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1.
Int J Antimicrob Agents ; 54(3): 361-366, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202926

RESUMO

Penicillinase-producing Neisseria gonorrhoeae (PPNG) expressing the TEM ß-lactamase variant TEM-135 are a global public-health concern as this variant requires only a single amino acid substitution to develop into an extended-spectrum ß-lactamase. The aim of this study was to investigate the epidemiology of blaTEM-135 in 505 N. gonorrhoeae isolates from Hangzhou, China, during the periods 2011-2012 and 2015-2017. Investigation by nitrocefin test and mismatch amplification PCR showed that 41.0% (207/505) of the isolates were PPNG, of which 37.2% (77/207) contained the blaTEM-135 gene. Further PCR-based plasmid typing showed that blaTEM-135 was predominantly expressed from the Asian plasmid (94%). PPNG isolates consisted of three major clusters, namely Asian plasmid/blaTEM-135 (34.8%), Asian plasmid/blaTEM-1 (32.4%) and African plasmid/blaTEM-1 (28.0%), which showed significant differences in penicillin minimum inhibitory concentrations (MICs) determined by the agar dilution method. Representative isolates were investigated by quantitative real-time PCR (plasmid copy number and blaTEM gene expression), western blot analysis (TEM levels and TEM stability) and in vivo ß-lactamase activity assays to elucidate the cause of the observed differences in penicillin MIC. Overall, isolates of the Asian plasmid/blaTEM-135 cluster showed the highest ß-lactamase activity, which was explained by higher blaTEM gene expression (Asian versus African plasmid) and higher TEM stability (TEM-135 versus TEM-1). In conclusion, the blaTEM-135 gene is commonly present on the Asian plasmid in PPNG isolates from Hangzhou. The PPNG isolate cluster containing the Asian plasmid and blaTEM-135 showed the highest penicillin MICs, which might explain its abundance in the Hangzhou population.


Assuntos
Perfilação da Expressão Gênica , Gonorreia/microbiologia , Neisseria gonorrhoeae/enzimologia , Plasmídeos/análise , beta-Lactamases/análise , beta-Lactamases/genética , Western Blotting , China/epidemiologia , Gonorreia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genética , Plasmídeos/classificação , Reação em Cadeia da Polimerase , Prevalência
2.
Front Immunol ; 10: 137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30787927

RESUMO

Control of the sexually transmitted infection gonorrhea is a major public health challenge, due to the recent emergence of multidrug resistant strains of Neisseria gonorrhoeae, and there is an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study, we evaluated the methionine sulfoxide reductase (MsrA/B) of N. gonorrhoeae as a potential vaccine candidate, in terms of its expression, sequence conservation, localization, immunogenicity, and the functional activity of antibodies raised to it. Gonococcal MsrA/B has previously been shown to reduce methionine sulfoxide [Met(O)] to methionine (Met) in oxidized proteins and protect against oxidative stress. Here we have shown that the gene encoding MsrA/B is present, highly conserved, and expressed in all N. gonorrhoeae strains investigated, and we determined that MsrA/B is surface is exposed on N. gonorrhoeae. Recombinant MsrA/B is immunogenic, and mice immunized with MsrA/B and either aluminum hydroxide gel adjuvant or Freund's adjuvant generated a humoral immune response, with predominantly IgG1 antibodies. Higher titers of IgG2a, IgG2b, and IgG3 were detected in mice immunized with MsrA/B-Freund's adjuvant compared to MsrA/B-aluminum hydroxide adjuvant, while IgM titers were similar for both adjuvants. Antibodies generated by MsrA/B-Freund's in mice mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity. Anti-MsrA/B was also able to functionally block the activity of MsrA/B by inhibiting binding to its substrate, Met(O). We propose that recombinant MsrA/B is a promising vaccine antigen for N. gonorrhoeae.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas , Metionina Sulfóxido Redutases/imunologia , Neisseria gonorrhoeae/imunologia , Animais , Antígenos de Bactérias/genética , Feminino , Imunogenicidade da Vacina , Metionina Sulfóxido Redutases/genética , Camundongos Endogâmicos BALB C , Neisseria gonorrhoeae/enzimologia , Proteínas Recombinantes/imunologia
3.
FASEB J ; 33(2): 2095-2104, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30260702

RESUMO

Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.


