RESUMEN
The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for ß-lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.
RESUMEN
We developed a multiplexed DNA microarray-based assay allowing identification of 12 causative agents of reproductive tract infections with the simultaneous detection of 47 genetic determinants of resistance to antimicrobial substances. The microarray was tested on 93 isolates of Neisseria gonorrhoeae, 32 isolates of Treponema pallidum and 29 samples of Ureaplasma spp./Mycoplasma spp. The N. gonorrhoeae isolates had multiple mutations in the penA, ponA, rpsJ, gyrA, parC, and mtrR genes; their prognostic value significantly increased when combinations of mutations were detected. In the analyzed T. pallidum isolates, single A2058G substitution in the 23S rRNA gene responsible for macrolide resistance was found. DNA sequences of Ureaplasma spp./Mycoplasma spp. were determined as wild type, which was not fully consistent with the results of analysis of their antimicrobial susceptibility.
Asunto(s)
Antibacterianos/farmacología , Gonorrea/diagnóstico , Técnicas de Diagnóstico Molecular , Sífilis/diagnóstico , Infecciones por Ureaplasma/diagnóstico , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Gonorrea/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Procedimientos Analíticos en Microchip , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Hibridación de Ácido Nucleico , Sífilis/microbiología , Treponema pallidum/genética , Treponema pallidum/aislamiento & purificación , Ureaplasma/genética , Ureaplasma/aislamiento & purificación , Infecciones por Ureaplasma/microbiologíaRESUMEN
Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8-36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Gonorrea/tratamiento farmacológico , Neisseria gonorrhoeae/genética , Cromosomas Bacterianos/genética , Fluoroquinolonas/uso terapéutico , Genotipo , Gonorrea/genética , Gonorrea/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/patogenicidad , Penicilinas/uso terapéutico , Fenotipo , Federación de Rusia , Tetraciclinas/uso terapéuticoRESUMEN
Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form.
Asunto(s)
Muramidasa/biosíntesis , Pichia , Fagos Pseudomonas/enzimología , Pseudomonas aeruginosa/virología , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae , Proteínas Virales/biosíntesis , Clonación Molecular , Expresión Génica , Genes Virales/fisiología , Muramidasa/genética , Fagos Pseudomonas/genética , Proteínas Recombinantes/genética , Proteínas Virales/genéticaRESUMEN
A method of monitoring the sequential events of IS481 transposition into the ctag site of bvg operon of Bordetella pertussis has been developed. Reproduction of virulent B. pertussis cells in vitro is accompanied by intrachromosomal site-specific IS481 transposition, which, in turn, results in inactivation of bvg operon of the causative agent and cell avirulent state. Avirulent bvg mutants of B. pertussis are incapable of intramolecular IS481 transposition. The frequency of the transposition increases when MgSO4 and nicotinic acid are present the culture medium. In the absence of these modulating factors. IS481 transposition along B. pertussis chromosome is inhibited but not arrested completely. Negative regulation of the bvg-repressed genes of B. pertussis seems to be a mechanism that controls bvg-dependent IS481 transposition.
Asunto(s)
Proteínas Bacterianas/genética , Bordetella pertussis/genética , Cromosomas Bacterianos/genética , Elementos Transponibles de ADN/genética , Sulfato de Magnesio/farmacología , Niacina/farmacología , Transactivadores/genética , Bordetella pertussis/crecimiento & desarrollo , Bordetella pertussis/patogenicidad , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genéticaRESUMEN
The d4 domain, a novel zinc finger-like structural motif, was first revealed in the rat neuro-d4 protein. Here we demonstrate that the d4 domain is conserved in evolution and that three related genes form a d4 family in the human genome. The human neuro-d4 is very similar to rat neuro-d4 at both the amino acid and the nucleotide levels. Moreover, the same splice variants have been detected among rat and human neuro-d4 transcripts. This gene has been localized on chromosome 19, and two other genes, members of the d4 family isolated by screening of the human genomic library at low stringency, have been mapped to chromosomes 11 and 14. The gene on chromosome 11 is the homolog of the ubiquitously expressed mouse gene ubi-d4/requiem, which is required for cell death after deprivation of trophic factors. A gene with a conserved d4 domain has been found in the genome of the nematode Caenorhabditis elegans. The conservation of d4 proteins from nematodes to vertebrates suggests that they have a general importance, but a diversity of d4 proteins expressed in vertebrate nervous systems suggests that some family members have special functions.
Asunto(s)
Genoma Humano , Familia de Multigenes/genética , Proteínas del Tejido Nervioso/genética , Factores de Transcripción , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 19/genética , Secuencia Conservada , Exones/genética , Humanos , Datos de Secuencia Molecular , Empalme del ARN , Ratas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
To isolate genes differentially expressed in the rat brain cortex and cerebellum, a subtractive cDNA cloning procedure requiring only small quantities of poly(A+) RNA, followed by differential screening, was used. Two novel genes, MK and 3L7, with cortex- and cerebellum-specific expression were identified. These genes displayed different expression patterns in the brain cortex, cerebellum, and hippocampus during postnatal development.
Asunto(s)
Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica , Animales , Clonación Molecular , ADN Complementario , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
To identify genes differentially expressed in the developing rat cerebellum, a subtractive cDNA cloning procedure, followed by differential screening, was used. Four novel genes, MB, MF, 3E7, and 3C6, the transcriptional activity of which changed by a factor of five during cerebellar development, were isolated. The genes obtained were differentially expressed in different regions of the central nervous system. Variations in the levels of corresponding transcripts in the brain cortex and cerebellum were also recorded during postnatal rat development.