Surge in Ceftriaxone-Resistant Neisseria gonorrhoeae FC428-Like Strains, Asia-Pacific Region, 2015-2022.

Ceftriaxone-resistant Neisseria gonorrhoeae FC428-like strains have disseminated across the Asia-Pacific region, with a continuous rise in prevalence during 2015-2022. To mitigate the effect of these strains, we advocate for enhanced molecular diagnostics, expanded surveillance networks, and a regionally coordinated effort to combat the global spread of FC428-like strains.

Bacitracin enhances ceftriaxone susceptibility of the high-level ceftriaxone-resistant gonococcal FC428 clone.

IMPORTANCE: Ceftriaxone-based antimicrobial therapies for gonorrhea are threatened by waning ceftriaxone susceptibility levels and the global dissemination of the high-level ceftriaxone-resistant gonococcal FC428 clone. Combination therapy can be an effective strategy to restrain the development of ceftriaxone resistance, and for that purpose, it is important to find an alternative antimicrobial to replace azithromycin, which has recently been removed in some countries from the recommended ceftriaxone plus azithromycin dual-antimicrobial therapy. Ideally, the second antimicrobial should display synergistic activity with ceftriaxone. We hypothesized that bacitracin might display synergistic activity with ceftriaxone because of their distinct mechanisms targeting bacterial cell wall synthesis. In this study, we showed that bacitracin indeed displays synergistic activity with ceftriaxone against Neisseria gonorrhoeae. Importantly, strains associated with the FC428 clone appeared to be particularly susceptible to the bacitracin plus ceftriaxone combination, which might therefore be an interesting dual therapy for further in vivo testing.

Th1-polarized MtrE-based gonococcal vaccines display prophylactic and therapeutic efficacy.

ABSTRACTGlobal dissemination of high-level ceftriaxone-resistant Neisseria gonorrhoeae strains associated with the FC428 clone poses a threat to the efficacy ceftriaxone-based therapies. Vaccination is the best strategy to contain multidrug-resistant infections. In this study, we investigated the efficacy of MtrE and its surface Loop2 as vaccine antigens when combined with a Th1-polarizing adjuvant, which is expected to be beneficial for gonococcal vaccine development. Using in vitro dendritic cell maturation and T cell differentiation assays, CpG1826 was identified as the optimal Th1-polarizing adjuvant for MtrE and Loop2 displayed as linear epitope (Nloop2) or structural epitope (Intraloop2) on a carrier protein. Loop2-based antigens raised strongly Th1-polarized and bactericidal antibody responses in vaccinated mice. Furthermore, the vaccine formulations provided protection against a gonococcal challenge in mouse vaginal tract infection model when provided as prophylactic vaccines. Also, the vaccine formulations accelerated gonococcal clearance when provided as a single therapeutic dose to treat an already established infection, including against a strain associated with the FC428 clone. Therefore, this study demonstrated that MtrE and Loop 2 are effective gonococcal vaccine antigens when combined with the Th1-polarizing CpG1826 adjuvant.

Identification of ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone and isolates harboring a novel mosaic penA gene in Chengdu in 2019-2020.

BACKGROUND: Antimicrobial resistance in gonorrhea has become a growing global public health burden. Neisseria gonorrhoeae isolates with resistance to ceftriaxone, the last remaining first-line option, represent an emerging threat of untreatable gonorrhea. METHODS: A total of ten ceftriaxone-resistant N. gonorrhoeae FC428 isolates and two isolates harboring a novel mosaic penA-232.001 allele from 160 gonococcal isolates in Chengdu in 2019-2020 was described in the present study. Multilocus sequence typing (MLST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were performed to characterize the isolates. Whole genome sequencing and maximum-likelihood method were performed to infer how the genetic phylogenetic tree of these isolates looks like. Recombination analysis was performed using the RDP4 software. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2100048771, registration date: 20210716). RESULTS: The genetic phylogeny showed that the ten FC428 isolates sporadically clustered into different phylogenetic clades, suggesting different introductions and local transmission of FC428. Two isolates showed close genetic relatedness to ceftriaxone-resistant clone A8806, which was only reported from Australia in 2013. Homologous recombination events were detected in penA between Neisseria gonorrhoeae and commensal Neisseria species (N. perflava and N. polysaccharea), providing evidence of commensal Neisseria species might serve as reservoirs of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains. CONCLUSIONS: Our results demonstrate further dissemination of FC428 in China and resurgence risks of sporadic ceftriaxone-resistant A8806 to become the next clone to spread.

