Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.

Next-Generation Sequencing for Characterizing Drug Resistance-Conferring Mycobacterium tuberculosis Genes from Clinical Isolates in the Ukraine.

The Ukraine ranks among the top 20 countries with the highest number of multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis cases in the world. However, little is known of the genetic diversity, i.e., resistance signatures, in clinical isolates from this region. We analyzed seven of most prevalent MDR/XDR antibiotic resistance-conferring genes from clinical isolates (n = 75) collected from geographically diverse Ukrainian oblasts and the southern Crimean peninsula. Genomic analysis revealed that 6 (8%) were sensitive, 3 (4%) were resistant to at least one antibiotic but were not MDR, 40 (53%) were MDR, and 26 (35%) were XDR. The majority of isolates (81%) were of the Beijing-like lineage. This is the first study to use next-generation sequencing (NGS) of clinical isolates from the Ukraine to characterize mutations in genes conferring M. tuberculosis drug resistance. Several isolates harbored drug resistance signatures that have not been observed in other countries with high-burden tuberculosis. Most notably, the absence of inhA gene promoter mutations, a diversity of mutation types in the rpoB resistance-determining region, and detection of heteroresistance provide a broader understanding of MDR/XDR from this area of the world.

Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array.

Enzootic pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. Most herds are vaccinated, but classical bacterin vaccines do not prevent colonization and it is not possible to detect flourishing M. hyopneumoniae infections in vaccinated herds since commonly used commercial ELISAs cannot differentiate infected from vaccinated animals. To solve this problem, new immunogenic proteins, up-regulated or solely expressed during infection, need to be identified. For this purpose a peptide-spot array was constructed which presents 105 potential linear B-cell epitopes identified by in silico analysis in 35 putative lipoproteins encoded on the genome of M. hyopneumoniae type strain 232. Subjecting this array to immunoblotting using porcine convalescent serum revealed a single strongly immunoreactive epitope on the Mhp366 protein which did not react with serum from bacterin-immunized pigs. In addition, it was not possible to detect Mhp366 in total cell lysates of in vitro grown M. hyopneumoniae strains, using a polyclonal rabbit serum raised against a recombinant GST-Mhp366 fusion protein. To investigate the possibility of using an Mhp366-based ELISA in the field for differentiating vaccinated herds with and without a flourishing infection it was shown that (i) homologues of the corresponding mhp366 gene were present in all 17 M. hyopneumoniae strains and porcine lung samples tested from different geographic origins and (ii) an ELISA based on epitope-specific synthetic peptides as solid phase antigen allowed a classification of field samples. Therefore, Mhp366 might be the first antigen identified which facilitates the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

Commissioning of the accelerator-recuperator for the FEL at the Siberian Center for Photochemical Research.

A 100 MeV eight-turn accelerator-recuperator intended to drive a high-power infrared free-electron laser (FEL) is currently under construction in Novosibirsk. The first stage of the machine includes a one-turn accelerator-recuperator that contains a full-scale RF system. It was commissioned successfully in June 2002.