Binding of PhoB to its targets <i>in vitro</i>.

<p>This figure shows the results of gel mobility shift assays for binding of PhoB-His fusion protein to the eight binding sites. The binding sites which are centered at their putative binding sites corresponding to their intergenic regions upstream of the <i>prpR</i>, <i>mipA</i>, <i>feaR</i>, <i>yedX</i>, <i>cusR</i>, <i>yegH</i>, <i>yhjC</i>, and <i>ydfH</i> genes. DNA fragments were incubated with 0, 5, 10, 15, 20, 25, 30, or 35 μM PhoB-His as indicated. Free DNA fragments and PhoB-His-DNA complexes are labeled as F and C respectively.</p

Novel findings of PhoB targets with differential expressions in the presence and the absence of PhoB activity.

<p>This figure shows the (A) ChIP-chip peaks and the (B) luminescence from reporter gene assay of PhoB novel targets which located in the promoter regions with differential expressions in MG1655 and MG1655_PhoB_KO. These targets are (i) <i>mipA</i>, (ii) <i>yedX</i>, (iii) <i>ydfH</i>, (iv) <i>cusC</i>, (v) <i>cusR</i>, (vi) <i>sbcD</i>, (vii) <i>yhjC</i>, (viii) <i>yegH</i>, (ix) <i>feaR</i>, and (x) <i>prpR</i>. (A) Expansion of PhoB binding peaks on the ten regions. The detected peaks were centered with 2000 bps flanking regions. The log₂ fold change (<i>y</i>-axis) represents the log₂ ratio (grey line) and the smoothed ratio (black line) of the normalized Cy5 signal (MG1655_PhoB_FLAG) divided by the normalized Cy3 signal (MG1655) after averaging our triplicate results. (B) Reporter gene assays were performed to further check the differential expression. Luminescence is expressed as relative light unit (RLU). The RLU was calculated by, first normalizing the light units to the optical densities of harvested cultures, then divided by the averaged light units from non-inserted pGL3 vector transformed MG1655 strain or MG1655_PhoB_KO strain (shown as ΔPhoB). The “*” sign indicates significant luciferase expression (p-value<0.05).</p

Bacterial strains used in this study.

<p>Abbreviations: Kmr, kanamycin resistance.</p

Cultivation of <i>E. coli</i> MG1655, MG1655_PhoB_FLAG, and MG1655_PhoB_KO strains under the phosphate limiting condition.

<p>The dashed lines indicate the phosphate consumption of MG1655 (filled circles), MG1655_PhoB_FLAG (filled triangles) and MG1655_PhoB_KO (hollow rectangles). The solid lines show the growth curves of the three strains. Bacterial cultures were harvested at an OD₆₀₀ value of 1.0 (indicated by the black arrow) for both the ChIP-chip and gene expression microarray experiments. For the reporter gene assay, transformed cells were also harvested at an OD₆₀₀ value of 1.0.</p

Table_1_Biofilm formation is not an independent risk factor for mortality in patients with Acinetobacter baumannii bacteremia.docx

In the past decades, due to the high prevalence of the antibiotic-resistant isolates of Acinetobacter baumannii, it has emerged as one of the most troublesome pathogens threatening the global healthcare system. Furthermore, this pathogen has the ability to form biofilms, which is another effective mechanism by which it survives in the presence of antibiotics. However, the clinical impact of biofilm-forming A. baumannii isolates on patients with bacteremia is largely unknown. This retrospective study was conducted at five medical centers in Taiwan over a 9-year period. A total of 252 and 459 patients with bacteremia caused by biofilm- and non-biofilm-forming isolates of A. baumannii, respectively, were enrolled. The clinical demographics, antimicrobial susceptibility, biofilm-forming ability, and patient clinical outcomes were analyzed. The biofilm-forming ability of the isolates was assessed using a microtiter plate assay. Multivariate analysis revealed the higher APACHE II score, shock status, lack of appropriate antimicrobial therapy, and carbapenem resistance of the infected strain were independent risk factors of 28-day mortality in the patients with A. baumannii bacteremia. However, there was no significant difference between the 28-day survival and non-survival groups, in terms of the biofilm forming ability. Compared to the patients infected with non-biofilm-forming isolates, those infected with biofilm-forming isolates had a lower in-hospital mortality rate. Patients with either congestive heart failure, underlying hematological malignancy, or chemotherapy recipients were more likely to become infected with the biofilm-forming isolates. Multivariate analysis showed congestive heart failure was an independent risk factor of infection with biofilm-forming isolates, while those with arterial lines tended to be infected with non-biofilm-forming isolates. There were no significant differences in the sources of infection between the biofilm-forming and non-biofilm-forming isolate groups. Carbapenem susceptibility was also similar between these groups. In conclusion, the patients infected with the biofilm-forming isolates of the A. baumannii exhibited different clinical features than those infected with non-biofilm-forming isolates. The biofilm-forming ability of A. baumannii may also influence the antibiotic susceptibility of its isolates. However, it was not an independent risk factor for a 28-day mortality in the patients with bacteremia.</p

