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Vol 32, No 4 (2017)
- Year: 2017
- Articles: 10
- URL: https://journal-vniispk.ru/0891-4168/issue/view/11065
Review
On the Origin and Distribution of Antibiotic Resistance: Permafrost Bacteria Studies
Abstract
The presented review summarizes the data obtained during the study of antibiotic-resistant bacteria isolated from the Arctic and Antarctic permafrost sediments. Comparative analysis of the molecular structure of the “ancient” environmental and modern clinical genes of resistance to β-lactams, streptomycin, spectinomycin, vancomycin, tetracycline, and sulfanilamides revealed a high level of their similarity, in some cases reaching 100%. The molecular structure of ancient bacterial plasmids carrying antibiotic resistance determinants was considered and compared with the plasmid structure of modern bacteria. A wide distribution of small plasmids among modern bacteria closely related to the ancient Acinetobacter plasmid carrying an autonomous streptomycin/spectinomycin resistance gene aadA27 has been revealed. The “ancient” integron structure, which agrees with the description of the hypothetical ancestor of the integrons of the subgroup aadA2, was described. By the examples of the Tn3 family transposons (Tn5393 and Tn21 subgroups) various mechanisms of formation of complex transposons simultaneously carrying several antibiotic-resistance genes were discussed. In particular, an important role of integrons in formation of complex transposons is demonstrated, and numerous cases of independent insertion of integrons with diverse cassette antibiotic-resistance genes into various base transposons of the Tn3 family are presented. Separately, the origin of the complex transposons of the Tn21 subgroup is discussed with the goal of revealing all the simple mobile elements, base ancestors for their formation, in permafrost. Together, the data presented are considered convincing evidence of the origin of both the clinical genes for antibiotic resistance and the mobile elements carrying them from the resistance determinants of bacteria inhabiting natural ecosystems.
169-179
Molecular Markers of Gliomas
Abstract
Gliomas are invasive recurrent brain tumors with high lethality. Gliomas have been morphologically divided into astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas. Gliomas are graded according to the criteria of the World Health Organization criteria as grade I (pilocytic astrocytomas), grade II (low-grade gliomas), grade III (high-grade gliomas), and grade IV (glioblastoma). The mutations of IDH1/2 and TP53 genes and MGMT methylation have been described as prognostic markers of glioma. The aim of the present review is to analyze research and experimental results (Scopus, Web of Science, Pubmed) concerning somatic mutations, aberrant regulation of gene expression of signal pathways, diagnostic and prognostic markers of glioma progression and characterizing various morphological types and grades of glioma. In particular, the specificities of medulloblastomas and ependymomas have been considered in the present review.
180-190
Experimental Works
Three Genetically Different Lineages of Yersinia pestis subsp. Microtus bv. Caucasica (0.PE2) Strains Circulate among Common Voles in Natural Plague Foci in the Caucasus
Abstract
Gram-negative bacteria Y. pestis subsp. pestis of 0.ANT-4.ANT, 1.ORI, and 2.MED SNP types are the cause of numerous epidemic outbreaks, epidemics, and three plague pandemics, claiming hundreds of millions of human lives. At the same time, strains of the microtus subspecies that belong to the 0.PE SNP type circulating in populations of different species of voles (Microtus spp.) are able to cause only extremely rare human infections that are not transmitted from person to person. It is suggested that the clinical form of the infection can develop only in individuals with impaired immune status. Strains of Y. pestis bv. caucasica (0.PE2), one of the most ancient phylogenetic groups of subsp. microtus, are isolated on the territory of the following natural foci: the Transcaucasian highland (including the Leninakan (4), Pre-Sevan (5), and Zanzegur- Karabakh (6)) mesofoci and the Dagestan-highland (39). In addition to the enumerated areas, similar strains of Y. pestis are isolated in the territories of the Pre-Araks focus (7) bordering the Transcaucasian highland one. Previously, we showed that passport data on the phenotypic differences of strains of the biovar caucasica, isolated from different foci, corresponded to their MLVA25, CRISPR, and DFR genotypes. In the present work, a comparative analysis of the clustering ability of MLVA25- and CRISPR-typing methods with the “gold standard” of phylogenetic studies—SNP typing—has been conducted on 21 strains of Y. pestis subsp. microtus bv. caucasica (0.PE2). The analysis of the obtained results confirms the existence of three clonal clusters of strains corresponding to the natural plague foci—39, 4, and the group of foci 5–7. Only the SNP-typing method made it possible to separate branch of focus 5 isolates from a group of strains isolated in foci 5–7. In addition, this method made it possible to reveal a greater genetic heterogeneity of strains from focus 39, in contrast to the strains of foci 4–7. When analyzing the genomes of Y. pestis strains isolated in the territory of focus 39, a deletion (~20 kb) was detected in the CRISPR region of the Ypb locus. The absence of this locus can serve as a marker for the determination of a given population of Y. pestis strains.
