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1.
Emerg Infect Dis ; 27(9): 2369-2378, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34424175

RESUMO

Azithromycin-nonsusceptible Neisseria gonorrhoeae strains are an emerging global public health threat. During 2015-2018, the prevalence of azithromycin-nonsusceptible gonococcal infection increased significantly in Argentina. To investigate the genomic epidemiology and resistance mechanisms of these strains, we sequenced 96 nonsusceptible isolates collected in Argentina during 2005-2019. Phylogenomic analysis revealed 2 main clades, which were characterized by a limited geographic distribution, circulating during January 2015-November 2019. These clades included the internationally spreading multilocus sequence types (STs) 1580 and 9363. The ST1580 isolates, which had MICs of 2-4 µg/mL, had mutations in the 23S rRNA. The ST9363 isolates, which had MICs of 2-4 or >256 µg/mL, had mutations in the 23S rRNA, a mosaic mtr locus, or both. Identifying the geographic dissemination and characteristics of these predominant clones will guide public health policies to control the spread of azithromycin-nonsusceptible N. gonorrhoeae in Argentina.


Assuntos
Azitromicina , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Argentina/epidemiologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana , Genômica , Neisseria gonorrhoeae/genética
2.
Euro Surveill ; 26(31)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34355690

RESUMO

We monitored antimicrobial susceptibility developments of Neisseria gonorrhoeae in Germany from January 2014 to May 2021. The proportion of isolates with azithromycin minimum inhibitory concentrations above the epidemiological cut-off increased substantially, from 1.3% in 2014 to 12.2% in 2020. Preliminary data from 2021 showed a further rise (January to May: 20.7%). Therefore, azithromycin as part of the recommended dual therapy in Germany for non-adherent patients is challenged. Antimicrobial susceptibility testing in clinical practice is crucial and continuous susceptibility surveillance indispensable.


Assuntos
Gonorreia , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Farmacorresistência Bacteriana , Alemanha/epidemiologia , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genética
3.
Beijing Da Xue Xue Bao Yi Xue Ban ; 53(4): 785-788, 2021 Aug 18.
Artigo em Chinês | MEDLINE | ID: mdl-34393245

RESUMO

OBJECTIVE: To investigate the value of clinical application of simultaneous amplification and testing of RNA (SAT-RNA) for detecting Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) by comparing with the polymerase chain reaction testing of DNA (PCR-DNA) method. METHODS: Specimens from both urethra swab and the first avoid urine which should be at least one hour after the previous urination were collected from 163 men who were scheduled for in vitro fertilization and embryo transfer (IVF-ET) treatment due to female factors at Center for Reproductive Medicine, Shengjing Hospital of China Medical University during the period of April 2016 to April 2017. Among the 163 men, 109 simultaneously provided semen that was collected after 3-7 days of sexual abstinence for the testing. Urine and semen specimens were detected for CT and UU with SAT-RNA, while urethra swab specimens were detected for CT and UU with standard PCR-DNA. Detection results of the SAT-RNA were compared with those of the PCR-DNA method. RESULTS: The positive rate of UU in the urethra swab detected with PCR-DNA and that of UU in the urine with SAT-RNA were 47.24% and 47.85%, respectively, and the coincidence rate was 93.25%. In addition, the positive and negative coincidence rates were 93.51% and 93.02%, respectively, and the concordance between the two methods was very good (Kappa=0.865). On the other hand, the positive rate of CT in the swab specimen tested with PCR-DNA was 3.07% and that of CT in urine with SAT-RNA was 4.29%, and the coincidence rate was 97.55%. Moreover, the positive and negative coincidence rates were 80.00% and 98.10%, respectively, and the concordance between the two methods was good (Kappa=0.654). Regarding SAT-RNA detection of UU in the urine and semen specimen of the 109 patients, the positive rates of UU in the urine and semen specimens were 50.46% and 44.95%, respectively; and the coincidence rate between the two specimens was 88.99%. In addition, the positive coincidence rate and the negative coincidence rate was 93.88% and 85.00%, respectively, and the concordance between the two specimens was good (Kappa=0.780). Similarly, SAT-RNA detection of CT in the urine and semen specimens showed the positive rate was 5.50% and 3.67%, respectively; and the two specimens showed 98.17% coincidence rate. The positive and negative coincidence rates were 100.00% and 98.10%, respectively, and the concordance was also good (Kappa=0.791). CONCLUSION: SAT-RNA detection of CT and UU in the urine specimen showed good concordance with the PCR-DNA detection of CT and UU in the urethra swab specimen. In addition, the concordance was also good between the urine and semen specimens detected with SAT-RNA. These results indicate that, as a less invasive and equally accurate procedure, SAT-RNA may be more suitable for clinical application.


