The authors would like to thank Dr. Olivia Steele-Mortimer for sharing pCHAR-Duo plasmid. This work was funded by the Italian Ministry of Health, grants PRC2019014 and PRC2021004.

<ol>
	<li>European Food Safety Authority &amp; European Centre for Disease Prevention and Control. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+European+Union+One+Health+2020+Zoonoses+Report.">The European Union One Health 2020 Zoonoses Report.</a> EFSA Journal. 19 (12), 6971 (2021).</li><li>Fattinger, S. A., Sellin, M. E., Hardt, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+effector+driven+invasion+of+the+gut+epithelium:+breaking+in+and+setting+the+house+on+fire.">Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire.</a> Current Opinion in Microbiology. 64, 9-18 (2021).</li><li>Knodler, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+of+invasive+Salmonella+via+bacterial-induced+extrusion+of+mucosal+epithelia.">Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (41), 17733-17738 (2010).</li><li>Fulde, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+SPI2+T3SS+mediates+transcytosis+in+polarized+enterocytes+in+vivo.">Salmonella SPI2 T3SS mediates transcytosis in polarized enterocytes in vivo.</a> Cell Host &amp; Microbe. , (2019).</li><li>Cooper, K. G., Chong, A., Starr, T., Finn, C. E., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictable,+tunable+protein+production+in+Salmonella+for+studying+host-pathogen+interactions.">Predictable, tunable protein production in Salmonella for studying host-pathogen interactions.</a> Frontiers in Cellular and Infection Microbiology. 7, 475 (2017).</li><li>Klein, J. A., Grenz, J. R., Slauch, J. M., Knodler, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+activity+of+the+Salmonella+invasion-associated+injectisome+reveals+its+intracellular+role+in+the+cytosolic+population.">Controlled activity of the Salmonella invasion-associated injectisome reveals its intracellular role in the cytosolic population.</a> mBio. 8 (6), 01931 (2017).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Ibarra, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Salmonella+pathogenicity+island+1+under+different+growth+conditions+can+affect+Salmonella-host+cell+interactions+in+vitro.">Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro.</a> Microbiology. 156 (4), 1120-1133 (2010).</li><li>Tambassi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+hilD+in+a+Salmonella+Derby+lineage+linked+to+swine+adaptation+and+reduced+risk+to+human+health.">Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health.</a> Scientific Reports. 10 (1), 21539 (2020).</li><li>Finn, C. E., Chong, A., Cooper, K. G., Starr, T., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+wave+of+Salmonella+T3SS1+activity+prolongs+the+lifespan+of+infected+epithelial+cells.">A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells.</a> PLoS Pathogens. 13 (4), 1006354 (2017).</li><li>Knodler, L. A., Nair, V., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+cytosolic+Salmonella+in+epithelial+cells.">Quantitative assessment of cytosolic Salmonella in epithelial cells.</a> PLoS One. 9 (1), 84681 (2014).</li><li>Chong, A., Starr, T., Finn, C. E., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Salmonella+Type+III+Secretion+System+1+in+bacterial+adaptation+to+the+cytosol+of+epithelial+cells.">A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.</a> Molecular Microbiology. 112 (4), 1270-1283 (2019).</li></ol>

The authors declare that they have no competing interests.

