The HDOH confirmed the identification of 61 isolates of N. gonorrhoeae, collected during February 2016–May 2016, and antimicrobial drug susceptibility testing was performed on all of them. Isolates were identified as N. gonorrhoeae by using the API NH test kit (bioMérieux, Marcy l’Etoile, France), and the MICs for azithromycin, ceftriaxone, and cefixime was assessed by Etest (bioMérieux) on GC II agar supplemented with 1% IsoVitaleX (bioMérieux). Etest carried out at the HDOH State Laboratories Division found that 8 N. gonorrhoeae isolates had extremely high MICs (>256 µg/mL) for azithromycin and MICs of 0.125–0.25 µg/mL for ceftriaxone and cefixime (Technical Appendix). The 8 isolates, which were collected from 7 patients and included 2 isolates (urethral and urine) from the same patient (GCWGS_0182 and GCWGS_0322), were sent to CDC for confirmatory testing using agar plate dilution (5).

All 61 N. gonorrhoeae isolates were sequenced (paired-end; 2 × 250-bp read length) on an Illumina MiSeq sequencer (Illumina Denmark ApS, Copenhagen, Denmark) at the HDOH State Laboratories Division. De novo assembly was conducted at CDC by using SPAdes 2.5.1 (http://www.cab.spbu.ru/software/spades), and the core genome single-nucleotide polymorphism alignment was generated by using Parsnp 1.2 (http://www.cbcb.umd.edu/software/harvest), with the FA19 genome (GenBank accession no. CP012026) as the reference. The maximum-likelihood phylogeny was reconstructed by using RAxML 8.0.0 (http://sco.h-its.org/exelixis/web/software/raxml) with 1,000 bootstrap replicates. Whole-genome sequencing data were also used to determine the multilocus sequence typing (MLST) and N. gonorrhoeae–multiantigen sequence typing (NG-MAST) allelic profiles for the targeted isolates.

Figure

Figure. Maximum-likelihood phylogeny of Neisseria gonorrhoeae samples (N = 62) collected in Hawaii during February–May 2016, 1 isolate collected in Hawaii in 2011, and 1 isolate collected in the United Kingdom in...

Results of the phylogenetic analysis indicated that the 8 isolates were closely related and formed a single clade (Figure) with 223 single-nucleotide polymorphism differences. MLST analysis revealed 1 unique profile, sequence type (ST) 1901 (Technical Appendix), which is a highly successful lineage associated with multidrug resistance that probably originated in Japan (6). The results of the NG-MAST analysis indicated that all 8 isolates shared 1 novel profile, ST14121. Epidemiologic investigations did not associate sexual network transmission among the 7 patients, although 2 patients reported sex with the same partner. However, the consistent MLST and NG-MAST profiles, in combination with the strongly supported clade, suggest the circulation of a single strain within the population.

To assess the contribution of known mutations to macrolide and cephalosporin resistance, we examined mutations in penA, ponA, mtrR, and 23S rRNA genes. Regarding azithromycin resistance, a deletion in the mtrR promoter associated with low-level resistance (7) and 4 mutated 23S rRNA copies with the A2059G mutation that confers high-level resistance (8) were identified in all 8 isolates. The ponA L421P mutation and mosaic penA alleles have been associated with reduced susceptibility to cephalosporins (7,9). The ponA L421P mutation was found in all 8 isolates; however, only the nonmosaic penA XVIII allele was detected.

The first N. gonorrhoeae isolate (H11S8) with high-level azithromycin resistance (HL-AziR) in the United States was identified in Hawaii in 2011 (10). More recently, Public Health England characterized 7 N. gonorrhoeae HL-AziR isolates that were collected in northern England during November 2014–March 2015 (11). Isolate H11S8 and those from England were more susceptible to ceftriaxone (MIC range 0.004–0.03 µg/mL) than the cluster of N. gonorrhoeae HL-AziR isolates identified in Hawaii. Genetic comparisons of the 2011 Hawaii isolate placed it in a distinct clade on the phylogenetic tree (Figure). The NG-MAST of H11S8 was ST649, and those from England were ST9768. Three HL-AziR N. gonorrhoeae strains were isolated in 2011 and 2012 in Sweden with slightly higher ceftriaxone MICs (range 0.032–0.064 µg/mL) and were identified as either NG-MAST ST285 or ST8727 (12).

All patients infected with the HL-AziR isolates in our study were successfully treated with 250 mg ceftriaxone plus 1 g azithromycin. In contrast, a recent pharyngeal N. gonorrhoeae isolate, resistant to azithromycin and ceftriaxone, was recovered from a patient in the United Kingdom following treatment with dual antimicrobial therapy of 500 mg ceftriaxone plus 1 g azithromycin (13). Although the isolate was genetically distinct from the 8 isolates in Hawaii, it was more closely related to those 8 isolates than to the other 53 contemporary isolates from Hawaii.

The combination of ceftriaxone and azithromycin remains the hallmark for the treatment of gonorrhea worldwide on the basis of surveillance data that monitors antimicrobial susceptibility (2,14,15). Slight fluctuations have been observed in ceftriaxone MICs, but rarely have isolates been recovered with a MIC >0.5 μg/mL. However, a growing body of evidence suggests that azithromycin is becoming less effective and should not be used as a monotherapeutic agent for gonorrhea. The observation of increased MICs for ceftriaxone and azithromycin in a cluster of strains from Hawaii might be the harbinger that the effectiveness of current treatment options will be challenged. It is critical that countries expand systematic surveillance for drug-resistant N. gonorrhoeae and that laboratories maintain culture capacity to support rapid response activities to confirm suspected treatment failures and mitigate transmission through contact tracing. Expansion of laboratory capacity to conduct genetic analysis in real time would further benefit clinicians and sexually transmitted disease public health programs by identifying novel mechanisms of resistance that could be used to develop nonculture antimicrobial resistance tests and rapidly identify resistant N. gonorrhoeae strains in sexual networks.