Objective This study aimed to evaluate the synergistic effect of berberine hydro-chloride on the antibacterial activity of penicillin against Penicillinase-Producing Neisseria gonor-rhoeae（PPNG）, offering new insights for the prevention and treatment of highly drug-resistant PPNG.Methods The minimum inhibitory concentration（MIC）of berberine hydrochloride com-bined with penicillin against PPNG strains were determined using microbroth dilution and checker-board assays, with fractional inhibitory concentration（FIC）indices calculated to assess antibacte-rial efficacy.TEM DNA mutations were detected via gene sequencing.TEM mRNA expression levels were measured by RT-qPCR.Penicillinase was extracted by ultrasonic disruption, and its activity was assessed using the dual-wavelength colorimetric Amplite assay kit.Results The MIC range of the berberine hydrochloride-only treatment against PPNG was 0.03～128 μg/mL. The MIC of penicillin decreased by ≥4-fold and the FIC index was ≤0.5 when berberine hydrochlo-ride was combined with penicillin against PPNG, demonstrating the synergistic antibacterial effects of these two compounds.The combined antibacterial strategy did not induce mutations in TEM genes, but it decreased penicillinase activity（P＜0.05）through downregulating the mRNA ex-pression of TEM（2-ΔΔCt＜1）.Conclusions Berberine hydrochloride not only directly inhibits PPNG growth but also restores penicillin sensitivity by down-regulating TEM gene expression and inhibiting penicillinase activity.These findings showed that berberine hydrochloride is an antibiotic adjuvant against PPNG.

BACKGROUND:Recovering clinical isolates of Treponema pallidum (Tp) can expedite the research on molecular epidemiology and vaccine development of syphilis. Nevertheless, obtaining numerous isolates through rabbit infectivity testing remains unsatisfactory, particularly in secondary syphilis cases. This study re-evaluates the reliability of orchitis as a phenotypic indicator for assessing Tp clinical strains during rabbit infectivity testing. METHODS:Treponemal and nontreponemal tests were used to analyze the correlation between serological titers and Tp burden. An optimal titer threshold was identified and applied to verify the maximum achievable Tp burden across different initial inoculation doses. A modified recovery protocol was developed for isolating Tp clinical strains from chancre exudates or whole blood, and its success rate was subsequently evaluated. RESULTS:We demonstrated that a Tp particle agglutination titer of 1:1280 reliably indicated the peak burden of Tp Nichols strain and clinical isolates during rabbit infectivity testing. To achieve stable recovery, the initial inoculum should be at least 105 organisms. Using this optimized protocol, we successfully recovered and maintained Tp clinical isolates from 82.69% (43/52) of syphilis patients overall. Notably, the success rate from whole blood samples of secondary syphilis cases increased markedly to 82.50% (33/40) despite many of these specimens testing negative for Tp DNA by quantitative polymerase chain reaction. CONCLUSIONS:This study shows that the pathogenicity of secondary syphilis blood is greater than previously recognized. Thus, monitoring Tp particle agglutination titers enables the recovery of Tp clinical isolates, even from specimens with extremely low bacterial burdens.

Female sex workers (FSWs) are at high risk of chlamydia infection, yet the seroprevalence among FSWs in China remains unclear. This study aimed to determine the seroprevalence of Chlamydia trachomatis and associated factors among FSWs in Guangdong Province, China. A cross-sectional study was conducted among FSWs in two cities in Guangdong Province. Participants provided serum and urine samples. Nucleic acid amplification test (NAAT) was used to detect Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in urine samples. Enzyme-linked immunosorbent assay (ELISA) was used to detect chlamydia IgG antibodies in serum samples. Seropositivity was defined by IgG-positive results. Current chlamydia infection was identified by a positive NAAT result, while prior infection was indicated by positive chlamydia IgG and negative NAAT results. Reinfection was defined by positive results for both NAAT and chlamydia IgG. Moreover, positive ELISA results were reclassified into two categories: DU/mL values ≥ 37.89 (the median) were classified as high-positive and DU/mL values > 11 to 37.89 as low-positive. Sociodemographic data, CT and NG testing, and paper questionnaires were collected through face-to-face interviews. Univariate and multivariable logistic regressions explored factors associated with current CT infection. A total of 435 serum and urine samples were analyzed. The median age of the participants was 32.0 (IQR: 27.0–37.0) years. Among the participants, 326 were CT IgG positive, resulting in an overall seroprevalence of 74.9

Knowledge of Treponema pallidum subspecies pallidum (TPA) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as the design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China, and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols- and three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Finally, we noted that BamA and FadL candidate ECL vaccinogens, previously shown to elicit antibodies that kill treponemes during in vitro cultivation, are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing the selection of vaccine targets.IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

