Neisseria gonorrhoeae and extended-spectrum cephalosporins in California: surveillance and molecular detection of mosaic penA

Abstract

BackgroundThe spread of Neisseria gonorrhoeae strains with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins is a major public health problem. While much work has been performed internationally, little is known about the genetics or molecular epidemiology of N. gonorrhoeae isolates with reduced susceptibility to extended-spectrum cephalosporins in the United States. The majority of N. gonorrhoeae infections are diagnosed without a live culture. Molecular tools capable of detecting markers of extended-spectrum cephalosporin resistance are needed.MethodsUrethral N. gonorrhoeae isolates were collected from 684 men at public health clinics in California in 2011. Minimum inhibitory concentrations (MICs) to ceftriaxone, cefixime, cefpodoxime and azithromycin were determined by Etest and categorized according to the U.S. Centers for Disease Control 2010 alert value breakpoints. 684 isolates were screened for mosaic penA alleles using real-time PCR (RTPCR) and 59 reactive isolates were subjected to DNA sequencing of their penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). To increase the specificity of the screening RTPCR in detecting isolates with alert value extended-spectrum cephalosporin MICs, the primers were modified to selectively amplify the mosaic XXXIV penA allele.ResultsThree mosaic penA alleles were detected including two previously described alleles (XXXIV, XXXVIII) and one novel allele (LA-A). Of the 29 isolates with an alert value extended-spectrum cephalosporin MIC, all possessed the mosaic XXXIV penA allele and 18 were sequence type 1407, an internationally successful strain associated with multi-drug resistance. The modified RTPCR detected the mosaic XXXIV penA allele in urethral isolates and urine specimens and displayed no amplification of the other penA alleles detected in this study.ConclusionN. gonorrhoeae isolates with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins are currently circulating in California. Isolates with the same NG-MAST ST, penA allele and extended-spectrum cephalosporin MICs have caused treatment failures elsewhere. The RTPCR assay presented here may be useful for the detection of N. gonorrheoae isolates and clinical specimens with reduced extended-spectrum cephalosporin MICs in settings where antimicrobial susceptibility testing is unavailable. In an era of increasing antimicrobial resistance and decreasing culture capacity, molecular assays capable of detecting extended-spectrum cephalosporin of resistance are essential to public health.

Failure of Azithromycin 2.0 g in the Treatment of Gonococcal Urethritis Caused by High-Level Resistance in California

“…A previous case of unsuccessful treatment with AZM was reported in Oregon in 2011, but the strain responsible for that case only displayed moderate AZM resistance (MIC 8–16 μg/mL). 2 Isolates with moderate AZM resistance have also been reported in California, 9 but CA-1461 is the first report of a strain with high-level AZM resistance in California. Fortunately, CA-1461 was fully susceptible to both cefixime (MIC 0.015 μg/mL) and ceftriaxone (MIC 0.008 μg/mL) and could therefore have been successfully treated with the CDC-recommended dual-therapy regimen, which includes ceftriaxone 250 mg IM.…”

Real-Time PCR Targeting the penA Mosaic XXXIV Type for Prediction of Extended-Spectrum-Cephalosporin Susceptibility in Clinical Neisseria gonorrhoeae Isolates

“…First, since the penA real-time PCR presented herein was designed to specifically detect the mosaic XXXIV type, the sensitivity of the assay would depend on the prevalence of penA mosaic XXXIV among isolates with MIC values in the alert range in the geographical areas in which the assay would be performed. In a recent genotypic surveillance study of clinical N. gonorrhoeae isolates in California, alert value extendedspectrum cephalosporin MICs were observed in 29/684 isolates, all of which carried mosaic XXXIV (26). Presuming that the panel of clinical isolates used to validate the mosaic XXXIV PCR served as an appropriate representation of the population of N. gonorrhoeae in the United States, the assay should perform well, with high sensitivity and specificity, as presented herein.…”

“…In order to determine the presence of penA mosaic XXXIV and other mosaic types with homologous sequences in the target region, which include X, XXVII, XXXVII, XLII, LI, LII, LIII, LIV, LV, and LVIII (determined in silico by performing BLAST [23] against a sequence database of penA mosaic patterns, courtesy of Magnus Unemo), some of which have been associated with reduced susceptibility to ESCs (24, 25), we designed a novel real-time PCR based on an existing TaqMan PCR assay (26). Modifications in primer and probe sequences were made to convert the assay to FRET probe format due to the incompatibility of the TaqMan assay with the previously validated FRET probebased gyrA genotyping assay (16) (see Table S1 in the supplemental material).…”

Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility

“…penA mosaicism, along with other point mutations in penA, helps N. gonorrhoeae to develop resistance against extendedspectrum cephalosporins like cefixime. [14][15][16][17][18][19][20][21][22][23][24][25][26][27][41][42][43] In this review, we describe molecular mechanisms of cefixime-decreased susceptibility in N. gonorrhoeae, summarise findings from published reports of various gene mutations contributing to that decreased susceptibility and make suggestions on how to develop a molecular-based cefixime susceptibility assay.…”