Assuntos
Aminopeptidases/antagonistas & inibidores , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Metionina/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Células Cultivadas , Colo do Útero/efeitos dos fármacos , Colo do Útero/metabolismo , Colo do Útero/microbiologia , Farmacorresistência Bacteriana Múltipla , Feminino , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/enzimologia
4.
Sex Transm Dis ; 46(1): e3-e4, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30216232

RESUMO

Gyrase A genotyping reliably predicts Neisseria gonorrhoeae susceptibility to ciprofloxacin. It is unknown whether concurrent infections at different anatomic sites harbor different susceptibility profiles. We found a 3.2% frequency of discordant gyrase A genotypes among concurrent but anatomically separate N. gonorrhoeae infections diagnosed at 2 laboratories in Los Angeles.


Assuntos
DNA Girase/genética , Genótipo , Gonorreia/microbiologia , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana , Humanos , Los Angeles , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Estudos Retrospectivos
5.
Infect Dis Obstet Gynecol ; 2018: 7263849, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30584266

RESUMO

Objectives: To identify the prevalence and the types of Neisseria gonorrhoeae (NG) resistance plasmids-mediated penicillin (PPNG) and tetracycline (TRNG), the ciprofloxacin resistance (CRNG), and related risk factors of each types of resistance. Methods: The beta-lactamase-producing plasmid types (Africa, Asia, and Toronto), tetM tetracycline resistance plasmid types (America and Dutch), and the determination of the Ser-91 mutation of GyrA were detected by specifics PCRs on 149 diagnosed NG positives samples followed by Hinf1 digestion for tetM and gyrA mutation. Results: 135 (90.1%) samples showed a profile of molecular resistance to at least one antibiotic with predominance of ciprofloxacin resistance. In fact, 36 (24.2%) and 69 (46.3%) cases harbored PPNG and TRNG, respectively, and 116 (77.9%) cases showed the mutation Ser-91 of GyrA (CRNG). From a total of 36 PPNG isolates, the Toronto, Asian, and Toronto/Asian types were detected in 13 (36.1%), 10 (27.8%), and 13 (36.1%) cases, respectively, whereas the African type was not detected. In addition, the American type of TRNG was detected in 92.8% (64/69) of cases, while the Dutch type was detected in 7.2% (5/69) of cases. The association of demographics and clinical variables with NG resistance to ciprofloxacin, penicillin, and tetracycline was studied and the risk factors have been determined. Conclusion: Resistance to penicillin, tetracycline, and ciprofloxacin among NG samples positives remained at high levels in Morocco as determined by molecular profile. So, the use of molecular tools for NG antimicrobial resistance detection can help in the management and spread limitation of this infection.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Adulto , Idoso , Feminino , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Marrocos/epidemiologia , Mutação , Neisseria gonorrhoeae/enzimologia , Resistência às Penicilinas/genética , Penicilinas/farmacologia , Plasmídeos/genética , Reação em Cadeia da Polimerase , Prevalência , Fatores R/genética , Resistência a Tetraciclina/genética , Adulto Jovem , beta-Lactamases/genética
6.
BMC Microbiol ; 18(1): 95, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30165819

RESUMO

BACKGROUND: The functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins. Until now, in vivo activity has only been shown for V.EcoKDcm - the only Vsr endonuclease in Escherichia coli. Additionally, the VSP system of E. coli is the only one for which interactions between proteins of the system have been demonstrated. Neisseria gonorrhoeae FA1090 is the first bacterium that we previously demonstrated to encode two active in vitro Vsr endonucleases: V.NgoAXIII and V.NgoAXIV. RESULTS: We elucidate the mutator phenotype of N. gonorrhoeae mutants with disrupted genes encoding V.NgoAXIII or V.NgoAXIV endonuclease. Furthermore, we investigate the interactions between gonococcal Vsr endonucleases and MutL and MutS proteins. The Vsr endonucleases physically interact with gonococcal MutL protein but not with MutS protein. In the presence of the MutL protein, the efficiency of DNA cleavage by both V.NgoAXIII and V.NgoAXIV endonucleases increases, resulting in a decrease in the amount of Vsr enzyme required to complete digestion of mismatched DNA. Both Vsr endonucleases are also stimulated in vitro by the MutL protein of E. coli. In turn, the gonococcal MutS protein hinders DNA cleavage by the Vsr endonucleases. However, this effect is overridden in the presence of MutL, and furthermore, the simultaneous presence of MutL and MutS causes an increase in the efficiency of DNA cleavage by the Vsr endonucleases compared to the reaction catalyzed by V.NgoAXIII or V.NgoAXIV alone. CONCLUSIONS: For the first time, interactions between proteins of the DNA repair system encoded by N. gonorrhoeae that are responsible for the correction of mismatches resulting from the 5-methylcytosine deamination were identified. The increase in activity of Vsr endonucleases in the presence of MutL protein could allow for reduced synthesis of the Vsr endonucleases in cells, and the susceptibility of gonococcal Vsr endonucleases on MutL protein of E. coli implies a universal mechanism of Vsr stimulation by MutL protein.