The way Salmonella colonizes intestinal epithelial cells, influences the infection outcome. Upon invasion, the cytosolic hyper-replication induces inflammation of the gut3, whereas vacuolar replication can lead to systemic spread4. Salmonella strains can vary in their ability to invade and replicate inside intestinal epithelial cells9. Indeed, Salmonella is an extremely diverse genus comprising more than 2,500 serovars, which have different abilities to cause disease. In addition, Salmonella is the most frequently reported cause of foodborne outbreaks in the European Union, indicating a large spread in the human population1. Therefore, the availability of reliable, high-throughput methods for the quantification of the intracellular phenotypes of Salmonella is of great importance to evaluate differences in virulence among large numbers of Salmonella strains and ultimately allow an accurate and strain-specific risk assessment of this pathogen.
The protocol described here measures the behavior of Salmonella inside epithelial cells in a fast and automated way. The infection of epithelial cells is performed in 96-well imaging microplates and an automated fluorescence microscope is used for image acquisition, making the protocol suitable for high-throughput applications. This protocol takes advantage of the pCHAR-Duo plasmid, which allows distinguishing vacuolar from cytosolic Salmonellae through the differential expression of two fluorescent reporters5. The intracellular phenotypes of Salmonella are often analyzed by manual scoring, a time-consuming procedure that is unsuitable for the analysis of large numbers of strains and cell cultures per strain and prone to operator&#39;s errors and inter-operator variation. To overcome these limitations, two complementary and automated image analyses were developed, the area analysis and the single-cell analysis. ImageJ, a freely available software, was used to develop the two analyses. In order to accelerate the protocol execution, ImageJ scripts for batch analysis of multiple acquisition files with no operator intervention are provided as supplemental files.
The area analysis was designed to quantify, in a few steps, the overall colonization of epithelial cells (infection ratio) and the&#160;hyper-replication level (hyper-replication ratio) through the measurement of the areas specifically occupied by the nuclei of epithelial cells and by Salmonellae expressing either mCherry or mCherry together with GFP. The area analysis is applied to images acquired at low magnification, where both vacuolar and hyper-replicating Salmonellae are in focus in the same z-plane, and a large number of epithelial cells is displayed per microscopic field, reducing the size and number of acquisition files. The area analysis allows for an automated, fast, and computationally-light analysis of large number of samples, making it suitable for high-throughput assays such as screening experiments.
The single-cell analysis was designed to quantify Salmonella phenotypes inside epithelial cells with single-cell resolution, obtained through cell segmentation and measurement of the cellular area and the percentage of area occupied by vacuolar and cytosolic hyper-replicating Salmonellae. The overall colonization of epithelial cells characterized through the area analysis is here broken down into three quantitative parameters, the percentage of infected cells, the mean vacuolar load, and the hyper-replication rate, allowing to evaluate and quantify the contribution of each phenotype to the overall colonization, therefore,&#160;complementing the results of area analysis. The greater details offered by the single-cell analysis come at the cost of slower image acquisition and analysis. In fact, in order to achieve single-cell resolution, image acquisition is performed at high magnification. This implies that multiple z-planes are needed to observe both vacuolar and cytosolic Salmonellae in focus and that the acquisition of a large number of fields per sample is needed to score a high number of epithelial cells, thus extending acquisition time in comparison with area analysis. Furthermore, the analysis of several large acquisition files is computationally demanding, thus requiring a suitable workstation (a 6-core, 32 GB RAM workstation was used). Therefore, single-cell analysis can be used as a stand-alone in limited throughput conditions or as a second-level method coupled to the area analysis to gain a deeper understanding of the Salmonella phenotypes inside epithelial cells.
The two complementary analyses were validated by using S. Tm, S. Derby wt, and S. Derby &#916;sipA, chosen because their behavior inside epithelial cells was already known to differ in terms of invasion or replication9,10. The results of the area and single-cell analyses show that the protocol allowed to quantitatively distinguish differences in intracellular Salmonella phenotypes in accordance with the characteristics of the tested strains. Furthermore, these results demonstrate that the single-cell analysis allows for quantification of the contribution of each intracellular phenotype (invasion, vacuolar load&#160;and cytosolic replication) to the overall colonization scored using the area analysis.
This protocol was applied here to study in vitro pathogenicity of different strains of Salmonella, but it can have other applications such as the study of random mutants to identify genes involved in the invasion and/or replication inside epithelial cells. In addition, the protocol can be adapted to analyze the behavior of Salmonella inside other cell lines than INT407 cells. It can also be used as the starting point to develop similar methods for studying cell-pathogen interaction of other intracellular microorganisms.

