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1.
BMC Infect Dis ; 20(1): 703, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32977759

RESUMO

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Neisseria gonorrhoeae/genética , Penicilinas/uso terapêutico , Plasmídeos/genética , Resistência a Tetraciclina/genética , Tetraciclina/uso terapêutico , DNA Bacteriano/genética , Feminino , Genótipo , Gonorreia/microbiologia , Humanos , Quênia/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/isolamento & purificação , Sequenciamento Completo do Genoma , beta-Lactamases/genética
2.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807804

RESUMO

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Assuntos
Proteínas de Bactérias/metabolismo , Colo do Útero/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Animais , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria gonorrhoeae/metabolismo , Óperon/genética , Regiões Promotoras Genéticas/genética
3.
BMC Infect Dis ; 20(1): 486, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641070

RESUMO

BACKGROUND: Asymptomatic screening for gonorrhoea in heterosexual men is currently not recommended in many countries including Australia, given the prevalence is relatively low in the heterosexual population. We aimed to determine the proportion of urethral gonorrhoea cases among heterosexual men attending a sexual health clinic that was asymptomatic and symptomatic, the time since last sexual contact to the onset of symptoms and the time to clinic presentation following the onset of symptoms. METHODS: This was a cross-sectional study that included heterosexual men aged 16 years or above attending the Melbourne Sexual Health Centre (MSHC) in Australia between August 2017 and August 2018. Gonorrhoea cases were diagnosed by nucleic acid amplification testing (NAAT) and/or culture. Descriptive analyses were conducted for all gonorrhoea cases including demographic characteristics, recent sexual practices, reported urethral symptoms and duration, sexual contact with a person diagnosed with gonorrhoea, investigations performed and laboratory results. RESULTS: There were 116 confirmed cases of urethral gonorrhoea in heterosexual men over the study period of which 6.0% (95% CI: 2.7-12.1%) were asymptomatic. Typical urethral discharge was present in 80.2% (95% CI: 71.9-86.5%) of men. The mean time between last sexual contact and the onset of symptoms was 7.0 days, and between the onset of symptoms to presentation to the clinic was 5.6 days. CONCLUSIONS: A small proportion of heterosexual men with urethral gonorrhoea do not have any symptoms. Heterosexual men with urethral symptoms usually seek for healthcare within a week, prompting rapid healthcare-seeking behaviour.


Assuntos
Infecções Assintomáticas/epidemiologia , Gonorreia/diagnóstico , Gonorreia/epidemiologia , Heterossexualidade , Neisseria gonorrhoeae/genética , Saúde Sexual , Doenças Uretrais/diagnóstico , Doenças Uretrais/epidemiologia , Adolescente , Adulto , Austrália/epidemiologia , Estudos Transversais , Gonorreia/microbiologia , Gonorreia/fisiopatologia , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico/métodos , Prevalência , Doenças Uretrais/microbiologia , Adulto Jovem
4.
BMC Infect Dis ; 20(1): 419, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546213

RESUMO

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos , Candida/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Infecções por Chlamydia/microbiologia , Reações Cruzadas , Feminino , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/genética , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Trichomonas vaginalis/genética
5.
BMC Infect Dis ; 20(1): 375, 2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32460721

