Whole-Genome Sequencing to Predict Antimicrobial Susceptibility Profiles in Neisseria gonorrhoeae.

BACKGROUND: Neisseria gonorrhoeae is a major public health problem due to increasing incidence and antimicrobial resistance. Genetic markers of reduced susceptibility have been identified; the extent to which those are representative of global antimicrobial resistance is unknown. We evaluated the performance of whole-genome sequencing (WGS) used to predict susceptibility to ciprofloxacin and other antimicrobials using a global collection of N. gonorrhoeae isolates. METHODS: Susceptibility testing of common antimicrobials and the recently developed zolifodacin was performed using agar dilution to determine minimum inhibitory concentrations (MICs). We identified resistance alleles at loci known to contribute to antimicrobial resistance in N. gonorrhoeae from WGS data. We tested the ability of each locus to predict antimicrobial susceptibility. RESULTS: A total of 481 N. gonorrhoeae isolates, collected between 2004 and 2019 and making up 457 unique genomes, were sourced from 5 countries. All isolates with demonstrated susceptibility to ciprofloxacin (MIC ≤0.06 µg/mL) had a wild-type gyrA codon 91. Multilocus approaches were needed to predict susceptibility to other antimicrobials. All isolates were susceptible to zoliflodacin, defined by an MIC ≤0.25 µg/mL. CONCLUSIONS: Single marker prediction can be used to inform ciprofloxacin treatment of N. gonorrhoeae infection. A combination of molecular markers may be needed to determine susceptibility for other antimicrobials.

Identification of plasmids in avian-associated Escherichia coli using nanopore and illumina sequencing.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project. RESULTS: Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected. CONCLUSIONS: Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.

High Prevalence of Macrolide and Fluoroquinolone Resistance-Mediating Mutations in Mycoplasma genitalium-Positive Urine Specimens From Saskatchewan.

BACKGROUND: Mycoplasma genitalium is an emerging, sexually transmitted infection, which is more prevalent than Chlamydia trachomatis in some regions. An increase in antibiotic resistance, that is, azithromycin and moxifloxacin, recommended for treating M. genitalium infections has been noted. This is the first detailed report on the prevalence of M. genitalium and its antimicrobial resistance in Saskatchewan, Canada. METHODS: Aptima urine specimens (n = 1977), collected for the diagnosis of C. trachomatis/Neisseria gonorrhoeae, were tested for M. genitalium using the Aptima M. genitalium assay (MG-TMA). Antimicrobial resistance was ascertained using polymerase chain reaction and DNA sequencing of 23S rRNA (azithromycin) and parC (moxifloxacin) from Aptima M. genitalium assay-positive specimens; mutations predictive of resistance were noted. RESULTS: The prevalence of M. genitalium was 9.6% (189/1977). Predicted resistance to azithromycin (substitutions at positions 2058/2059 in 23S rRNA) was observed in 63.6% (70/110) of the specimens tested, whereas resistance to moxifloxacin (S83I in ParC) was observed in 10.6% (9/85) of the specimens. Mutations in both 23S rRNA and ParC were observed in 2.12% (4/189) of the specimens. Women aged 20 to 24 years had the highest prevalence (18.3%, P < 0.001), and in females, M. genitalium was significantly associated with C. trachomatis or N. gonorrhoeae/C. trachomatis (P < 0.001) coinfection. The prevalence of M. genitalium (9.6%) in the province of Saskatchewan was higher than that of the other 2 bacterial sexually transmitted infections (N. gonorrhoeae (3.09%) and C. trachomatis (6.85%). CONCLUSIONS: The prevalence of M. genitalium (9.6%) and associated resistance to azithromycin (63.6%) in Saskatchewan high, suggesting that empiric azithromycin therapy may not be adequate for treating M. genitalium infections.

Genomic Analysis Reveals Antibiotic-Susceptible Clones and Emerging Resistance in Neisseria gonorrhoeae in Saskatchewan, Canada.