Salmonella is the most frequently reported bacterial agent causing outbreaks of foodborne disease in the European Union1. The primary pathological manifestation of Salmonella infection is enteritis, which is the result of the pathogen behavior in the gut following ingestion and the consequent local inflammatory response2. However, Salmonella can also disseminate to extra-intestinal sites and cause systemic infection, especially in immunocompromised individuals. The type of interaction between Salmonella and the intestinal epithelium conditions the outcome of the infection. Once in the gut lumen, Salmonella invades and replicates inside intestinal epithelial cells. At the intracellular level, Salmonella can present two different replication phenotypes, the cytosolic hyper-replication and the intravacuolar slow replication within Salmonella-containing vacuoles (SCVs). The cytosolic hyper-replication induces inflammatory host cell death and Salmonella extrusion into the gut lumen3; the vacuolar replication leads to a trans-epithelium penetration and systemic spread4. Therefore, the extent of invasion and vacuolar vs. cytosolic replication influences the course of infection.
The genus Salmonella is very diverse, including thousands of serotypes with different host-ranges and abilities to cause disease. For example, S. Typhimurium is defined as a generalist serovar, because it infects multiple unrelated hosts, and represents one of the major causes of human salmonellosis. Differently, S. Derby is considered a swine-adapted serovar, as it is mostly isolated from the swine, but it is also reported in the top five of the serovars responsible for human infection1. However, knowledge about the bacterial behavior inside the epithelial cells is essentially limited to the study of a few reference strains, as S. Typhimurium SL1344, that do not represent the vast natural diversity of Salmonella pathogenicity. Characterizing the interaction of different strains of Salmonella with epithelial cells would contribute to understanding their different pathogenicity. For this reason, a high-throughput fluorescence microscopy-based protocol was developed to analyze the intracellular behavior of a large number of strains in a fast and largely automated way. In this protocol, infection of epithelial cells was performed in 96-well imaging plates and image acquisition was made using an automated fluorescence microscope. The pCHAR-Duo plasmid was used to observe the invasion and replication phenotypes of Salmonella inside epithelial cells through fluorescent microscopy5. This plasmid carries the gene encoding the red fluorescent reporter mCherry, constitutively expressed by all the transformed bacterial cells, and the gene encoding the green fluorescent reporter GFP, whose expression is activated by glucose-6-phosphate present exclusively in the cytosol of eukaryotic cells and absent in SCVs. Therefore, the plasmid allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP reporters.
The vacuolar and cytosolic bacteria on microscopy images are commonly quantified by manual scoring6, but this is a time-consuming method that limits analysis to a small number of samples. Therefore, two complementary and automated image analyses were developed-area analysis and single-cell analysis-using ImageJ7, a freely available image analysis software. The area analysis measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication by using data of areas occupied by epithelial cells, red and green Salmonellae in each acquired microscopy image. This method can be applied to images acquired at low magnification; therefore, it allows to score a high number of epithelial cells with few images, shortening the acquisition time. The single-cell analysis uses cell segmentation to determine the percentage of infected cells, the mean vacuolar load, and the percentage of infected cells undergoing cytosolic hyper-replication with single-cell resolution.
In this protocol, all steps of the image analysis are described in detail to be performed manually, but the same analysis can be automated by our specifically designed ImageJ scripts. These scripts also allow to run batch analyses to automatically analyze multiple images and thus speed up the execution of the method.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well imaging microplate</td>
			<td>Eppendorf</td>
			<td>30741030</td>
			<td>Cell culture and infection</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>59349</td>
			<td>Bacteria culture</td>
		</tr>
		<tr>
			<td>Axio observer inverted microscope</td>
			<td>ZEISS</td>
			<td></td>
			<td>Automated fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiocam 305 Mono</td>
			<td>ZEISS</td>
			<td></td>
			<td>Microscope Camera</td>
		</tr>
		<tr>
			<td>Breathable sealing membrane</td>
			<td>Sigma-Aldrich</td>
			<td>Z380059</td>
			<td>Infection assay</td>
		</tr>
		<tr>
			<td>Colibr&#236; 5/7</td>
			<td>ZEISS</td>
			<td></td>
			<td>Led light source</td>
		</tr>
		<tr>
			<td>Collagen I rat tail</td>
			<td>Life Technologies</td>
			<td>A1048301</td>
			<td>Collagen coating</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td>Cell staining</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Cell culture and infection</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G12664</td>
			<td>Infection assay</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Carlo Erba</td>
			<td>401391</td>
			<td>Collagen coating</td>
		</tr>
		<tr>
			<td>HCS CellMask Blue</td>
			<td>Invitrogen</td>
			<td>H32720</td>
			<td>Cell staining</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)</td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Minimum Essential Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M5650-500ML</td>
			<td>Cell culture and infection</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4%</td>
			<td>Invitrogen</td>
			<td>FB002</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>PanReac AppliChem</td>
			<td>131494.1211</td>
			<td>PBS preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>60220</td>
			<td>PBS preparation</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>PanReac AppliChem</td>
			<td>131659.1214</td>
			<td>Bacteria culture and PBS preparation</td>
		</tr>
		<tr>
			<td>Sodium phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>71640</td>
			<td>PBS preparation</td>
		</tr>
		<tr>
			<td>Tissue Culture Flask 25 cm2 plug seal screw cap</td>
			<td>Euroclone</td>
			<td>ET7025</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Biorad</td>
			<td>1610407</td>
			<td>Cell staining</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Cell culture and infection</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042B</td>
			<td>Bacteria culture</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Biolife</td>
			<td>4122202</td>
			<td>Bacteria culture</td>
		</tr>
	</tbody>
</table>