RESUMO

BACKGROUND: Sexually transmitted infections (STIs) cause a major public health problem that affect both men and women in developing and developed countries. The aim of the study was to estimate the prevalence of 11 STIs among women who voluntarily participated in the study, while seeking gynecological checkup. The existence of an association between the presence of pathogens and symptoms and various sociodemographic risk factors was assessed. METHODS: A total of 505 vaginal and cervical specimens were collected from women above 18 years of age, with or without symptoms related to gynecological infections. Nucleic acid was extracted and samples were tested by real-time PCR for the following pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis, Candida albicans and Human Papillomavirus (HPV). Positive HPV samples underwent genotyping using a microarray system. RESULTS: Of the 505 samples, 312 (62%) were screened positive for at least one pathogen. Of these, 36% were positive for Gardnerella vaginalis, 35% for Ureaplasma parvum, 8% for Candida albicans, 6.7% for HPV, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis, 2% for Trichomonas vaginalis, 0.8% for Chlamydia trachomatis, 0.4% for Mycoplasma girerdii, 0.2% for Mycoplasma genitalium and 0.2% for Neisseria gonorrhoeae. Lack of symptoms was reported in 187 women (37%), among whom 61% were infected. Thirty-four samples were HPV positive, with 17 high risk HPV genotypes (HR-HPV); the highest rates being recorded for types 16 (38%), 18 (21%) and 51 (18%). Out of the 34 HPV positives, 29 participants had HR-HPV. Association with various risk factors were reported. CONCLUSIONS: This is the first study that presents data about the presence of STIs among women in Lebanon and the MENA region by simultaneous detection of 11 pathogens. In the absence of systematic STI surveillance in Lebanon, concurrent screening for HPV and PAP smear is warranted.


Assuntos
Doenças Sexualmente Transmissíveis/epidemiologia , Adulto , Colo do Útero/microbiologia , Colo do Útero/parasitologia , Colo do Útero/virologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Estudos Transversais , Feminino , Gardnerella vaginalis/genética , Gardnerella vaginalis/isolamento & purificação , Humanos , Líbano/epidemiologia , Masculino , Epidemiologia Molecular , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Mycoplasma hominis/genética , Mycoplasma hominis/isolamento & purificação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Fatores de Risco , Doenças Sexualmente Transmissíveis/microbiologia , Doenças Sexualmente Transmissíveis/parasitologia , Doenças Sexualmente Transmissíveis/virologia , Trichomonas vaginalis/genética , Trichomonas vaginalis/isolamento & purificação , Ureaplasma/genética , Ureaplasma/isolamento & purificação , Vagina/microbiologia , Vagina/parasitologia , Vagina/virologia , Esfregaço Vaginal , Adulto Jovem
6.
Proc Natl Acad Sci U S A ; 117(21): 11692-11702, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393643

RESUMO

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.


Assuntos
Proteínas de Bactérias , Endopeptidases , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Mutação/genética , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Conformação Proteica , Vibrio cholerae/enzimologia , Vibrio cholerae/genética
7.
Sex Transm Infect ; 96(6): 402-407, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32447324

RESUMO

OBJECTIVES: Test of cure (TOC) for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) infection is an important tool in the public health management of STIs. However, there are limited data about the optimal time to perform TOC using nucleic acid amplification tests (NAATs) for NG and CT infections. A study was performed to assess the feasibility of a larger study to determine the optimal time to TOC using NAATS. METHODS: The Sexually Transmitted Bacteria Reference Unit at Public Health England undertook testing of gonococcal and chlamydial nucleic acids within neat urine stored in different conditions over 25 days to provide evidence of the stability of the nucleic acid prior to recruitment. Individuals diagnosed with uncomplicated NG or CT infection were recruited from three sexual health clinics. Individuals were asked to return nine self-taken samples from the site of infection over a course of 35 days. Survival analyses of time to first negative NAAT result for NG and CT infection and univariate regression analysis of factors that affect time to clearance were undertaken. RESULTS: At room temperature, chlamydial DNA in urine is stable for up to 3 weeks and gonococcal DNA for up to 11 days. We analysed data for 147 infections (81 NG and 66 CT). The median time to clearance of infection was 4 days (IQR 2-10 days) for NG infection and 10 days (IQR 7-14 days) for CT infection. Vaginal CT infections took longer to clear (p=0.031). NG infection in men who have sex with men took longer to clear (p=0.052). CONCLUSION: Chlamydial and gonococcal nucleic acids are stable in urine before addition of preservatives, longer than recommended by the manufacturer. The TOC results suggest that it may be possible to undertake TOC for NG and CT infections earlier than current guidelines suggest and that anatomical site of infection may affect time to clearance of infection.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/tratamento farmacológico , Gonorreia/diagnóstico , Gonorreia/tratamento farmacológico , Adulto , Idoso , Azitromicina/uso terapêutico , Ceftriaxona/uso terapêutico , Chlamydia trachomatis/genética , Doxiciclina/uso terapêutico , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/genética , Técnicas de Amplificação de Ácido Nucleico , Faringite/diagnóstico , Faringite/tratamento farmacológico , Proctite/diagnóstico , Proctite/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Resultado do Tratamento , Uretrite/diagnóstico , Uretrite/tratamento farmacológico , Vulvovaginite/diagnóstico , Vulvovaginite/tratamento farmacológico , Adulto Jovem
8.
APMIS ; 128(7): 465-475, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32441045

