Molecular epidemiology and molecular mechanisms of antimicrobial resistance in Neisseria gonorrhoeae in China : implications for disease control

Gonorrhea, caused by the human pathogen Neisseria gonorrhoeae, is a severe public health problem worldwide with more than 82 million new infections each year. N. gonorrhoeae is transmitted by sexual contact and primarily causes urogenital mucosal infections in men and women. Left untreated, this infection may cause severe complications, especially in females. Eye infections of the newborn can occur. Gonorrhea infections enhance HIV transmission. The highly prevalent antibiotic resistance and the emergence of new drug resistances render treatment of the infections increasingly difficult. Close monitoring of antimicrobial susceptibility of this pathogen is crucial, and enhanced knowledge of molecular mechanisms of gonococcal antimicrobial resistance is urgently needed. There are no vaccines available against N. gonorrhoeae. Control of gonorrhea relies on comprehensive strategies which can be better formulated by understanding, at molecular levels, how N. gonorrhoeae is transmitted in communities. My research aimed to illustrate the severe burden of antimicrobial resistance in N. gonorrhoeae temporally and geographically in China and to reveal the molecular mechanisms of antibiotic resistance particularly the development of reduced susceptibility to ceftriaxone in N. gonorrhoeae isolates. To determine specific strain distributions, N. gonorrhoeae isolates were characterized using molecular typing methods such as a modified porB-based typing scheme and the N. gonorrhoeae Multi-Antigen Typing (NG-MAST) method, compared to traditional epidemiological approaches. The ultimate goal was to provide information for better formulating disease control strategies for gonorrhea. In this research, male patients with gonorrhea and their sex partners were recruited in Shanghai (2005 and 2008) and in Urumchi (2007-2008), China. Epidemiological information pertaining to sexual contacts was collected. N. gonorrhoeae isolates were investigated for their antimicrobial susceptibility. Molecular mechanisms of antimicrobial resistance were explored by analysis of potential resistant determinants (gyrA, parC, porB, mtrR, ponA and penA). The molecular data were combined with bioinformatic analysis and traditional epidemiological data. High percentages of N. gonorrhoeae isolates (11% - 19% in Shanghai, 4.5% in Urumchi) exhibited reduced susceptibility to ceftriaxone (MICs = 0.125-0.25 mg/L), the first line drug recommended for the treatment of gonorrhea in China. The majority of isolates (>98%) were susceptible to spectinomycin, an alternative regimen for gonorrhea treatment; however, the proportion of isolates having intermediate levels of susceptibility increased from 1.9% in 2005 to 9.9% in 2008. The majority of isolates tested were resistant to penicillin (80% - 93%), tetracycline (56% - 65%) and ciprofloxacin (98% - 100%). Plasmid-mediated resistance in N. gonorrhoeae isolates were highly prevalent (51% - 79%) in Shanghai and Urumchi. Analysis of 60 clinical isolates revealed that reduced susceptibility to ceftriaxone is mediated by porB1b allele and is associated with specific mutations in penicillin binding protein 2 and in the DNA binding and dimerization domains of MtrR. Penicillin binding protein 1 is not involved in reduced susceptibility to ceftriaxone. Although mutation patterns in quinolone resistant determinant regions (QRDRs) varied, the majority of ciprofloxacin resistant isolates had double mutations in GyrA (S91F and D95G/A/N) and most isolates also carried a S87R/N mutation in ParC. The presence of mutations in the QRDR of ParC is correlated with elevated ciprofloxacin MICs. A modified porB-based molecular typing scheme was developed and involved ~82% of the DNA sequence of gonococcal porB. This typing method proved to have high discriminatory ability (index of discrimination = 0.93 – 0.96), and was cost effective and easy to perform as compared to the NG-MAST analysis. Using the modified porB-based typing method, N. gonorrhoeae isolates were reliably differentiated, and transmission clusters were identified. Molecular epidemiology using the porB-based method confirmed direct sexual connections and identified sexual networks otherwise unrevealed by the patient self-reporting or traditional case-tracing methods

Influence of Conserved and Hypervariable Genetic Markers on Genotyping Circulating Strains of Neisseria gonorrhoeae

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced

The Cytoplasmic Domain of MUC1 Induces Hyperplasia in the Mammary Gland and Correlates with Nuclear Accumulation of β-Catenin

MUC1 is an oncoprotein that is overexpressed in up to 90% of breast carcinomas. A previous in vitro study by our group demonstrated that the cytoplasmic domain of MUC1 (MUC1-CD), the minimal functional unit of MUC1, contributes to the malignant phenotype in cells by binding directly to β-catenin and protecting β-catenin from GSK3β-induced degradation. To understand the in vivo role of MUC1-CD in breast development, we generated a MUC1-CD transgenic mouse model under the control of the MMTV promoter in a C57BL/6J background, which is more resistant to breast tumor. We show that the expression of MUC1-CD in luminal epithelial cells of the mammary gland induced a hyperplasia phenotype characterized by the development of hyper-branching and extensive lobuloalveoli in transgenic mice. In addition to this hyperplasia, there was a marked increase in cellular proliferation in the mouse mammary gland. We further show that MUC1-CD induces nuclear localization of β-catenin, which is associated with a significant increase of β-catenin activity, as shown by the elevated expression of cyclin D1 and c-Myc in MMTV-MUC1-CD mice. Consistent with this finding, we observed that overexpression of MUC1-C is associated with β-catenin nuclear localization in tumor tissues and increased expression of Cyclin D1 and c-Myc in breast carcinoma specimens. Collectively, our data indicate a critical role for MUC1-CD in the development of mammary gland preneoplasia and tumorigenesis, suggesting MUC1-CD as a potential target for the diagnosis and chemoprevention of human breast cancer

Comparison of Neisseria gonorrhoeae Multiantigen Sequence Typing and porB Sequence Analysis for Identification of Clusters of N. gonorrhoeae Isolates▿ †

porB DNA sequence analysis and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) methods were compared for their abilities to discriminate strains and to identify epidemiologically congruent pairs of N. gonorrhoeae. Both methods provided high-level discrimination of strains. NG-MAST further differentiated large porB-based clusters. However, considerations of cost suggest that porB DNA sequence analysis is a useful tool for preliminary molecular analysis of the epidemiology of N. gonorrhoeae

Dual inhibition of vacuolar ATPase and TMPRSS2 is required for complete blockade of SARS-CoV-2 entry into cells

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.This research was funded (SI and RAM) from Fast Grants, part of the Emergent VenturesProgram at the Mercatus Centre at George Mason University, with support from Thistledown Foundation. This research was supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant 790012 (ER). This work was further supported by the CIFAR Azrieli Global Scholar program (JPJ), the Ontario Early Researcher Award program (JPJ) and the Canada Research Chair program (JPJ). The Synergy Neo2 Multi636 Mode Assay Microplate Reader instrument was accessed at the Structural and Biophysical Core Facility, The Hospital for Sick Children, supported by the Canada Foundation for Innovation and Ontario Research FundN

Behavioral and Socioeconomic Risk Factors Associated with Probable Resistance to Ceftriaxone and Resistance to Penicillin and Tetracycline in <i>Neisseria gonorrhoeae</i> in Shanghai

<div><p>Globally, incidence of <i>Neisseria gonorrhoeae</i> infection is once again the highest of the bacterial sexually transmitted infections. The bacterium can produce serious complications in those infected, and emerging resistance to third generation cephalosporins could usher in an era of potentially untreatable gonorrhea. This research aimed to identify risk factors for antibiotic resistant gonorrhea infection among clients at a Shanghai sexually transmitted infection clinic over two time periods, 2004–2005 and 2008–2011. Demographic and risk factor behavior data, and biological samples for antimicrobial resistance analysis, were collected. Statistical models were built to identify risk factors associated with probable resistance to ceftriaxone and resistance to penicillin and tetracycline. High levels of ciprofloxacin resistance (98%) in our sample precluded examining its risk factors; all isolates were susceptible to spectinomycin. Overall (P<0.001), chromosomal (P<0.001), and plasmid-mediated (P = 0.01) penicillin resistance decreased from the first to second period of the study. For tetracycline, chromosomal resistance decreased (P = 0.01) and plasmid-mediated resistance increased (P<0.001) between the first and second periods of study. In multi-level multivariable regression models, male gender (P = 0.03) and older age (P = 0.01) were associated with increased minimum inhibitory concentrations to ceftriaxone. Male gender (P = 0.03) and alcohol use (P = 0.02) were associated with increased odds of overall tetracycline resistance. Male gender was associated with increased odds of chromosomally-mediated tetracycline resistance (P = 0.04), and alcohol use was associated with increased odds of plasmid-mediated tetracycline resistance (P = 0.02). Additionally, individuals in middle-salary categories were found to have lower odds of plasmid-mediated resistance to tetracycline compared with those in the lowest salary category (P≤0.02). This study is one of the first to use multilevel analysis to consider the association between risk factors for gonorrhea infections and mechanisms of resistance to individual antibiotics. Such information is urgently needed to combat the growing threat of untreatable gonorrhea.</p></div

Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using nonspecific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high-throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.This research was funded (S.I. and R.A.M.) from Fast Grants, part of the Emergent Ventures Program at the Mercatus Centre at George Mason University, with support from Thistledown Foundation. This research was supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant 790012 (E.R.). This work was further supported by the CIFAR Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Award program (J.-P.J.) and the Canada Research Chair program (J.-P.J.). The Synergy Neo2 Multi-Mode Assay Microplate Reader instrument was accessed at the Structural and Biophysical Core Facility, The Hospital for Sick Children, supported by the Canada Foundation for Innovation and Ontario Research Fund.Peer reviewe

Results of multivariable analysis of factors associated with resistance to penicillin based on MIC classification and type of penicillin resistance based on molecular data (odds ratios (OR) and 95% confidence intervals (95% CI)).

<p>Phase of study was the only significant predictor of penicillin outcomes.</p