Emergence of Neisseria gonorrhoeae Clone with Reduced Susceptibility to Sitafloxacin in China: An In Vitro and Genomic Study

Drug-resistant Neisseria gonorrhoeae poses an urgent threat to public health. Recently, sitafloxacin, a new-generation fluoroquinolone, has shown high in vitro activity against drug-resistant N. gonorrhoeae. However, data on its effectiveness in clinical isolates remains limited. In this study, we collected 507 N. gonorrhoeae isolates from 21 hospitals in Shanghai, China, during 2020 and 2021. Antimicrobial susceptibility testing revealed that sitafloxacin minimum inhibitory concentrations (MICs) exhibited a bimodal distribution, ranging from <0.004 to 2 mg/L. The MIC50 and MIC90 for sitafloxacin were 0.125 mg/L and 0.5 mg/L, respectively, which are 32 and 16 times lower than those for ciprofloxacin (4 mg/L and 8 mg/L, respectively). Sitafloxacin demonstrated high in vitro activity against isolates resistant to either ceftriaxone, azithromycin, or both. Notably, among the isolates with reduced sitafloxacin susceptibility (MIC ≥ MIC90), 83.7% (36/43) were identified as sequence type (ST) 8123. Further phylogenetic analysis showed that ST8123 has evolved into two subclades, designated as subclade-I and subclade-II. A majority of the isolates (80%, 36/45) within subclade-I exhibited reduced susceptibility to sitafloxacin. In contrast, all isolates from subclade-II were found to be susceptible to sitafloxacin. Subsequent genomic investigations revealed that the GyrA-S91F, D95Y, and ParC-S87N mutations, which were exclusively found in ST8123 subclade-I, might be linked to reduced sitafloxacin susceptibility. Our study reveals that sitafloxacin is a promising antibiotic for combating drug-resistant N. gonorrhoeae. However, caution is advised in the clinical application of sitafloxacin for treating N. gonorrhoeae infections due to the emergence of a clone exhibiting reduced susceptibility.


Introduction
Gonorrhea remains a major public health challenge, with an estimated 82 million cases reported worldwide in 2020 among adults [1].The rapid emergence and spread of drug-resistant Neisseria gonorrhoeae have exacerbated the situation, prompting the WHO to classify drug-resistant N. gonorrhoeae as a high-priority pathogen [2].Currently, ceftriaxone is the last-resort antibiotic for empirical gonorrhea treatment in most countries, with dual therapy including ceftriaxone and azithromycin recommended for co-infections with pathogens like Chlamydia trachomatis and Ureaplasma urealyticum [3,4].A global study of antimicrobial resistance in N. gonorrhoeae collected in 2017-2018 found that azithromycin resistance was increasing, and resistance to ceftriaxone and cefixime continued to emerge in multiple counties [5].A recent study showed that the prevalence of ceftriaxone-resistant N. gonorrhoeae approximately tripled in China during 2017−2022 [6].The rising incidence of resistance to ceftriaxone and azithromycin [7,8] necessitates the exploration of new drugs and treatment strategies to combat drug-resistant N. gonorrhoeae.
Earlier investigations have shown that mutations in the gyrA and parC genes, specifically, GyrA-S91F, D95G/A/N, and ParC-S87R/N, D86N, S88P, may contribute to the reduced susceptibility of sitafloxacin; however, all these mutations only increase the MICs of sitafloxacin in N. gonorrhoeae to less than 0.5 mg/L [15,16].Our understanding of gyrA and parC mutations that confer sitafloxacin resistance is still limited.Furthermore, the knowledge of sitafloxacin-resistant clones is also lacking.
In this study, we collected 507 N. gonorrhoeae isolates from 21 hospitals across different regions of Shanghai, China, during 2020 and 2021.Our genomic and phylogenomic analyses identified a clone with reduced susceptibility to sitafloxacin, belonging to the multi-locus sequence type (ST) 8123, within this collection.This reduced susceptibility was potentially determined by the GyrA-S91F, D95Y, and ParC-S87N mutations.Further analysis, incorporating public genome data, revealed that ST8123 strains harboring the GyrA-S91F, D95Y, and ParC-S87N mutations had already appeared in Vietnam in 2012, China in 2013, and the UK in 2014.These findings highlight the early dissemination of a sitafloxacin susceptibility reduced N. gonorrhoeae clone prior to the widespread adoption of sitafloxacin, underscoring the urgent need for continuous surveillance of these N. gonorrhoeae strains in clinical settings.