Assuntos
Infecções por Chlamydia , Infertilidade Masculina , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/genética , Feminino , Humanos , Masculino , Neisseria gonorrhoeae/genética , Reação em Cadeia da Polimerase , Ureaplasma urealyticum/genética
4.
mBio ; 12(4): e0047321, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34281392

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic is a challenge for ongoing efforts to combat antimicrobial-resistant (AMR) bacterial infections. As we learn more about COVID-19 disease and drug stewardship evolves, there is likely to be a lasting impact of increased use of antimicrobial agents and antibiotics, as well as a lack of consistent access to health care across many populations. Sexually transmitted infections have been underreported during the pandemic and are often caused by some of the most drug-resistant pathogens. In their recent article in mBio, Parzych et al. (E. M. Parzych, S. Gulati, B. Zheng, M. A. Bah, et al., mBio 12:e00242-21, 2021, https://doi.org/10.1128/mBio.00242-21) focus on protection against Neisseria gonorrhoeae infection via in vivo delivery of an antigonococcal DNA-encoded antibody that has been modified for increased complement activation. Nucleic acid approaches are highly adaptable and could be tremendously beneficial for personalized strategies to combat AMR pathogens.


Assuntos
Antibacterianos/uso terapêutico , Gestão de Antimicrobianos/métodos , COVID-19/patologia , Farmacorresistência Bacteriana Múltipla/genética , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , Gonorreia/tratamento farmacológico , Acesso aos Serviços de Saúde , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Medicina de Precisão , SARS-CoV-2
5.
Nat Commun ; 12(1): 3801, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155204

RESUMO

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935-1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.


Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Genes Bacterianos/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Alelos , Resistência às Cefalosporinas/efeitos dos fármacos , Resistência às Cefalosporinas/genética , Variação Genética , Genoma Bacteriano/genética , Gonorreia/epidemiologia , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/isolamento & purificação , Filogenia , Recombinação Genética , Análise de Sequência de DNA , Estados Unidos/epidemiologia
6.
Western Pac Surveill Response J ; 12(1): 17-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34094619

RESUMO

Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health and is of particular concern in the Western Pacific Region, where the incidence of gonorrhoea is high. The Antimicrobial Resistance Surveillance Program (ARSP) has been capturing information on resistant gonorrhoea since 1996, but genomic epidemiology studies on this pathogen are lacking in the Philippines. We sequenced the whole genomes of 21 N. gonorrhoeae isolates collected in 2013-2014 by ARSP. The multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance and relatedness among the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined. Ten of 21 isolates were resistant to penicillin, ciprofloxacin and tetracycline, due mainly to the presence of the blaTEM gene, the S91F mutation in the gyrA gene and the tetM gene, respectively. None of the isolates was resistant to ceftriaxone or cefixime. The concordance between phenotypic and genotypic resistance was 92.38% overall for five antibiotics in four classes. Despite the small number of isolates studied, they were genetically diverse, as shown by the sequence types, the N. gonorrhoeae multiantigen sequence typing types and the tree. Comparison with global genomes placed the Philippine genomes within global lineage A and led to the identification of an international transmission route. This first genomic survey of N. gonorrhoeae isolates collected by ARSP will be used to contextualize prospective surveillance. It highlights the importance of genomic surveillance in the Western Pacific and other endemic regions for understanding the spread of drug-resistant gonorrhoea worldwide.