RESUMO

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major health threat compromising the gonorrhoea treatment globally. AMR surveillance including whole genome sequencing (WGS)-based epidemiology provides ideal resolution to identify and describe AMR gonococcal clones, AMR determinants and populations, which can inform management guidelines and antimicrobial stewardship policies. Our aims were to, for the first time, elucidate the WGS-based epidemiology and characterize AMR determinants of gonococcal strains spreading in Ukraine, 2013-2018. Gonococcal isolates (n = 150) from Ternopil and Dnipro, Ukraine (2013-2018), were subjected to AMR testing (Etest) for eight antimicrobials and WGS. Overall, 11.3% of isolates were resistant to ciprofloxacin, 6.0% to tetracycline, and 0.7% to benzylpenicillin. No isolates were resistant to azithromycin, spectinomycin, ceftriaxone, or cefixime, but one isolate was bordering resistance to both cephalosporins. Twenty-five MLST STs, 50 NG-MAST STs, and 34 NG-STAR types were identified. The phylogenomic analysis revealed six main clusters, mostly associated with the internationally described multidrug-susceptible gonococcal lineage. Resistance to ciprofloxacin was associated with GyrA S91F and ParC S87R mutations; tetracyclines with rpsJ V57M and tetM; penicillins with mosaic penA-34.001 and ß-lactamase; mtrR; PorB1b G101D, and PBP1 L421P mutations. One isolate of the multidrug-resistant NG-MAST ST1407, MLST ST1901 was found, which was bordering resistance to ceftriaxone and cefixime. The antimicrobial susceptibility of gonococcal strains spreading in Ternopil and Dnipro, Ukraine, in 2013-2018 was surprisingly high. Continued and expanded gonococcal AMR surveillance, ideally including WGS, in Ukraine is essential. This could inform action plans and public health policies to control the spread of AMR gonococcal strains in Ukraine.


Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Farmacorresistência Bacteriana/genética , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Neisseria gonorrhoeae/classificação , Filogenia , Fatores de Tempo , Ucrânia , Sequenciamento Completo do Genoma , Adulto Jovem
9.
J Vis Exp ; (157)2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32202529

RESUMO

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.


Assuntos
Regulação Bacteriana da Expressão Gênica , Metais/metabolismo , Metais/farmacologia , Neisseria gonorrhoeae/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Humanos , Ferro/metabolismo , Ligantes , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Zinco/metabolismo
11.
APMIS ; 128(6): 440-444, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32202687

RESUMO

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection globally, and nucleic acid amplification tests (NAATs) are recommended for highly sensitive and specific diagnosis. In early 2019, the Finnish new variant of Chlamydia trachomatis (FI-nvCT) was identified. The FI-nvCT has a C1515T mutation in the 23S rRNA gene, making it escaping detection in the Aptima Combo 2 (AC2; Hologic) NAAT, and the FI-nvCT has been subsequently reported in Sweden and Norway. In the present study, we investigated the presence of the FI-nvCT and other AC2 diagnostic-escape CT mutants in July-September 2019 in Denmark. The FI-nvCT was present but rare in Denmark. However, another AC2 diagnostic-escape CT mutant (with a 23S rRNA G1523A mutation) was found to be widespread across Denmark, accounting for 95% (76/80) of AC2 diagnostic-escape nvCT samples from five Danish CT-diagnostic laboratories. This nvCT-G1523A has previously only been detected in one single sample in the United Kingdom and Norway, respectively. It is vital to monitor the continued stability of the NAAT targets in local, national and international settings and monitor as well as appropriately analyse incidence, unexplained shifts in diagnostics rates and/or annual collections of samples diagnosed as negative/equivocal using NAATs with different target(s). Furthermore, diagnostic CT NAATs with dual target sequences are crucial, and fortunately, an updated Hologic AC2 assay including one additional target sequence is in advanced development.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Ribossômico 23S/genética , Análise de Sequência de RNA/métodos , Infecções por Chlamydia/microbiologia , Dinamarca/epidemiologia , Feminino , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Masculino , Neisseria gonorrhoeae/genética , RNA Bacteriano/genética , Sensibilidade e Especificidade
12.
Emerg Microbes Infect ; 9(1): 517-519, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32116136