Increase in Multidrug Resistant Neisseria gonorrhoeae FC428-Like Isolates Harboring the Mosaic penA 60.001 Gene, in Nanjing, China (2017-2020).

Background: Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China. Objective: To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features. Methods: N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS). Results: Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city's N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates. Conclusion: penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.

Multicentre Clinical Evaluation of a Molecular Diagnostic Assay to Identify Neisseria gonorrhoeae Infection and Detect Antimicrobial Resistance.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (N. gonorrhoeae) is an urgent threat to public health, with the emergence of highly resistant strains such as the FC428 clone. This study aimed to evaluate the high-resolution melting assay of N. gonorrhoeae AMR (HRM-NG-AMR) for diagnosing N. gonorrhoeae infection and detecting extended-spectrum cephalosporins and azithromycin resistance. METHODS: A multicentre collection of 1488 samples, including 770 isolates and 718 urogenital swabs, was used to evaluate the performance of the HRM-NG-AMR assay. The presence of N. gonorrhoeae was confirmed by culture. Minimum inhibitory concentrations of antibiotics against the tested isolates were determined using the agar dilution method. RESULTS: Regarding N. gonorrhoeae identification, HRM-NG-AMR had a sensitivity of 95.15% (95% CI 91.65-97.28) and a specificity of 96.44% (95% CI 94.17-97.89) using culture as standard. Regarding AMR detection, the specificity ranged from 96.29% (95% CI 94.57-97.50) for cefixime to 99.52% (95% CI 98.68-99.85) for azithromycin. Additionally, the sensitivity ranged from 31.34% (95% CI 20.87-43.97) for azithromycin to 79.10% (95% CI 63.52-89.42) for ceftriaxone. It was determined that 664 of 672 (98.81%) and 615 of 672 (91.52%) N. gonorrhoeae isolates were susceptible to ceftriaxone and cefixime, respectively, by detecting non-mosaic penA. Lastly, 40 genotypic FC428-related strains with the penA-60.001 allele were accurately identified. CONCLUSIONS: The HRM-NG-AMR assay showed promising diagnostic performance for detecting N. gonorrhoeae infection and predicting AMR. This study aimed to evaluate the application of this assay in the clinical setting to enhance AMR surveillance and treatment intervention.

Emergence and Genomic Characterization of Neisseria gonorrhoeae Isolates with High Levels of Ceftriaxone and Azithromycin Resistance in Guangdong, China, from 2016 to 2019.

Currently, antibiotic resistance (especially ceftriaxone and azithromycin dual resistance) in Neisseria gonorrhoeae is the main obstacle affecting the efficacy of treatment. As analysis of drug sensitivity, molecular features, and dissemination of dual-resistant strains is important for gonococcal prevention and control, MIC, genotyping, and genome analysis were conducted to reveal the molecular characteristics and phylogeny of N. gonorrhoeae isolates. During 2016 to 2019, 5 out of 4,113 strains were defined as dual-resistant clones, with ceftriaxone MICs of 0.25 to ≥1 mg/L and azithromycin MICs of 2 to ≥2,048 mg/L. In particular, two strains with a ceftriaxone MIC above 0.5 mg/L were characterized as penA-60.001 FC428-related clones, and two isolates with a high-level azithromycin MIC above 1,024 mg/L featuring a 23S rRNA mutation were identified. Furthermore, phylogenetic analysis confirmed that the dual-resistant strains were closer to the evolutionary origin of F89 in France, global FC428-related clones, and high-level dual-resistant clones in Australia and the United Kingdom. Dual-resistant strains, including FC428-related clones and high-level azithromycin-resistant clones, have circulated in Guangdong, China. The ability of laboratories to perform real-time drug susceptibility and genetic analyses should be strengthened to monitor the spread of threatening strains. IMPORTANCE Here, we report five sporadic dual-resistant isolates, including FC428-related ceftriaxone-resistant clones with MICs of ≥0.5 mg/L and high-level azithromycin resistance with MICs of ≥1,024 mg/L. This study highlights that dual-resistant clones with the same evolutionary origin as FC428, A2735, and F89 have circulated in Guangdong, China, which suggests that the capacity for antibiotic resistance testing and genome analysis should be strengthened in daily epidemiological surveillance.

Antimicrobial Activity of Auranofin, Cannabidivarin, and Tolfenamic Acid against Multidrug-Resistant Neisseria gonorrhoeae.

Alternative antimicrobial therapies are urgently required for the multidrug-resistant bacterial pathogen Neisseria gonorrhoeae, for which currently ceftriaxone is the only remaining recommended first-line therapy. Repurposing of drugs that are approved for other clinical applications offers an efficient approach for development of alternative antimicrobial therapies. Auranofin, cannabidivarin, and tolfenamic acid were recently identified to display antimicrobial activity against N. gonorrhoeae. Here, we investigated their activity against a collection of 575 multidrug-resistant clinical isolates. All three compounds displayed consistent antimicrobial activity against all isolates, including against strains associated with the high-level ceftriaxone-resistant FC428 clone, with both the mode and MIC90 for auranofin of 0.5 mg/L, while both the mode and MIC90 for cannabidivarin and tolfenamic acid were 8 mg/L. Correlations between MICs of ceftriaxone and auranofin, cannabidivarin or tolfenamic acid were low, indicating that development of cross-resistance is unlikely. Furthermore, antimicrobial synergy analysis between ceftriaxone and auranofin, cannabidivarin, or tolfenamic acid by determination of the fractional inhibitory concentration index (FICI) resulted in an interpretation of indifference. Finally, time-kill analyses showed that all three compounds are bactericidal against both the N. gonorrhoeae ATCC 49226 reference strain and an FC428-associated clinical isolate, with particularly cannabidivarin displaying rapid bactericidal activity. Overall, auranofin, cannabidivarin, and tolfenamic acid displayed consistent antimicrobial activity against multidrug-resistant N. gonorrhoeae, warranting further exploration of their suitability as alternative antimicrobials for treatment of gonococcal infections. IMPORTANCE Neisseria gonorrhoeae is a major public health concern because of the high incidence of gonorrhea and the increasingly limited options for antimicrobial therapy. Strains associated with the FC428 clone are a particular concern because they have shown global dissemination and they display high-level resistance against the currently recommended ceftriaxone therapy. Therefore, development of alternative antimicrobial therapies is urgently required to ensure treatment of gonorrhea remains available in the future. Repurposing of clinically approved drugs could be a rapid approach for the development of such alternative antimicrobials. In this study, we showed that repurposing of auranofin, cannabidivarin, and tolfenamic acid for antimicrobial therapy of gonorrhea deserves further clinical explorations because these compounds displayed consistent antimicrobial activity against a large collection of contemporary multidrug-resistant gonococcal isolates that included strains associated with the FC428 clone.

Markedly Increasing Antibiotic Resistance and Dual Treatment of Neisseria gonorrhoeae Isolates in Guangdong, China, from 2013 to 2020.

The emergence of multidrug resistance in Neisseria gonorrhoeae is concerning, especially the cooccurrence of azithromycin resistance and decreased susceptibility to extended-spectrum cephalosporin. This study aimed to confirm the antibiotic resistance trends and provide a solution for N. gonorrhoeae treatment in Guangdong, China. A total of 5,808 strains were collected for assessment of antibiotic MICs. High resistance to penicillin (53.80 to 82%), tetracycline (88.30 to 100%), ciprofloxacin (96 to 99.8%), cefixime (6.81 to 46%), and azithromycin (8.60 to 20.03%) was observed. Remarkably, spectinomycin and ceftriaxone seemed to be the effective choices, with resistance rates of 0 to 7.63% and 2.00 to 16.18%, respectively. Moreover, the rates of azithromycin resistance combined with decreased susceptibility to ceftriaxone and cefixime reached 9.28% and 8.64%, respectively. Furthermore, genotyping identified NG-STAR-ST501, NG-MAST-ST2268, and MLST-ST7363 as the sequence types among representative multidrug-resistant isolates. Evolutionary analysis showed that FC428-related clones have spread to Guangdong, China, which might be a cause of the rapid increase in extended-spectrum cephalosporin resistance currently. Among these strains, the prevalence of N. gonorrhoeae was extremely high, and single-dose ceftriaxone treatment might be a challenge in the future. To partially relieve the treatment pressure, a susceptibility test for susceptibility to azithromycin plus extended-spectrum cephalosporin dual therapy was performed. The results showed that all the representative isolates could be effectively killed with the coadministration of less than 1 mg/liter azithromycin and 0.125 mg/liter extended-spectrum cephalosporin, with a synergistic effect according to a fractional inhibitory concentration (FIC) of <0.5. In conclusion, dual therapy might be a powerful measure to treat refractory N. gonorrhoeae in the context of increasing antibiotic resistance in Guangdong, China.