Observation of Substrate Orientation-Dependent Oxygen Defect Filling in Thin WO_3−δ/TiO₂ Pulsed Laser-Deposited Films with in Situ XPS at High Oxygen Pressure and Temperature

Substoichiometric tungsten oxide films of approximately 10 nm thickness deposited with pulsed laser ablation on single-crystal TiO₂ substrates with (001) and (110) orientation show defect states near the Fermi energy in the valence-band X-ray photoelectron spectroscopy (XPS) spectra. The spectral weight of the defect states is particularly strong for the film grown on the (001) surface. In situ XPS under an oxygen pressure of 100 mTorr shows that the spectral weight of the defect states decreases significantly at 500 K for the film on the (110) substrate, whereas that of the film grown on the (001) substrate remains the same at a temperature up to 673 K. Furthermore, diffusion of titanium from the substrate to the film surface is observed on the (110) substrate, as is evidenced by the sudden appearance of the Ti 2p core level signature above 623 K and below 673 K. The film grown on the (001) surface does not show such an interdiffusion effect, which suggests that the orientation of the substrate can have a significant influence on the high-temperature integrity of the tungsten oxide films. Quantitative analysis of the O 1s core level XPS spectra shows that chemisorbed water from sample storage under ambient conditions is desorbed during heating under oxygen exposure

Tandem duplication of the <i>bla</i>_NDM-1 gene in pKPX-1 and pECX-1.

<p>(<b>A</b>) Diagrammatic representation of the analysis of <i>bla</i>_NDM-1 copy number by Southern blot. The probe is shown with a red arrow, and the tandem duplication of the 8588-bp repeat is indicated by the bracket. The asterisks indicate the methylated <i>Nru</i>I sites. The sizes of <i>Bam</i>HI or <i>Hind</i>III digested fragments depend on the copy number of the repeat. The pound sign indicates 79.1 kb and 71.8 kb for <i>Bam</i>HI and <i>Hind</i>III restrictions, respectively, when there are 8 copies of the tandem repeat, as in the case of pECX-1. (<b>B</b>) Sequencing read distribution and Southern analysis of the <i>bla</i>_NDM-1 region for pECX-1. The upper panel shows the relative coverage depth of the repeat region and its flanking sequences. The average coverage of <i>bla</i>_NDM-1 is 7–8 fold of those sequences of the immediately adjacent regions, suggesting that there are eight copies of the repeat. As shown in the lower panel, Southern analysis confirms this model of tandem duplication. (<b>C</b>) <i>Bla</i>_NDM-1 copy number variation detected by the Southern analysis. Sequence depth of the region revealed an average of 3–4 copies of the repeat sequence in pKPX. <i>Bam</i>HI and <i>Hind</i>III digestion gave a series of ladder bands, corresponding to different copy numbers of the repeat. By contrast, <i>Avr</i>II and <i>Nru</i>I both deliberated a single major band of 8.6 kb, representing the unit length of the tandem repeats.</p

Sequence analysis of KPX plasmids.

<p>Two circular sequences are shown for the organization of pKPX-1 (<b>A</b>) and pKPX-2 (<b>B</b>). Mapping shotgun sequencing reads of pECX-1 to the pKPX-1 is indicated by the red half-circle. A large part of the plasmid, corresponding to the nucleotide positions 23,125 to 145,377 of pKPX-1, was not found in pECX-1. Only the part on the left side, totaling 128,191-bp, is retained. Two genes encoding chloramphenicol and amikacin resistance were identified by functional library screening. Their positions in the deleted region are indicated. Nucleotides are numbered according to the replication origin. Genes are color coded: yellow, β-lactamase; red, antimicrobial resistance associated; blue, plasmid replication and partitioning; black, transposases or IS elements; and white, other coding sequences of miscellaneous features. The arrows on the open reading frames (ORFs) indicate the gene orientation. Gene clusters involved in gene transfer or mobility are marked in green. <i>Xba</i>I and <i>Avr</i>II restriction sites are shown inside the circle.</p

Restriction analysis of the KPX plasmids by PFGE.

<p>Plasmid DNA isolated from the KPX isolate was analyzed using pulsed field gel electrophoresis (PFGE). The restriction patterns of the plasmid DNA were compared to those of pECX-1 and pECX-2 in <i>Escherichia coli</i>. The size of the DNA markers is shown in kilobases (kb) on the left side.</p

Antimicrobial resistance determinants in pKPX-1 and pKPX-2.

*<p>Nonfunctional; ^† Deleted in pECX-1.</p><p>Abbreviations: AAC, aminoglycoside acetyltransferase; APH, aminoglycoside phosphotransferase; ANT, aminoglycoside nucleotdyltransferase; Rmt, 16S rRNA methyltransferase; Qnr, quinolone resistance protein; TetA, tetracycline efflux protein; Cat, chloramphenicol acetyltransferase; ARR-2, rifampin ADP-ribosyltransferase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; Mph, macrolide phosphotransferase.</p