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The growing antimicrobial resistance (AMR) in Neisseria gonorrhoeaeis a serious global threat to gonococcal therapy. Molecular typing is an ideal tool to reveal the association between specific genotypes and resistance phenotypes that provide effective data for tracking the transmission of resistant clones of N. gonorrhoeae. In our study, we aimed to describe the molecular epidemiology of AMR and the distribution of resistance-associated genotypes in Shenzhen, China, during 2014 to 2018.

Antimicrobial resistance in Neisseria gonorrhoeaepersists as a major public health concern globally. We developed and evaluated a multiplex assay that relied on high-resolution melting (HRM) technology as a rapid, simple, and cost-effective method for simultaneously detecting and identifying different molecular markers associated with extended-spectrum cephalosporins (ESCs) and azithromycin (AZM) resistance in N. gonorrhoeae. Forty-eight well-characterized N. gonorrhoeaeclinical isolates were selected for initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the antimicrobial resistance determinants related to ESCs and AZM resistance. Specificity and cross-reactivity were assessed by testing 15 nongonococcal strains. Then, the assay was validated on 218 archived DNA specimens that had been sequenced using whole-genome sequencing technology. Compared with whole-genome sequencing, these assays had a sensitivity of 98.6%, with a specificity of 99.2%. For further validation of the assay's performance, a total of 338 samples (156 clinical isolates and 182 clinical specimens) were screened using the multiplex HRM assay. The results showed good concordance with the results of PCR sequencing. Given its rapidity (within 90 minutes), ease of performing, and low cost (<$1.00 per sample), this method may be applied to large-scale epidemiologic programs for increasing surveillance of ESCs and AZM resistance in N. gonorrhoeae.

Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission, timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance (AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer (Oxford Nanopore Technologies (ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrRwhich results in a frame-shift, premature stop codon, and truncated peptide. In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeaestrains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N. gonorrhoeaecan be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new AMR determinants.