The MIC Distributions of Sitafloxacin and Ciprofloxacin in 507 N. gonorrhoeae Clinical Isolates
The MICs for ciprofloxacin against the 507 N. gonorrhoeae clinical isolates varied widely, from <0.004 to >8 mg/L, with a resistance rate of 99.4% (504/507).In contrast, sitafloxacin MICs ranged from <0.004 to 2 mg/L, with MIC 50 and MIC 90 values of 0.125 mg/L and 0.5 mg/L, respectively.These values are 32-and 16-fold lower than those for ciprofloxacin (4 mg/L and 8 mg/L, respectively), suggesting that sitafloxacin exhibits superior in vitro activity against N. gonorrhoeae.A total of 43 isolates had sitafloxacin MIC values equal to or greater than MIC 90 and were defined as sitafloxacin susceptibility reduced strains.A notable bimodal distribution was observed in the sitafloxacin MICs, with peaks at 0.125 mg/L and 1 mg/L (Figure 1), indicating a subpopulation with reduced susceptibility to sitafloxacin within our collection.Given that ceftriaxone and azithromycin are the primary antibiotics for N. gonorrhoeae treatment, we also assessed sitafloxacin's effectiveness against isolates resistant to ceftriaxone (n = 57), azithromycin (n = 95), and both (dualresistant) (n = 11).The MIC 50 /MIC 90 values of sitafloxacin against these resistant isolates were 0.125/0.5 mg/L, 0.125/0.25 mg/L, and 0.125/0.25 mg/L, respectively, mirroring those of the overall population.Nonetheless, the sitafloxacin MICs for these resistant isolates were higher compared to that of the population (Table 1), suggesting potential cross-resistance to sitafloxacin, ceftriaxone, and azithromycin.isolates were higher compared to that of the population (Table 1), suggesting potential cross-resistance to sitafloxacin, ceftriaxone, and azithromycin.

N. gonorrhoeae Strains with Reduced Sitafloxacin Susceptibility Were Isolated from Multiple Regions in Shanghai
To explore the geographical spread of N. gonorrhoeae strains with reduced susceptibility to sitafloxacin, we analyzed the 507 strains based on their isolation locations.Figure 2 illustrates that strains exhibiting reduced susceptibility were found across various regions, with rates fluctuating between 0% and 19.8%.Notably, no susceptibility reduced strain was detected in Baoshan, the northern region of Shanghai.Meanwhile, Jiading, another northern area, had four such strains.A significant proportion of strains with reduced susceptibility were identified in the southern regions (Songjiang [n = 8], Jingshan [n = 3], and Pudong [n = 3]) and the downtown area (n = 25).This indicates that sitafloxacin susceptibility reduced N. gonorrhoeae strains are widely disseminated across Shanghai.

N. gonorrhoeae Strains with Reduced Sitafloxacin Susceptibility Were Isolated from Multiple Regions in Shanghai
To explore the geographical spread of N. gonorrhoeae strains with reduced susceptibility to sitafloxacin, we analyzed the 507 strains based on their isolation locations.Figure 2 illustrates that strains exhibiting reduced susceptibility were found across various regions, with rates fluctuating between 0% and 19.8%.Notably, no susceptibility reduced strain was detected in Baoshan, the northern region of Shanghai.Meanwhile, Jiading, another northern area, had four such strains.A significant proportion of strains with reduced susceptibility were identified in the southern regions (Songjiang [n = 8], Jingshan [n = 3], and Pudong [n = 3]) and the downtown area (n = 25).This indicates that sitafloxacin susceptibility reduced N. gonorrhoeae strains are widely disseminated across Shanghai.

