First Study on Antimicrobial Susceptibility and Molecular Epidemiology of Neisseria gonorrhoeae Isolates from Infertile Women.

Gonorrhea poses a major threat to female reproductive health globally. 1 According to the World Health Organization (WHO), there were an estimated 82.4 million new cases of gonorrhea globally in 2020, 2 with a prevalence of 0.9% among women. 3 While gonorrhea in women can manifest as cervicitis or urethritis, nearly 50% are asymptomatic. 4 Without effective treatment, Neisseria gonorrhoeae can ascend from the ectocervix to the fallopian tubes, thereby causing pelvic inflammatory disease, which may lead to serious reproductive complications such as infertility and ectopic pregnancy. 5–8 Effective treatment of gonorrhea is increasingly complicated by the growing antimicrobial resistance in N. gonorrhoeae . 9 Owing to the widespread resistance in N. gonorrhoeae to all previously recommended antibiotics and the absence of new therapeutic alternatives, the cephalosporin ceftriaxone now serves as the cornerstone for first-line gonorrhea treatment in most countries. 10 Unfortunately, the utility of this option is now being compromised by growing documentation of ceftriaxone-resistant N. gonorrhoeae strains on a global scale. 11–15 Moreover, clinical cases of treatment failures with ceftriaxone monotherapy and ceftriaxone–azithromycin dual therapy have been reported in several countries. 16–20 Collectively, these threats are raising concerns that gonorrhea may evolve into an prolonged or even incurable infection, thereby heightening the risk of long-term reproductive complications such as female infertility. 21 Antimicrobial resistance surveillance in N. gonorrhoeae is essential to guide effective recommendations for gonorrhea treatment. 22 Although gonorrhea prevalence is considerably higher among infertile populations than among the general population (global estimate: 2.2% vs 0.8%, respectively), 3 , 23 data regarding the antimicrobial resistance profiles of N. gonorrhoeae isolates from infertile populations remain scarce. This study aimed to characterize the antimicrobial resistance profiles and analyze the molecular epidemiology of clinical isolates of N. gonorrhoeae obtained from infertile women in China between 2018 and 2022. The clinical characteristics of infertile women with gonorrhea are also described.

Between January 2018 and September 2022, a total of 145,168 infertile women screened for  N. gonorrhoeae , and 106 (0.07%) infertile women had culture-verified gonorrhea (annual distribution: 30/38,527 in 2018, 26/34,132 in 2019, 17/25,999 in 2020, 20/27,127 in 2021, and 13/19,383 in 2022). The median patient age was 30 years (IQR 26–34 years) ( Table 1 ). Among them, 87 (82.1%) were asymptomatic, whereas 19 (17.9%) showed symptoms of gonorrhea, such as mucopurulent cervicitis or vaginal discharge. The primary cause of infertility was tubal-factor infertility (49.1%), followed by mixed or unexplained causes (31.1%) and male-factor infertility (7.5%). Table 1 Clinical Characteristics of Infertile Women with Gonorrhea (n=106) Characteristic Age (years), n (%)  <30 51 (48.1)  30–34 33 (31.1)  35–37 6 (5.7)  ≥38 16 (15.1) Age (years), median (IQR) 30 (26–34) Symptoms of gonorrhea, n (%)  Mucopurulent cervicitis/vaginal discharge 19 (17.9)  Asymptomatic 87 (82.1) Cause of infertility, n (%)  Tubal factor 52 (49.1)  Mixed and unexplained infertility 33 (31.1)  Male factor 8 (7.5)  Endometriosis 7 (6.6)  Ovarian factor 6 (5.7) Year of isolation, n (%)  2018 30 (28.3)  2019 26 (24.5)  2020 17 (16.0)  2021 20 (18.9)  2022 (Jan–Sep) 13 (12.3) Note : IQR, 25–75% interquartile range. Clinical Characteristics of Infertile Women with Gonorrhea (n=106) Note : IQR, 25–75% interquartile range. Table 2 presents antimicrobial susceptibility profiles of the 106 N. gonorrhoeae isolates from 