Shanghai Neisseria gonorrhoeae Isolates Exhibit Resistance to Extended-Spectrum Cephalosporins and Clonal Distribution

The emergence of Neisseria gonorrhoeae strains with resistance (R) to extended-spectrum cephalosporins (ESCsR) represents a public health threat of untreatable gonococcal infections. This study was designed to determine the prevalence and molecular mechanisms of ESCR of Shanghai N. gonorrhoeae isolates. A total of 366 N. gonorrhoeae isolates were collected in 2017 in Shanghai. Susceptibility to ceftriaxone (CRO), cefixime (CFM), azithromycin (AZM), ciprofloxacin (CIP), spectinomycin, penicillin, and tetracycline was determined using the agar dilution method. A subset of 124 isolates was subjected to phylogenetic analysis for nine antimicrobial resistance-associated genes, i.e., penA, porB, ponA, mtrR, 23S rRNA, gyrA, parC, 16S rRNA, and rpsE. Approximately 20.0% of the isolates exhibited CFMR [minimum inhibitory concentration (MIC) >0.125 mg/L], and 5.5% were CROR (MIC > 0.125 mg/L). In total, 72.7% of ESCR isolates were clonal and associated with mosaic penA 10 and 60 alleles. Non-mosaic penA 18 allele and substitutions of PenA A501T, G542S, and PorB1b G213S/Y were observed in non-clonal ESCR. Approximately 6.8% of the isolates showed AZM MIC above the epidemiological cutoff (ECOFF, 1 mg/L), were associated with 23S rRNA A2059G mutation, and did not exhibit clonal distribution. Almost all isolates were CIPR (resistance to ciprofloxacin) and associated with GyrA-91/92 and ParC-85/86/87/88/89/91 alterations. Isolates with ParC S88P substitution were clustered into the ESCR clade. The Shanghai isolates exhibited a high level of ESCR and distinct resistant patterns.


INTRODUCTION
Neisseria gonorrhoeae is the causative agent of gonorrhea. The World Health Organization (WHO) estimated that N. gonorrhoeae causes more than 86.9 million new infections worldwide annually (World Health Organization [WHO], 2018). Meanwhile, gonococcal antimicrobial resistance (AMR) continues to spread worldwide and could lead to a pandemic of extensively drug-resistant gonococci (World Health Organization [WHO], 2018). Of particular concern is the fact that ESC RS [reduced susceptibility to the extended-spectrum cephalosporins (ESCs), i.e., cefixime (CFM), and ceftriaxone (CRO)], which is the first-line empirical treatment for N. gonorrhoeae infections (Unemo and Shafer, 2014;Wi et al., 2017), is becoming widely spread. According to the WHO Global Gonococcal Antimicrobial Surveillance Programme (GASP), in 2016, about one-third of the participating countries reported that ≥5% of isolates are resistant to ESCs (CRO and/or CFM), and half reported ≥5% resistance to azithromycin (AZM R ). Of the 59 countries reporting ciprofloxacin resistance (CIP R ), 95% reported ≥5% resistance and 17% reported >90% resistance (Wi et al., 2017;World Health Organization [WHO], 2018). In China, from 2013 to 2016, high prevalence of decreased susceptibility to CRO (CRO RS ) (9.7-12.2%, MIC ≥ 0.125 mg/L) and AZM R (18.6%, MIC ≥ 1.0 mg/L) has been reported (Yin et al., 2018). In Shanghai, the proportion of CRO RS (MIC ≥ 0.125 mg/L) ranged from 7 to 13% during 1988-2013 (Gu et al., 2014).
Drug-resistant N. gonorrhoeae has been attributed to several molecular mechanisms. The primary mechanism for ESC R (resistance to ESC) is mutations of the penA gene (encodes penicillin (PEN)-binding protein 2, PBP2, PenA), including a recombinant mosaic allele from commensal Neisseria (Ameyama et al., 2002;Lee et al., 2010). Mutations in the Mtr repressor genes mtrR and porB have been shown to contribute to ESC R (Barry and Klausner, 2009;Unemo and Shafer, 2014). Loci involved in other AMR include mutations of 23S rRNA (Ng et al., 2002) and mtrR (Zarantonelli et al., 1999) for AZM R , mutations in gyrA and parC for CIP R (Yang et al., 2006), and mutations in 16S rRNA and rpsE for spectinomycin (SPT) (Galimand et al., 2000;Unemo et al., 2013). Currently, the identified resistance determinants do not fully account for the observed drug resistance, and thus, other factors may be involved (Unemo and Shafer, 2014).
Genetic analysis has provided insight into outbreaks and transmission networks for several pathogens with greater resolution than traditional methods (Diep, 2013). Using genetic methods, researchers found that ESC RS in Canada first emerged from a group of diverse isolates in the 1990s with non-mosaic penA alleles, followed in 2000/2001 with the mosaic penA 10 allele and then in 2007 with the mosaic penA 34 allele (Demczuk et al., 2015). ESC RS strains in the United States are mainly clonal and associated with the mosaic penA 34 allele and derivatives, whereas AZM R strains have arisen through multiple mechanisms and show limited clonal spread (Grad et al., , 2016. To date, reported cases of CRO R are sporadic, except for the FC428 strain, which was first identified in Japan in 2015 and has since then been observed in other countries (Lahra et al., 2018;Lee et al., 2019).
The objectives of this study were to assess whether nine loci can increase the resolution of genetic analysis and to determine the association of genetic characterization and ESC R phenotypes in N. gonorrhoeae isolates in Shanghai. This is the first indepth genomic analysis based on nine AMR-associated loci in N. gonorrhoeae in a high-level AMR setting. This study provides solid information on the molecular mechanisms and genetic characteristics of AMR in N. gonorrhoeae in Shanghai.