Whole-genome sequencing was used to identify mutations in antibiotic resistance-conferring genes to compare susceptibility predictions with MICs and to ascertain strain types in 99 isolates of Neisseria gonorrhoeae Genotypes associated with susceptibility, as well as MIC creep or emerging resistance, were noted. Phylogenomic analysis revealed three distinctive clades and putative gonococcal transmission linkages involving a tetracycline-resistant N. gonorrhoeae outbreak and the clonal spread of susceptible isolates in men.

Gen2EpiGUI: User-Friendly Pipeline for Analyzing Whole-Genome Sequencing Data for Epidemiological Studies of Neisseria gonorrhoeae.

We have developed a graphical user interface for our Gen2Epi computational pipeline named Gen2EpiGUI. A total of 594 published whole-genome sequence datasets of Neisseria gonorrhoeae were used to validate the program. Gen2Epi facilitates an understandable analysis of N. gonorrhoeae whole-genome sequence data for users with limited bioinformatics skills.

CcpN: a moonlighting protein regulating catabolite repression of gluconeogenic genes in Bacillus subtilis also affects cell length and interacts with DivIVA.

CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.

Antimicrobial resistance genetic factor identification from whole-genome sequence data using deep feature selection.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to global public health because it makes standard treatments ineffective and contributes to the spread of infections. It is important to understand AMR's biological mechanisms for the development of new drugs and more rapid and accurate clinical diagnostics. The increasing availability of whole-genome SNP (single nucleotide polymorphism) information, obtained from whole-genome sequence data, along with AMR profiles provides an opportunity to use feature selection in machine learning to find AMR-associated mutations. This work describes the use of a supervised feature selection approach using deep neural networks to detect AMR-associated genetic factors from whole-genome SNP data. RESULTS: The proposed method, DNP-AAP (deep neural pursuit - average activation potential), was tested on a Neisseria gonorrhoeae dataset with paired whole-genome sequence data and resistance profiles to five commonly used antibiotics including penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime. The results show that DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae, and also provide a list of candidate genomic features (SNPs) that might lead to the discovery of novel AMR determinants. Logistic regression classifiers were built with the identified SNPs and the prediction AUCs (area under the curve) for penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime were 0.974, 0.969, 0.949, 0.994, and 0.976, respectively. CONCLUSIONS: DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae. It also provides a list of candidate genes and intergenic regions that might lead to novel AMR factor discovery. More generally, DNP-AAP can be applied to AMR analysis of any bacterial species with genomic variants and phenotype data. It can serve as a useful screening tool for microbiologists to generate genetic candidates for further lab experiments.

Gen2Epi: an automated whole-genome sequencing pipeline for linking full genomes to antimicrobial susceptibility and molecular epidemiological data in Neisseria gonorrhoeae.

BACKGROUND: Recent adva1nces in whole genome sequencing (WGS) based technologies have facilitated multi-step applications for predicting antimicrobial resistance (AMR) and investigating the molecular epidemiology of Neisseria gonorrhoeae. However, generating full scaffolds of N. gonorrhoeae genomes from short reads, and the assignment of molecular epidemiological information (NG-MLST, NG-MAST, and NG-STAR) to multiple assembled samples, is challenging due to required manual tasks such as annotating antimicrobial resistance determinants with standard nomenclature for a large number of genomes. RESULTS: We present Gen2Epi, a pipeline that assembles short reads into full scaffolds and automatically assigns molecular epidemiological and AMR information to the assembled genomes. Gen2Epi is a command-line tool integrating third-party software and tailored specifically for N. gonorrhoeae. For its evaluation, the Gen2Epi pipeline successfully assembled the WGS short reads from 1484 N. gonorrhoeae samples into full-length genomes for both chromosomes and plasmids and was able to assign in silico molecular determinant information to each dataset automatically. The assemblies were generated using raw as well as trimmed short reads. The median genome coverage of full-length scaffolds and "N" statistics (N50, NG50, and NGA50) were higher than, or comparable to, previously published results and the scaffolding process improved the quality of the draft genome assemblies. Molecular antimicrobial resistant (AMR) determinants identified by Gen2Epi reproduced information for the 1484 samples as previously reported, including NG-MLST, NG-MAST, and NG-STAR molecular sequence types. CONCLUSIONS: Gen2Epi can be used to assemble short reads into full-length genomes and assign accurate molecular marker and AMR information automatically from NG-STAR, NG-MAST, and NG-MLST. Gen2Epi is publicly available under "CC BY-NC 2.0 CA" Creative Commons licensing as a VirtualBox image containing the constituent software components running on the LINUX operating system (CentOS 7). The image and associated documentation are available via anonymous FTP at ftp://www.cs.usask.ca/pub/combi or ftp://ftp.cs.usask.ca/pub/combi.

