The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo.

Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt - an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

Contribution of BlaA and BlaB β-Lactamases to Antibiotic Susceptibility of Yersinia enterocolitica Biovar 1B▿

Highly pathogenic Yersinia enterocolitica biovar 1B produces two distinct β-lactamases, BlaA and BlaB. Mutants of a representative biovar 1B isolate were constructed and evaluated to determine the extent of limitation of susceptibility to broad-spectrum β-lactam antibiotics by BlaA and BlaB. The results demonstrated that BlaA, a class A enzyme, plays a significant role in limiting susceptibility to penicillins and cephalosporins. The contribution of BlaB, a class C enzyme, was less profound and was limited primarily to cephalosporin susceptibility

The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo.

Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt - an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice

Resistance Determinants and Mobile Genetic Elements of an NDM-1-Encoding <i>Klebsiella pneumoniae</i> Strain

<div><p>Multidrug-resistant <i>Enterobacteriaceae</i> are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-lactamase NDM-1 is rapidly spreading. We present the complete genome of <i>Klebsiella pneumoniae</i> ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight β-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the <i>cps-lps</i> polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne <i>bla</i>_CTX-M-15 was transposed recently to the chromosome by IS<i>Ecp1</i>. None of the eleven full copies of IS<i>26</i>, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS<i>26</i> as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence IS<i>Kpn21</i> of the IS<i>NCY</i> family, suggesting that it uses the two-step transposition mechanism of IS<i>3</i>. Robust genome-based phylogeny showed that a unified <i>Klebsiella</i> cluster contains <i>Enterobacter aerogenes</i> and <i>Raoultella</i>, suggesting the latter genus should be abandoned.</p></div

Learned phyloblocks identify a new island and the highly variable capsular polysaccharide and lipopolysaccharide synthesis gene cluster (<i>cps-lps</i>).

<p>Nonubiquity phyloblocks: those missing in at least one of the 11 reference chromosomes. Complex phyloblocks: those requiring more than one gain/loss event to reconcile the phylotype with the genome tree of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone-0099209-g001" target="_blank">Fig. 1</a>. As a percentage of their combined 411 kbp, the learned phyloblocks mapped either to the training islands (81.9%), the two newly indicated regions (12.0%), insertion sequences (2.1%), or to small scattered regions that did not show hallmarks of islands (4.0%). Red segments: the 11 final islands (including a tandem array of Kpn21L and Kpn11L). Circles, the two newly indicated regions.</p

<i>Klebsiella</i> phylogeny.

<p>Tree for 108 genomes based on a 2.93-Mbp alignment, rooted at the midpoint of the outgroup (Ecl/Yre) branch. Nodes with <30% bootstrap support were combined forming the multifurcated dashed line; otherwise support values are shown only when <100%. Brackets: Kpn multilocus sequence type (ST). Inset: enlargement of the “core Kpn” phylogeny. Kpn2146 falls in a clade containing fellow ST11 strains Kpn JM45 and Kpn HS11286 and a tight clade (circled) of ST258 and ST512 strains. The ST258/ST512 clade is heavily sequenced, and represented here with only five of its most diverse members. Bold: complete genomes used for phyloblocks analysis. Species name abbreviations: Kpn, <i>K. pneumoniae</i>; Ksp, <i>K. sp</i>.; Kpl, <i>K. cf. planticola</i>; Kox, <i>K. oxytoca</i>; Kva, <i>K. variicola</i>; Eae, <i>Enterobacter aerogenes</i>; Ecl, <i>E. cloacae</i>; Ror, <i>Raoultella ornithinolytica</i>; Yre, <i>Yokanella regensburgei</i>.</p

Replicon copy numbers.

a<p>Unique 21-mers were identified for each replicon, and counted among MiSeq reads.</p>b<p>Mean 21-mer coverages were very similar to median values, and were normalized to that of the chromosome (which had 68.8 occurrences per 21-mer).</p

pKpn2146b.

<p>Key, color coding of genes, mobile elements, and unique regions and juxtapositions, with additional colors for non-gene features. Inner ring, representative long matches to other plasmids. Innermost black arrows, recent recombination events. HR, homologous recombination; abR, antibiotic resistance.</p

Enzymes, efflux pumps, and mutations expected to confer resistance to antibiotics of clinical relevance^a.

a<p>Excluding the resistance enzyme for bleomycin, an antibiotic used clinically only as an antitumor agent.</p>b<p>Variant number from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone-0099209-t001" target="_blank">Table 1</a> of Ramirez <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099209#pone.0099209-Ramirez2" target="_blank">[37]</a> is used to distinguish the AAC(6′)-Ib variants.</p>c<p>Two silent differences between the two copies.</p>d<p>Probable efflux substrates identified from literature sources including ARDB; the substrates list is not comprehensive and in many cases has been deduced from organisms other than <i>K. pneumoniae</i>.</p>e<p>Mdt genes are scattered over the chromosome.</p