Synthesis, characterization and complex evaluation of antibacterial activity and cytotoxicity of new arylmethylidene ketones and pyrimidines with camphane skeletons

The synthesis of 20 arylidenecamphors and 15 pyrimidines with camphane skeleton is described in the current report. A modified method for preparation of sterically hindered 2- aminopyrimidines in two steps was demonstrated. The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed different MIC values (up to 0.91 μM for ketone 39). Compound 35 demonstrated moderate (8.23 μM), but sustainable activity toward a collection of drug-resistant M. tuberculosis strains. Many of the compounds (especially among 2-aminopyridines 42–56) exhibited good to excellent activity against different strains of pathogenic bacteria and fungi (MIC up to 0.60 μM for compound 50), compared with reference antibiotics. Many of the newly designed compounds possess also in vitro cytotoxicity.This study was supported by: Bulgarian National Science Fund- project KP-06-H39/7 and Spanish Ministry of Science, Innovation and Universities- Grant RTI2018-094629-BI00. MEDINA’s authors disclosed the receipt of financial support from Fundación MEDINA, a public-private partnership of Merck Sharp and Dohme de EspañaS.A./Universidad de Granada/Junta de Andalucía

Genome-Wide Mycobacterium tuberculosis Variation (GMTV) Database: A New Tool for Integrating Sequence Variations and Epidemiology

Background Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome. Description Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes. Conclusions Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains

Evaluation of New Variable-Number Tandem-Repeat Systems for Typing Mycobacterium tuberculosis with Beijing Genotype Isolates from Beijing, China▿

The newly proposed variable-number tandem-repeat (VNTR) typing system, which includes a basic 15-locus set and a high-resolution 24-locus set (P. Supply et al., J. Clin. Microbiol. 44:4498-4510, 2006), demonstrated a high power for the discrimination of Mycobacterium tuberculosis isolates collected worldwide. To evaluate its ability to differentiate the Beijing genotype strains from the Beijing area in China, 72 isolates with typical Beijing or Beijing-like spacer oligonucleotide typing profiles were subjected to typing with the VNTR system (24 loci) and typing by restriction fragment polymorphism analysis with IS6110 (IS6110-RFLP). Compared to the “old” 12-locus VNTR typing method, use of the 15- and 24-locus systems had a dramatically improved power to discriminate the Beijing genotype strains. A subtle difference in the Hunter-Gaston discriminatory index (HGI) between the 15-locus and the 24-locus systems resulted from only one locus, Mtub29. However, the VNTR-based clusters could be further differentiated by IS6110-RFLP (HGI by IS6110 RFLP, 0.999), although in one case an IS6110 cluster was subdivided by the 15-locus VNTR system. In this sense, use of the newly proposed 15-locus VNTR system along with the Mtub29 locus can serve as a first-line typing method for the epidemiological study of M. tuberculosis isolates in Beijing, while secondary typing of clustered strains by IS6110-RFLP is still required

Russian "successful" clone B0/W148 of Mycobacterium tuberculosis Beijing genotype: a multiplex PCR assay for rapid detection and global screening

Submitted by Sandra Infurna ([email protected]) on 2016-10-11T12:03:41Z No. of bitstreams: 1 lia_gomes_etal_IOC_2011.pdf: 354512 bytes, checksum: 03de1c7e6ff32ced2ca65fe09a996128 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-10-11T12:28:20Z (GMT) No. of bitstreams: 1 lia_gomes_etal_IOC_2011.pdf: 354512 bytes, checksum: 03de1c7e6ff32ced2ca65fe09a996128 (MD5)Made available in DSpace on 2016-10-11T12:28:20Z (GMT). No. of bitstreams: 1 lia_gomes_etal_IOC_2011.pdf: 354512 bytes, checksum: 03de1c7e6ff32ced2ca65fe09a996128 (MD5) Previous issue date: 2012St. Petersburg Pasteur Institute. Laboratory of Molecular Microbiology. St. Petersburg, Russia.St. Petersburg Pasteur Institute. Laboratory of Molecular Microbiology. St. Petersburg, Russia.St. Petersburg Pasteur Institute. Laboratory of Molecular Microbiology. St. Petersburg, Russia.The Research Institute of Phthisiopulmonology. Laboratory of Microbiology. St. Petersburg, Russia.National Key Discipline of Paediatrics. Key Laboratory of Major Diseases in Children (Capital Medical University). Ministry of Education, Beijing Pediatric Research Institute. Beijing Children’s Hospital affiliated to Capital Medical University, Beijing, China.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.National Key Discipline of Paediatrics. Key Laboratory of Major Diseases in Children (Capital Medical University). Ministry of Education, Beijing Pediatric Research Institute. Beijing Children’s Hospital affiliated to Capital Medical University, Beijing, China.The Research Institute of Phthisiopulmonology. Laboratory of Microbiology. St. Petersburg, Russia.We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a "successful" clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis

Rapid Detection of the Mycobacterium tuberculosis Beijing Genotype and Its Ancient and Modern Sublineages by IS6110-Based Inverse PCR

The Mycobacterium tuberculosis Beijing genotype strains appear to be hypervirulent and associated with multidrug-resistant tuberculosis. Therefore, the development of a both rapid and simple method to detect the M. tuberculosis Beijing genotype is of clinical interest per se. Previously, we described a simple and fast approach to detect the Beijing genotype based on IS6110 inverse-PCR typing. Here, we evaluated this method against a large, diverse, and recent collection of strains. The study sample included 866 M. tuberculosis strains representing but not limited to the regions in Russia, Europe, and East Asia where the Beijing genotype is endemic. Based on a spoligotyping method, 408 strains were identified as Beijing genotypes; they were additionally subdivided into ancient and modern sublineages based on the analysis of the NTF locus. All strains were further subjected to the IS6110-based inverse PCR. All of the Beijing genotype strains were found to have identical two-band (ancient sublineage) or three-band (modern sublineage) profiles that were easily recognizable and distinct from the profiles of the non-Beijing strains. Therefore, we suggest using IS6110-based inverse-PCR typing for the correct identification of the Beijing genotype and its major sublineages. The method is fast and inexpensive and does not require additional experiments but instead is implemented in the routine typing method of M. tuberculosis

Insight into Population Structure and Drug Resistance of Pediatric Tuberculosis Strains from China and Russia Gained through Whole-Genome Sequencing

This study aimed to determine phenotypic and genotypic drug resistance patterns of Mycobacterium tuberculosis strains from children with tuberculosis (TB) in China and Russia, two high-burden countries for multi/extensively-drug resistant (MDR/XDR) TB. Whole-genome sequencing data of M. tuberculosis isolates from China (n = 137) and Russia (n = 60) were analyzed for phylogenetic markers and drug-resistance mutations, followed by comparison with phenotypic susceptibility data. The Beijing genotype was detected in 126 Chinese and 50 Russian isolates. The Euro-American lineage was detected in 10 Russian and 11 Chinese isolates. In the Russian collection, the Beijing genotype and Beijing B0/W148-cluster were dominated by MDR strains (68% and 94%, respectively). Ninety percent of B0/W148 strains were phenotypically pre-XDR. In the Chinese collection, neither of the Beijing sublineages was associated with MDR/pre-XDR status. MDR was mostly caused by low fitness cost mutations (rpoB S450L, katG S315T, rpsL K43R). Chinese rifampicin-resistant strains demonstrated a higher diversity of resistance mutations than Russian isolates (p = 0.003). The rifampicin and isoniazid resistance compensatory mutations were detected in some MDR strains, but they were not widespread. The molecular mechanisms of M. tuberculosis adaptation to anti-TB treatment are not unique to the pediatric strains, but they reflect the general situation with TB in Russia and China

Unusual Large-Scale Chromosomal Rearrangements in <i>Mycobacterium tuberculosis</i> Beijing B0/W148 Cluster Isolates

<div><p>The <i>Mycobacterium tuberculosis</i> (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.</p></div

Genome rearrangements' representation for W-148 Progenitor I like H37Rv, W-148 Progenitor II and W-148 genomes.

<p>Each local collinear block (LCB) I–V is represented by a different color. Upside-down blocks (LCBs II and IV) represent the location of the reverse strand, which means an inversion has occurred. Asterisk indicates a terminus of a replication site. Terminus of a replication site was calculated based on GraphDNA (GC-skew mode) software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971-Thomas1" target="_blank">[19]</a>.</p

Southern blot analysis of H37Rv and SP21 MTB strains.

<p>Genomic DNA was digested with <i>Mlu</i>I and hybridized with the fluorescent labeled probes obtained by amplification. The probes are listed at the top of the lanes (from A to H). (A) Hybridization patterns of H37Rv strain. The order of probes corresponds to the order of complementary sequence sites in the genome of H37Rv. (B) Hybridization pattern of SP21 strains. The order of probes corresponds to the order of complementary sequence sites in the genome of H37Rv. (C) Hybridization patterns of SP21 strain. The order of probes is rearranged in accordance with the expected order of complementary sequence sites in the inverted genome (Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084971#pone.0084971.s005" target="_blank">Text S1</a>). The merged bands from probes complementary to the boundaries of recombination junctions are boxed. M, marker strain Mt14323 (Mycobacterial Reference Laboratory, National Public Health Institute (Turku, Finland)).</p