Neisseria gonorrhoeae Isolate Collection and Antimicrobial Susceptibility Testing
Neisseria gonorrhoeae isolates were collected from male patients with uncomplicated urogenital gonorrhea (symptoms may include pain or a burning sensation when urinating, a greater frequency or urgency of urination, and abnormal urinary discharge) at the Shanghai Skin Disease Hospital in conjunction with the China GASP. Patient consent was obtained, and ethics approval was received from the Shanghai Skin Disease Hospital. The first 30 N. gonorrhoeae isolates of each month in 2017 (except for 36 isolates collected in July to avoid recovery failure, making a total of 366 isolates) were used in this study. Basic demographic data of all patients were collected. The median age was 34 years (range: 18-69). Of the 366 subjects, 363 (99.2%) were ethnic Han. All patients were heterosexual. A majority of the patients had abnormal urinary discharge (98.9%). Approximately 16.4% of the patients had previous history of gonorrhea, and 12.8% received antibiotic treatment in the past month. One isolate was collected from one patient. Briefly, one urogenital specimen was collected using sterile Dacron swab and streaked on Thayer-Martin (T-M) medium (Oxoid; GuangZhou LOSO Science, Ltd.) supplemented with 1% IsoVitaleX (Oxoid; GuangZhou LOSO Science, Ltd.). N. gonorrhoeae was identified using criteria that included an oxidase test, Gram staining, and glucose utilization test (WHO Western Pacific Gonococccal Antimicrobial Surveillance Programme, 2008). One identified N. gonorrhoeae isolate for each patient was collected and stored at −70 • C. Minimum inhibitory concentrations (MICs) for seven antimicrobials, including PEN, tetracycline (TET), ciprofloxacin (CIP), azithromycin (AZM), CFM, CRO, and SPT, were determined using the agar dilution method. Antimicrobial agents were purchased from Shanghai ANPEL Scientific Instrument, Co., Ltd. (Shanghai, China; distributors of Sigma-Aldrich, United States). Each MIC determination was performed in duplicate, and N. gonorrhoeae ATCC 49226 strain was used as a reference strain. Antimicrobial susceptibility testing results were interpreted using the EUCAST (2020) breakpoints.