RESUMO

We report a recent (2018) gonorrhoeal urethritis caused by a multidrug-resistant Neisseria gonorrhoeae strain in China. The isolated N. gonorrhoeae strain from a male and female pair expressed high-level resistance to spectinomycin (SPC), azithromycin, and other antibiotics but was sensitive to ceftriaxone. The SPC high-level resistance (MIC = 2048 mg/L) was due to a small deletion in rspE that caused two amino acid changes in ribosomal protein S5.


Assuntos
Farmacorresistência Bacteriana Múltipla , Neisseria gonorrhoeae/efeitos dos fármacos , Espectinomicina/farmacologia , China , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Fenótipo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
13.
Nat Microbiol ; 5(3): 455-464, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32042129

RESUMO

Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empirical antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could affect patient treatment and outcomes. Here, we present a method called 'genomic neighbour typing' for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both Streptococcus pneumoniae and Neisseria gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in the determination of resistance within 10 min (91% sensitivity and 100% specificity for S. pneumoniae and 81% sensitivity and 100% specificity for N. gonorrhoeae from isolates with a representative database) of starting sequencing, and within 4 h of sample collection (75% sensitivity and 100% specificity for S. pneumoniae) for clinical metagenomic sputum samples. This flexible approach has wide application for pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment.


Assuntos
Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Genômica , Bases de Dados Factuais , Humanos , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Fenótipo , Sensibilidade e Especificidade , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação
14.
BMC Genomics ; 21(1): 116, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32013864

RESUMO

BACKGROUND: Multidrug-resistant Neisseria gonorrhoeae strains are prevalent, threatening gonorrhoea treatment globally, and understanding of emergence, evolution, and spread of antimicrobial resistance (AMR) in gonococci remains limited. We describe the genomic evolution of gonococci and their AMR, related to the introduction of antimicrobial therapies, examining isolates from 1928 (preantibiotic era) to 2013 in Denmark. This is, to our knowledge, the oldest gonococcal collection globally. METHODS: Lyophilised isolates were revived and examined using Etest (18 antimicrobials) and whole-genome sequencing (WGS). Quality-assured genome sequences were obtained for 191 viable and 40 non-viable isolates and analysed with multiple phylogenomic approaches. RESULTS: Gonococcal AMR, including an accumulation of multiple AMR determinants, started to emerge particularly in the 1950s-1970s. By the twenty-first century, resistance to most antimicrobials was common. Despite that some AMR determinants affect many physiological functions and fitness, AMR determinants were mainly selected by the use/misuse of gonorrhoea therapeutic antimicrobials. Most AMR developed in strains belonging to one multidrug-resistant (MDR) clade with close to three times higher genomic mutation rate. Modern N. gonorrhoeae was inferred to have emerged in the late-1500s and its genome became increasingly conserved over time. CONCLUSIONS: WGS of gonococci from 1928 to 2013 showed that no AMR determinants, except penB, were in detectable frequency before the introduction of gonorrhoea therapeutic antimicrobials. The modern gonococcus is substantially younger than previously hypothesized and has been evolving into a more clonal species, driven by the use/misuse of antimicrobials. The MDR gonococcal clade should be further investigated for early detection of strains with predispositions to develop and maintain MDR and for initiation of public health interventions.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Evolução Molecular , Genômica/métodos , Genótipo , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/isolamento & purificação , Filogenia , Sequenciamento Completo do Genoma/métodos
15.
Biochemistry ; 59(10): 1104-1112, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32078293