106 infertile women (one isolate per patient). For ceftriaxone, the observed MIC range was 0.008–0.25 mg/L, with the MIC 50 and MIC 90 recorded at 0.03 mg/L and 0.125 mg/L, respectively. According to resistance criteria, 2.8% (3/106) of the isolates demonstrated resistance to ceftriaxone (MIC >0.125 mg/L), while 16.0% (17/106) exhibited decreased susceptibility to ceftriaxone (defined as MIC ≥0.125 mg/L). Regarding azithromycin, the MIC distribution ranged from 0.06 to >8 mg/L, with MIC 50 and MIC 90 values of 0.5 mg/L and 1 mg/L. Resistance to azithromycin (MIC ≥1 mg/L) occurred in 19.8% (21/106) of isolates. Notably, 2.8% (3/106) of the isolates displayed a combined phenotype of decreased susceptibility to ceftriaxone and resistance to azithromycin. In addition, widespread resistance to other antibiotics was observed, with all isolates (100%) being resistant to ciprofloxacin, 98.1% to tetracycline, and 79.3% to penicillin, of which 32.7% were lactamase-producing N. gonorrhoeae . Consequently, resistance to three or more antibiotics was observed in 83 isolates (78.3%): one isolate was resistant to five antibiotics, 17 were resistant to four antibiotics, and 65 to three antibiotics. All isolates were susceptible to spectinomycin. Table 2 MICs of 106 Neisseria gonorrhoeae Isolates from Infertile Women to Six Antibiotics Antibiotic Range of MICs (mg/L) MIC 50 (mg/L) MIC 90 (mg/L) Distribution (%) S DS R Ceftriaxone ≤0.008–0.25 0.03 0.125 84.0 13.2 2.8 Azithromycin 0.06–>8 0.5 1 80.2 NA 19.8 Ciprofloxacin 1–>8 >8 >8 0 NA 100 Tetracycline 0.5–>16 8 >16 1.9 NA 98.1 Penicillin 0.06–>8 4 >8 0.9 19.8 79.3 Spectinomycin 8–32 16 16 100 NA 0 Note : MIC 50/90 , minimum concentration inhibiting 50%/90% of isolates. Abbreviations : S, susceptible; DS, decreased susceptibility; R, resistant; NA, not applicable. MICs of 106 Neisseria gonorrhoeae Isolates from Infertile Women to Six Antibiotics Note : MIC 50/90 , minimum concentration inhibiting 50%/90% of isolates. Abbreviations : S, susceptible; DS, decreased susceptibility; R, resistant; NA, not applicable. Among the 106  N. gonorrhoeae  isolates, 83 distinct STs were identified by NG-MAST analysis, including 36 new STs that had not been previously recorded. The most prevalent STs were ST5061 (six isolates), followed by ST4539 (five isolates), with ST1791, ST11123, and ST17105 each comprising three isolates. In addition, eight STs contained two isolates, and 70 STs were represented by a single isolate ( Table 3 ). The 17 isolates with decreased susceptibility to ceftriaxone were classified into 17 distinct STs, including six new STs. Similarly, the 21 azithromycin-resistant isolates were classified into 20 distinct STs (including 11 new STs), with only ST5061 containing two isolates. Overall, molecular typing revealed no clear association between specific STs and resistance patterns. Table 3 NG-MAST ST and Ceftriaxone/Azithromycin MIC for 106 Neisseria gonorrhoeae Isolates from Infertile Women ST Number of Isolates Number of Isolates with Ceftriaxone MIC (mg/L) Number of Isolates with Azithromycin MIC (mg/L) 0.25 0.125 0.06 0.03 0.015 ≤0.008 >8 4 2 1 0.5 0.25 0.125 5061 6 0 0 1 3 2 0 0 0 0 3 0 3 0 4539 5 0 1 2 1 1 0 0 0 0 0 0 4 1 1791 3 0 1 1 0 0 1 0 0 0 0 2 1 0 11,123 3 0 0 0 2 0 1 0 0 0 0 1 1 1 17,105 3 0 0 0 2 1 0 0 0 1 0 2 0 0 STs represented by two isolates (n=8) 16 0 2 4 7 1 2 0 0 2 2 5 6 1 Unique ST (n=70) 70 3 10 15 22 14 6 2 2 2 7 23 26 8 Total no. of isolates 106 3 14 23 37 19 10 2 2 5 12 33 41 11 Abbreviations : NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; ST, sequence type; MIC, minimum inhibitory concentration. NG-MAST ST and Ceftriaxone/Azithromycin MIC for 106 Neisseria gonorrhoeae Isolates from Infertile Women Abbreviations : NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; ST, sequence type; MIC, minimum inhibitory concentration.