Phylogenetic Analysis
The sequences of all nine loci were concatenated for each strain. IQ-TREE (v1.6.12) (Nguyen et al., 2015) was used for constructing maximum-likelihood phylogenies with 1,000 bootstraps, and the best-fit model was autodetected. Phylogenies were assessed using midpoint rooting. Phylogenetic clades were determined by cluster analysis using ClusterPicker (Ragonnet-Cronin et al., 2013) with the following settings: initial and main support thresholds of 90 and genetic distance threshold of 4.5. Phylogenies and metadata (including MICs, AMR phenotypes, and molecular profiles associated with AMR) were visualized in FigTree 2 and phandango (Hadfield et al., 2017).

Statistical Analysis
The χ 2 test was used to identify AMR determinants associated with CFM R , CRO R , and MICs above ECOFF for AZM using R (version 3.4.1). Multiple linear regression analysis was performed using R (version 3.4.1), to determine the relationship of log 10 (CRO/CFM/AZM MICs) or CIP MIC intervals as the dependent variable to the presence of gene mutations.

Antimicrobial Susceptibility of N. gonorrhoeae Isolates
Among the 366 N. gonorrhoeae isolates, 5.5% of the isolates were CRO R (MICs > 0.125 mg/L), and 18.6% of isolates had CRO MICs ≥ 0.125 mg/L ( Table 1 and Supplementary  Table S1). About 19.4% of the 366 isolates were CFM R (MICs > 0.125 mg/L). About 6.8% of the isolates showed AZM MIC above the epidemiological cutoff (ECOFF, 1 mg/L), and 99.5% of the isolates were CIP R . The percentages of PEN R and TET R were 82.5% and 60.9%, respectively. One isolate was SPT R . Demographic/clinical information including age, ethnicity, abnormal urinary discharge, previous history of gonorrhea, and antibiotic use in the past month was not associated with resistance to CFM, CRO, and AZM (Supplementary Table S2).
Supplementary Table S3 shows that 24.0% (88/366) of the sequenced isolates exhibited multidrug-resistant (MDR) phenotypes (resistance to ESC or AZM plus resistance to at least two other antimicrobials) (Martin et al., 2019). Among these phenotypes, ESC-associated phenotypes accounted for 17.7%, and AZM-associated phenotype accounted for 6.3%. Extensively drug-resistant phenotypes (resistance to ESC and AZM plus resistance to at least two other antimicrobials) (Martin et al., 2019)

Mosaic penA and Substitutions in PenA and Association With NG-STAR Types
Approximately 76.2% of the CFM R isolates (16/21) had mosaic penA alleles, and only 2.9% of the CFM S isolates (3/103) possessed mosaic penA alleles ( Table 2). Mosaic penA 10 and 60 alleles and substitutions in the mosaic penA coding region such as D101E and A549T were significantly associated with CFM R . Specifically, 10 out of 11 (90.9%) penA-10.001 isolates were CFM R , and four out of four (100%) penA-60.001 isolates were CFM R . Substitutions of F374V, H541N, P552V, KI555QV, I566V, and A574V were also statistically associated with CFM R .
Mosaic penA alleles were detected in 37.5% of CRO R isolates and in 13.8% of CRO S isolates. Only the mosaic penA 60 allele was significantly associated with CRO R ( Table 2). PenA substitutions F374V and A501T showed significantly higher frequencies in CRO R isolates than in CRO S isolates.
The metadata of four mosaic penA 60 isolates are listed in Table 3. Demographic and clinical information revealed that four patients were young (age range: 18-44), all of them had abnormal urinary discharge, and none reported previous history of gonorrhea or any antibiotic use in the past month. Three of four mosaic penA 60 isolates were NG-STAR genotype 233, whereas one was NG-STAR genotype 1143. Four penA 60 isolates had the same pattern of PenA substitutions, which contained A311V and T483S alterations, the key CRO R substitution. All penA 60 isolates have identical ponA, mtrR, 23S rRNA, gyrA, parC, 16S rRNA, and rpsE patterns and different PorB substitutions. Additional MICs and the molecular profiles of four isolates are summarized in Table 3.

mtrR Gene and Promoter
MtrR G45D and mtrR promoter -35A were significantly lower in CFM R isolates than in CFM S isolates. MtrR A40D and T86A were significantly associated with CFM R . No mtrR mutations was found to be associated with CRO R .