191-195
Multilocus Sequence Analysis of “Atypical” Borrelia burgdorferi sensu lato Isolated in Russia
Abstract
Seventeen isolates of Borrelia burgdorferi sensu lato with an unclear genetic characteristic isolated in Russia from various species of Ixodes ticks (I. persulcatus, I. ricinus, and I. pavlovskyi) have been studied by multilocus sequencing analysis. According to the results of typing, these isolates were identified as B. bavariensis (n = 7), B. garinii (n = 6), B. finlandensis (n = 3), and B. spielmanii (n = 1), that is, species that can cause ixodid tick-borne borreliosis diseases in human beings. The allelic polymorphism of five genes (rrs, fla, groEL, recA, and ospA) and spacer rrfA–rrlB of B. bavariensis and B. garinii isolates was analyzed in comparison with their diversity in the International Nucleotide Sequence Database Collaboration.
196-203
The “Protein Corona” of Silver-Sulfide Nanoparticles Obtained Using Gram-Negative and -Positive Bacteria
Abstract
A comparative analysis of the amount and composition of the proteins adsorbed onto the surface of silver-sulfide (NpAg2S) nanoparticles obtained by biosynthesis using bacteria was carried out for the first time. These were the gram-negative bacteria Shewanella oneidensis MR-1 and Escherichia coli K12, as well as the gram-positive bacterium Bacillus subtilis 168. The biosynthesis of NpAg2S was carried out in nutrient broth with 1 mM of AgNO3 and Na2S2O3 · 5H2O salts in the presence of bacterial cells under aerobic conditions. Analysis of NpAg2S by transmission electron microscopy showed that the particles were spherical and had an average diameter of 8 ± 2 nm for S. oneidensis MR-1 and E. coli K12 and 10 ± 3 nm for B. subtilis 168. It was found that the highest amount of protein was sorbed on NpAg2S, when the strain B. subtilis 168 was used, and the smallest amount was sorbed by E. coli. The main proteins adsorbed on NpAg2S were determined by the MALDI TOF/TOF method, and the heterogeneity of the protein coating was revealed. The lowest heterogeneity of proteins on the surface of nanoparticles is observed in the case of B. subtilis (only one protein, flagellin, predominates); the highest heterogeneity of proteins was found on nanoparticles obtained using S. oneidensis MR-1. All proteins covering the surface of NpAg2S were shown to be outer-membrane or cytoplasmic-membrane proteins of the studied bacteria. The composition of the protein coating of nanoparticles is individual and constant for each bacterial strain used. The values of the ζ-potential and the effective diameter of nanoparticles were shown to differ depending on the “protein corona” of the strain that was used to obtain NpAg2S. The characteristic of the “protein corona” of biologically obtained nanoparticles is an important and necessary condition for their practical application.
204-211
Change in Oncolytic Activity of Sendai Virus during Adaptation to Cell Cultures
Abstract
This work is devoted to the study of the correlation between mutational variability and cytotoxic properties of the oncolytic Sendai virus when using different culture systems. We have shown that, as a result of 12 consecutive passages in the allantoic fluid of chick embryos, Sendai virus accumulates in it in high titers without changing the RNA genomic sequence and cytotoxic properties in relation to melanoma (Mel8) and human glioma cells (U87MG). However, for the drugs obtained in this system, there is a problem of allergenicity and standardization. The solution could be an adaptation of the virus to cell culture. We used cell cultures 4647 (African green monkey kidney cells) and 293 (human embryonic kidney cells) that are certified for vaccine production in Russia. It turned out that in the course of the reproduction of the virus in these cell cultures during 21–25 passages, accumulation of mutations with the highest density of nonsynonymous substitutions in the F and HN genes, which encode the surface proteins of the virion and ensure its infectivity, takes place. The accumulation of mutations leads to a significant decrease in the cytotoxic activity of Moscow strain of Sendai virus in respect of melanoma and human glioma cells. Reverse passaging on chick embryos of the Sendai virus, adapted to cells 4647, provides partial elimination of adaptive mutations and restoration of oncolytic activity of the virus. Mutations acquired during adaptation to cells 293 are not eliminated in the process of tenfold reverse passaging in chick embryos and the oncolytic activity of the virus does not recover. Thus, the stability of the Sendai virus genome is important for the implementation of its oncolytic potential, and this must be taken into account when choosing a technique for the production of antitumor drugs based on this virus.
