Emergence and Characterization of a Ceftriaxone-Resistant Neisseria gonorrhoeae FC428 Clone Evolving Moderate-Level Resistance to Azithromycin in Shenzhen, China

Introduction

With the emerging resistance of N. gonorrhoeae to nearly all antibiotics, effective antimicrobials for gonorrhoea have become increasingly scarce, including first-line dual therapy with ceftriaxone (CRO) and azithromycin (AZM) recommended by WHO.¹ To date, the MDR N. gonorrhoeae isolates have been reported in Ireland,² Denmark,³ UK⁴ and Australia.⁵ In China, N. gonorrhoeae isolates with decreased susceptibility or resistance to both CRO and AZM have been reported,^6–8 and here in Guangdong Province (South China), we describe a ceftriaxone-resistant N. gonorrhoeae FC428 clone with a higher level of macrolide resistance than previously reported.

The patient was a heterosexual male in his late twenties. He visited the sexually transmitted diseases clinic in Shenzhen Center for Chronic Disease Control in August, 2020 with urethritis symptoms. He reported this was his third infection, and all infections were due to sexual intercourse with commercial sex workers. N. gonorrhoeae (isolate YL201) was cultured from urethral secretions.

The minimal inhibitory concentrations (MICs) of the isolate were determined using E-TEST method, and the results were interpreted in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org) interpretative criteria. YL201 showed resistance to CRO (MIC: 0.75 mg/L) and AZM (MIC: 12 mg/L), but was susceptible to spectinomycin (MIC: 12 mg/L) (Table 1).

Table 1 Phenotypic Characteristics and Molecular Characteristics of Isolates Related to the FC428 Clone

Whole genome sequencing of YL201 was performed using Illumina HiSeq X Ten and Oxford Nanopore MinION sequencer. N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST) and N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) were confirmed using Sanger sequencing. The NG-MAST type was novel with porB-3101 and tbpB-752. The MLST type was ST1600, identical with SRRSH214 and SRRSH229 identified in Hangzhou.⁷ Results of antimicrobial susceptibility testing showed that the three isolates with MLST_ST1600 have higher MIC for ceftriaxone than most strains with MLST_ST1903 (Table 1). This finding indicates that although isolates harbor identical penA mosaic allele, their MIC values may differ. Such variation can be explained by penA-60.001 allele recombined into isolates with certain MLST types associated with CRO decreased susceptibility, and in this case, recombination events happening in MLST_ST1600 isolates may contribute to a higher MIC value. According to our previous study,⁸ MLST_ST7363 is associated with decreased ceftriaxone susceptibility. Moreover, phylogenetic analysis showed that MLST_ST7363 isolates (SC18-68) were clustered with MLST_ST1600 isolates, and that they share 6 identical loci with each other. Therefore, considering the genomic similarity between isolates with the two MLST STs, and the fact that MLST_ST7363 is a common ST in Shenzhen, the expansion of penA-60.001 allele to MLST_ST7363 isolates may have already happened and resulted in elevated MIC values. YL201 had the NG-STAR type of ST2238, containing a mosaic penA-60.001 allele with key resistance-mediating amino acid substitutions A311V and T483S, as well as G545S, I312M and V316T, which is typical characteristics of FC428 clone. YL201 has different NG-STAR type with SRRSH214 and SRRSH229 (ST2238 versus ST2208). The reason for this difference is that YL201 harbours four copies of the 23S rRNA C2611T mutation, while SRRSH214 and SRRSH229 with wild type 23S rRNA allele. Compared with wild-type, four copies of 23S rRNA C2611T mutation increased MICs 40–120-fold,⁷ conferring moderate-level resistance to azithromycin.

Raw short-reads or draft genome assemblies of worldwide FC428-related strains were analysed to infer the phylogeny of YL201. A concatenate superset of SNPs relative to NCCP11945 was generated as previously described.⁹ Based on the genome-wide SNP sites, a maximum likelihood tree was built using PhyML 3.0¹⁰ and the substitution model was automatically selected using SMS (http://www.atgc-montpellier.fr/phyml/).¹¹ According to the phylogeny, YL201, SC18-68, SRRSH214 and SRRSH229 formed a clade (Figure 1), indicating FC428 clones originated from distinct regions have undergone further transmission in China. To date, all isolates within this clade have MLST STs different from ST1903, which may confer a higher MIC for ceftriaxone. In future, novel identified isolates belonging to this clade may present similar features. Additionally, including YL201, genomes of FC428-related strains were compared using BLAST Ring Image Generator (BRIG) and showed high similarities in genome structure without large insertions or deletions (Figure 2). Illumina and Nanopore sequencing data of YL201 have been stored in NCBI short read archive under BioProject PRJNA560592.

Figure 1 Maximum-likelihood tree based on 13,236 SNPs extracted from whole-genome sequences. FA1090 was placed as the outgroup. STs, antimicrobial resistance determinants and antimicrobial susceptibility are also shown. For YL201 and A2543, they contain four copies of the 23S rRNA C2611T and A2059G mutation respectively. Isolate YL201 described in this study is shown in red. The color coding of AMR phenotype and AMR-related alleles are indicated in the columns on the bottom left. Abbreviations: R, resistance; S susceptibility; HLR, High-level resistance; WT, Wild type.

Figure 2 Comparison of YL201 and other 14 strains genomes in the phylogenetic tree. FA1090 (GenBank: AE004969.1) genome was used as the reference. The outermost ring indicates YL201. BLASTn matches with less than 30% identity appear as blank spaces (gaps) in each ring.

In conclusion, we have identified an MDR N. gonorrhoeae isolate in Shenzhen China with resistance to CRO and moderate-level resistance to AZM. The findings demonstrated that FC428 clones have undergone further transmission in China, and during the spread, they have presented more extensive and concerning AMR characteristics. More importantly, as a major port with a large floating population, combined with our previous baseline data, we consider Shenzhen possesses the conditions for further transmission of FC428 clones, thus increasing the severity of gonococcal resistance. Therefore, regional surveillance should be highlighted to understand the transmission of emerging gonococcal drug-resistant clones.