Assuntos
Endodesoxirribonucleases/metabolismo , Proteínas MutL/metabolismo , Proteínas MutS/metabolismo , Neisseria gonorrhoeae/enzimologia , 5-Metilcitosina/metabolismo , Proteínas de Bactérias , Clivagem do DNA , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA , Endodesoxirribonucleases/genética , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Escherichia coli , Proteínas de Escherichia coli , Proteínas MutL/genética , Proteínas MutS/genética , Mutação , Neisseria gonorrhoeae/genética , Especificidade por Substrato
7.
Artigo em Inglês | MEDLINE | ID: mdl-30249694

RESUMO

We evaluated microbiological correlates for the successful treatment of Neisseria gonorrhoeae isolates from a phase 2 study of gepotidacin, a novel triazaacenaphthylene antibacterial, for therapy of uncomplicated urogenital gonorrhea. Culture, susceptibility testing, genotypic characterization, and frequency of resistance (FoR) were performed for selected isolates. Microbiological success was defined as culture-confirmed eradication of N. gonorrhoeae Against 69 baseline urogenital isolates, gepotidacin MICs ranged from ≤0.06 to 1 µg/ml (MIC90 = 0.5 µg/ml). For gepotidacin, the ratio of the area under the free-drug concentration-time curve to the MIC (fAUC/MIC) was associated with therapeutic success. Success was 100% (61/61) at fAUC/MICs of ≥48 and decreased to 63% (5/8) for fAUC/MICs of ≤25. All 3 isolates from microbiological failures were ciprofloxacin resistant, had a baseline gepotidacin MIC of 1 µg/ml, and carried a preexisting ParC D86N mutation, a critical residue for gepotidacin binding. In a test-of-cure analysis, the resistance to gepotidacin emerged in 2 isolates (MICs increased ≥32-fold) with additional GyrA A92T mutations, also implicated in gepotidacin binding. Test-of-cure isolates had the same sequence type as the corresponding baseline isolates. For 5 selected baseline isolates, all carrying a ParC D86N mutation, the in vitro FoR to gepotidacin was low (10-9 to 10-10); the resistant mutants had the same A92T mutation as the 2 isolates in which resistance emerged. Five participants with isolates harboring the ParC D86N mutation were treatment successes. In summary, fAUC/MICs of ≥48 predicted 100% microbiological success, including 3 isolates with the ParC D86N mutation (fAUC/MICs ≥ 97). Pharmacokinetic/pharmacodynamic determinations may help to evaluate new therapies for gonorrhea; further study of gepotidacin is warranted. (This study has been registered at ClinicalTrials.gov under identifier NCT02294682.).


Assuntos
Acenaftenos/farmacocinética , Antibacterianos/farmacocinética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Gonorreia/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Neisseria gonorrhoeae/efeitos dos fármacos , Acenaftenos/sangue , Acenaftenos/farmacologia , Administração Oral , Adulto , Antibacterianos/sangue , Antibacterianos/farmacologia , Área Sob a Curva , Técnicas de Tipagem Bacteriana , Hemocultura , Ciprofloxacino/uso terapêutico , DNA Topoisomerase IV/metabolismo , Esquema de Medicação , Feminino , Expressão Gênica , Gonorreia/sangue , Gonorreia/microbiologia , Gonorreia/patologia , Compostos Heterocíclicos com 3 Anéis/sangue , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutação , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Resultado do Tratamento
8.
J Biol Chem ; 293(43): 16559-16571, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30181210

RESUMO

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dissulfetos/metabolismo , Neisseria gonorrhoeae/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Dissulfetos/química , Modelos Moleculares , Oxirredução , Domínios Proteicos
9.
Phys Rev Lett ; 121(11): 118102, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30265121

RESUMO

Bacteria can adjust the structure of colonies and biofilms to enhance their survival rate under external stress. Here, we explore the link between bacterial interaction forces and colony structure. We show that the activity of extracellular pilus motors enhances local ordering and accelerates fusion dynamics of bacterial colonies. The radial distribution function of mature colonies shows local fluidlike order. The degree and dynamics of ordering are dependent on motor activity. At a larger scale, the fusion dynamics of two colonies shows liquidlike behavior whereby motor activity strongly affects surface tension and viscosity.


