Digerente uccelli

<div><p>We investigated the streptomycin-induced stress response in <i>Salmonella enterica</i> serovars with a laser optical sensor, BARDOT (<b>ba</b>cterial <b>r</b>apid <b>d</b>etection using <b>o</b>ptical scattering <b>t</b>echnology). Initially, the top 20 <i>S</i>. <i>enterica</i> serovars were screened for their response to streptomycin at 100 μg/mL. All, but four <i>S</i>. <i>enterica</i> serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125–250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25–5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25–50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of <i>Salmonella</i> grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (<i>aadA</i>), aminoglycoside efflux pump (<i>aep</i>), multidrug resistance subunit <i>acrA</i>, and ribosomal protein S12 (<i>rpsL</i>), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in <i>Salmonella</i> in conjunction with the molecular and immunoassay methods.</p></div

Immunoassays to monitor the streptomycin induced expression of chaperonin GroEL in <i>S</i>. Typhimurium.

<p><b>(A)</b> ELISA with protein fractions from cell lysate and cell wall/membrane (2 μg/well) of <i>S</i>. Typhimurium grown in the presence (25 and 50 μg/mL) or absence of streptomycin was performed. The mouse anti-GroEL mAb (1:2000), HRP-conjugated anti-mouse antibody (1:4000 dilutions; Jackson Immunologicals), and ortho-phenylenediamine (OPD, Sigma-Aldrich) were used in the assay. Data represent, fold change in absorbance values compared to the control. <b>(B)</b> Western blot showing over expression of GroEL in the cell fractions after immunoprobing with anti-GroEL mAb (1:1000), HRP-conjugated anti-mouse antibody (1:5000 dilutions; Jackson Immunologicals), and chemiluminescence substrate Lumiglo (Cell signaling, Danvers, MA).</p

Differences in the scatter patterns calculated in terms of true negative after <i>S</i>. <i>enterica</i> serovar Typhimurium colonies grown in the presence and absence (control) of different concentration of streptomycin.

<p>^aBrain-heart infusion agar (BHIA) supplemented with streptomycin 25 μg/mL (BHIA+Strep25), or 50 μg/mL (BHI+Strep50). Values in a row marked with alphabets (A, B, C) indicate significant difference at <i>p</i> < 0.05.</p><p>Differences in the scatter patterns calculated in terms of true negative after <i>S</i>. <i>enterica</i> serovar Typhimurium colonies grown in the presence and absence (control) of different concentration of streptomycin.</p

Protein analysis, mass spectrometry and qRT-PCR analysis.

<p><b>(A)</b> SDS-PAGE (7.5%-acrylamide)-analysis of protein preparations from whole cell lysate and cell wall/outer membrane. The bottom panel shows quantitative estimation of a protein band (pixel intensity) from the marked lanes using the ImageJ software. Protein bands marked with dashed arrow (1, 2, and 3) were analyzed by MALDI-TOF MS to be GroEL. <b>(B)</b> qRT-PCR showing fold difference in the <i>groEL</i> gene expression in <i>S</i>. Typhimurium strains grown in BHI broth supplemented with streptomycin (25 and 50 μg/mL). Bars marked with the letters (a and b) denote significant difference at <i>p</i> < 0.05.</p

Scatter pattern of <i>S</i>. Typhimurium in the presence and absence of streptomycin in BHI agar (BHIA) and error matrix used for analyzing the scatter patterns.

<p><b>(A)</b> Scatter patterns of <i>S</i>. Typhimurium (13ENT1277, 13ENT1140, 13ENT1288, 13ENT0899) colonies grown on BHIA with or without streptomycin (Strep, 25 and 50 μg/mL) for 10–16 h to achieve a desired colony diameter (1.2 ± 0.1 mm). Phase-contrast microscopic images of individual cells of <i>S</i>. Typhimurium obtained from the colony grown under different condition at 1000X (scale bar, 5 μm) to compare the effect of streptomycin on cell size. Two independent experiments were performed to obtain at least 60 scatter images (30 images/experiment) for each strain. Colony profile (diameter) was measured at 100X magnification. Scatter images for BHIA (Control), BHIA+Strep25, and BHIA+Strep50 belongs to Group-A, B and C, respectively. <b>(B)</b> Error matrix grid (2 x 2) to calculate true negative values described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135035#pone.0135035.t001" target="_blank">Table 1</a></b>. The separate groups of scatter images were acquired from: ST-library containing independent control (without antibiotic) data set; Group-A contains colonies grown on BHIA without antibiotic (BHIA); Group-B contains colonies grown on BHIA supplemented with 25 μg/mL streptomycin (BHI+Strep25); Group-C contains colonies grown on BHIA supplemented with 50 μg/mL streptomycin (BHIA+Strep50). The scatter images in the ST-library were matched with the scatter images of other three groups (A, B, and C) separately in 2 x 2 matrix, to calculate the true negatives. <b>(C)</b> Colony profile, scatter pattern and surface plots for the streptomycin-sensitive strains <i>S</i>. Enteritidis PT21 and <i>S</i>. Mississippi E345 grown on BHIA (control) and BHIA supplemented with sub-inhibitory concentrations of streptomycin, 1.25 μg/mL (Strep1.25), 2.5 μg/mL (Strep2.5), and 5 μg/mL (Strep5). Surface plots were constructed for the scatter images using NIH ImageJ software based analysis of non-RGB images for grayscale values (x-, y-axis: 992 pixels; z-axis, indicates pixel intensity at the scale of 0–255). Scatter patterns were captured at definite time when the colony size reached 1.2 ± 0.1 mm diameter after incubation on respective streptomycin concentration on BHIA plates.</p

MALDI-TOF mass spectrometry based identification of differentially expressed protein bands from <i>S</i>. <i>enterica</i> serovar Typhimurium after treatment with streptomycin.

