Dissemination of Neisseria gonorrhoeae with decreased susceptibility to extended-spectrum cephalosporins in Southern China, 2021: a genome-wide surveillance from 20 cities.

BACKGROUND: Antimicrobial resistance (AMR) of untreatable gonococcal infection is an emerging threat, especially in Guangdong, a prosperous province in Southern China. METHODS: N.gonorrhoeae was isolated from 20 cities in Guangdong and determined antimicrobial susceptibility. Through whole-genome sequencing (WGS), multilocus sequence typing (MLST), N.gonorrhoeae multiantigen sequence typing (NG-MAST), and N.gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were obtained based on the PubMLST database ( https://pubmlst.org/ ). Phylogenetic analysis was used for dissemination and tracking analysis. RESULTS: Antimicrobial susceptibility was performed on 347 isolates, and 50 isolates were identified as decreased susceptibility (DS) to cephalosporins. Of which 16.0% (8/50) were ceftriaxone DS, 38.0% (19/50) were cefixime DS, and 46.0% (23/50) were both ceftriaxone and cefixime DS. In all, the dual-resistant rate of the cephalosporin-DS isolates was 96.0% for penicillin and 98.0% for tetracycline-resistant, and 10.0% (5/50) were resistant to azithromycin. All cephalosporin-DS isolates were resistant to ciprofloxacin but sensitive to spectinomycin. The predominant MLSTs were ST7363 (16%, 8/50), ST1903 (14%, 7/50), ST1901 (12%, 6/50), and ST7365 (10%, 5/50). Besides some isolates that failed genotyping (NA), NG-STAR ST1143 (n = 6) and NG-MAST ST17748 (n = 4) were the most prevalent. Twelve isolates with mosaic penA-60.001 allele retained the most elevated cephalosporin MIC (Minimum Inhibitory Concentration). Phylogenetic analysis revealed that epidemic penA-60.001 clones, either domestic or foreign, had spread to nine cities in Guangdong, and 9/12 clones were from the Pearl River Delta region. CONCLUSIONS: N. gonorrhoeae with cephalosporins-DS was extensively disseminated in Guangdong, Southern China, requiring strict surveillance.

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.

Development and application of Cas13a-based diagnostic assay for Neisseria gonorrhoeae detection and azithromycin resistance identification.

BACKGROUND: Gonorrhoea, caused by Neisseria gonorrhoeae, has spread worldwide. Strains resistant to most antibiotics, including ceftriaxone and azithromycin, have emerged to an alarming level. Rapid testing for N. gonorrhoeae and its antimicrobial resistance will therefore contribute to clinical decision making for early diagnosis and rational drug use. METHODS: A Cas13a-based assay (specific high-sensitivity enzymatic reporter unlocking; SHERLOCK) was developed for N. gonorrhoeae detection (porA gene) and azithromycin resistance identification (A2059G, C2611T). Assays were evaluated for sensitivity with purified dsDNA and specificity with 17 non-gonococcal strains. Performance of SHERLOCK (porA) was compared with Roche Cobas 4800 using 43 urine samples. Identification of azithromycin resistance mutations (A2059G, C2611T) was evaluated using a total of 84 clinical isolates and 18 urine samples. Lateral flow was tested for this assay as a readout tool. Moreover, we directly assayed 27 urethral swabs from patients with urethritis to evaluate their status in terms of N. gonorrhoeae infection and azithromycin resistance. RESULTS: The SHERLOCK assay was successfully developed with a sensitivity of 10 copies/reaction, except 100 copies/reaction for A2059G, and no cross-reaction with other species. Comparison of the SHERLOCK assay with the Cobas 4800 revealed 100% concordance within 18 positive and 25 negative urine samples. Of the 84 isolates, 21 strains with azithromycin resistance mutations were distinguished and further verified by sequencing and MIC determination. In addition, 62.96% (17/27) strains from swab samples were detected with no mutant strains confirmed by sequencing. CONCLUSIONS: The SHERLOCK assay for rapid N. gonorrhoeae detection combined with azithromycin resistance testing is a promising method for application in clinical practice.

Immunophenotypic variations in syphilis: insights from Mendelian randomization analysis.

Background: Infection with Treponema pallidum instigates complex immune responses. Prior research has suggested that persistent Treponema pallidum infection can manipulate host immune responses and circumvent host defenses. However, the precise role of immune cells in Treponema pallidum infection across different stages remains a contentious issue. Methods: Utilizing summary data from genome-wide association studies, we employed a two-sample Mendelian randomization method to investigate the association between 731 immunophenotypes and syphilis. Syphilis was categorized into early and late stages in this study to establish a more robust correlation and minimize bias in database sources. Results: Our findings revealed that 33, 36, and 27 immunophenotypes of peripheral blood were associated with syphilis (regardless of disease stage), early syphilis and late syphilis, respectively. Subsequent analysis demonstrated significant variations between early and late syphilis in terms of immunophenotypes. Specifically, early syphilis showcased activated, secreting, and resting regulatory T cells, whereas late syphilis was characterized by resting Treg cells. More B cells subtypes emerged in late syphilis. Monocytes in early syphilis exhibited an intermediate and non-classical phenotype, transitioning to classical in late syphilis. Early syphilis featured naive T cells, effector memory T cells, and terminally differentiated T cells, while late syphilis predominantly presented terminally differentiated T cells. Immature myeloid-derived suppressor cells were evident in early syphilis, whereas the dendritic cell immunophenotype was exclusive to late syphilis. Conclusion: Multiple immunophenotypes demonstrated associations with syphilis, showcasing substantial disparities between the early and late stages of the disease. These findings hold promise for informing immunologically oriented treatment strategies, paving the way for more effective and efficient syphilis interventions.