Assuntos
Fímbrias Bacterianas/fisiologia , Modelos Biológicos , Neisseria gonorrhoeae/fisiologia , Adenosina Trifosfatases/metabolismo , Fímbrias Bacterianas/metabolismo , Neisseria gonorrhoeae/enzimologia
10.
Biochem Biophys Res Commun ; 503(3): 1993-1999, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30093108

RESUMO

Neisseria gonorrhoeae, an obligate human pathogen, is a leading cause of communicable diseases globally. Due to rapid development of drug resistance, the rate of successfully curing gonococcal infections is rapidly decreasing. Hence, research is being directed toward finding alternative drugs or drug targets to help eradicate these infections. 4-Hydroxy-tetrahydrodipicolinate reductase (DapB), an important enzyme in the meso-diaminopimelate pathway, is a promising target for the development of new antibiotics. This manuscript describes the first structure of DapB from N. gonorrhoeae determined at 1.85 Å. This enzyme uses NAD(P)H as cofactor. Details of the interactions of the enzyme with its cofactors and a substrate analog/inhibitor are discussed. A large scale bioinformatics analysis of DapBs' sequences is also described.


Assuntos
NADP/metabolismo , Neisseria gonorrhoeae/enzimologia , Coenzimas/metabolismo , Modelos Moleculares , NADP/química , Conformação Proteica , Especificidade por Substrato
11.
J Phys Chem B ; 122(36): 8526-8536, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30114369

RESUMO

The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys-Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Domínio Catalítico/genética , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Mutação , Neisseria gonorrhoeae/enzimologia , Maleabilidade , Conformação Proteica , Engenharia de Proteínas , Temperatura
13.
J Antimicrob Chemother ; 73(8): 2064-2071, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29726994

RESUMO

Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae. Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-α by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined. Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at ≥5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-α probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor. Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that warrants further investigation particularly owing to the rise in MDR gonorrhoea.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Monócitos/citologia , Monócitos/microbiologia , Neisseria gonorrhoeae/enzimologia , Células THP-1 , Fator de Necrose Tumoral alfa/imunologia
14.
Ann Lab Med ; 38(4): 324-330, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29611382

RESUMO

BACKGROUND: We investigated the molecular epidemiological characteristics and antimicrobial susceptibility pattern of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates to monitor the change in distribution of bla(TEM) in Korea. METHODS: We collected 804 PPNG isolates from diverse hospitals and clinics mainly located in Seoul, Korea, over a period of 11 years (2005-2015). Isolate susceptibility to seven antimicrobials was determined using the agar dilution test. The molecular epidemiological characteristics of the isolates were determined by Sanger sequencing of bla(TEM), N. gonorrhoeae multiantigen sequence typing (NG-MAST) and plasmid typing. RESULTS: Among 72 fully sequenced PPNG isolates, sixteen (22.2%) possessed TEM-135. All TEM-135 isolates had a common silent mutation (c.18C>T), which was previously unreported. We observed a pattern of continuous increase in the number of TEM-135 isolates since 2012. The median and 90% minimum inhibitory concentration of azithromycin were substantially lower in the TEM-135 group than in the non-PPNG and TEM-1 groups. All TEM-135 isolates showed different NG-MAST types and predominantly harbored Toronto/Rio (75%) plasmids. A comprehensive comparative analysis of PPNG with TEM-135 according to NG-MAST, plasmid type, and year of isolation revealed a wide distribution. CONCLUSIONS: The proportion of TEM-135 PPNG has continuously increased since 2012, in association with clonal spread. The difference at position 18 of the TEM-135 sequence can be interpreted as the existence of multiple clonal complexes. The possibility that TEM-135 was acquired via foreign plasmids requires careful follow-up and continuous monitoring of TEM-135 to ascertain whether it constitutes a step towards evolutionary change.