Assuntos
Gonorreia/microbiologia , Neisseria gonorrhoeae/genética , Genômica , Gonorreia/epidemiologia , Humanos , Neisseria gonorrhoeae/isolamento & purificação , Filipinas/epidemiologia
7.
Sex Transm Dis ; 48(8): 536-541, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34110758

RESUMO

BACKGROUND: Investigation was undertaken to determine the genetic relatedness of Neisseria gonorrhoeae (NG) isolates of young (<25 years) heterosexuals of a potential outbreak from October 2017 to March 2019 in South-Limburg, the Netherlands. METHODS: Data and residual sample material of routine diagnostics were retrieved for outbreak cases (78/81), young heterosexuals at baseline (January 2016 to September 2017, n = 30), and men who have sex with men (2018, n = 47). Total DNA was isolated, and NG was genotyped using culture-free NG multiantigen sequence typing. Sanger sequence data were used to construct a phylogenetic tree. Cases of outbreak clusters were geographically mapped, and descriptive analyses were performed on patient characteristics, comparing these clusters. RESULTS: Outbreak investigation showed 81 cases of young heterosexuals between October 2017 and March 2019 (4.5 per month) compared with 30 between January 2016 and September 2017 (1.4 per month), which was considered as baseline. Culture-independent genotyping of NG was performed to assess the genetic relatedness, as only 21 outbreak cases were culture confirmed. This revealed 3 independent outbreak clusters G2 (n = 18), G13113 (n = 11), and GNewST (n = 24). None of the clusters were geographically linked or introduced by bridging with men who have sex with men networks. Number of sex partners reported by men and Chlamydia trachomatis coinfection were associated with clusters G2 and GNewST, respectively. CONCLUSIONS: Culture-independent typing proved to be essential to identify the 3 outbreak clusters. However, targeted interventions were difficult because information on sex partners was limited. Therefore, prospective culture-independent typing could be used for early outbreak detection and aid in transmission prevention.


Assuntos
Gonorreia , Minorias Sexuais e de Gênero , Surtos de Doenças , Genótipo , Gonorreia/epidemiologia , Heterossexualidade , Homossexualidade Masculina , Humanos , Masculino , Neisseria gonorrhoeae/genética , Países Baixos/epidemiologia , Filogenia , Estudos Prospectivos
8.
BMC Infect Dis ; 21(1): 559, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34118893

RESUMO

BACKGROUND: Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are significant public health concerns globally. Nearly no gonococcal AMR data are available from Central Asia, and no data from Kyrgyzstan has been published. We examined, for the first time, AMR and molecular epidemiology of N. gonorrhoeae isolates cultured in Kyrgyzstan in 2012 and 2017, in order to inform refinements of the Kyrgyz national gonorrhoea management guidelines. METHODS: N. gonorrhoeae isolates cultured in 2012 (n = 84) and 2017 (n = 72) in Kyrgyzstan were examined. MICs of nine antimicrobials were determined using Etest and, where available, clinical breakpoints from the EUCAST were applied. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. RESULTS: The overall resistance levels were high to ciprofloxacin (88.5%), tetracycline (56.9%), benzylpenicillin (39.1%), and kanamycin (4.7%). Resistance to cefixime (0.6%, n = 1 isolate), azithromycin (0.6%, n = 1), and gentamicin (0.6%, n = 1) was rare. No resistance to ceftriaxone or spectinomycin was found. However, the proportion of isolates with decreased susceptibility (MIC = 0.125 mg/L) to ceftriaxone and cefixime was 12.8 and 11.5%, respectively. Gonococcal isolates were assigned 69 sequence types, of which 52 (75.4%) were new. CONCLUSIONS: The gonococcal population in Kyrgyzstan in 2012 and 2017 showed a high genetic diversity. Ceftriaxone, 500-1000 mg, in combination with azithromycin 2 g or doxycycline, particularly when chlamydial infection has not been excluded, should be recommended as empiric first-line treatment. Spectinomycin 2 g could be an alternative treatment, and given with azithromycin 2 g if pharyngeal gonorrhoea has not been excluded. Fluoroquinolones, aminoglycosides, benzylpenicillin, or tetracyclines should not be used for empiric treatment of gonorrhoea in Kyrgyzstan. Timely updating and high compliance to national gonorrhoea treatment guidelines based on quality-assured AMR data is imperative. Expanded and improved gonococcal AMR surveillance in Kyrgyzstan is crucial.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Gonorreia/microbiologia , Neisseria gonorrhoeae , Humanos , Quirguistão , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem Molecular , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética
9.
Sex Transm Dis ; 48(8S): S78-S87, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33993166