Neutrophil CD64 index as a potential blood biomarker for the diagnosis of neurosyphilis in secondary and tertiary syphilis: A retrospective study.

Objective: To examine the correlation of neutrophil CD64 (nCD64) index with neurosyphilis (NS) across different stages of syphilis. Methods: A total of 1243 syphilis patients at different stages (344 of primary, 385 of secondary, and 514 of tertiary) included in this study were divided into NS and non-NS (NNS). Correlations of nCD64 index with currently used syphilis biomarkers were explored using Spearman correlation test. Relationships between nCD64 index and NS at different stages were investigated by stratified analysis and restricted cubic spline model. The diagnostic performance of nCD64 index for NS was assessed by receiver operating characteristic (ROC) curve. Results: Significant statistical correlations of nCD64 index with cerebrospinal fluid (CSF) NS indicators were found in secondary and tertiary syphilis. Increased nCD64 index was associated with increased risk of NS in secondary and tertiary syphilis. ROC analysis values further confirmed the diagnostic potential of nCD64 index for NS. Marked decrease of nCD64 index was observed in NS patients after effective antisyphilitic treatments. Conclusions: The nCD64 index may help to the diagnosis of NS in secondary and tertiary syphilis.

penA profile of Neisseria gonorrhoeae in Guangdong, China: Novel penA alleles are related to decreased susceptibility to ceftriaxone or cefixime.

BACKGROUND: Resistance to extended-spectrum cephalosporins (ESCs) has become a public health concern with the spread of Neisseria gonorrhoeae and increasing antimicrobial resistance. Mutation of penA, encoding penicillin-binding protein 2, represents a mechanism of ESC resistance. This study sought to assess penA alleles and mutations associated with decreased susceptibility (DS) to ESCs in N. gonorrhoeae. MATERIALS AND METHODS: In 2021, 347 gonococci were collected in Guangdong, China. Minimum inhibitory concentations (MICs) of ceftriaxone and cefixime were determined, and whole-genome sequencing and phylogenetic analysis were performed. Multi-locus sequence typing (MLST) and conventional resistance determinants such as penA, mtrR, PonA and PorB were analysed. penA was genotyped and sequence-aligned using PubMLST. RESULTS: Genome-wide phylogenetic analysis revealed that the prevalence of DS to ESCs was highest in Clade 11.1 (100.0%), Clade 2 (66.7%) and Clade 0 (55.7%), and the leading cause was strains with penA-60.001 or new penA alleles in clades. The penA phylogenetic tree is divided into two branches: non-mosaic penA and mosaic penA. The latter contained penA-60.001, penA-10 and penA-34. penA profile analysis indicated that A311V and T483S are closely related to DS to ESCs in mosaic penA. The new alleles NEIS1753_2840 and NEIS1753_2837 are closely related to penA-60.001, with DS to ceftriaxone and cefixime of 100%. NEIS1753_2660, a derivative of penA-10 (A486V), has increased DS to ceftriaxone. NEIS1753_2846, a derivative of penA-34.007 (G546S), has increased DS to cefixime. CONCLUSION: This study identified critical penA alleles related to elevated MICs, and trends of gonococcus-evolved mutated penA associated with DS to ESCs in Guangdong.

Dissemination and genome analysis of high-level ceftriaxone-resistant penA 60.001 Neisseria gonorrhoeae strains from the Guangdong Gonococcal antibiotics susceptibility Programme (GD-GASP), 2016-2019.

Background: After Neisseria gonorrhoeae FC428 was first found in Japan, ceftriaxone-resistant strains disseminated globally, and the gonococcal resistance rate increased remarkably. Epidemiological investigations are greatly significant for the analysis of antimicrobial resistance (AMR) trends, molecular features and evolution. Objectives: To clarify the AMR trend from 2016-2019 and reveal the molecular characteristics and evolution of ceftriaxone-resistant penA 60.001 isolates. Methods: The minimum inhibitory concentrations (MICs) of antibiotics against 4113 isolates were detected by the agar dilution method. N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST) and N.gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were used to identify the sequence types. Genome analysis was conducted to analyze resistance genes, virulence factors, and evolutionary sources. Results: Isolates with decreased ceftriaxone susceptibility have increased from 2.05% (2016) to 16.18% (2019). Six ceftriaxone-resistant isolates possessing penA 60.001 appeared in Guangdong Province, and were resistant to ceftriaxone, penicillin, tetracycline, ciprofloxacin and cefixime, but susceptible to azithromycin and spectinomycin. Single-nucleotide polymorphisms (SNPs) in the porB gene were the major cause of different NG-MAST types. ST1903 was the main NG-STAR genotype and only strain-ZH545 was ST7365, with molecular features consistent with the MICs. Furthermore, different MLSTs suggested diverse evolutionary sources. Genome analysis revealed a set of virulence factors along with the resistance genes "penA" and "blaTEM-1B". Half of penA 60.001 strains were fully mixed with global FC428-related strains. Conclusions: Global FC428-related clones have disseminated across Guangdong, possibly causing decreased ceftriaxone susceptibility. Enhanced gonococcal surveillance will help elucidate the trajectory of transmission and curb further dissemination.

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.