RESUMEN
An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos Bacterianos/análisis , Técnicas para Inmunoenzimas/normas , Proteínas Citotóxicas Formadoras de Poros/análisis , Yersinia pestis/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Humanos , Hibridomas/inmunología , Immunoblotting , Límite de Detección , Ratones , Ratones Endogámicos BALB C , Peste/diagnóstico , Peste/microbiología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Yersinia pestis/química , Yersinia pestis/inmunología , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/aislamiento & purificaciónRESUMEN
The spread of the New Delhi metallo-beta-lactamase (NDM-1), a plasmid-borne enzyme conferring bacterial resistance to any known beta-lactam antibiotics, represents the global health threat. There is an urgent need to develop the efficient NDM-1 inhibitors of various mode of action thereby necessitating structural studies of the enzyme as well as analysis of the secretion pathway and localization of the protein. The recombinant full-length NDM-1 is produced in E. coli in the inactive form and is mostly accumulated in the inclusion bodies. The secreted recombinant NDM-1 forms are several N-terminally truncated species. The robust expression system capable of high-level production of the full-length NDM-1 and derivatives thereof is required to obtain NDM-1 in the quantities necessary for drug discovery, diagnostics, and research purposes. Therefore, we developed a new system that utilizes antibiotic pressure to select E. coli producing increased quantity of soluble NDM-1 and showed that an increase in the NDM-1 solubility occurs in the bacterial clones producing increased amounts in the chaperones.
Asunto(s)
Antibacterianos/uso terapéutico , Chaperonas Moleculares/biosíntesis , Resistencia betalactámica/genética , beta-Lactamasas/biosíntesis , Antibacterianos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Pruebas de Sensibilidad Microbiana , Chaperonas Moleculares/genética , Plásmidos , Inhibidores de beta-Lactamasas , beta-Lactamasas/genética , beta-Lactamas/uso terapéuticoRESUMEN
Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means ofenvironmental monitoring. Aptamers can be regarded as fully synthetic analogs of antibodies. At the same time, certain properties ofaptamers render them superior to antibodies in terms of development of new diagnostic and monitoring systems that combine high sensitivity and specificity with high reproducibility and inexpensive manufacturing. In particular, the aptamers tailored to bind biomolecules and live cells can be employed in solving the problem of combining short analysis time with high sensitivity and specificity in detection of pathogenic bacteria. The present review summarizes the current state of the techniques developed for aptamer-based detection of bacteria and their components and discusses the potential of their practical application.
Asunto(s)
Aptámeros de Nucleótidos/química , Infecciones Bacterianas/diagnóstico , Animales , HumanosRESUMEN
Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.
Asunto(s)
Proteínas Bacterianas/genética , Catalasa/genética , Análisis Mutacional de ADN/métodos , Mycobacterium tuberculosis/genética , Ácidos Nucleicos/química , Oligonucleótidos/química , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Codón , Cartilla de ADN/química , Farmacorresistencia Microbiana/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Nucleicos Heterodúplex , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
AIM: Certification of V. cholerae strains stored at State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk. MATERIALS AND METHODS: 50 V. cholerae strains were studied. Real-time PCR with primers to genes ctxA, ctxB, ace, zot, tcpA, toxR, hlyA, rtxC, rfbO1, ompU, ompW was used. RESULTS: Membership of the studied strains in V. cholerae species was confirmed by molecular-biological methods. 46 strains belong to O1 serogroup, 42 of those - E1 Tor toxigenic, having all the virulence genes and 4 non-toxigenic strains. A strain had ace, zot, tcpA, toxR, rtxC, hlyA, ompU genes. 2 strains had ace, toxR, rtxC, hlyA genes, a strain had only toxR gene which is a global regulatory gene. 2 of the 4 serogroup O1 strains were non-toxigenic and had all the virulence genes (ctxA, ctxB, ace, zot, tcpA, toxR, rtxC, hlyA, ompU). 1 non-toxigenic strain has ace, zot, toxR, hlyA, ompUgenes, and the other - toxR, hlyAgenes. CONCLUSION: Genome certificates of all the V. cholerae strains stored in State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk were created. Markers of epidemic threat - ctxA, ctxB, tcpA, toxR and additional virulence genes were determined.