Characteristic Genotypes in N. gonorrhoeae SPT R Isolates
There was only one SPT R isolate identified. This SPT R isolate was the only strain that harbored 16S rRNA C1192U mutation in our dataset (Figure 1), which has earlier been reported to be associated with SPT R . The K26E substitution in RpsE was not detected. 2 | Molecular profiles associated with resistance to cefixime, ceftriaxone, and azithromycin in N. gonorrhoeae.

All isolates
Non-mosaic penA isolates All isolates Molecular markers

Clonal Distribution of N. gonorrhoeae ESC R Isolates by Phylogenetic Analysis
Phylogenetic analysis of nine genes was performed. Compared to a seven-gene phylogeny (Supplementary Figure 1), the inclusion of two SPT R genes (16S rRNA and rpsE) did not change the structure or resolution of the phylogeny. Cluster analysis results showed that a nine-gene phylogeny had 14 clusters, while a sevengene phylogeny had 16 clusters. Most ESC R strains were classified as one clade that had the mosaic penA alleles (Figure 1, clade A). NG-STAR ST-233, ST-348, and ST-90 belonged to clade A. Approximately 76% (16 of 21) of CFM R were included in clade A (Figure 1). CRO R appeared sporadically across the phylogeny. Four of the six ESC R that did not possess a mosaic penA allele harbored a penA 18 allele and was classified into clade C (Figure 2). The penA 18 allele consisted of the A501T and G542S double substitutions.
Multiple linear regression analysis revealed that the mosaic penA, PenA A501T/V, and PorB1b G213S/Y substitutions were strongly associated with increased MICs of CRO or CFM (Supplementary Table S4).

Phylogenetic Analysis of N. gonorrhoeae Isolates With MICs Above AZM ECOFF
Isolates with MICs above AZM ECOFF appeared sporadically across the phylogenetic tree (Figure 1) and were highly associated with the 23S rRNA A2059G mutation (Table 2 and Figure 1). All NG-STAR ST-202 isolates harbored the 23S rRNA A2059G mutation. Multiple linear regression analysis indicated that the 23S rRNA A2059G mutation was strongly associated with increased AZM MICs (Supplementary Table S5).

Analysis of N. gonorrhoeae CIP R Isolates
The 124 isolates included 123 CIP R and 1 CIP S . Substitutions at GyrA-91 and GyrA-95 were highly predictive of the resistant phenotype (Figure 3) Table S6).