Assuntos
Gonorreia/diagnóstico , beta-Lactamases/genética , Antibacterianos/farmacologia , Azitromicina/farmacologia , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana , Gonorreia/epidemiologia , Gonorreia/microbiologia , Humanos , Incidência , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/isolamento & purificação , Plasmídeos/genética , Plasmídeos/metabolismo , República da Coreia/epidemiologia
15.
FEBS Lett ; 592(9): 1473-1483, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29665008

RESUMO

The Neisseria gonorrhoeae bacterial cytochrome c peroxidase plays a key role in detoxifying the cells from H2 O2 by reducing it to water using the lipid-modified azurin, LAz, a small type 1 copper protein, as electron donor. Here, the interaction between these two proteins was characterized by steady-state kinetics, two-dimensional NMR and molecular docking simulations. LAz is an efficient electron donor capable of activating this enzyme. This electron transfer complex is weak with a hydrophobic character, with LAz binding close to the electron transferring heme of the enzyme. The high catalytic rate (39 ± 0.03 s-1 ) is explained by the LAz pre-orientation, due to a positive dipole moment, and by the fast-dynamic ensemble of orientations, suggested by the small chemical shifts.


Assuntos
Azurina/química , Azurina/metabolismo , Citocromo-c Peroxidase/metabolismo , Lipídeos/química , Neisseria gonorrhoeae/enzimologia , Transporte de Elétrons , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Solubilidade
17.
Sex Transm Dis ; 45(5): 312-315, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29465687

RESUMO

BACKGROUND: The emergence and spread of antimicrobial-resistant (AMR) Neisseria gonorrhoeae (NG) is a major public health concern. In the era of nucleic acid amplifications tests, rapid and accurate molecular approaches are needed to help increase surveillance, guide antimicrobial stewardship, and prevent outbreaks. METHODS: Residual urethral swabs, collected prospectively in the Baltimore City Health Department during a 6-month period, were analyzed by real-time polymerase chain reaction assays for NG DNA and AMR determinants to fluoroquinolones, penicillin, and extended-spectrum cephalosporins. RESULTS: N. gonorrhoeae DNA was detected in 34.8% (73/210) of samples, including 67.3% (68/101) of the swabs that had been previously identified as NG positive by culture. Markers associated with decreased susceptibility to fluoroquinolones were detected in 22.4% of the polymerase chain reaction NG-positive samples. The rate of penicillinase-producing NG was very low (1.6%), and no markers associated with decreased susceptibility to extended-spectrum cephalosporins were detected in this cohort of men using the AMR assays herein described. CONCLUSIONS: Detection of molecular markers associated with AMR in NG can be performed directly from residual clinical samples, although the recovery rate of adequate DNA for molecular testing from these samples can be suboptimal. A high number of samples with mutations associated with decreased susceptibility to fluoroquinolones were identified.


Assuntos
Farmacorresistência Bacteriana/genética , Marcadores Genéticos , Gonorreia/microbiologia , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Baltimore/epidemiologia , Estudos de Coortes , DNA Bacteriano/genética , Fluoroquinolonas/farmacologia , Gonorreia/epidemiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/enzimologia , Penicilinase/biossíntese , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Uretra/microbiologia
18.
Sex Transm Dis ; 45(2): 87-91, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29329176

RESUMO

BACKGROUND: Novel approaches to combating drug-resistant Neisseria gonorrhoeae infections are urgently needed. Targeted therapy with ciprofloxacin has been made possible by a rapid assay for genotyping the gyrase A (gyrA) gene; a nonmutated gene reliably predicts susceptibility to ciprofloxacin. METHODS: We determined the costs of running the gyrA assay, 500 mg of ciprofloxacin, 250 mg of ceftriaxone injection, and 1000 mg of azithromycin. Cost estimates for gyrA testing included assay reagents and labor. Cost estimates for ceftriaxone included medication, injection, administration, supplies, and equipment. We measured the cost of using the gyrA assay and treatment based on genotype using previously collected data over a 13-month period between November 2015 and November 2016 for all N. gonorrhoeae cases diagnosed at UCLA. We subsequently developed 3 cost models, varying the frequency of testing and prevalence of N. gonorrhoeae infections with ciprofloxacin-resistant or genotype-indeterminate results. We compared those estimates with the cost of recommended 2-drug therapy (ceftriaxone and azithromycin). RESULTS: Based on a 65.3% prevalence of cases with ciprofloxacin-resistant or genotype indeterminate N. gonorrhoeae infections when running an average of 1.7 tests per day, the per-case cost of gyrA genotyping and targeted therapy was US $197.19. The per-case cost was US $155.16 assuming a 52.6% prevalence of ciprofloxacin-resistant or genotype-indeterminate infections when running an average of 17 tests per day. The per-case cost of 2-drug therapy was US $142.75. CONCLUSIONS: Direct costs of gyrA genotyping and targeted ciprofloxacin therapy depend on the prevalence of ciprofloxacin-resistant or genotype-indeterminate infections and testing frequency.