Neisseria gonorrhoeae FC428 Subclone, Vietnam, 2019-2020.

Among 114 clinical Neisseria gonorrhoeae isolates collected in Vietnam during 2019-2020, we detected 15 of subclone sequence type 13871 of the FC428 clonal complex. Fourteen sequence type 13871 isolates with mosaic penA allele 60.001 were ceftriaxone or cefixime nonsusceptible, and 3/14 were azithromycin nonsusceptible. Emergence of this subclone threatens treatment effectiveness.

Impact of the gonococcal FC428 penA allele 60.001 on ceftriaxone resistance and biological fitness.

Global dissemination of the Neisseria gonorrhoeae ceftriaxone-resistant FC428 clone jeopardizes the currently recommended ceftriaxone-based first-line therapies. Ceftriaxone resistance in the FC428 clone has been associated with the presence of its mosaic penA allele 60.001. Here we investigated the contribution penA allele 60.001 to ceftriaxone resistance and its impact on biological fitness. Gonococcal isolates expressing penA allele 60.001 and mosaic penA allele 10.001, which is widespread in the Asia-Pacific region and associated with reduced susceptibility to ceftriaxone and cefixime, were genetic engineered to exchange their penA alleles. Subsequent antimicrobial susceptibility analyses showed that mutants containing penA 60.001 displayed 8- to 16-fold higher ceftriaxone and cefixime minimal inhibitory concentrations (MICs) compared with otherwise isogenic mutants containing penA 10.001. Further analysis of biological fitness showed that in vitro liquid growth of single strains and in the competition was identical between the isogenic penA allele exchange mutants. However, in the presence of high concentrations of palmitic acid or lithocholic acid, the penA 60.001-containing mutants grew better than the isogenic penA 10.001-containing mutants when grown as single strains. In contrast, the penA 10.001 mutants outcompeted the penA 60.001 mutants when grown in competition at slightly lower palmitic acid or lithocholic acid concentrations. Finally, the penA 60.001 mutants were outcompeted by their penA 10.001 counterparts for in vivo colonization and survival in a mouse vaginal tract infection model. In conclusion, penA allele 60.001 is essential for ceftriaxone resistance of the FC428 clone, while its impact on biological fitness is dependent on the specific growth conditions.

Multidrug-resistant Neisseria gonorrhoeae isolate, belonging to the internationally spreading Japanese FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, Ireland, August 2018.

We describe a multidrug-resistant Neisseria gonorrhoeae urethritis case with ceftriaxone resistance and azithromycin intermediate resistance in a heterosexual man in Ireland, August 2018. Whole-genome sequencing showed that the isolate IR72 belongs to the internationally spreading multidrug-resistant ceftriaxone-resistant FC428 clade, initially described in Japan in 2015. IR72 was assigned MSLT ST1903, NG-MAST ST17842 and NG-STAR type 1133, including the ceftriaxone resistance-mediating penA-60.001. Global awareness of spreading ceftriaxone-resistant gonococcal strains that threaten recommended dual therapies is essential.

Direct Detection of penA Gene Associated with Ceftriaxone-Resistant Neisseria gonorrhoeae FC428 Strain by Using PCR.

The ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone was first observed in Japan in 2015, and in 2017, it was documented in Denmark, Canada, and Australia. Here, we describe a PCR for direct detection of the penA gene associated with this strain that can be used to enhance surveillance activities.