RESUMO

BACKGROUND: The prevalence of Neisseria gonorrhoeae (GC) isolates with elevated minimum inhibitory concentrations to various antibiotics continues to rise in the United States and globally. Genomic analysis provides a powerful tool for surveillance of circulating strains, antimicrobial resistance determinants, and understanding of transmission through a population. METHODS: Neisseria gonorrhoeae isolates collected from the US Gonococcal Isolate Surveillance Project in 2018 (n = 1479) were sequenced and characterized. Whole-genome sequencing was used to identify sequence types, antimicrobial resistance profiles, and phylogenetic relationships across demographic and geographic populations. RESULTS: Genetic characterization identified that (1) 80% of the GC isolates were represented in 33 multilocus sequence types, (2) isolates clustered in 23 major phylogenetic clusters with select phenotypic and demographic prevalence, and (3) common antimicrobial resistance determinants associated with low-level or high-level decreased susceptibility or resistance to relevant antibiotics. CONCLUSIONS: Characterization of this 2018 Gonococcal Isolate Surveillance Project genomic data set, which is the largest US whole-genome sequence data set to date, sets the basis for future prospective studies, and establishes a genomic baseline of GC populations for local and national monitoring.


Assuntos
Anti-Infecciosos , Gonorreia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Genômica , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genética , Filogenia , Estudos Prospectivos , Estados Unidos/epidemiologia
10.
Sci Transl Med ; 13(593)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980576

RESUMO

Effective treatment of sexually transmitted infections (STIs) is limited by diagnostics that cannot deliver results rapidly while the patient is still in the clinic. The gold standard methods for identification of STIs are nucleic acid amplification tests (NAATs), which are too expensive for widespread use and have lengthy turnaround times. To address the need for fast and affordable diagnostics, we have developed a portable, rapid, on-cartridge magnetofluidic purification and testing (PROMPT) polymerase chain reaction (PCR) test. We show that it can detect Neisseria gonorrhoeae, the pathogen causing gonorrhea, with simultaneous genotyping of the pathogen for resistance to the antimicrobial drug ciprofloxacin in <15 min. The duplex test was integrated into a low-cost thermoplastic cartridge with automated processing of penile swab samples from patients using magnetic beads. A compact instrument conducted DNA extraction, PCR, and analysis of results while relaying data to the user via a smartphone app. This platform was tested on penile swab samples from sexual health clinics in Baltimore, MD, USA (n = 66) and Kampala, Uganda (n = 151) with an overall sensitivity and specificity of 97.7% (95% CI, 94.7 to 100%) and 97.6% (95% CI, 94.1 to 100%), respectively, for N. gonorrhoeae detection and 100% concordance with culture results for ciprofloxacin resistance. This study paves the way for delivering accessible PCR diagnostics for rapidly detecting STIs at the point of care, helping to guide treatment decisions and combat the rise of antimicrobial resistant pathogens.


Assuntos
Gonorreia , Doenças Sexualmente Transmissíveis , Baltimore , Gonorreia/diagnóstico , Gonorreia/tratamento farmacológico , Humanos , Neisseria gonorrhoeae/genética , Sensibilidade e Especificidade , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/tratamento farmacológico , Uganda
11.
ACS Infect Dis ; 7(6): 1833-1847, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33980014

RESUMO

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.


