RESUMO
We applied complex genetic analysis for evaluation of tetracycline-resistance markers in 129 clinical strains of Neisseria gonorrhoeae from Central, Privolzhskii, and Siberian regions. For detection of mutations in rpsJ gene and MtrRCDE locus we first used minisequence reaction followed by identification of products by MALDI-TOF mass spectrometry. The incidence of detection of resistance markers among the analyzed strains were: tetM--3.1%, mutations in genes rpsJ--82.2%, penB--62.8%, and mtrR--54.3%. The analyzed genetic markers were not detected in 17.5% strains. tetM gene was detected in only 12.5% strains from the Central Region. No differences were revealed in regional distribution of other genotypes. Genotypes tetM(pres), rpsJ(mut), mtrR(mut), and rpsJ(mut), penB(mut), mtrR(mut) reliably predict tetracycline resistance. Microbiological and genetic testing of tetracycline resistance yielded similar results.
Assuntos
Antibacterianos/uso terapêutico , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Resistência a Tetraciclina/genética , Tetraciclina/uso terapêutico , Antibacterianos/farmacologia , Marcadores Genéticos , Genótipo , Gonorreia/microbiologia , Humanos , Mutação , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/patogenicidade , Federação Russa , Tetraciclina/farmacologiaRESUMO
New fluoroquinolones with higher antipneumococcal activity are considered promising in the treatment of respiratory tract infections. Still, their wide use in clinical practice is connected with possible selection and rapid distribution of the resistance, requiring constant monitoring. Development of resistance to fluoroquinolones results from step-wise accumulation of mutations in the genes of DNA-gyrase and topoisomerase IV, the mutations of the first step being not always accompanied by a significant increase of the MIC of the new fluoroquinolones. Therefore, to detect the first signs of the resistance development, it is necessary not only to detect the susceptibility of the circulating Streptococcus pneumoniae strains phenotypically, but also to detect the genetic changes. In the present study the minisequent reaction followed by detection of the reaction products by MALD-ToF mass-spectrometry was used to reveal the mutations in the genes of the fluoroquinolone targets of 38 S. pneumoniae strains with different levels of the resistance to ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin. In the strains with high resistance to all the three fluoroquinolones (MIC 4-16 mcg/ml) there were detected mutations in GyrA (Ser81Tyr or Glu85Zys) and as well in ParC (Ser79Phe or Ser79Tyr). In the strains resistant to ofloxacin and ciprofloxacin (MIC 4-8 mcg/ml) with preserved susceptibility to levofloxacin and moxifloxacin, the mutations were detected only in GyrA (Ser114Gly). In the moderately resistant strains (MICs 4 and 2-4 mcg/ml respectively for ofloxacin and ciprofloxacin) there were detected the known mutations in ParC (Ser79Tyr or Ser79Phe or Asp83Tyr) and in GyrB (Glu475Lys) as well as the earlier not described mutations in ParE (ins Asn381a) and in Gyr B (Thr329Ala or Va1355Ile). The described method can be used in mass screening of S. pneumoniae strains for the presence of mutations in the genes of the fluoroquinolone targets.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Proteínas de Bactérias/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Testes de Sensibilidade Microbiana , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptococcus pneumoniae/genéticaRESUMO
We carried out complex genetic analysis of clinical samples containing N. gonorrhoeae DNA, the genotype and profile of drug resistance of this agent were evaluated. Changes in genes responsible for the formation of N. gonorrhoeae resistance to penicillins, fluoroquinolones, and spectinomycin were detected during minisequencing with subsequent MALDI-TOF mass spectrometry. The sensitivity of gonococcus was evaluated directly in the clinical sample without culturing.
Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Feminino , Fluoroquinolonas/farmacologia , Genes Bacterianos , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutação , Neisseria gonorrhoeae/genética , Resistência às Penicilinas/genética , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A complex method for detection of genetic markers of N. gonorrhoeae resistance to penicillin was developed. Mutations in penA and ponA genes were detected by minisequencing reaction with subsequent detection of reaction products by MALDI-TOF mass spectrometry. This approach was tested on 31 clinical strains of N. gonorrhoeae with minimum inhibitory concentration of penicillin from 0.03 to 8 microg/ml and higher. Mutations in penA and ponA genes in moderately resistant strains were shown (minimum inhibitory concentration up to 0.5 microg/ml) and mutations in penA, ponA, and penB genes in resistant strains (minimum inhibitory concentration more than 1.0 microg/ml). beta-Lactamase genes were detected in 4 strains with high resistance (minimum inhibitory concentration 4-8 and more microg/ml). Correlation between microbiological resistance and presence of respective mutations in the studied locuses was detected.
Assuntos
Proteínas de Bactérias/genética , Neisseria gonorrhoeae/genética , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica/genética , Primers do DNA , Marcadores Genéticos/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Penicilinas/toxicidade , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases/genéticaRESUMO
For many known mechanisms of the drug resistance in microorganisms are described genetic markers (specific changes in the genome of microorganism, in the majority of the cases representing single nucleotide polymorphism). The search for the new methods, which make possible to identify single nucleotide changes with the greater effectiveness and at smaller prime is actual for the solution of the problem of the identification of the resistant strains. In this work a new approach of the determination of single nucleotide polymorphisms is proposed. It is based on the reactions of mini-sequencing and/or sequencing with the subsequent Matrix-Assisted Laser Desorption/Ionisation Time Of Flight Mass-Spectrometry (MALDI-TOF MS) of the reaction products. The approach was tested on a clinical group of Neisseria gonorrhoeae strains to investigate specific single nucleotide polymorphisms in genes gyrA and parC (the genetic markers of the bacterium fluoroquinolone resistance). The results of the nucleotide polymorphism deter- mination was completely agreed with the data, obtained earlier with the use of a "gold standard" (sequencing with the classical gel-electrophoresis separation of the reaction products). There is specific interest in the method of sequencing of the short DNA sequences using MALDI-TOF MS. The new high-throughput approach of the single nucleotide polymorphisms determination in bacterial genes considerably increases the effectiveness of the methods of microorganism's identification, genotyping and determining the genetic markers of the drug resistance.
Assuntos
DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas , Genes Bacterianos/genética , Neisseria gonorrhoeae/genética , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
To detect point mutations A2115C, A2143G/C, and A2143G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.
Assuntos
Antibacterianos , Claritromicina , Farmacorresistência Bacteriana/genética , Helicobacter pylori/genética , Mutação Puntual , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Antibacterianos/farmacologia , Claritromicina/farmacologia , Genes Bacterianos/genética , Genes de RNAr/genética , HumanosRESUMO
Fluoroquinolones still belong to the drugs of choice in the treatment of uncomplicated gonorrhea. At the same time, there have been more data on the spreading N. gonorrhoeae strains resistant to fluoroquinolones. A variety of mechanisms, like modification of the target of antibiotic's action (point mutations in genes gyrA and parC), a decreasing permeability of the bacterial cell membrane (amino-acid changes Por protein) and a growing efflux of antibiotic (mutations in the promoter or in the coding region of mtrR) mediate in the shaping resistance of the drugs. The MIC values for four fluoroquinolone-series antibiotics were determined and the gyrA, parC, por and mtrR genes were examined for resistance-responsible mutations in 32 studied clinical strains of N. gonorrhoeae. Strains with high resistance to fluoroquinolones were detected; 3 of them had no common changes in GyrA or ParC, however, amino acid changes and mutations were detected in Por protein and promoter or gene mtrR encoding region, respectively. The paper contains priority data on the detection (in Russia) of N. gonorrhoeae strains with high resistance to fluoroquinolones. Involvement of different mechanisms in the process of resistance shaping is discussed. The results are of practical importance for planning the antibacterial therapy of gonorrhoeae; they point out the need in regional testing of resistance in the N. gonorrhoeae population encountered in Russia.
Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , DNA Topoisomerases Tipo II/genética , Resistência Microbiana a Medicamentos/genética , Fluoroquinolonas/uso terapêutico , Gonorreia/tratamento farmacológico , Humanos , Cetona Oxirredutases/antagonistas & inibidores , Cetona Oxirredutases/genética , Testes de Sensibilidade Microbiana , Moscou , Mutação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Regiões Promotoras Genéticas , Piruvato Sintase , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Inibidores da Topoisomerase IIRESUMO
Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.
Assuntos
Genoma Viral , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Substituição de Aminoácidos , DNA Complementar/genética , DNA Viral/genética , Humanos , Filogenia , Reação em Cadeia da Polimerase , Federação Russa , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/classificação , Síndrome Respiratória Aguda Grave/virologiaRESUMO
OBJECTIVES: During a longitudinal study of the prevalence of antimicrobial resistance in Neisseria gonorrhoeae, a number of high-level fluoroquinolone-resistant isolates were obtained from the sexually transmitted diseases clinic in the Moscow region in 2002. The aim of the present study was to determine the molecular mechanisms of resistance and to assess the clonal relationship of these strains METHODS: For the 32 clinical strains of N. gonorrhoeae studied, the MIC values were determined for four fluoroquinolones. The gyrA, parC, por and mtrR genes were studied for the presence of mutations associated with fluoroquinolone resistance. RESULTS: We detected strains of N. gonorrhoeae showing high-level resistance to fluoroquinolones (21 strains, with MICs 1-32 mg/L). Mutations in gyrA and parC known to cause fluoroquinolone resistance were detected in a majority of strains. There were four strains (among 21) without known changes in gyrA and parC. However, amino acid changes in the Por protein and mutations in the promoter or encoding region of the mtrR gene were detected in three of them. One strain had no alteration in gyrA, parC, por or mtrR. CONCLUSIONS: The present study documents the first case of fluoroquinolone-resistant N. gonorrhoeae in Russia.
Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Humanos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Mutação , Neisseria gonorrhoeae/isolamento & purificação , Federação RussaRESUMO
Genetic polymorphism of Russian population of N. gonorrhoeae was detected and a system for genotyping of its clinical strains was introduced into practice. Comparative analysis of the prevalence of N. gonorrhoeae genotypes in Russia and abroad was carried out. For adaptation of the methods of molecular typing of N. gonorrhoeae strains and its approbation on clinical strains isolated in Russia 41 clinical strains of N. gonorrhoeae were typed. The predominance of PIB serovar (83%) was demonstrated.
Assuntos
Técnicas de Tipagem Bacteriana , Epidemiologia Molecular , Neisseria gonorrhoeae/classificação , Genótipo , Gonorreia/diagnóstico , Gonorreia/epidemiologia , Humanos , Neisseria gonorrhoeae/genética , Federação Russa/epidemiologiaRESUMO
On the basis of statistical analysis of chest wounds in 349 patients the authors have determined the "risk zone". Wounds in this zone have the greatest probability to injure the heart. The zone is restricted by the II-VIII ribs on the left and III-VIII ribs on the right, left medioaxillary and right parasternal lines.
Assuntos
Traumatismos Cardíacos/diagnóstico , Diagnóstico por Computador , Traumatismos Cardíacos/patologia , Humanos , Probabilidade , Ferimentos Penetrantes/diagnóstico , Ferimentos Penetrantes/patologiaRESUMO
Forty observations of damaged hearts and pericardium in thoracoabdominal wounds were studied. All the patients were operated on. Causes of diagnostic errors and erroneous choice of surgical methods are analyzed. It is found that 61% of patients with heart damage in thoracoabdominal wounds are admitted to hospitals and given urgent surgical aid. Fifteen patients were examined in the long-term postoperative period.