DISCUSSION
A large proportion of N. gonorrhoeae isolates in Shanghai in 2017 exhibited resistance to ESCs. Approximately 19.4% of 366 N. gonorrhoeae isolates were CFM R (MICs > 0.125 mg/L), and 40.7% of the isolates had CFM MICs ≥ 0.125 mg/L. About 5.5% of the isolates were CRO R (MICs > 0.125 mg/L), and 18.0% of isolates had CRO MICs ≥ 0.125 mg/L. About 6.8% of the isolates had MICs above AZM ECOFF. One isolate was SPT R . N. gonorrhoeae CFM R isolates exhibited clonal distribution of one cluster containing mosaic penA alleles, whereas CRO R isolates appeared sporadically across the phylogeny.
The resistant percentages of N. gonorrhoeae isolates to CRO, CFM, or AZM in Shanghai exceeded the WHO cutoff of 5%, indicating a need to review recommended treatments (World Health Organization [WHO], 2012). Over 18.0% of N. gonorrhoeae isolates had CRO MICs ≥ 0.125 mg/L, higher than that reported in previous years in Shanghai and other places in China (Gu et al., 2014;Yin et al., 2018) as well as several other countries such as 0.1% in the United States in 2013 and 2014 , 1.8% in Canada in 2016 (Martin et al., 2019), and 10.7% in Japan in 2012-2013 (Hamasuna et al., 2015). The proportion of N. gonorrhoeae CFM R isolates (MICs > 0.125 mg/L, 19.4%) in Shanghai in 2017 was much higher than that in the United States (0.4-0.8% in 2013-2014) , Europe (1.7-2.0% in 2014-2015) (Cole et al., 2017), and Canada (0.3% in 2016) (Martin et al., 2019). These findings indicate that unlike those in the United States, European countries, Japan, and Canada, CRO and CFM may need to be reviewed as a treatment for gonorrhea in Shanghai. N. gonorrhoeae isolates in Shanghai remain susceptible to SPT (Yang et al., 2006), suggesting that SPT may have potential as a first-line therapy for the treatment of uncomplicated urogenital gonorrhea in Shanghai. SPT is available in China. However, SPT is not suitable for the treatment of pharyngeal gonorrhea, as its efficacy rate is approximately 80% (Moran and Levine, 1995). Furthermore, SPT R isolates have been reported in several countries such as the Netherlands, the Philippines, South Korea, and the United Kingdom (Unemo and Shafer, 2014). There is concern that drug resistance would be rapidly selected when SPT is introduced as a first-line monotherapy. Therefore, SPT should be considered as a first-line treatment in combination with CRO or AZM in Shanghai.

Neisseria gonorrhoeae ESC R Isolates Tend to Be Clonal
Previous studies have shown that N. gonorrhoeae CFM R isolates in Japan (Yahara et al., 2018) and ESC RS isolates in the United States (Grad et al., , 2016, Europe (Chisholm et al., 2013), and Canada (Demczuk et al., 2015) are predominantly clonal and associated with the mosaic penA allele. We also found that N. gonorrhoeae ESC R isolates were predominantly clonal in this study. In addition, we observed that ESC R isolates without the penA mosaic alleles were distributed sporadically across the phylogenetic tree, which is also concordant with a previous report (Grad et al., 2016). In our study, of the six ESC R isolates that did not possess the mosaic penA allele, four contained the penA 18 allele that included the PenA A501T and G542S double substitutions. However, ESC RS lineages in Canada were associated with non-mosaic penA 12 and 13 alleles (Demczuk et al., 2015), while ESC RS isolates with non-mosaic penA reported in the United States have sporadically emerged even in the penA gene phylogeny (Grad et al., 2016). The sample size of N. gonorrhoeae isolates in this study could be expanded, and whole-genome sequencing should be examined to further confirm this difference.
Mosaic penA Alleles Are Associated With N. gonorrhoeae ESC R and NG-STAR Clusters Mosaic penA alleles have been associated with N. gonorrhoeae ESC RS (Ameyama et al., 2002;Lee et al., 2010). We observed that ESC R is highly associated with mosaic penA 10, whereas reports in the United States and Canada indicated that N. gonorrhoeae ESC RS is highly associated with mosaic penA 34 (Grad et al., , 2016Demczuk et al., 2015). All of the N. gonorrhoeae isolates with an NG-STAR ST-348 genotype (n = 5) contained the mosaic penA 10 allele. Several mosaic penA alleles (penA 60, penA 71, and penA 34) are associated with ESC R in this study. penA 60 is significantly associated with both CRO R and CFM R and occurs in a single cluster; thus, it is of great concern when it spreads. None of the carriers of the penA 60 isolates reported a previous history of gonorrhea or any antibiotic use in the past month, which indicates that they were recently infected with penA 60 ESC R strains. It is important to monitor the clonal expansion of penA 60 ESC R strains to contain its spread. The reported CRO-resistant cluster FC428 has a mosaic penA 60 genotype with a NG-STAR sequence type ST-233 (Lee et al., 2019). Three of the four penA 60 N. gonorrhoeae isolates also have an NG-STAR ST-233. Links between the penA 60 isolates in this study and FC428 strains remain to be elucidated.