Most of the N. gonorrhoeae Strains with Reduced Susceptibility to Sitafloxacin Belonged to the ST8123 Clone
In our study, we sequenced all 507 N. gonorrhoeae isolates and constructed a minimum spanning tree (MST) based on the multi-locus sequence typing (MLST) (Figure 3).This MST underscored the genetic diversity within our collection, identifying 59 known sequence types (STs) and nine new STs.Predominant among these were ST7363 (n = 88) and ST8123 (n = 66), together representing 30.4% of all isolates.We also identified ST1901 (n = 21) and ST1903 (n = 9), known globally for their resistance to cefixime and ceftriaxone.Notably, a significant majority of the isolates with reduced susceptibility to sitafloxacin (83.7%, 36/43) were classified as ST8123, which exhibited MIC 50 /MIC 90 values of 0.5/2 mg/L, significantly higher than those found in ST7363, ST1901, and ST1903 (Table 2).The presence of reduced susceptibility in other minor STs, such as ST1580, ST1582, and ST14422, was also noted.The pronounced prevalence of reduced sitafloxacin susceptibility within ST8123 suggests a potential underlying mechanism contributing to this phenomenon.
Significantly, subclade-I was marked by a higher incidence of reduced sitafloxacin susceptibility.About 80% of the isolates (36/45) presented MICs > 0.25 mg/L, and 20% (9/45) had MICs between 0.125 mg/L and 0.25 mg/L, excluding strains without MIC data (n = 13).Conversely, in subclade-II, 85.7% of strains (18/21) had MICs within the 0.125 to 0.25 mg/L range, and 14.3% (3/21) exhibited MICs <0.125 mg/L, excluding strains without MIC data (n = 15).It is noteworthy that all subclade-I strains harbored the GyrA-S91F, D95Y, and ParC-S87N mutations, indicative of reduced susceptibility, except for one with a ParC-S87C mutation.On the other hand, subclade-II predominantly comprised strains with the GyrA-S91F, D95A, and ParC-S87R mutations or wild-type gyrA and parC sequences.Furthermore, the strains within subclade-I were genetically closer to each other, suggesting that this group, characterized by reduced sitafloxacin susceptibility, has recently emerged.These findings collectively underscore that the spread and prevalence of ST8123 subclade-I have significantly contributed to the development of sitafloxacin susceptibility reduced N. gonorrhoeae in the population.

quences.
Furthermore, the strains within subclade-I were genetically closer to each other, suggesting that this group, characterized by reduced sitafloxacin susceptibility, has recently emerged.These findings collectively underscore that the spread and prevalence of ST8123 subclade-I have significantly contributed to the development of sitafloxacin susceptibility reduced N. gonorrhoeae in the population.