212-217
New Mutations in Ciprofloxacin Resistant Strains of Pseudomonas aeruginosa Isolated from Guilan Province, Northern Iran
Abstract
It seems that high resistance to ciprofloxacin can simultaneously be the result of mutation in several genes such as topoisomerase enzymes and negative regulatory genes implicated in the expression of efflux pump systems in Guilan Province.
218-223
miR-17 and miR-20a Expression in IL-2 Signaling Pathway in Jurkat T Cells
Abstract
Background: the miR-17-92 cluster is known as an oncogene (oncomiR-1) overexpressed in most cancers. However, recent studies have shown that these miRNAs were downregulated in some cancers. Thus, some or all members of miR-17-92 cluster may have dual activity: apoptosis induction or inhibition. Objectives: For a better understanding of miR-17-92 cluster activities, we focused on the function of mir-17 and mir-20a as two members of miR-17-92 cluster in Jurkat cells activated by phytohemagglutinin (PHA). Materials and Methods: Jurkat cells were stimulated by PHA for 24 h. P-lenti-mico-GFP plasmids containing miR-17 or miR-20a and an empty vector (blank) were transfected into activated Jurkat cells using Lipofectamine 2000 reagent. Cell viability was measured using XTT assay after transfection. Putative targets of these miRNAs were predicted by Targetscan and miRWalk algorithms in JAK/STAT and PI3K/AKT signaling pathways. Then the expression of several putative targets was evaluated by quantitative RT-PCR. Results: Transfection of mir-17 and mir-20a decreased the proliferation in activated Jurkat cells. In silico investigations revealed that mir-17 and mir-20a potentially target JAK1, AKT1 and AKT3. Q-RT-PCR analysis illustrated that these targets were downregulated in Jurkat cells after transfection with miR-17 and miR-20a. Conclusions: According to our findings, it seems that mir-17 and mir-20a expression, two members of miR-17-92 cluster, is dependent on cell condition and cell type; as a result, their ectopic expression in activated Jurkat cells may lead to cell death.
224-229
In silico Prediction and in vitro Verification of a Novel Multi-Epitope Antigen for HBV Detection
Abstract
After years of ongoing endeavors for HBV infection prognosis, diagnosis and treatment, it still remains a major health problem worldwide. About 400 million chronic carriers and an annual death rate as high as one million reflects the seriousness of the problem. Developing novel and more effective diagnostic strategies, using in silico approaches and their subsequent empirical verification, will be helpful in providing for blood supply safety, therapeutics efficacy and disease activity assessment. Exploiting various in silico tools a novel multiepitope detection construct was designed which was consisted of eight linked linear immunodominant HBV epitopes. The designed antigen was expressed in Escherichia coli as the host. The detection capability of the designed antigen was tested using Chemiluminescent immunoassay method. Chemiluminescent immunoassay on the expressed antigen revealed that the product may be a credible candidate for simultaneous detection of three main HBV antibodies. All three test samples in two concentrations indicated lower RLU/s in comparison to the positive control which was the direct consequence of HBV antibody detection by the designed antigen. In the present study, employing bioinformatics tools paved the way for rational design of multiepitope antigen in a more cost effective, intelligent and knowledge-based method. The obtained results could be construed as a primary proof of concept that the in silico predictions could be used as primary steps of the biological studies and their subsequent empirical conduction.
230-240
Mutations in Hotspot Regions of ERG11 Gene in Fluconazole Resistant Isolates of Candida albicans in Guilan Province, Northern Iran
Abstract
Our study indicates that the level of fluconazole resistance in C. albicans strains is high in Guilan province and other drugs should be used in resistant infections. It seems that missense mutations in four isolates play role in azole resistance. However in three isolates premature stop codon may be involved in high dose resistance. And it is suggested that in fourth isolates another mechanisms introduce increase of resistance dose in combination with missense mutation in ERG11. Results of this study suggest that in patients by high dose of resistance do not use azole because of mutations that decrease azole effects.