Novel PorB1b Substitutions Associated With N. gonorrhoeae ESC R
In this study, we found that in contrast to CFM R , CRO R is apparently associated with PorB1b substitutions other than mosaic penA or PenA substitutions. This is concordant with the results of a previous study that CRO is more severely affected by PorB1b than CFM (Unemo and Shafer, 2014), suggesting that either CFM does not readily diffuse into the periplasm through PorB1b or such diffusion is not altered by the porB determinant (Unemo and Shafer, 2014). To our knowledge, our study is the first to report that PorB1b substitutions T87A, T89S, S213S/Y, Q214L, and G259A are associated with CRO R . Similar to a previous report, although certain mutations in porB can contribute to N. gonorrhoeae resistance, most mutations in porB do not (Goire et al., 2014). In vitro selection by introducing porB mutations into CRO S isolates should be considered in the future to confirm the role of these mutations in the formation of CRO R (Johnson et al., 2014). Previous studies have reported substitutions at amino acid positions 120 and 121 in putative loop 3 of PorB1b, which reduce the permeability of ESCs (Olesky et al., 2002). Interestingly, none of the substitutions detected in the present study are situated in any loops of PorB1b, and whether these substitutions could perturb protein structure remains unknown. Electrophysiological and biochemical studies of PorB1b proteins to reveal the mechanism of CRO R conferred by these substitutions are warranted.

ParC S88P Substitution Clustered in ESC R Clade
We noticed that among the 14 isolates with ParC S88P substitution, nine were clustered in the ESC R clade (clade A), suggesting that this substitution may be associated with ESC R .
Intriguingly, it was reported that various MDR bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extendedspectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae, and ESBL-producing Escherichia coli were demonstrated to have been selected by favorable fitness balance associated with highlevel resistance to fluoroquinolones, principally attained by the mutations of some serine residues in gyrA and parC/grlA (Fuzi et al., 2017(Fuzi et al., , 2020. The association of the ParC S88P substitution and that of other QRDR serine replacements with fitness gain, the promotion of particular clades, and the acquisition of the MDR phenotype warrant further investigation.

Limitations
This study only investigated a small percentage of N. gonorrhoeae isolates in Shanghai, with a total of 5,711 reported cases in this city in 2017. However, it is representative of the institution where the isolates were collected. A study with a larger sample size is required to extrapolate a broader strain distribution and to provide convincing evidence for the clonal distribution of ESC R with mosaic penA alleles and the sporadic distribution of ESC R with non-mosaic penA alleles. Specimens were obtained only from male patients, which may cause a higher proportion of CRO-resistant isolates, as reported by a Chinese national surveillance (Yin et al., 2018). Transmission of gonorrhea via different behaviors may result in infection of other mucosal sites, and isolates from other sites may exhibit different AMR phenotypes and genotypes. In the future, whole-genome sequencing should be considered to examine the population structure in Shanghai N. gonorrhoeae isolates, which would provide a significantly higher resolution for phylogenetic reconstruction.

CONCLUSION
This study observed a high percentage of N. gonorrhoeae isolates with reduced susceptibility to ESCs in Shanghai in 2017. Phylogenetic analysis of resistance determinants revealed that CFM R isolates tend to be clonal. Mosaic penA alleles and certain substitutions in PenA and PorB1b are associated with N. gonorrhoeae ESC R . CRO and CFM may need to be reviewed as treatment for gonorrhea in Shanghai. Monitoring clonal expansion and development of novel antimicrobials for gonorrhea treatment are urgently needed.

DATA AVAILABILITY STATEMENT
The sequence data generated for this study has been submitted to GenBank and accession numbers can be found in the article.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Shanghai Skin Disease Hospital. The patients/participants provided their written informed consent to participate in this study.