RESUMO

Neisseria gonorrhoeae possesses a programmed recombination system that allows the bacteria to alter the major subunit of the type IV pilus, pilin or PilE. An alternate DNA structure known as a guanine quadruplex (G4) is required for pilin antigenic variation (pilin Av). The G-C base pairs within the G4 motif are required for pilin Av, but simple mutation of the loop bases does not affect pilin Av. We show that more substantial changes to the loops, in both size and nucleotide composition, with the core guanines unchanged, decrease or abrogate pilin Av. We investigated why these loop changes might influence the efficiency of pilin Av. RecA is a recombinase required for pilin Av that can bind the pilE G4 in vitro. RecA binds different G4 structures with altered loops with varied affinities. However, changes in RecA binding affinities to the loop mutants do not absolutely correlate with the pilin Av phenotypes. Interestingly, the yeast RecA ortholog, Rad51, also binds the pilE G4 structure with a higher affinity than it binds single-stranded DNA, suggesting that RecA G4 binding is conserved in eukaryotic orthologs. The thermal stability the pilE G4 structure and its loop mutants showed that the parental G4 structure had the highest melting temperature, and the melting temperature of the loop mutants correlated with pilin Av phenotype. These results suggest that the folding kinetics and stability of G4 structures are important for the efficiency of pilin Av.


Assuntos
Proteínas de Fímbrias/metabolismo , Neisseria gonorrhoeae/genética , Variação Antigênica/genética , Pareamento de Bases/genética , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Quadruplex G , Guanina/metabolismo , Mutação/genética , Neisseria gonorrhoeae/imunologia , Ligação Proteica/genética , Recombinação Genética/genética
16.
Gastroenterology ; 158(6): 1546-1547, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017908

Assuntos
Síndrome de Imunodeficiência Adquirida/complicações , Coinfecção/diagnóstico , Granuloma/diagnóstico , Proctite/diagnóstico , Doenças Sexualmente Transmissíveis/diagnóstico , Síndrome de Imunodeficiência Adquirida/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida/imunologia , Antibacterianos/administração & dosagem , Antivirais/administração & dosagem , Ceftriaxona/administração & dosagem , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Coinfecção/tratamento farmacológico , Coinfecção/imunologia , Coinfecção/microbiologia , Colonoscopia , Citomegalovirus/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Doxiciclina/administração & dosagem , Quimioterapia Combinada/métodos , Granuloma/tratamento farmacológico , Granuloma/imunologia , Granuloma/microbiologia , Humanos , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Proctite/tratamento farmacológico , Proctite/imunologia , Proctite/microbiologia , Reto/diagnóstico por imagem , Reto/microbiologia , Reto/patologia , Doenças Sexualmente Transmissíveis/tratamento farmacológico , Doenças Sexualmente Transmissíveis/imunologia , Doenças Sexualmente Transmissíveis/microbiologia , Resultado do Tratamento , Valganciclovir/administração & dosagem
17.
Mikrobiyol Bul ; 54(1): 135-143, 2020 Jan.
Artigo em Turco | MEDLINE | ID: mdl-32050884