automated analysis of intracellular phenotypes of salmonella using imagej

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Infection of epithelial cells with Salmonella strains 
This protocol was developed to analyze the cellular invasion and the cytosolic replication (Figure 1A) vs vacuolar load&#160;(Figure 1B) of Salmonella inside epithelial cells. The protocol was validated by using the three following Salmonella strains, S. Typhimurium SL1344 reference strain (S. Tm), S. Derby ER1175 wildtype (S. Derby wt) and the isogenic mutant of S. Derby ER1175 without sipA gene (S. Derby &#916;sipA). S. Derby ER1175 strain was isolated from swine and belongs to the IZSLER surveillance collection of Salmonella isolates. The strains were selected in order to represent the phenotype diversity covered by the protocol. In particular, these strains were chosen because their behavior inside epithelial cells was already known to differ in terms of invasion or replication9; therefore, they were valuable controls to test whether the protocol allowed to quantitatively distinguish differences in intracellular Salmonella phenotypes. In particular, S. Tm and S. Derby wt were included because we had previously demonstrated that S. Tm has higher invasion and intracellular replication efficiency than S. Derby wt9 while S. Derby &#916;sipA was added as a hyper-replication impaired strain since the virulence effector SipA plays a crucial role in the onset of hyper-replication10,11. It was previously reported for S. Tm that cytosolic replication starts&#160;4 h post-invasion, and then the cytosolic population rapidly hyper-replicates to fill the epithelial cell by 8 h11,12. Consistently, 8 h long infection was suitable to observe and quantify the cytosolic hyper-replication phenotype also in S. Derby wt and S. Derby &#916;sipA.
Area analysis 
The area analysis in step 4.1 provides a measurement of the overall colonization of epithelial cells by Salmonella (infection ratio), together with a measurement of the hyper-replication (hyper-replication ratio). In the area analysis workflow described in Figure 2, the random noise is reduced, and then channels are split and processed independently. A threshold is set for each channel in order to exclude the background from the area measurement. Then, the area occupied by thresholded pixels is measured for each channel. The output of area analysis is a table reporting, for each acquisition file, the extension of the areas occupied by epithelial cell nuclei (blue channel), intracellular mCherry-expressing Salmonellae (red channel), and cytosolic hyper-replicating Salmonellae that express GFP (green channel) along with mCherry. The infection ratio is calculated by dividing the area occupied by mCherry-expressing Salmonellae by the area occupied by host cell nuclei. The results of the tested strains showed that S. Tm displays a significantly higher infection ratio compared to both S. Derby strains, as expected (Figure 3A). Therefore, these results demonstrate the efficacy of area analysis in detecting differences in the ability of Salmonella strains to colonize epithelial cells. Salmonella hyper-replication ratio is measured by dividing the area occupied by GFP-expressing Salmonellae by the area occupied by intracellular mCherry-expressing Salmonellae. Consistently with the role of SipA in determining hyper-replication, a significantly reduced hyper-replication ratio for S. Derby &#916;sipA strain was measured compared to S. Derby wt, (Figure 3B). No hyper-replication difference was observed between S. Tm and S. Derby wt. Overall, area analysis effectively revealed different hyper-replication levels among the assayed strains.
Single-cell analysis 
The single-cell analysis allows quantifying Salmonella invasion and vacuolar load vs. cytosolic replication inside epithelial cells with single-cell resolution. As described in step 4.2, for each acquisition file, blue, red, and green channels were processed independently (Figure 4). Specifically, the blue channel, corresponding to epithelial cells, was processed in step 4.2.3 to obtain cell segmentation. Then, segmented cells were added to the Region of Interest (ROI) Manager to obtain a list of ROIs, corresponding to all segmented cells uniquely labeled with y-x coordinates. In order to remove out-of-focus pixels of GFP- expressing hyper-replicating Salmonellae, the pixels of the green channel were subtracted from those of the red channel. The output table of single cell analysis reports, for each ROI, the area and the percentage of the ROI&#39;s area occupied by Salmonellae expressing the mCherry constitutive reporter only or the GFP cytosol-responsive reporter. The percentage of the cell&#39;s area occupied by mCherry-only expressing Salmonellae was processed in step 4.2.4 to calculate the percentage of infected cells and the mean vacuolar load (Figure 5A).&#160;Analysis of the vacuolar load showed that the S. Derby strains generate a mean vacuolar load significantly lower than S. Tm (Figure 5B). Conversely, only a slight and not significant reduction of the percentage of infected cells was observed in S. Derby strains compared to S. Tm (Figure 5A). The percentage of the cell&#39;s area occupied by GFP-expressing Salmonellae was processed in step 4.2.5 to obtain the hyper-replication rate. No difference was observed between S. Tm and S. Derby wt, while S. Derby &#916;sipA displayed a significant decrease of hyper-replication, consistent with the result of the area analysis (Figure 5C)&#160;and the role of SipA in inducing hyper-replication.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64263/64263fig1v2.jpg" /> 