A ß-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly.

BACKGROUND: Seven structurally related ß-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903. OBJECTIVES: To compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 ß-lactamases. METHODS: N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. ß-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays. RESULTS: N. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow ß-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 ß-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours. CONCLUSIONS: This unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 ß-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5'-carboxyfluorosceinsuccidyl ester.

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. RESULTS: We observed that 20.3% (+/- 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. CONCLUSIONS: Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.

Antimicrobial Resistance in Neisseria gonorrhoeae: Proceedings of the STAR Sexually Transmitted Infection-Clinical Trial Group Programmatic Meeting.

The goal of the Sexually Transmitted Infection Clinical Trial Group's Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, nonprofit and industry to discuss the current state of research, gaps and challenges in research and technology and priorities and new directions to address the continued emergence of multidrug-resistant NG infections. Topics discussed at the meeting, which will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG; (ii) the US National Strategy for combating antimicrobial-resistant bacteria; (iii) surveillance needs, challenges, and novel technologies; (iv) plasmid-mediated and chromosomally mediated mechanisms of AMR in NG; (v) novel therapeutic (eg, sialic acid analogs, factor H [FH]/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (eg, peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives, and collaborations across academic and commercial research and public health programs.

World Health Organization Global Gonococcal Antimicrobial Surveillance Program (WHO GASP): review of new data and evidence to inform international collaborative actions and research efforts.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a serious public health problem, compromising the management and control of gonorrhoea globally. Resistance in N. gonorrhoeae to ceftriaxone, the last option for first-line empirical monotherapy of gonorrhoea, has been reported from many countries globally, and sporadic failures to cure especially pharyngeal gonorrhoea with ceftriaxone monotherapy and dual antimicrobial therapies (ceftriaxone plus azithromycin or doxycycline) have been confirmed in several countries. In 2018, the first gonococcal isolates with ceftriaxone resistance plus high-level azithromycin resistance were identified in England and Australia. The World Health Organization (WHO) Global Gonococcal Antimicrobial Surveillance Program (GASP) is essential to monitor AMR trends, identify emerging AMR and provide evidence for refinements of treatment guidelines and public health policy globally. Herein we describe the WHO GASP data from 67 countries in 2015-16, confirmed gonorrhoea treatment failures with ceftriaxone with or without azithromycin or doxycycline, and international collaborative actions and research efforts essential for the effective management and control of gonorrhoea. In most countries, resistance to ciprofloxacin is exceedingly high, azithromycin resistance is present and decreased susceptibility or resistance to ceftriaxone has emerged. Enhanced global collaborative actions are crucial for the control of gonorrhoea, including improved prevention, early diagnosis, treatment of index patient and partner (including test-of-cure), improved and expanded AMR surveillance (including surveillance of antimicrobial use and treatment failures), increased knowledge of correct antimicrobial use and the pharmacokinetics and pharmacodynamics of antimicrobials and effective drug regulations and prescription policies (including antimicrobial stewardship). Ultimately, rapid, accurate and affordable point-of-care diagnostic tests (ideally also predicting AMR and/or susceptibility), new therapeutic antimicrobials and, the only sustainable solution, gonococcal vaccine(s) are imperative.

Association of Neisseria gonorrhoeae genogroups and specific PBP2/MtrR/PorB mutation patterns with susceptibility to penicillin in a susceptible gonococcal population.

Objectives: To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB). Methods: The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs. Results: The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05). Conclusions: Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens.

High levels of susceptibility to new and older antibiotics in Neisseria gonorrhoeae isolates from Saskatchewan (2003-15): time to consider point-of-care or molecular testing for precision treatment?