Discussion
The rising prevalence of drug-resistant N. gonorrhoeae significantly complicates gonorrhea management and narrows the therapeutic options available [17].Sitafloxacin, known for its broad antibacterial spectrum, emerges as a promising treatment alternative.As of 2017, sitafloxacin has been recommended in Japan as a primary treatment for chlamydial urethritis and a secondary option for non-chlamydial, non-gonococcal urethritis [18].Moreover, since 2019, China has approved sitafloxacin for treating urinary tract infections and pneumonia when caused by susceptible strains [19].Recent studies, including ours, have confirmed sitafloxacin's high in vitro effectiveness against drug-resistant strains of N. gonorrhoeae.Our analysis across 507 N. gonorrhoeae isolates from Shanghai, China, has pinpointed a sitafloxacin susceptibility reduced clone, already spreading across multiple countries before sitafloxacin became widely used.This finding stresses the critical need for vigilant surveillance of sitafloxacin susceptibility in clinical settings.
In our investigation, the MIC 50 and MIC 90 values of sitafloxacin for N. gonorrhoeae, estimated at 0.125 mg/L and 0.5 mg/L, respectively, are consistent with reports from Japan and Sweden [16,20], confirming sitafloxacin's potent in vitro activity against N. gonorrhoeae within China as well.Differently from other studies, however, our research revealed a bimodal distribution in sitafloxacin MICs, signifying a notable number of isolates with MICs exceeding the MIC 90 .This suggests the existence of a subpopulation with reduced susceptibility to sitafloxacin.Our subsequent phylogenomic analysis accurately identified this subpopulation as belonging to a specific subclade of ST8123 N. gonorrhoeae, which is prevalent across Shanghai.Notably, although reports of ST8123 on an international scale are rare, it is the most common sequence type in Shenzhen, a major southern coastal city in China, from 2014 to 2018 [21].This finding implies the spread of a sitafloxacin susceptibility reduced N. gonorrhoeae clone within China, especially in its foremost coastal cities.
Earlier investigations have pinpointed mutations in the gyrA and parC genes, specifically, GyrA-S91F, D95G/A/N, and ParC-S87R/N, D86N, S88P, as primary contributors to the reduced susceptibility of sitafloxacin, albeit maintaining MICs below 0.5 mg/L [15,16].Consistent with these findings, 89.3% of the isolates in our study harbored these mutations, aligning with the observed MICs under 0.5 mg/L.Notably, our research goes a step further by uncovering a novel mutation combination: GyrA-S91F, D95Y, and ParC-S87N.This combination was associated with reduced susceptibility to sitafloxacin, reflected by MICs exceeding 1 mg/L.This suggests that the GyrA-D95Y mutation might significantly impair sitafloxacin's binding efficacy, underscoring a potentially critical mechanism for diminished drug effectiveness.
Pharmacokinetic studies have shown that a single oral dose of sitafloxacin, at 100 mg or 200 mg, can attain maximum serum concentrations of 1.62 mg/L and 2.73 mg/L, respectively, with the drug's mean half-life ranging between 5.80 to 6.28 h [22].These pharmacokinetic properties suggest that a 200 mg dose of sitafloxacin could maintain serum concentrations above 0.5 mg/L for approximately 12 h.This duration and concentration level could be sufficient to effectively target gonococcal strains exhibiting MICs under 0.5 mg/L.Nonetheless, strains harboring the GyrA-S91F, D95Y, and ParC-S87N mutations, which indicate reduced susceptibility, might necessitate higher or repeated dosing of sitafloxacin for successful eradication.
MLST clones such as ST7363, ST1901, and ST1903, known for their resistance to cefixime and ceftriaxone, have been identified globally [21,[23][24][25][26] and linked to treatment failures, particularly concerning dual therapy with ceftriaxone and azithromycin in the UK [27].In our analysis, these sequence types were present, displaying sitafloxacin MICs within the ranges of 0.015 to 0.25 mg/L for ST7363 and ST1901, and 0.03 to 0.125 mg/L for ST1903, all of which are beneath the achievable serum concentrations post-administration.This positions sitafloxacin as a potentially effective alternative for managing infections resistant to or in cases of allergy to ceftriaxone.Of note, the close phylogenetic relationship between ST7363 and ST8123 raises concerns over the potential development of dual resistance under the selective pressure of widespread sitafloxacin use.Thus, ongoing surveillance for drug-resistant N. gonorrhoeae remains essential to thwart the rise and dissemination of multi-drug resistant strains.

N. gonorrhoeae Strains Isolation
A total of 507 N. gonorrhoeae isolates were consecutively collected from patients who attended STD clinics and dermatology departments across eight different regions of Shanghai within the Shanghai Gonococcal Resistance Surveillance Programme (SH-GRSP) during 2020 and 2021.Neither the gender nor the patients' symptomatic state was presented in this study.The isolates were cultured on Thayer-Martin (TM) agar (Comagal, Shanghai, China) and incubated at 37 • C in a humidified atmosphere containing 5% CO 2 .For preservation, the isolates were suspended in tryptic soy broth (BD, Franklin Lakes, NJ, USA) with glycerol and stored at −80 • C until further use.

Antimicrobial Susceptibility Testing
Ciprofloxacin (Sigma, Darmstadt, Germany), sitafloxacin (MeilunBio, Dalian, China), ceftriaxone (China National Institutes for Drug control, Beijing, China), and azithromycin (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) powders were bought from manufacturers.The minimum inhibitory concentrations (MICs) for the isolates against these antibiotics were determined via the agar dilution method, following the Clinical and Laboratory Standards Institute (CLSI) guidelines (www.clsi.org,accessed on 1 July 2023).For each testing run, the N. gonorrhoeae reference strain ATCC 49226 served as a quality control.Resistance breakpoints for ciprofloxacin, ceftriaxone, and azithromycin were established at ≥1 mg/L, ≥0.125 mg/L, and ≥1 mg/L, respectively, in line with the WHO Global Gonococcal Antimicrobial Surveillance Programme (GASP).As there is currently no defined resistance breakpoint for sitafloxacin, MICs equal to or greater than the MIC 90 were considered indicative of reduced susceptibility.