Asunto(s)
Proteínas Fimbrias/genética , Islas Genómicas/genética , Vibrio cholerae , Virulencia/genética , Toxina del Cólera/genética , Cartilla de ADN , Genes Reguladores , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Vibrio cholerae/clasificación , Vibrio cholerae/genética , Vibrio cholerae/patogenicidadRESUMEN
The isoniazid resistance of mycobacteria tuberculosis (MBT) is associated with point mutations in the codon 315 of katG gene of MBT. The two PCR-techniques for detection of point mutations in codon 315 have been developed. A use of two sets of primers comprising the additional competitive blocking primer with 3'-terminal phosphate group (in order to eliminate unspecific amplification) allowed to identify most frequent point mutations AGC-->ACC and AGC-->AGA in the codon 315. PCR with a set of two primers one of which contained 5 LNA-monomers allowed to discriminate between wild type and isoniazid-resistant MBT isolates; any of 6 known mutations in codon 315 of kafG gene being detected. A structure and purity of the LNA-containing oligonucleotides (length of 17 nucleotides) was characterized by MALDI-TOF mass spectrometry; the duplex formed by two LNA-containing complementary oligonucleotides (length of 17 b.p.) was anlyzed by thermal denaturation.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Isoniazida/farmacología , Mycobacterium tuberculosis/genética , Mutación Puntual , Humanos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
The optimal parameters of the Alamar Blue test have been determined to detect the antituberculous activity of the chemical compounds under study. The duration of mycobacterial cell incubation before addition of Alamar Blue is 24 hours; that is 17 hours for both H37Ra and H37Rv M. tuberculosis. A method has been devised to evaluate the bactericidal/bacteriostatic activity of the chemical compounds. A thoroughly characterized collection of clinical M. tuberculosis strains that differ in drug sensitivity has been created. A procedure has been developed to reveal the activity of the chemical compounds, by applying mono and multiresistant M. tuberculosis strains. Variability in the growth rate for the clinical strains of mycobacterial cultures is shown. A method has been devised to evaluate the toxicity of the chemical compounds for eukaryotic cells.
Asunto(s)
Antituberculosos/farmacología , Diseño de Fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Composición de Medicamentos , Humanos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/microbiologíaAsunto(s)
Infecciones por Mycobacterium , Mycobacterium/aislamiento & purificación , Brotes de Enfermedades , Salud Global , Humanos , Morbilidad/tendencias , Infecciones por Mycobacterium/epidemiología , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/prevención & controlRESUMEN
The critical concentration of canamycin for the MGIT Bactec 960 system, which has been determined on a panel of the tho--roughly characterized genetically heterogeneous clinical Mycobacterium tuberculosis isolated in the Central Region of Russia, is equal to 1.25 microg/ml.
Asunto(s)
Antibacterianos/administración & dosificación , ADN Bacteriano/análisis , Kanamicina/administración & dosificación , Pruebas de Sensibilidad Microbiana/instrumentación , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis Pulmonar/microbiologíaRESUMEN
We and other authors have recently shown that the pattern of the immune response to components of anthrax, the Bacillus anthracis lethal toxin, is complex. In addition to neutralizing antibodies, the antitoxin antibody pool contains antibodies enhancing the toxin lethal action. We mapped the epitopes in the protective antigen that are responsible for the induction of both antibody types. In this study, we obtained new data on the cytotoxicity of the B. anthracis lethal toxin toward the J774 A.1 cell line in the presence of monoclonal antibodies to various domains of the protective antigen and the lethal factor. The role of the Fc fragment of immunoglobulins in enhancing the lethal toxin action was shown. These results may serve as a basis for the development of a new generation vaccine for anthrax.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Exotoxinas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Línea Celular , Mapeo Epitopo , Fragmentos Fc de Inmunoglobulinas/inmunología , RatonesRESUMEN
Two strains of Legionella isolated from patient and hot water supply system during outbreak in Sverdlovsk region in 2007 were studied. Using genetic analysis methods (genes sequencing, VNTR-typing and PCR-based study of omp 28 gene), it was shown that tested strains are pathogenic but do not belong to one genetic group and epidemic cluster. Performed immunochemical analysis confirmed that both isolated strains belong to Legionella serogroup 1.
Asunto(s)
Brotes de Enfermedades , Legionella/clasificación , Legionelosis/epidemiología , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Cartilla de ADN , Genes Bacterianos/genética , Humanos , Immunoblotting , Legionella/genética , Legionella/inmunología , Legionelosis/microbiología , Lipopolisacáridos/análisis , Lipopolisacáridos/inmunología , Repeticiones de Minisatélite/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Federación de Rusia/epidemiología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Población Urbana , Microbiología del Agua , Abastecimiento de AguaRESUMEN
Description of the pyrazinamide-resistant clinical strains of M. tuberculosis derived from sputum of patients treated in TB clinics in Tula was made (June, 2001 - July, 2002). It was demonstrated that 30.3% (n = 91) strains were resistant to pyrazinamide. It was found out that these strains were resistant to other antituberculosis drugs in most cases. The method of PCR-sequencing was used to find the mutations in the gene pncA determining resistance to pyrazinamide. 44 different types of mutations localized in 28 codons were detected. The predominance of the mutations in 57 (13.2%), 63 (7.7%), 97 (7.7%), 12 (6.6%), 103 (6.6%) codons and in -11 (6.6%) promoter ofp ncA was observed in the pyrazinamide-resistant strains. Several new mutations determining resistance of the clinical strains of M. tuberculosis to pyrazinamide were described. A high correlation between resistance of mycobacteria to pyrazinamide and activity of pyrazinamidase was observed.