RESUMO

Sexually transmitted infections (STIs) are important as a public health problem all over the world. There are some difficulties in prevention and control programs of STIs due to clinical and laboratory diagnostic problems.The most common STIs are Chlamydia trachomatis infections, trichomoniasis and gonorrhea. The study aimed to investigate the direct microscopic examination, culture and polymerase chain reaction (PCR) tests in the diagnosis of Trichomonas vaginalis infection; to determine other microbiological agents that may cause vaginal discharge and to evaluate the various social variables in women with vaginal discharge admitted to the outpatient clinic of Obstetrics and Gynecology in Akdeniz University Hospital. Two hundred and fifteen patients were enrolled in the study. The socio-demographic features of the patients were recorded. Vaginal/endocervical swab specimens taken from patients were evaluated by microscopic examination. Swab specimens were inoculated into blood agar, MacConkey agar and chocolate agar for bacterial culture. Modified Trichosel broth with 5% horse blood (Becton Dickinson, USA) was used for Trichomonas spp. culture. The presence of C.trachomatis, Neisseria gonorrhoeae, and T.vaginalis in swab samples were investigated by multiplex PCR assay (BD Max CT/GC/TV, Becton Dickinson, USA). At least one pathogen was detected among 65 (30.3%) samples. T.vaginalis was detected by microscopic examination and PCR in four of 215 (1.9%) patients. Existence of yeast morphology was observed in 21 (9.8%) specimens by microscopic examination. Twenty four (11.2%) patients were diagnosed as bacterial vaginosis microscopically according to Nugent score system. Candida species grew in 32 (14.9%) and Streptococcus agalactiae grew in 2 (0.9%) of the specimens. C.trachomatis was detected in 2 (0.9%) samples and N.gonorrhoeae in 1 (0.5%) sample by PCR. In this study, 95.3% of the patients were married and 96.7% had only one sexual partner in the mean time. The rate of detection of pathogens were statistically higher in women who have had two or more pregnancies (p<0.05). In our study, T.vaginalis together with N.gonorrhoeae and C.trachomatis were investigated by PCR method in women with vaginal discharge. The use of multiplex PCR test allowed simultaneous investigation of multiple pathogens in the patient samples.


Assuntos
Infecções por Chlamydia , Técnicas e Procedimentos Diagnósticos , Gonorreia , Tricomoníase , Vaginite por Trichomonas , Técnicas de Cultura de Células , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas e Procedimentos Diagnósticos/normas , Feminino , Gonorreia/diagnóstico , Humanos , Microscopia/normas , Reação em Cadeia da Polimerase Multiplex , Neisseria gonorrhoeae/genética , Gravidez , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Vaginite por Trichomonas/diagnóstico , Trichomonas vaginalis/genética
18.
PLoS One ; 15(2): e0228467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040516

RESUMO

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.


Assuntos
Neisseria meningitidis/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Uretrite/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Reações Falso-Positivas , Gonorreia/diagnóstico , Gonorreia/epidemiologia , Gonorreia/microbiologia , Gonorreia/urina , Humanos , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Doenças Bacterianas Sexualmente Transmissíveis/diagnóstico , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Doenças Bacterianas Sexualmente Transmissíveis/urina , Estados Unidos/epidemiologia , Uretra/microbiologia , Uretra/patologia , Uretrite/diagnóstico , Urinálise/métodos , Sequenciamento Completo do Genoma , Adulto Jovem
19.
BMC Infect Dis ; 20(1): 110, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033533

RESUMO

BACKGROUND: Men who have sex with men (MSM) are disproportionally affected by sexually transmitted infections (STI). STI are often extragenital and asymptomatic. Both can delay diagnosis and treatment. Approval of HIV pre-exposure prophylaxis (PrEP) might have influenced sexual behaviour and STI-prevalence of HIV- MSM. We estimated STI-prevalence and risk factors amongst HIV- and HIV+ MSM in Germany to plan effective interventions. METHODS: We conducted a nationwide, cross-sectional study between February and July 2018. Thirteen MSM-friendly STI-practices screened MSM for Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Neisseria gonorrhea (NG), and Trichomonas vaginalis (TV) using self-collected rectal and pharyngeal swabs, and urine samples. APTIMA™ STI-assays (Hologic™ Inc., San Diego, USA) were used for diagnostics, and samples were not pooled. We collected information on socio-demographics, HIV-status, clinical symptoms, sexual behaviour within the last 6 months, and PrEP use. We combined HIV status and PrEP use for defining risk groups, and used directed acyclic graphs and multivariable logistic regression to identify risk factors for STI. RESULTS: Two thousand three hundred three MSM were included: 50.5% HIV+, median age 39 [18-79] years. Median number of male sex partners within the last 6 months was five. Sex without condom was reported by 73.6%, use of party drugs by 44.6%. 80.3% had a STI history, 32.2% of STI+ MSM reported STI-related symptoms. 27.6% of HIV- MSM used PrEP. Overall STI-prevalence was 30.1, 25.0% in HIV-/PrEP- MSM (CT:7.2%; MG:14.2%; NG:7.4%; TV:0%), 40.3% in HIV-/PrEP+ MSM (CT:13.8%; MG:19.4%; NG:14.8%; TV:0.4%), and 30.8% in HIV+ MSM (CT:10.1%; MG:18.4%; NG:8.6%; TV:0.1%). Being HIV+ (OR 1.7, 95%-CI 1.3-2.2), using PrEP (OR 2.0, 95%-CI 1.5-2.7), having > 5 sex partners (OR:1.65; 95%-CI:1.32-2.01.9), having condomless sex (OR:2.11.9; 95%-CI:1.65-2.86), and using party drugs (OR:1.65; 95%-CI:1.32-2.0) were independent risk factors for being tested positive for at least one STI. CONCLUSIONS: We found a high STI-prevalence in MSM in Germany, especially in PrEP users, frequently being asymptomatic. As a relevant proportion of PrEP users will not use a condom, counselling and comprehensive STI screening is essential and should be low threshold and preferably free of cost. Counselling of PrEP users should also address use of party drugs.