Statistical Analysis
Rank Sum Tests in R were used to test the difference of MICs between ceftriaxoneresistant isolates, ceftriaxone-susceptible isolates, azithromycin-resistant isolates, azithromycinsusceptible isolates, ceftriaxone and azithromycin-resistant isolates, and all the isolates.Rank Sum Tests were used to test the difference in MICs between different ST clones.A p-value less than 0.05 was considered statistically significant.

Conclusions
In conclusion, our study contributes significant insights into the effectiveness of sitafloxacin against N. gonorrhoeae clinical isolates within China.While sitafloxacin demonstrated high efficacy against the majority of tested isolates, our analysis has unveiled the emergence and spread of a ST8123 subclade exhibiting reduced susceptibility to sitafloxacin.These findings underscore the need for cautious application of sitafloxacin in the clinical treatment of gonorrhea, highlighting the importance of ongoing surveillance of drugresistant gonococcal strains.

Figure 1 .
Figure 1.MIC distribution of sitafloxacin (green) and ciprofloxacin (red) against 507 N. gonorrhoeae isolates.MIC50, MIC where 50% of isolates inhibited.MIC90, MIC where 90% of isolates inhibited.MIC50 and MIC90 of each drug were marked by the dashed lines.The number of isolates is indicated on the top of the bars.

Figure 1 .
Figure 1.MIC distribution of sitafloxacin (green) and ciprofloxacin (red) against 507 N. gonorrhoeae isolates.MIC 50 , MIC where 50% of isolates inhibited.MIC 90 , MIC where 90% of isolates inhibited.MIC 50 and MIC 90 of each drug were marked by the dashed lines.The number of isolates is indicated on the top of the bars.

Figure 3 .
Figure 3. Minimum spanning tree of 507 N. gonorrhoeae based on the allelic profiles of the 68 MLST STs isolated.The ST labels in red are major clones of N. gonorrhoeae.

Figure 3 .Table 2 .
Figure 3. Minimum spanning tree of 507 N. gonorrhoeae based on the allelic profiles of the 68 MLST STs isolated.The ST labels in red are major clones of N. gonorrhoeae.Table 2. The sitafloxacin MIC ranges of isolates with different STs.

Figure 4 .
Figure 4. Distribution of MIC values of sitafloxacin in 507 N. gonorrhoeae isolates with different combinations of GyrA and ParC mutations.Only combinations observed in at least 5 isolates are shown.Dashed horizontal lines on the violin plots mark the MIC50 and MIC90 for sitafloxacin.Colored dots inside violins represent different MIC ranges.Barplots on the top show the number of isolates with each mutation combination.Combinations of GyrA and ParC mutations were indicated in the grid below by black circles connected vertically.Horizontal thick grey lines connect combinations of mechanisms that share an individual determinant.Barplots on the right show the number of isolates with each mutation.

Figure 4 .
Figure 4. Distribution of MIC values of sitafloxacin in 507 N. gonorrhoeae isolates with different combinations of GyrA and ParC mutations.Only combinations observed in at least 5 isolates are shown.Dashed horizontal lines on the violin plots mark the MIC50 and MIC90 for sitafloxacin.Colored dots inside violins represent different MIC ranges.Barplots on the top show the number of isolates with each mutation combination.Combinations of GyrA and ParC mutations were indicated in the grid below by black circles connected vertically.Horizontal thick grey lines connect combinations of mechanisms that share an individual determinant.Barplots on the right show the number of isolates with each mutation.

Figure 5 . 5 .
Figure 5. Phylogenic analysis of globally distributed 94 ST8123 N. gonorrhoeae strains.Bars from the inner to outer are isolation years, countries, MIC group of sitafloxacin, GyrA mutations, and ParC Figure 5. Phylogenic analysis of globally distributed 94 ST8123 N. gonorrhoeae strains.Bars from the inner to outer are isolation years, countries, MIC group of sitafloxacin, GyrA mutations, and ParC mutations, respectively.Names in red are isolates sequenced in this study (n = 66), while others are from public databases (n = 28).

Table 1 .
The sitafloxacin MIC ranges of isolates resistant or susceptible to ceftriaxone or (and) azithromycin.

Table 1 .
The sitafloxacin MIC ranges of isolates resistant or susceptible to ceftriaxone or (and) azithromycin.

Table 2 .
The sitafloxacin MIC ranges of isolates with different STs.