Assuntos
Antibacterianos/uso terapêutico , Ciprofloxacino/uso terapêutico , DNA Girase/genética , Farmacorresistência Bacteriana , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/enzimologia , Azitromicina/uso terapêutico , California/epidemiologia , Ceftriaxona/uso terapêutico , Custos e Análise de Custo , DNA Girase/efeitos dos fármacos , Genótipo , Técnicas de Genotipagem/economia , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/efeitos dos fármacos , Estudos Retrospectivos
19.
J Glob Antimicrob Resist ; 13: 91-93, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29229280

RESUMO

OBJECTIVES: The aim of this study was to determine the antimicrobial susceptibility of Neisseria gonorrhoeae isolates in the Lao People's Democratic Republic (Laos). METHODS: A total of 165 gonococcal isolates (1.3%) were obtained from 12 281 genital samples routinely submitted to a diagnostic laboratory in Vientiane, Laos, between 2011 and 2015. Susceptibility to five antibiotics was determined by the standard disk diffusion method for 158 of the isolates. RESULTS: Rates of resistance to penicillin (by ß-lactamase production), tetracycline and ciprofloxacin were 89.9%, 99.4% and 84.8%, respectively. All isolates were susceptible to ceftriaxone and spectinomycin. CONCLUSIONS: The situation in Laos is similar to that in neighbouring countries; this fortunately means that the latest Lao national guidelines for treating gonorrhoea should still be effective.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Neisseria gonorrhoeae/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ceftriaxona/farmacologia , Criança , Pré-Escolar , Ciprofloxacino/farmacologia , Feminino , Gonorreia/epidemiologia , Gonorreia/microbiologia , Humanos , Laos/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Neisseria gonorrhoeae/enzimologia , Adulto Jovem , beta-Lactamases/biossíntese
20.
Microb Drug Resist ; 24(1): 30-34, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28581359

RESUMO

Emergence of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally, and new antimicrobials for treatment of gonorrhea are imperative. In this study, the in vitro activity of sitafloxacin, a fluoroquinolone mainly used for respiratory tract or urogenital infections in Japan, and additional newer generation fluoroquinolones were determined against ciprofloxacin-resistant N. gonorrhoeae isolates. Minimum inhibitory concentrations (MICs) of ciprofloxacin, levofloxacin, moxifloxacin, sitafloxacin, pazufloxacin, and tosufloxacin against 47 N. gonorrhoeae isolates cultured in 2009 in Japan were determined by agar dilution method. The quinolone resistance-determining region (QRDR) of gyrA and parC was sequenced. The in vitro potency of sitafloxacin was substantially higher compared with all other tested fluoroquinolones. The MICs of sitafloxacin ranged from 0.03 to 0.5 mg/L for 35 ciprofloxacin-resistant N. gonorrhoeae isolates (ciprofloxacin MICs from 2 to 32 mg/L). No identified mutations in GyrA and ParC QRDR resulted in higher sitafloxacin MIC than 0.5 mg/L. Sitafloxacin had a high activity against N. gonorrhoeae isolates, including strains with mutations in DNA gyrase and topoisomerase IV, resulting in high-level resistance to ciprofloxacin and all other newer generation fluoroquinolones examined. However, it was still to a lower extent affected by GyrA and ParC QRDR mutations resulting in sitafloxacin MICs of up to 0.5 mg/L. This indicates that sitafloxacin should not be considered for empirical first-line monotherapy of gonorrhea. However, sitafloxacin could be valuable in a dual antimicrobial therapy and for cases with ceftriaxone resistance or allergy.


Assuntos
Antibacterianos/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Ciprofloxacino/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , Expressão Gênica , Gonorreia/microbiologia , Humanos , Japão , Levofloxacino/farmacologia , Testes de Sensibilidade Microbiana , Moxifloxacina , Naftiridinas/farmacologia , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Oxazinas/farmacologia , Análise de Sequência de DNA
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