Extracellular GSH compromises the interaction of <i>B. cenocepacia</i> with mucociliary-differentiated CF bronchial epithelial cells. Panel A.

<p>MIP (Maximum Intensity Projection) from confocal system acquisition (Olympus IX 81 inverted microscope, software FV 1000) of monolayers infected with <i>B. cenocepacia</i> LMG 16656 for 3 hours, in absence (<b>upper</b>) or in presence (<b>bottom</b>) of 10 mM extracellular GSH. Cells were washed, fixed and permeabilized as described in Materials and Methods. Bacteria (red) and zona occludens (green) were detected using specific antibodies (R418 and anti-ZO1, respectively). Bar = 20 µm. <b>Panel B.</b> Invasion of mucociliary-differentiated CF bronchial epithelial cells by <i>B. cenocepacia</i> LMG 16656 in absence or in presence of GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayers. The reported values represent the mean ± SD obtained by measuring LMG 16656 invasion ability from six individual cultures. (*p = 0.01; #p = 0.002; **p<0.05).</p

Extracellular GSH modifies <i>B. cenocepacia</i> LMG 16656 adhesion, but not intracellular replication. Panel A.

<p>Effect of extracellular GSH on <i>B. cenocepacia</i> LMG 16656 adhesion to 9HTEo- and CFTE29o- cells. Bars indicate the % of the bacteria adhering to cells with respect to the bacteria added to the cell monolayer. Results are the average ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (*p<0.05; ** p<0.01). <b>Panel B.</b> Effect of 10 mM GSH on the total number (adherent + intracellular bacteria) of <i>B. cenocepacia</i> LMG 16656 recovered after 3 hours of 9HTEo- epithelial cell infection. Results are shown as % of total bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (***p<0.0001). <b>Panel C. </b><i>B. cenocepacia</i> LMG 16656 replication within 9HTEo- cells. Fold replication values were determined by dividing the intracellular bacterial load at 1, 2.5 and 4 h post-infection by that determined after 30 minutes of infection. Results are the mean ± SD of three independent experiments.</p

Effect of extracellular GSH on pro-inflammatory cytokines expression stimulated by <i>B. cenocepacia</i> LMG 16656.

<p>9HTEo-, CFTE29o- cells were incubated in presence or absence of 10 mM extracellular GSH and infected with <i>B. cenocepacia</i> LMG 16656 (10 CFU/cell) in presence of 0 and 10 mM GSH for 3 hours. The expression of IL-8, TNF-α and IL-1β was analyzed by RT-PCR. Data are from a typical experiment out of three giving qualitatively similar results. Each data point is the average of three independent measures on each sample.</p

Cytofluorimetric analysis of surface thiols.

<p>After an incubation in presence or absence of 10 mM extracellular GSH, cells were treated with 10 µM Alexa fluor C₅-maleimide to label surface free thiols and then analyzed by FACScalibur system, as described in Materials and Methods. The histograms are from a typical experiment out of three giving essentially identical results.</p

Extracellular GSH decreases <i>B. cenocepacia</i> invasion. Panel A.

<p>Invasion of 9HTEo- and CFTE29o- by <i>B. cenocepacia</i> LMG 16656 was assayed in presence of 0, 0.1, 1 and 10 mM GSH. The % of invasion indicates the ratio between the number of intracellular bacteria recovered from infected cells with respect to the bacteria added to the cell monolayer. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion in three independent experiments. Asterisks denote statistically significant results (* p<0.05; ** p<0.01 and *** p<0.0001, respectively). White bar: untreated cells; grey bar: cells treated with 0.1 mM GSH; dotted bar: cells treated with 1 mM GSH; black bar: cells treated with 10 mM GSH. <b>Panel B.</b> 9HTEo- and CFTE29o- were pretreated with 0, 0.1, 1 and 10 mM GSH for 3 hours at 37°C, washed to remove GSH and then used in the infection assay. Results, which are expressed as the mean ± SD of intracellular bacteria isolated from cells pretreated with GSH with respect to untreated cells, are the average of three independent experiments (**p<0.01; ***p<0.0001). Bar colors are as in panel A. <b>Panel C.</b> Invasion of C38 and IB3-1 by <i>B. cenocepacia</i> LMG 16656 in presence of 0 and 10 mM GSH. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion ability in three independent experiments (*** p<0.0001). White bars: control cells; black bars: cells treated with 10 mM GSH. <b>Panel D.</b> Invasion of 9HTEo-, CFTE29o-, C38 and IB3-1 by <i>B. cenocepacia</i> 6L in presence of 0 and 10 mM GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD obtained by measuring 6L invasion ability in three independent assays (** p<0.01). White bars: control cells; black bars: cells treated with 10 mM GSH.</p

DataSheet8_A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-.docx

<p>Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.</p

DataSheet6_A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-.XLSX

<p>Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.</p

DataSheet7_A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-.docx

<p>Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.</p

DataSheet9_A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-.XLSX

<p>Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.</p

DataSheet1_A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-.XLSX

<p>Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.</p