Assuntos
Proteínas de Bactérias , Proteínas de Membrana Transportadoras , Neisseria gonorrhoeae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Neisseria gonorrhoeae/genética
12.
Methods Mol Biol ; 2302: 101-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877625

RESUMO

Membrane proteins serve essential roles in all aspects of life and make up roughly one-third of all genomes from prokaryotes to eukaryotes. Their responsibilities include mediating cell signaling, nutrient import, waste export, cellular communication, trafficking, and immunity. For their critical role in many cellular processes, membrane proteins serve as targets for up to 50% of drugs currently on the market and remain primary targets in new therapeutics being developed. Despite their importance and abundance in nature, only ~1% of structures in the Protein Data Bank are of transmembrane proteins. This discrepancy can be directly attributed to the biochemical properties of membrane proteins and the difficulty in producing sufficient yields for structural studies or the difficulty in growing well-ordered crystals. Here, we present methods from our work that outline our general pipeline from cloning to structure determination of membrane proteins, with a focus on using X-ray crystallography, which still yields ~90% of all structures being deposited into the Protein Data Bank.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Neisseria gonorrhoeae/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Bases de Dados de Proteínas , Modelos Moleculares , Neisseria gonorrhoeae/genética , Dobramento de Proteína , Estrutura Secundária de Proteína
13.
Mol Biol (Mosk) ; 55(2): 289-304, 2021.
Artigo em Russo | MEDLINE | ID: mdl-33871442

RESUMO

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli , Trifosfato de Adenosina , DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA , Proteínas MutL/genética , Proteínas MutL/metabolismo , Neisseria gonorrhoeae/genética
14.
J Antimicrob Chemother ; 76(7): 1769-1775, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33930160

RESUMO

OBJECTIVES: To investigate the spread of ceftriaxone-resistant Neisseria gonorrhoeae lineages similar to strains H041 (2009) and FC428 (2015), we characterized 55 strains collected in 2013 from hospitals across Japan. METHODS: Susceptibility testing and whole-genome sequencing. RESULTS: Susceptibility rates were 58% for cefixime and 98% for ceftriaxone. The 55 strains were whole-genome sequenced and classified into nine MLST-STs. MLST-ST1901 was the most prevalent (n = 19) followed by MLST-ST7363 (n = 12) and MLST-ST7359 (n = 11). The most prevalent penA [encoding penicillin binding protein 2 (PBP2)] mosaic types, based on the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) scheme, were 10.001 (n = 20) followed by 34.001 (n = 13). The H041 and FC428 strains were not detected; however, a single ceftriaxone-resistant strain (TUM15748) with a MIC of 0.5 mg/L ceftriaxone was identified. The TUM15748 strain belonged to MLST-ST7359 and N. gonorrhoeae multiantigen sequence typing-ST6771, and had a novel PBP2 (PBP2TUM15748, penA type 169.001). The amino acid sequence of PBP2TUM15748 showed partial similarity to that of PBP2 from N. gonorrhoeae GU140106 and commensal Neisseria perflava and Neisseria cinerea. Natural transformation and recombination experiments using full-length TUM15748 penA showed that the ceftriaxone MICs of transformants increased 16-fold or more compared with the parental ceftriaxone-susceptible recipient strain (NG9807, belonging to MLST-ST7363). No ceftriaxone-resistant MLST-ST7359 strains have previously been reported. CONCLUSIONS: We showed here that a ceftriaxone-susceptible lineage acquired a mutant PBP2 mosaic type, integrating partial PBP2 sequences from commensal Neisseria species, resulting in the emergence of ceftriaxone-resistant strains.


Assuntos
Gonorreia , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ceftriaxona/farmacologia , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria , Neisseria gonorrhoeae/genética
15.
J Clin Microbiol ; 59(5)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33658263

RESUMO

Resistance-guided therapy (RGT) for gonorrhea may reduce unnecessary use of broad-spectrum antibiotics. When reflexed from the Aptima Combo 2 assay, the ResistancePlus GC assay demonstrated 94.8% sensitivity and 100.0% specificity for Neisseria gonorrhoeae detection. Of the 379 concordant N. gonorrhoeae-positive samples, 86.8% were found to possess the gyrA S91F mutation, which was highly predictive for ciprofloxacin resistance and stable across 3,144 publicly available N. gonorrhoeae genomes. Our work supports the feasibility of implementing RGT for gonorrhea into routine molecular workflows.


Assuntos
Gonorreia , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Gonorreia/diagnóstico , Gonorreia/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genética , Reflexo
16.
Mol Biotechnol ; 63(6): 491-501, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33763825

RESUMO

2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2R,3R)-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2R,3R)-2,3-BD or meso-2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3R/3S)-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2R,3R)-2,3-BD (Km = 0.16 mM, kcat/Km = 673 s-1 · mM-1) and meso-BD (Km = 0.72 mM, kcat/Km = 165 s-1 · mM-1). Moreover, it could also reduce (3R/3S)-acetoin with a Km of 0.14 mM and a kcat/Km of 885 s-1 · mM-1. The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Gonorreia/enzimologia , Neisseria gonorrhoeae/genética , Acetoína/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos/genética , Butileno Glicóis/metabolismo , Clonagem Molecular , Escherichia coli/genética , Gonorreia/microbiologia , Humanos , Cinética , NAD/genética , Neisseria gonorrhoeae/enzimologia , Especificidade por Substrato , Zinco/química
17.
Nucleic Acids Res ; 49(7): 4155-4170, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33784401

RESUMO

Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5'-MCRTRCRN4YGYAYGK-3'. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.


Assuntos
Proteínas de Bactérias , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Neisseria gonorrhoeae , Proteínas Repressoras , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Mutação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
18.
Nat Commun ; 12(1): 1799, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741965

RESUMO

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.


Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Células CACO-2 , Feminino , Gonorreia/microbiologia , Gonorreia/prevenção & controle , Humanos , Camundongos , Neisseria gonorrhoeae/genética , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
19.
J Antimicrob Chemother ; 76(7): 1759-1768, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33739419

RESUMO

BACKGROUND: Emerging resistance to cephalosporins in Neisseria gonorrhoeae (Ng) is a major public health threat, since these are considered antibiotics of last resort. Continuous surveillance is needed to monitor the circulation of resistant strains and those with reduced susceptibility. OBJECTIVES: For the purpose of epidemiological surveillance, genomic population analysis was performed on Ng isolates from Amsterdam with a focus on isolates with reduced susceptibility to ceftriaxone. METHODS: WGS data were obtained from 318 isolates from Amsterdam, the Netherlands between 2014 and 2019. Isolates were typed according to MLST, Ng Multi-Antigen Sequence Typing (NG-MAST) and Ng Sequence Typing for Antimicrobial Resistance (NG-STAR) schemes and additional resistance markers were identified. Phylogenetic trees were created to identify genetic clusters and to compare Dutch and non-Dutch MLST7827 isolates. RESULTS: MLST7363 and MLST1901 were the predominant strains having reduced susceptibility to ceftriaxone during 2014-16; MLST7827 emerged and dominated during 2017-19. NG-STAR38 and NG-MAST2318/10386 were predominant among MLST7827 isolates. MLST7827 reduced susceptibility isolates carried a non-mosaic 13.001 penA allele with an A501V mutation and porB1b G120K/A121D mutations, which were lacking in susceptible MLST7827 isolates. Phylogenetic analysis of all publicly available MLST7827 isolates showed strong genetic clustering of Dutch and other European MLST7827 isolates. CONCLUSIONS: MLST7827 isolates with reduced ceftriaxone susceptibility have emerged during recent years in Amsterdam. Co-occurrence of penA A501V and porB1b G120K/A121D mutations was strongly associated with reduced susceptibility to ceftriaxone. Genetic clustering of Dutch and other European MLST7827 isolates indicates extensive circulation of this strain in Europe. Close monitoring of the spread of this strain having an alarming susceptibility profile is needed.


Assuntos
Gonorreia , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Cefalosporinas/farmacologia , Células Clonais , Farmacorresistência Bacteriana/genética , Europa (Continente) , Genômica , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/genética , Países Baixos/epidemiologia , Filogenia
20.
J Antimicrob Chemother ; 76(7): 1752-1758, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33760080

RESUMO

BACKGROUND: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. OBJECTIVES: To characterize the genetic pathways leading to high-level azithromycin resistance. METHODS: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. RESULTS: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. CONCLUSIONS: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.


Assuntos
Azitromicina , Neisseria gonorrhoeae , Antibacterianos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Mutação , Neisseria gonorrhoeae/genética , RNA Ribossômico 23S/genética
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