Asunto(s)
Amidohidrolasas/genética , Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Pirazinamida/farmacología , Humanos , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Regiones Promotoras Genéticas , Esputo/microbiologíaRESUMEN
The correlation of the Beijing/W and LAM strains with multiple drug resistance was studied. The transmissibility of the multidrug-resistant strains of these families was demonstrated.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/transmisión , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/transmisión , Genotipo , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Prisioneros , Federación de RusiaRESUMEN
Deletions were analyzed in the genomes of four Mycobacterium tuberculosis strains isolated from the sputum of patients living in the Central Region of Russia. The strains of the Beijing family were found to have deletions affecting 40 open reading frames (ORF) and to amount to 0.7% of the genome H37Rv. Genome deletions in a strain from the Haarlem family affected 20 ORF and accounted for 0.26% of the genome H37Rv. Six of the eight deletions were located at the site of preferred cloning on the insertion element IS6110. Prophage phiRv1-associated deletion sequence 149 is the only common to the strains of both families. The deletion ends were evenly distributed among the intergenic and coding chromosome regions with an insignificant preference of the latter. The authors revealed a new deletion in strain 1540 belonging to the Haarlem family and a two-component deletion in the region RvD2. The deletions detected in the genomes of Russian Beijing strains were typical of strains from South-East Asian countries.
Asunto(s)
Eliminación de Gen , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/microbiología , Cartilla de ADN/genética , HumanosRESUMEN
The review of scientific periodicals relevant to research on the mycobacteria of the tuberculosis complex with the use of modern methods of molecular biology is presented. The main mechanisms of the intraspecific variability of mycobacteria and the updated view on the tuberculosis complex mycobacteria evolution are described.
Asunto(s)
Mycobacterium/genética , Tuberculosis/microbiología , Animales , Evolución Molecular , Variación Genética , Genoma Bacteriano , Humanos , Mycobacterium/clasificación , Especificidad de la EspecieRESUMEN
The Mycobacterium tuberculosis strains of genotype A1 (LAM family, VNTR profile 222222) are often resulted from people with pulmonary tuberculosis, who live in Central Russia. Among strains of this family, drug-resistant strains, including those with simultaneous resistance to isoniazid and rifampicin (MDR), are common. The strains of the genotype A1 tend to spread as clones.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Antituberculosos/farmacología , ARN Polimerasas Dirigidas por ADN , Femenino , Genotipo , Humanos , Isoniazida/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Above two million people annually die worldwide of tuberculosis. There has been a serious uptrend in the tuberculosis morbidity observed during the recent decade in Russia. The growing prevalence of drug-resistant strains of pathogens is highly dangerous. A timely diagnosis reduces significantly the risk of epidemics. The use of molecular methods cuts the time of tuberculosis diagnosis, including its resistant forms, from several weeks to several days. The presented survey contains a discussion of modem methods used for identification of Mycobacterium tuberculosis resistance to the main anti-TB drugs.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Alelos , ADN Bacteriano/análisis , ADN Bacteriano/genética , Humanos , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Análisis de Secuencia , Tuberculosis/tratamiento farmacológicoRESUMEN
One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.
Asunto(s)
Genes Bacterianos , Infecciones por Mycobacterium/microbiología , Mycobacterium/aislamiento & purificación , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , Secuencia de Bases , Humanos , Mycobacterium/genética , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
Anthrax belongs to highly dangerous infections of man and animals. No effective treatment methods for pulmonary types of the disease have been yet developed. The existing anthrax vaccines were designed decades ago and need improvement to fit the large-scale vaccination of population. At the same time, the immunological properties of the anthrax vaccine main component, i.e. of the protective agent, have been poorly studied. We obtained, within the present case study, a panel of mouse monoclonal antibodies to the protective agent and investigated the properties of the highest-affine panel representatives. An unusual phenomenon was detected, which is related with enhancement of the anthrax toxin action on the mouse macrophage-like cell-line in presence of the 1F2 monoclonal antibody. The remaining analyzed antibodies, i.e. 6G8 and 6G7, were found to neutralize effectively the toxin action. The enhancing and neutralizing antibodies were proven to be specific to different domains of the protective antigen and to recognize epitopes in its composition. The antibody-mediated enhancement of the anthrax lethal action is a convincing argument for further development of a new-generation anthrax vaccine. Definition of the linear antigen determinants for neutralizing antibodies in the protective antigens is an important step in the development of the next-generation anthrax vaccine.