<p>^aSample consists of Coommassie blue stained protein band that were sequenced at Applied Biomics (Hayward, CA).</p><p>^bProtein fractions (whole cell lysate and cell wall) were collected from <i>S</i>. Typhimurium cell that were grown in BHI broth supplemented with streptomycin at the concentration of 25 μg/mL (Strep25) and 50 μg/mL (Strep50).</p><p>^cPeptide sequence of the band generated by MALDI-TOF MS were compiled and further matched with NCBI protein database. Accession number represents the matched protein.</p><p>^dRepresent properties of identified protein; MW: molecular weight; PI: isoelectric point; C.I.: confidence interval.</p><p>MALDI-TOF mass spectrometry based identification of differentially expressed protein bands from <i>S</i>. <i>enterica</i> serovar Typhimurium after treatment with streptomycin.</p

Schematic for capturing scatter pattern of colony with optical sensor and selection of optimal media.

<p><b>(A)</b> Schematics of BARDOT to acquire scatter patterns of colonies. <b>(B)</b> Evaluation of different non-selective agar media to capture scatter pattern of <i>Salmonella enterica</i> serovar Typhimurium 13ENT0899 colonies. Brain–heart infusion agar (BHIA); Luria–Bertani agar (LBA); Nutrient agar (NA); Tryptic soy agar (TSA). Scatter patterns were captured when the colony diameter reached to 1.2 ± 0.1 mm after 10–12 h of incubation.</p

Quantitative reverse transcription PCR (qRT-PCR) to quantify streptomycin resistance-related gene expression in <i>S</i>. <i>enterica</i> serovars in the presence of streptomycin.

<p>Data showing fold difference in the expression of genes coding for (<b>A</b>) aminoglycoside adenyltransferase, <i>aadA</i>, (<b>B</b>) aminoglycoside efflux pump, <i>aep</i>, (<b>C</b>) multidrug efflux pump subunit, <i>acrA</i>, and (<b>D</b>) ribosomal S12 protein, <i>rpsL</i> (<i>strA</i>) after the <i>S</i>. Typhimurium cells were grown in BHI broth supplemented with different concentrations (25, 50 μg/mL) of streptomycin (Strep). Bars marked with *, **, *** are significantly different at <i>p</i> < 0.05.</p

Visible-Light-Induced Acetalization of Aldehydes with Alcohols

In this work, we have achieved a simple and general method for acetalization of aldehydes by means of a photochemical reaction under low-energy visible light irradiation. A broad range of aromatic, heteroaromatic, and aliphatic aldehydes have been protected under neutral conditions in good to excellent yields using a catalytic amount of Eosin Y as the photocatalyst. Our visible light mediated acetalization strategies are successful for more challenging acid-sensitive aldehydes and sterically hindered aldehydes. Notably, this protocol is chemoselective to aldehydes, while ketones remain intact

Image_1_Tunicamycin Mediated Inhibition of Wall Teichoic Acid Affects Staphylococcus aureus and Listeria monocytogenes Cell Morphology, Biofilm Formation and Virulence.TIF

<p>The emergence of bacterial resistance to therapeutic antibiotics limits options for treatment of common microbial diseases. Subinhibitory antibiotics dosing, often aid in the emergence of resistance, but its impact on pathogen’s physiology and pathogenesis is not well understood. Here we investigated the effect of tunicamycin, a cell wall teichoic acid (WTA) biosynthesis inhibiting antibiotic at the subinhibitory dosage on Staphylococcus aureus and Listeria monocytogenes physiology, antibiotic cross-resistance, biofilm-formation, and virulence. Minimum inhibitory concentration (MIC) of tunicamycin to S. aureus and L. monocytogenes was 20–40 μg/ml and 2.5–5 μg/ml, respectively, and the subinhibitory concentration was 2.5–5 μg/ml and 0.31–0.62 μg/ml, respectively. Tunicamycin pre-exposure reduced cellular WTA levels by 18–20% and affected bacterial cell wall ultrastructure, cell membrane permeability, morphology, laser-induced colony scatter signature, and bacterial ability to form biofilms. It also induced a moderate level of cross-resistance to tetracycline, ampicillin, erythromycin, and meropenem for S. aureus, and ampicillin, erythromycin, vancomycin, and meropenem for L. monocytogenes. Pre-treatment of bacterial cells with subinhibitory concentrations of tunicamycin also significantly reduced bacterial adhesion to and invasion into an enterocyte-like Caco-2 cell line, which is supported by reduced expression of key virulence factors, Internalin B (InlB) and Listeria adhesion protein (LAP) in L. monocytogenes, and a S. aureus surface protein A (SasA) in S. aureus. Tunicamycin-treated bacteria or the bacterial WTA preparation suppressed NF-κB and inflammatory cytokine production (TNFα, and IL-6) from murine macrophage cell line (RAW 264.7) indicating the reduced WTA level possibly attenuates an inflammatory response. These results suggest that at the subinhibitory dosage, tunicamycin-mediated inhibition of WTA biosynthesis interferes with cell wall structure, pathogens infectivity and inflammatory response, and ability to form biofilms but promotes the development of antibiotic cross-resistance.</p