OBJECTIVES: The antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Saskatchewan was determined retrospectively (2003-15) to ascertain temporal trends to both current and older antimicrobials used for treatment. METHOD: The agar dilution method was used to test the antimicrobial susceptibilities of 685 isolates to seven antibiotics. RESULTS: Over the period, only three (0.4%) gonococcal isolates had reduced susceptibility to cefixime and/or ceftriaxone. All isolates were susceptible to spectinomycin. Over 95% of the isolates tested were susceptible to azithromycin except in 2010 and 2013 (27.6% and 7.2% resistant, respectively). One isolate was resistant to both azithromycin and cefixime. Ciprofloxacin resistance was seen in < 5% of isolates prior to 2010, but in > 5% thereafter. From 2006 to 2012, and in 2015, penicillin resistance was detected in < 5% (0%-4.0%) of isolates, but in > 5% for the rest of the study period. Tetracycline resistance remained >5% (11.8%-89.1%) throughout the study. Plasmid-mediated resistance to tetracycline fluctuated between 0% and 17.5% of isolates tested. Four isolates were MDR and two isolates were XDR. CONCLUSIONS: N. gonorrhoeae isolates were largely susceptible (∼85%) to antibiotics no longer recommended for treatment, such as penicillin and ciprofloxacin. Gonorrhoea in Saskatchewan is primarily (>95%) diagnosed by nucleic acid amplification testing, which does not permit antimicrobial susceptibility testing. The development of molecular testing, or point-of-care tests, to evaluate antimicrobial susceptibility, would enhance knowledge of true levels of resistance and allow discretion as to whether older but still effective antibiotics could be used in individual patient care.

Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Latin American and Caribbean countries, 2013-2015.

OBJECTIVES: A Neisseria gonorrhoeae antimicrobial susceptibility quality control comparison programme was re-established in Latin America and the Caribbean to ensure antimicrobial susceptibility data produced from the region are comparable nationally and internationally. METHODS: Three panels, consisting of N. gonorrhoeae isolates comprising reference strains and other characterised isolates were sent to 11 participating laboratories between 2013 and 2015. Antimicrobial susceptibilities for these isolates were determined using agar dilution, Etest or disc diffusion methods. Modal minimum inhibitory concentrations (MICs) for each panel isolate/antibiotic combination were calculated. The guidelines of the Clinical and Laboratory Standards Institute were used for interpretations of antimicrobial susceptibility. The agreement of MICs with the modal MICs was determined for each of the participating laboratories as well as for each of the antibiotics tested. RESULTS: Five of 11 laboratories that participated in at least one panel had an overall average agreement between participants' MIC results and modal MICs of >90%. For other laboratories, agreements ranged from 60.0% to 82.4%. The proportion of agreement between interpretations for all the antibiotics, except penicillin and tetracycline, was >90%. The percentages of agreement between MIC results and their modes for erythromycin, spectinomycin, cefixime and azithromycin were >90%. Tetracycline, ceftriaxone and ciprofloxacin agreement ranged from 84.5% to 89.1%, while penicillin had 78.8% agreement between MICs and modal MICs. CONCLUSIONS: The participating laboratories had acceptable results, similar to other international quality assurance programmes. It is important to ensure continuation of the International Gonococcal Antimicrobial Susceptibility Quality Control Comparison Programme to ensure that participants can identify and correct any problems in antimicrobial susceptibility testing for N. gonorrhoeae as they arise and continue to generate reproducible and reliable data.

Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action.

In a Policy Forum, Teodora Wi and colleagues discuss the challenges of antimicrobial resistance in gonococci.

Multidrug-resistant gonorrhea: A research and development roadmap to discover new medicines.

Emilie Alirol and colleagues discuss the development of new treatments for gonorrhea.

Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status.

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in gyrA of N. gonorrhoeae The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 N. gonorrhoeae isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeae species isolates. The performance of the primers was validated using genomic DNA from 100 different N. gonorrhoeae isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeae isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the N. gonorrhoeae isolates and 23 non-N. gonorrhoeae isolates. These primers detected N. gonorrhoeae and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status.