Brucella microti: the genome sequence of an emerging pathogen

<p>Abstract</p> <p>Background</p> <p>Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel <it>Brucella </it>species, <it>Brucella microti</it>, was determined. <it>B. microti </it>is a member of the genus <it>Brucella </it>within the <it>Alphaproteobacteria</it>, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other <it>Brucella </it>species, <it>B. microti </it>is a fast growing and biochemically very active microorganism with a phenotype more similar to that of <it>Ochrobactrum</it>, a facultative human pathogen. The atypical phenotype of <it>B. microti </it>prompted us to look for genomic differences compared to other <it>Brucella </it>species and to look for similarities with <it>Ochrobactrum</it>.</p> <p>Results</p> <p>The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of <it>B. microti </it>is almost identical to that of <it>Brucella suis </it>1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in <it>B. microti</it>. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between <it>B. microti </it>and <it>Ochrobactrum anthropi </it>but impaired in other sequenced <it>Brucella</it>. The most noticeable difference between <it>B. microti </it>and other <it>Brucella </it>species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of <it>B. microti</it>.</p> <p>Conclusion</p> <p>Contrary to expectations from the phenotypic analysis, the genome sequence of <it>B. microti </it>is highly similar to that of known <it>Brucella </it>species, and is remotely related to the one of <it>O. anthropi</it>. How the few differences in gene content between <it>B. microti </it>and <it>B. suis </it>1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the <it>Brucella </it>genus. The genome sequence of <it>B. microti </it>will serve as a model for differential expression analysis and complementation studies. Our results also raise some concerns about the importance given to phenotypical traits in the definition of bacterial species.</p

Phorbol-Ester Mediated Suppression of hASH1 Synthesis: Multiple Ways to Keep the Level Down

Human achaete-scute homolog-1 (hASH1), encoded by the human ASCL1 gene, belongs to the family of basic helix-loop-helix transcription factors. hASH1 and its mammalian homolog Mash1 are expressed in the central and peripheral nervous system during development, and promote early neuronal differentiation. Furthermore, hASH1 is involved in the specification of neuronal subtype identities. Misexpression of the transcription factor is correlated with a variety of tumors, including lung cancer and neuroendocrine tumors. To gain insights into the molecular mechanisms of hASH1 regulation, we screened for conditions causing changes in hASH1 gene expression rate. We found that treatment of human neuroblastoma-derived Kelly cells with phorbol 12-myristate 13-acetate (PMA) resulted in a fast, strong and long-lasting suppression of hASH1 synthesis. Reporter gene assays with constructs, in which the luciferase activity was controlled either by the ASCL1 promoter or by the hASH1 mRNA untranslated regions (UTRs), revealed a mainly UTR-dependent mechanism. The hASH1 promoter activity was decreased only after 48 h of PMA administration. Our data indicate that different mechanisms acting consecutively at the transcriptional and post-transcriptional level are responsible for hASH1 suppression after PMA treatment. We provide evidence that short term inhibition of hASH1 synthesis is attributed to hASH1 mRNA destabilization, which seems to depend mainly on protein kinase C activity. Under prolonged conditions (48 h), hASH1 suppression is mediated by decreased promoter activity and inhibition of mRNA translation

Yersinia pestis DNA from Skeletal Remains from the 6(th) Century AD Reveals Insights into Justinianic Plague.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics

Improvement of an invA-based PCR for the specific detection of Salmonella typhimurium in organs of pigs

The aim of this study was to investigate the suitability of the invA-based polymerase chain reaction (PCR) assay for the specific detection of Salmonella in organs of experimentally infected pigs and to compare these results to classical bacterial culture. While the PCR conditions specified in the Deutsche Industrie Norm , DIN I 0135 (§ 35 LMBG, 1999), revealed various unspecific amplification products, modifications of the PCR conditions allowed the specific amplification of the invA fragment from inner organs. The modified PCR assay correlates excellently with cultivation results (as required by DIN Norm 6579) and enables the detection of Salmonella within 48 hours with equal sensitivity compared to routine cultivation

Influence of long-time transportation stress on re-activation of Salmonella Typhimurium DT104 in experimentally infected pigs

In this study a Salmonella Typhimurium infection model in swine was used in order to investigate the influence of pre-mortal stress induced by long time period transportation on the re-activation of Salmonella in experimentally infected pigs. Salmonella free pigs were exposed to a highly virulent strain of Salmonella Typhimurium DT I 04 by direct intragastrical administration. Clinical parameters were monitored and the shedding rate in faeces was qualitatively and quantitatively determined by standard bacteriological procedures for 2 1 days. The distribution of the challenge organism in 14 different internal organs of transported and nontransported animals was determined. All infected animals developed clinical signs of salmonellosis 12 to 24 hours post infection. About 88 to 100 % of the fecal samples were culture-positive up to post exposure day 6, and then varied from 71 to 92 % until slaughter, respectively. At necropsy S. typhimurium was recovered most frequently from caecum and ileocolic lymph nodes (83 %), colon (79 %), pa latine tonsils (71 %) and mandibular lymph nodes (62.5 %). A negative impact of transportation stress on the shedding rate and the genera l condition of the animals was observed

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

BACKGROUND: Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. RESULTS: A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. CONCLUSIONS: Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed

Differential phenotyping of Brucella species using a newly developed semi-automated metabolic system

<p>Abstract</p> <p>Background</p> <p>A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus <it>Brucella </it>and their differentiation into species and biovars.</p> <p>Results</p> <p>A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of <it>Brucella </it>and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "<it>Brucella </it>identification and typing" plate (Micronaut™) was designed and re-tested in 113 <it>Brucella </it>isolates and a couple of closely related bacteria.</p> <p><it>Brucella </it>species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of <it>Brucella </it>isolates to the species level could be achieved. The separation of <it>B. canis </it>from <it>B. suis </it>bv 3, however, failed. At the biovar level, <it>B. abortus </it>bv 4, 5, 7 and <it>B. suis </it>bv 1-5 could be discriminated with a specificity of 100%. <it>B. melitensis </it>isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars.</p> <p>Conclusions</p> <p>The comprehensive testing of metabolic activity allows cluster analysis within the genus <it>Brucella</it>. The biotyping system developed for the identification of <it>Brucella </it>and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.</p

Повышение эффективности производственной деятельности на предприятии

Выпускная квалификационная работа 62 с, 6 рис., 11 табл., 30 источников, 2 прил. Объектом исследования является холдинг DI-Group включающий в себе деятельность предприятия ООО «Монета». Предметом исследования является эффективность внедрения инструментов бережливого производства, применяемых в процессе сборки автоматов по изготовлению сувенирных монет и жетонов «Монетный аттракцион». Цель работы − повышение эффективности процесса производства автоматов «Монетный аттракцион» с помощью внедрения инструментов бережливого производства. В процессе исследования изучалась деятельность ООО «Монета» посредством проведения хронометража всех операций производства, а также посредством изучения литературы, рассматривались применяющие раннее технологии в производстве.Final qualifying work 62, 6 fig., 11 tab., 30 sources, 2 adj. The object of this study is holding DI-Group includes a company's activity of "Coin". The subject of study is the effectiveness of the implementation of lean manufacturing tools used during assembly machines for the production of souvenir coins and tokens "Mint attraction." Purpose - improving the efficiency of the production process "Mint attraction" machines through the implementation of lean manufacturing tools. The study investigated the activity of LLC "Coin" by means of the timing of production operations as well as through the study of literature, we considered applying early technology in production