Atypical Salmonella enterica serovars in murine and human infection models: Is it time to reassess our approach to the study of salmonellosis?

<div> <p>Nontyphoidal <i>Salmonella </i>species<i> </i>are globally disseminated pathogens and the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using <i>in vivo</i> murine models and cell lines typically challenged with <i>Salmonella</i> Typhimurium. Although<i> </i>serovars Enteritidis and Typhimurium are responsible for the most<i> </i>of human infections reported to the CDC, several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical <i>S. enterica</i> serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage<i> </i>model of infection is unsuitable for inferring human relevant differences in nontyphoidal <i>Salmonella </i>infections<i> </i>whereas differentiated human THP-1 macrophages allowed these isolates to be further characterised in a more relevant, human context.</p></div

Relative expression of outer membrane proteins, regulators and inner membrane transporter genes.

<p>Data from three independent total mRNA extractions of <i>E. coli</i> AG100 physiologically adapted to increasing concentrations of TET compared to its parental non-induced strain grown in the absence of TET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000365#s3" target="_blank">Materials and Methods</a>. A ratio of 1 corresponds to no alterations in expression compared with untreated control cells. Values were corrected for standard deviation range.</p

Relative quantification of the expression level of the protease genes.

<p>Data from three independent total mRNA extractions of <i>E. coli</i> AG100 physiologically adapted to increasing concentrations of TET compared to its parental non-induced strain grown in absence of TET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000365#s3" target="_blank">Materials and Methods</a>. A ratio of 1 corresponds to no alterations in expression compared with untreated control cells. Values were corrected for standard deviation range.</p

Relative quantification of phagosomal acidification in <i>M. bovis</i> BCG-GFP-infected macrophages stained with LysoTracker Red and analysed by flow cytometry.

<p> Human macrophages were infected with <i>M</i>. <i>bovis</i> BCG-GFP, treated with verapamil (VP), thioridazine (TZ), chlorpromazine (CPZ), flupenthixol (FPX), and haloperidol (HAL). Data was analysed by flow cytometry using an easyCyte^™ 5HT flow cytometer. A) Bars graph: quantification of the increase on the overall number of LysoTracker Red stained vesicles per cell (average fluorescence intensity); B) cytograms for (i), unstained and non-treated infected macrophages; (ii), stained and non-treated infected macrophages; and (iii) to (vii), stained and infected macrophages treated with VP, TZ, CPZ, FPX and HAL. VP was tested at 10 μg/ml; TZ at 2.5 μg/ml; CPZ, HAL and FPX at 1.25 μg/ml. Significance of the results was tested using Student’s t-test and were considered highly significant, ***<i>P</i> <0.001.</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.</p

Tetracycline activation cascade of <i>E. coli</i> resistance physiological adaptation.

<p>Broken arrows indicate the activation in 1 and 2 over-expression of specific gene (direct TET pressure effect), in 3, the regulation by induced regulators (second level of control), in 4 the effect of activated genes coding for membrane proteins (third level of effect). Thick arrows (5) illustrate the effect of over-production of OMPs and proteases.</p

Primers used in this study.

<p>Primers used in this study.</p

Immunodetection of the outer membrane proteins.

<p>The detections were carried out using antisera prepared against OmpC porin (A), OmpF porin (B), antigenic peptide located inside the internal loop 3 porin (C), OmpA (D) and OmpX (E) respectively. Immunodetection were carried out with total cell extracts from non-induced AG100 (1, 2) and 10 mg/mL TET-induced (3, 4) strains grown in LB and MH media (odd and even lanes respectively).</p

Determination of cathepsin B activity.

<p> Cathepsin B activity was evaluated in human monocyte-derived macrophages infected and non-infected with <i>M</i>. <i>tuberculosis</i> H37Rv and non-treated and treated with verapamil (VP), thioridazine (TZ), chlorpromazine (CPZ), flupenthixol (FPX) and haloperidol (HAL). VP was tested at 10 μg/ml; TZ at 2.5 μg/ml; CPZ at 1.25 μg/ml; HAL at 1.25 μg/ml; FPX at 1.25 μg/ml. The results were considered highly significant, **<i>P</i> < 0.01.</p