Assuntos
Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/genética , Gonorreia/epidemiologia , Infecções por HIV/epidemiologia , Homossexualidade Masculina , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/genética , Neisseria gonorrhoeae/genética , Profilaxia Pré-Exposição , Minorias Sexuais e de Gênero , Adolescente , Adulto , Idoso , Chlamydia trachomatis/isolamento & purificação , Preservativos , Aconselhamento , Estudos Transversais , Alemanha/epidemiologia , Gonorreia/diagnóstico , Gonorreia/microbiologia , Infecções por HIV/prevenção & controle , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Mycoplasma genitalium/isolamento & purificação , Neisseria gonorrhoeae/isolamento & purificação , Prevalência , Fatores de Risco , Comportamento Sexual , Parceiros Sexuais , Adulto Jovem
20.
Lancet Infect Dis ; 20(4): 478-486, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31978353

RESUMO

BACKGROUND: Characterising sexual networks with transmission of sexually transmitted infections might allow identification of individuals at increased risk of infection. We aimed to investigate sexual mixing in Neisseria gonorrhoeae transmission networks between women, heterosexual men, and men who report sex with men (MSM), and between people with and without HIV. METHODS: In this cross-sectional observational study, we whole-genome sequenced N gonorrhoeae isolates from the archive of the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP).w Isolates that varied by five single nucleotide polymorphisms or fewer were grouped into clusters that represented sexual networks with N gonorrhoeae transmission. Clusters were described by gender, sexual risk group, and HIV status. FINDINGS: We sequenced 1277 N gonorrhoeae isolates with linked clinical and sociodemographic data that were collected in five clinics in England during 2013-16 (July 1 to Sept 30 in 2013-15; July 1 to Sept 9 in 2016). The isolates grouped into 213 clusters. 30 (14%) clusters contained isolates from heterosexual men and MSM but no women and three (1%) clusters contained isolates from only women and MSM. 146 (69%) clusters comprised solely people with negative or unknown HIV status and seven (3%) comprised only HIV-positive people. 60 (28%) clusters comprised MSM with positive and negative or unknown HIV status. INTERPRETATION: N gonorrhoeae molecular data can provide information indicating risk of HIV or other sexually transmitted infections for some individuals for whom such risk might not be known from clinical history. These findings have implications for sexual health care, including offering testing, prevention advice, and preventive treatment, such as HIV pre-exposure prophylaxis. FUNDING: National Institute for Health Research Health Protection Research Unit; Wellcome; Public Health England.


Assuntos
Gonorreia/epidemiologia , Gonorreia/transmissão , Infecções por HIV , Neisseria gonorrhoeae , Filogenia , Sequenciamento Completo do Genoma , Adulto , Estudos Transversais , Inglaterra/epidemiologia , Feminino , Gonorreia/microbiologia , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Heterossexualidade/estatística & dados numéricos , Homossexualidade Masculina/estatística & dados numéricos , Humanos , Masculino , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/genética , Parceiros Sexuais , Adulto Jovem
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