Background:Neurosyphilis, caused by Treponema pallidum invading the nervous system, can lead to severe neurological complications across all stages of syphilis. Misdiagnosis is common, exacerbated by overlapping psychiatric conditions and diagnostic limitations. This study aims to improve the understanding and management of neurosyphilis in psychiatric settings to enhance diagnostic accuracy and treatment efficacy. Methods:A retrospective study used data from the Chinese Case Report System (CCRS) spanning 2014-2018. Four municipal psychiatric hospitals in Guangdong were chosen based on syphilis rates and psychiatric patient volumes. Sociodemographic data, syphilis history, symptoms, diagnostic and treatment details, and laboratory results were reviewed. The Brief Psychiatric Rating Scale (BPRS) assessed psychopathology symptoms. Treatment efficacy was evaluated using BPRS scores before and after standard treatment. Diagnoses followed national guidelines, with statistical analyses performed using logistic regression and t-tests. Results:Out of 69,436 psychosis patients screened, 1588 were diagnosed with syphilis, with 262 (16.5%) of these cases identified as neurosyphilis. Syphilis prevalence mildly declined from 2.8% (2014) to 2.0% (2016), while neurosyphilis cases increased marginally from 0.34% (2014) to 0.39% (2018). Confirmatory Cerebrospinal Fluid (CSF) tests were conducted in only 30.2% of neurosyphilis cases. Standard therapy was administered to 66.8% of patients, with significantly better outcomes in confirmed cases (p < 0.001). Conclusions:Diagnosing and treating neurosyphilis in Guangdong's psychiatric hospitals remains challenging. Accurate diagnosis and standardized treatment protocols are essential to effectively manage both syphilis and associated mental health complications caused by neurosyphilis.

OBJECTIVES:This study aimed to investigate the molecular characteristics of predominant epidemic clusters of FC428-like N. gonorrhoeae in Guangdong, China in 2022. METHODS:Minimum inhibitory concentrations (MICs) were determined for N. gonorrhoeae isolates collected from the Guangdong Gonococcal Antimicrobial Surveillance Program. N. gonorrhoeae multiantigen sequence typing (NG-MAST), antimicrobial resistance sequence typing (NG-STAR), multilocus sequence typing (MLST) sequence types (STs), and penA alleles were determined by whole-genome sequencing. The isolates were further characterised by phylogenetic analysis. RESULTS:A total of 537 N. gonorrhoeae isolates were analysed, molecular analysis revealed that 7.8% of the isolates carried penA 60.001 allele, which was highly resistant to ceftriaxone (88.1%) and cefixime (100.0%). The predominant STs in penA 60.001 isolates were MLST ST1903 (23,54.8%), ST7365 (11,26.2%), NG-STAR ST1143 (13,31.0%), ST233 (4,9.5%), ST1133 (4,9.5%), and NG-MAST ST22261 (10,23.8%). Among 537 isolates, of which 11.2% were resistant to ceftriaxone and 19.6% to cefixime. In western Guangdong, resistance to ceftriaxone and cefixime reached 17.3% and 26.9%, respectively. The most predominant types among ceftriaxone-resistant isolates are genetically closer to FC428 isolates, and differ from those among cefixime-resistant isolates. Phylogenetic analysis revealed that the Guangdong FC428-like isolates from 2021 to 2022 spread across the whole phylogenetic tree, but the majority were clustered within a distinct evolutionary clade. CONCLUSIONS:FC428-like isolates in Guangdong formed a unique evolutionary clade with high cephalosporin resistance. These findings highlight the need to revise national gonorrhea treatment guidelines and prioritise the development of new antimicrobials.

The prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) is a crucial public health concern because of its resistance to penicillin and cephalosporins. From 2013 to 2022, a total of 1,748 N. gonorrhoeae isolates from Guangdong, China, were examined for their antibiotic susceptibility and molecular epidemiological characteristics. PPNG prevalence increased markedly from 37.25% to 63.87%. This increase was accompanied by a shift in predominant plasmid types carried by PPNG isolates: the rate of PPNG isolates carrying the Africa-type plasmid increased from 18.42% to 91.55%, whereas the rate of isolates carrying the Asia-type plasmid decreased from 81.58% to 7.58%. The prevalence of blaTEM-135, which is linked to cephalosporin resistance, declined from 52.63% to 4.37%, whereas that of blaTEM-1 increased from 47.37% to 86.88%, and new blaTEM variants emerged (10.99% by 2022). Most blaTEM-1 (88.26%) and new blaTEM alleles (83.70%) were associated with the Africa-type plasmid, whereas 86.79% of blaTEM-135 alleles were linked to the Asia-type plasmid. Resistance to ceftriaxone was higher in the Asia-type group (11.67%) than in the Africa-type, Toronto/Rio-type and non-PPNG groups. Genotyping identified diverse sequence types (STs) among PPNGs, in which MLST ST7363, NG-STAR ST2477, NG-MAST ST17748, and NG STAR CC1124 were predominant. This study underscores the rising prevalence of PPNG in Guangdong driven by clonal expansion and changing plasmid dynamics, affecting cephalosporin resistance and highlighting the need for continued surveillance and research into effective treatment strategies.