From January 2018 to September 2022, infertile women seeking reproductive treatment at the Reproductive and Genetic Hospital of CITIC-Xiangya were enrolled. Infertility was clinically defined as the absence of conception after regular unprotected sexual intercourse over a period of at least 12 months. 24 All infertile women were routinely screened for sexually transmitted infections (STIs), including gonorrhea. In brief, endocervical swabs from infertile women were inoculated into a selective gonococcal agar medium and cultured at 35–37°C in 4–6% CO 2 for 24 h. Presumptive gonococcal colonies were initially characterized using Gram staining and oxidase tests. Definitive confirmation was subsequently achieved using the VITEK NH system (BioMerieux). Verified isolates were preserved in Microbank TM (Pro-Lab Diagnostics, Canada) and stored at –80°C until use. The United States Pharmacopeia (USP) reference standard powders of penicillin, ceftriaxone, tetracycline, azithromycin, ciprofloxacin, and spectinomycin were provided by the China Gonococcal Resistance Surveillance Program (China-GRSP). Antimicrobial susceptibility testing (agar dilution method) for  N. gonorrhoeae  isolates was performed according to WHO guidelines. 25 The MIC was determined as the minimum concentration at which no visible growth was observed. Interpretative criteria were based on EUCAST v. 13.1, except for decreased susceptibility to ceftriaxone and resistance to azithromycin, which were adopted from WHO criteria. 25 According to EUCAST, ceftriaxone resistance was defined as MIC >0.125 mg/L. In addition, following WHO criteria for surveillance purposes, decreased susceptibility to ceftriaxone was defined as MIC ≥0.125 mg/L (including resistant isolates). The specific breakpoints (in mg/L) for other antibiotics were as follows: azithromycin resistant (≥1), ciprofloxacin resistant (>0.06), tetracycline resistant (>1), penicillin resistant (>1), and spectinomycin resistant (>64). Reference strains (WHO P, G, and K) were included in each batch for quality control. β-Lactamase-producing N. gonorrhoeae (PPNG) was identified using the nitrocefin paper test (Oxoid, UK). NG-MAST was employed for the molecular typing of N. gonorrhoeae isolates, which defines the sequence type (ST) number according to specific combinations of porB and tbpB alleles. In brief, genomic DNA was extracted from the isolates using a commercial kit (TAKARA Bio). Amplification and sequencing of porB and tbpB allele was conducted using previously published primers and conditions. 26 STs of isolates were determined by submitting the sequences of porB and tbpB allele into the NG-MAST database v. 2.0 ( http://pubmlst.org/neisseria/ ).

In summary, this study provides the first characterization of antimicrobial resistance in N. gonorrhoeae isolates obtained from infertile Chinese women. We found a lower prevalence of resistance to ceftriaxone (2.8%), but a high prevalence of resistance to azithromycin (19.8%) and decreased susceptibility to ceftriaxone (16%), providing compelling evidence for recommending ceftriaxone monotherapy over ceftriaxone–azithromycin dual therapy for gonorrhea treatment in an infertile female population. These findings underscore the need for sustained gonococcal antimicrobial resistance surveillance targeting the general female population to curb the transmission of N. gonorrhoeae -resistant strains and protect female reproductive health.

The updated WHO Bacterial Priority Pathogens List (2024) retained antibiotic-resistant  N. gonorrhoeae  in the “high” priority group, underscoring its global public-health urgency. 27 The surveillance of antimicrobial resistance in N. gonorrhoeae is essential for curbing transmission of resistant strains and reducing long-term gonococcal complications. 5 To our knowledge, this study represents the first characterization of antimicrobial resistance in N. gonorrhoeae isolates from infertile women in China, conducted as part of the China-GRSP (2019–2022). The Western Pacific Region, including China, faces persistent challenges with antimicrobial resistance to N. gonorrhoeae , particularly to ceftriaxone. 11 , 28–31 During the study period, a total of 106  N. gonorrhoeae  isolates were obtained from 145,168 infertile women screened, and antimicrobial susceptibility testing was performed on all isolates. The prevalence of ceftriaxone resistance was 2.8%, remaining below the WHO 5% threshold for first-line empirical therapy, 1 , 10 and supporting the continued application of ceftriaxone monotherapy (1 g intravenous or intramuscular) in this population. Besides, this study observed a 16% rate of decreased susceptibility to ceftriaxone among  N. gonorrhoeae  isolates from infertile women, which underscores the need for sustained resistance surveillance in this specific population. Following the emergence of N. gonorrhoeae  strains with high-level ceftriaxone resistance a decade ago, 15 , 32 ceftriaxone–azithromycin dual therapy has become the first-line treatment for gonorrhea in many countries. 10 , 33 , 34 However, we found azithromycin resistance in 19.8% of the isolates, with 2.8% exhibiting resistance to azithromycin and decreased susceptibility to ceftriaxone, suggesting that ceftriaxone–azithromycin dual therapy is unsuitable for gonorrhea treatment in this population. Indeed, in line with the rapidly growing gonococcal azithromycin resistance in recent years, 35 the USA and UK have returned to ceftriaxone monotherapy for first-line gonorrhea treatment. 36–38 Finally, the prevalence of penicillin, tetracycline, and ciprofloxacin resistance has reached 79.3%, 98.1%, and 100%, respectively, indicating that these antibiotics are not effective for gonorrhea treatment. 10 An ascending infection of  N. gonorrhoeae  from the lower to the upper female reproductive tract may lead to infertility. 8 The growing antimicrobial resistance in N. gonorrhoeae may exacerbate this process by increasing the risk of treatment failure and persistent infection. 10 , 39 Therefore, we hypothesized that  N. gonorrhoeae  isolates from infertile women might exhibit a higher prevalence of ceftriaxone resistance. However, because isolates in our study were collected only from the cervix without assessment of ascending infection, and information on co-infections or prior STI history (both known contributors to infertility) were not systematically collected, our study was not designed to establish causality between current gonococcal infection and infertility. Unexpectedly, the prevalence of ceftriaxone-resistant N. gonorrhoeae isolates among infertile women in our study (2.8%, 3/106, 2018–2022) was lower than that reported a contemporaneous study from the same geographic region (Changsha, China) among the general population (7.8%, 8/103, 2018–2021). 40 Several factors may account for this difference. First, most women in our cohort were asymptomatic (82.1%) and thus less likely to have used antibiotics before sample collection, potentially reducing selective pressure for resistance. 41 Second, male sex has been associated with higher ceftriaxone MIC values, 42 and the general population study likely included a larger proportion of male patients, whereas our study exclusively enrolled women. Third, the small number of ceftriaxone-resistant isolates in our study (n=3) limits the precision of the resistance estimate and warrants cautious interpretation. NG-MAST is widely used for the molecular typing of N. gonorrhoeae . 43 , 44 Among the 106 isolates, NG-MAST identified 83 distinct sequence types, indicating a high level of genetic heterogeneity. All 17 isolates demonstrating decreased susceptibility or resistance to ceftriaxone were widely distributed across different STs (including six new STs) rather than clustered in a dominant lineage. This pattern suggests multiple independent origins of ceftriaxone resistance in N. gonorrhoeae , consistent with other China-GRSP studies. 45