinants for public health purposes when viable isolates are not required have been described (22,23).…”