Figure 1: Salmonella cytosolic hyper-replication and vacuolar load. Representative images of (A) the Salmonella hyper-replication&#160;and (B) the vacuolar load&#160;acquired at high magnification (40x) are shown. Panel B shows the different focus planes of cells containing hyper-replicating Salmonellae (z:7/8), compared to non-hyper-replicating Salmonellae (z:4/8). White scale bars are 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64263/64263fig02.jpg" /> 
Figure 2: Workflow of the area analysis. The workflow of the area analysis is shown for a representative low magnification (10x) acquisition of INT407 cells infected for 8 h with Salmonella carrying the pCHAR-Duo plasmid (MOI 100). First, the random noise is reduced, and then the channels are split into C1, C2, and C3 and processed independently. A threshold is set for each channel in order to exclude the background from the area of measurement. Then, the area occupied by the thresholded pixels only-corresponding to cell nuclei in C1, all intracellular Salmonellae in C2, and cytosolic Salmonellae in C3-is measured for each channel. The images analyzed here are the results of four tiles acquired at 10x magnification fused together by stitching. White scale bars are 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64263/64263fig03.jpg" /> 
Figure 3: Results of the area analysis. The results of the area analysis are shown. (A) Infection ratio is calculated by dividing the area occupied by mCherry-expressing Salmonellae (C2 channel), representing all the intracellular bacteria, by the area occupied by host cell nuclei (C1 channel). (B) Hyper-replication ratio was measured by dividing the area occupied by GFP-expressing Salmonellae (C3 channel), representing cytosolic hyper-replicating bacteria only, by the area occupied by all intracellular mCherry-expressing Salmonellae. Each dot represents a replicate. The analysis was conducted on three biological replicates, each tested in triplicate. The black lines indicate the mean values. The significance was calculated using a two-tailed t-test, and p values are reported. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64263/64263fig04.jpg" /> 
Figure 4: Workflow of the single-cell analysis. The workflow of the single-cell analysis is shown for a representative high magnification acquisition (40x) of INT407 cells infected for 8 h with Salmonella carrying the pCHAR-Duo plasmid (MOI 100). First, channels are split into C1, C2, and C3 and processed independently. Blue channel (C1), corresponding to epithelial cells, is processed to obtain cell segmentation, and then each segmented cell is defined as a Region of Interest (ROI) uniquely labeled with y-x coordinates. The red channel (C2) is processed by subtracting the green channel (C3) to remove out-of-focus cytosolic hyper-replicating Salmonellae, leaving all vacuolar Salmonellae expressing mCherry only, and then random noise was reduced, and the threshold is set to exclude the background from measurements. Finally, the area occupied by vacuolar Salmonellae for each ROI is measured. The green channel (C3), corresponding to total cytosolic GFP-expressing Salmonellae, is processed similarly. The images analyzed here are the results of 16 tiles fused together by stitching. White scale bars are 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64263/64263fig05.jpg" /> 
Figure 5: Results of the single-cell analysis. The results of the single-cell analysis are shown. (A) The percentage of infected cells is obtained by dividing the number of cells with a percentage of area occupied by mCherry-only expressing Salmonellae&#62; 0.2 by the total number of cells. (B) The mean vacuolar load was obtained by calculating the mean percent area occupied by mCherry-only expressing Salmonellae in the infected cells. (C) The hyper-replication rate was calculated by dividing the number of cells with a percentage of area occupied by GFP-expressing Salmonellae&#8805;20% by the total number of infected cells. Data from three to four biological replicates tested in triplicate are reported. The bars indicate the standard error of measurement. The significance was calculated using a two-tailed t-test, and p values are reported. <a href="https://www.jove.com/files/ftp_upload/64263/64263fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Area analysis script. <a href="https://www.jove.com/files/ftp_upload/64263/REVIEWED_Area analysis script.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 2: Single-cell analysis script. <a href="https://www.jove.com/files/ftp_upload/64263/REVIEWED_Single-cell analysis script.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ at JoVE.com

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

automated-analysis-intracellular-phenotypes-salmonella-using

Research

JoVE Journal

Immunology and Infection

2.7K Views.  Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER). Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Video: Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...