Chlamydia trachomatis, the most prevalent bacterial agent of sexually transmitted infections, poses a significant threat to reproductive health. The release of progeny through the orchestrated lysis of host cells plays a crucial role for the development of new infections, though the underlying molecular mechanisms remaining largely unexplored. In this study, we identified a novel mechanism by which Chlamydia induces host cell ferroptosis to facilitate its progeny release. This process involves the degradation of the host protein SLC7A11 by the chlamydial protease-like activity factor (CPAF), resulting in glutathione depletion and subsequent cell death characterized by lipid peroxidation. Infection with a CPAF-deficient strain fails to induce host cell ferroptosis. Notably, inhibiting ferroptosis by vitamin E reduces the Chlamydia burden in low genital tract of mice and trends toward attenuation of pathology. These findings provide new insights into the conserved survival strategies of Chlamydia and understanding of its pathogenesis.

The rising incidence of gonorrhea presents a global public health challenge, emphasizing the importance of accurate Neisseria gonorrhoeae detection for effective diagnosis, treatment and prevention. This study assessed the performance of sexually transmitted disease laboratories for N. gonorrhoeae detection in Guangdong, southern China. A questionnaire about N. gonorrhoeae detection and external quality assessments (EQAs) with five samples for culture and five for nucleic acid amplification tests (NAATs) were conducted annually from 2020 through 2022. The questionnaire was responded to by 1047 laboratories, with the results showing that Gram staining and culture were most commonly used by 44.3% and 40.4% of the laboratories, respectively, whereas NAATs were adopted by only 11.7%. The number of clinical samples tested for N. gonorrhoeae increased from 536,369 in 363 laboratories in 2020 to 1,093,147 in 582 laboratories in 2022 (P > 0.05), with an overall male-to-female testing ratio of 1:3.4 and a positive ratio of 3.6:1. The EQA results revealed a concordance rate of 90.8% for gonococcal culture and 93.1% for NAATs. NAATs demonstrated a decreasing detection rate (from 100.0% to 84.5%) with reducing gonococcal DNA copies and 89.0% specificity with the interference-negative sample. The overall concordance rates of four widely used commercial NAAT kits ranged from 95.7% to 88.5%. Gram staining and culture were widely used to detect N. gonorrhoeae in Guangdong. NAAT has long been recognized as a highly sensitive and specific method for detecting gonorrhea and should be more widely implemented in China.

BACKGROUND:The global resurgence of syphilis necessitates vaccine development. METHODS:We collected ulcer exudates and blood from 17 participants with primary syphilis (PS) and skin biopsies and blood from 51 patients with secondary syphilis (SS) in Guangzhou, China, for Treponema pallidum subsp pallidum (TPA) quantitative polymerase chain reaction, whole genome sequencing (WGS), and isolation of TPA in rabbits. RESULTS:TPA DNA was detected in 15 of 17 ulcer exudates and 3 of 17 blood PS specimens. TPA DNA was detected in 50 of 51 SS skin biopsies and 27 of 51 blood specimens. TPA was isolated from 47 rabbits with success rates of 71% (12/17) and 69% (35/51), respectively, from ulcer exudates and SS bloods. We obtained paired genomic sequences from 24 clinical samples and corresponding rabbit isolates. Six SS14- and 2 Nichols-clade genome pairs contained rare discordances. Forty-one of the 51 unique TPA genomes clustered within SS14 subgroups largely from East Asia, while 10 fell into Nichols C and E subgroups. CONCLUSIONS:Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS blood, with TPA isolation in more than two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development.

BACKGROUND:The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS:In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS:We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION:Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING:US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.

Background The indiscriminate use of antibiotics has accelerated antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG), emphasising the need to follow treatment guidelines. This study aimed to assess the rate of adherence to standard treatment among patients with gonorrhoea and identify influencing factors.Methods A survey was conducted in Guangdong province, China, involving uncomplicated gonorrhoea cases registered in the Chinese Information System for Disease Control and Prevention. Data on demographic characteristics and medical information were collected to determine the standard treatment rate, defined as the proportion of patients receiving treatment according to national guidelines (ie, a single dose of ceftriaxone 250 mg, spectinomycin 2 g, cefotaxime 1 g or other third-generation cephalosporins). Medication choices were documented. χ² tests and multilevel logistic regression were used to analyse factors associated with standard treatment.Results The survey included 2424 patients with gonorrhoea from 59 hospitals. The standard treatment rate was 30.7% (743/2424), with 36.2% for females and 29.6% for males. Common reasons for substandard treatment included the use of non-guideline medications (42.3%, 710/1681) and incorrect dosing (36.2%, 605/1681). Factors associated with the standard treatment rate included gender, address, educational level, department, physicians’ training, number of diagnosed gonorrhoea cases and hospital level.Conclusion The standard treatment rate for gonorrhoea in Guangdong province, China, is below expectations. Comprehensive measures, such as establishing a goal-directed monitoring system and implementing promotional activities, are needed to improve adherence to treatment guidelines.

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 sequencealigned 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 gonococcusevolved mutated penA associated with DS to ESCs in Guangdong. (c) 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY -NC -ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )