Name: a high-throughput platform for the screening of salmonella spp./shigella spp.
Uploaded: 2018-11-07T14:00:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about A High-throughput Platform for the Screening of Salmonella spp./Shigella spp. at JoVE.com

This work was supported by the Science and Technology Program of Zhuhai, China (grant number 20171009E030064), the Science and Technology Program of Guangdong, China (grant number 2015A020211004) and the Science and Technology Program of General Administration of Quality Supervision, Inspection and Quarantine of the People&#39;s Republic of China (grant number 2016IK302, 2017IK224).

The protocol follows the guidelines set by the human research ethics committee of Zhuhai International Travel Healthcare Center. Please use standard sterile operation during the experiment.
1. Culture Media Composition and Preparation
<ol>
	<li>Prepare Nutrient Broth: Dissolve 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.1% glucose in H2O, adjust pH to 7.5 and autoclave it in 121 &#176;C for 15 min.</li>
	<li>Prepare Selenite Cystine medium: Dissolve 0.5% peptone, 0.4% lactose, 1%Na2HCO3, 0.4% sodium hydrogen selenite, 0.001% L-Cystine in H2O, adjust pH to 7.0 and boil it for 5 min.</li>
	<li>Prepare the Xylose, Lysine, Deoxycholate agar (XLD) plate: Dissolve 0.3% yeast extract, 0.5% L-lysine, 0.375% xylose, 0.75% lactose, 0.75% sucrose, 0.5% sodium chloride, 0.008% phenol red, 0.68% sodium thiosulfate, 0.08% ammonium ferric citrate, 0.25% sodium deoxycholate, 1.5% agar in H2O, and adjust pH to 7.4. Then boil it for 5 min and pour it into 90 mm plates.</li>
	<li>Prepare the Salmonella chromogenic agar plate: Dissolve 1.5% agar, 0.7% peptone and yeast extract, 1.29% selective reagent in H2O. Then boil it for 5 min and pour it into 90 mm plates.</li>
	<li>Prepare the Nutrient agar plate: Dissolve 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.5% agar in H2O, and adjust pH to 7.3. Then autoclave it in 121 &#176;C for 15 min and pour it into 90 mm plates.</li>
	<li>Prepare the MacConkey agar (MAC) plate: Dissolve 2% peptone, 1% lactose, 0.5% sodium chloride, 0.5% OX Bile Salt, 0.0025% Neutral Red, 1.5% agar, 0.0001% crystal violet in H2O, and adjust pH to 7.2. Then autoclave it in 115 &#176;C for 20 min and pour it into 90 mm plates.</li>
</ol>
2. Real-time PCR
<ol>
	<li>Sample collection
	<ol>
		<li>Insert an anal swab into the patient&#39;s anus 3-5 cm deep, and rotate it 360&#176; around.</li>
		<li>Put the anal swab into a sterile collection tube. Mark sample ID.</li>
		<li>Send the sample to laboratory as soon as possible. 
		NOTE: Samples could be stored at 4 &#176;C for no more than 24 h.</li>
	</ol>
	</li>
	<li>Pre-enrichment
	<ol>
		<li>Add 3 mL of Nutrient Broth into each sample in the collection tube.</li>
		<li>Incubate at 36 &#176;C for 6 h in an incubator.</li>
	</ol>
	</li>
	<li>Sample mixing (optional)
	<ol>
		<li>Collect 100 &#181;L of each pre-enrichment culture and mix 8-10 samples of 1 specimen into a 1.5 mL tube if there are more than 10 samples.</li>
		<li>Mark properly.</li>
	</ol>
	</li>
	<li>DNA extraction
	<ol>
		<li>Centrifuge the pre-enrichment culture at 800 x g for 2 min to allow large particles to settle down, and transfer the supernatant to a new tube and centrifuge at 12,000 x g for 5 min. Discard the supernatant by aspiration.</li>
		<li>Add 100 &#181;L of DNA extraction solution (0.01 M pH 8.0 Tris-EDTA, 0.01% Nonidet P 40 (NP40)) to the pellet. Vortex vigorously for 1 min.</li>
		<li>Boil at 100 &#176;C for 5 min on a dry bath.</li>
		<li>Centrifuge at 12,000 x g for 5 min. Collect the supernatant using a new tube, which will be the template for the succeeding real-time PCR analysis.</li>
	</ol>
	</li>
	<li>Real-time PCR
	<ol>
		<li>Setup the reaction mixture as follow: for each sample, add 12.5 &#181;L of 2x reaction mixture, 0.4 &#181;M of each primer, 0.2 &#181;M of each probe (sequences in Table 1)27, 5 &#181;L of the template as prepared in step 2.4.4, and use ddH2O to add up to 25 &#181;L total volume.</li>
		<li>Setup the cycling program as follow: 95 &#176;C for 3 min, followed by 40 cycles of 95 &#176;C for 15 s, 55 &#176;C for 30 s, 72 &#176;C for 34 s. Collect fluorescent signals from 6-carboxy-fluorescein (FAM) and Hexachlorofluorescein (HEX) channels at the elongation step (72 &#176;C) automatically by the fluorescent real-time PCR machine.</li>
		<li>Perform real-time PCR on a fluorescent real-time PCR machine, according to instructions of the instrument.</li>
		<li>Go to Step 4 directly and issue negative reports if negative results occur on the FAM/HEX channels, which means that samples are negative for Salmonella spp./Shigella spp.</li>
		<li>Go to step 3.1 and/or 3.2 if positive results occur on the HEX and/or FAM channels, which means that the sample may be positive for Salmonella spp. and/or Shigella spp., respectively. 
		NOTE: If sample mixing has been performed in step 2.3, then real-time PCR on individual samples which made up the positive one should be performed to screen out the real positive sample.</li>
	</ol>
	</li>
</ol>
3. Guided Culture
<ol>
	<li>Salmonella spp. PCR positive sample
	<ol>
		<li>Selective culture in medium
		<ol>
			<li>Add 100 &#181;L of pre-enrichment culture into 5 mL of Selenite Cystine medium in a test tube. Incubate at 36 &#176;C for 18&#8211;24 h in an incubator.</li>
		</ol>
		</li>
		<li>Separating culture on plate
		<ol>
			<li>Collect one loop of the culture with a micro-loop and spread onto a XLD plate or Salmonella chromogenic agar plate. Incubate at 36 &#176;C for 18-24 h in an incubator.</li>
		</ol>
		</li>
		<li>Biochemical identification
		<ol>
			<li>Select suspicious colony on XLD plate (pink colony with/without dark heart; dark colony; yellow colony with/without dark heart) or Salmonella chromogenic agar plate (purple or prunosus colony, smooth and round) (Figure 2).</li>
			<li>Subject suspicious colony for biochemical identification on automated microbial identification system, according to instructions of the instrument.</li>
		</ol>
		</li>
		<li>Serological characterization
		<ol>
			<li>O antigen characterization
			<ol>
				<li>Add one drop of the O antigen polyvalent sera onto a clean slide.</li>
				<li>Collect one loop of the colony with a micro-loop and grind in the sera.</li>
				<li>Go to step 3.1.4.1.4 if it looks like flowing sand, which means that the colony is reactive to the sera (Figure 3). Otherwise, go to step 3.1.4.1.5.</li>
				<li>Use O antigen monovalent sera to repeat steps 3.1.4.1.1 to 3.1.4.1.3 until the specific O antigen is characterized.</li>
				<li>Use Vi sera to repeat steps 3.1.4.1.1 to 3.1.4.1.3. Collect those Vi sera reactive colonies in a tube and boil at 100 &#176;C for 5 min in a dry bath. Centrifuge at 12,000 x g for 5 min. Collect the pellet and repeat steps 3.1.4.1.1 to 3.1.4.1.4 until specific O antigen is characterized.</li>
			</ol>
			</li>
			<li>H antigen characterization
			<ol>
				<li>Add one drop of the H antigen polyvalent sera onto a clean slide.</li>
				<li>Collect one loop of the colony with a micro-loop and grind in the sera.</li>
				<li>Go to step 3.1.4.2.4 if it looks like flowing sand, which means that the colony is reactive to the sera.</li>
				<li>Use H antigen monovalent sera to repeat steps 3.1.4.2.1 to 3.1.4.2.3 until specific H antigen is characterized. 
				NOTE: Sometimes serum induction may be needed to characterize the second phase H antigen. If so, then the following optional steps should be performed.</li>
				<li>(Optional) Add one drop of specific H antigen sera onto Nutrient agar plate. Wait until all the sera are absorbed.</li>
				<li>(Optional) Collect one loop of the colony with a micro-loop and spread onto the plate where specific H antigen sera are absorbed. Incubate at 36 &#176;C for 18-24 h in an incubator.</li>
				<li>(Optional) Use H antigen monovalent sera to repeat steps 3.1.4.2.1 to 3.1.4.2.3 until second phase H antigen is characterized.</li>
				<li>Go to step 4.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Shigella spp. PCR positive sample
	<ol>
		<li>Separating culture on plate
		<ol>
			<li>Collect one loop of the pre-enrichment culture with a micro-loop and spread onto a XLD plate or MAC plate. Incubate at 36 &#176;C for 18&#8211;24 h in an incubator.</li>
		</ol>
		</li>
		<li>Biochemical identification
		<ol>
			<li>Collect suspicious colony on XLD plate (smooth, round, transparent and red colony) or MAC plate (smooth, round, transparent and colorless colony with 2-3 mm in diameter; Shigella sonnei may be bigger and turn to light pink as the elongation of incubation time) (Figure 4).</li>
			<li>Subject suspicious colony for biochemical identification on automated microbial identification system, according to instructions of the instrument.</li>
		</ol>
		</li>
		<li>Serological characterization
		<ol>
			<li>Add one drop of the Shigella spp. polyvalent sera onto a clean slide.</li>
			<li>Collect one loop of the colony with a micro-loop and grind in the sera.</li>
			<li>Go to step 3.2.3.4 if it looks like flowing sand, which means that the colony is reactive to the sera.</li>
			<li>Use Shigella spp. monovalent sera to repeat steps 3.2.3.1 to 3.2.3.3 until specific Shigella spp. is characterized.</li>
			<li>Go to step 4.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Report
<ol>
	<li>Issue positive or negative reports according to the above results.</li>
</ol>

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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=Three-hour+molecular+detection+of+Campylobacter,+Salmonella,+Yersinia,+and+Shigella+species+in+feces+with+accuracy+as+high+as+that+of+culture.">Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture.</a> Journal of Clinical Microbiology. 48 (8), 2929-2933 (2010).</li><li>Eriksson, E., Aspan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+culture,+ELISA+and+PCR+techniques+for+salmonella+detection+in+faecal+samples+for+cattle,+pig+and+poultry.">Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry.</a> BMC Veterinary Research. 3, 21 (2007).</li><li>Dutta, S., 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=Sensitivity+and+performance+characteristics+of+a+direct+PCR+with+stool+samples+in+comparison+to+conventional+techniques+for+diagnosis+of+Shigella+and+enteroinvasive+Escherichia+coli+infection+in+children+with+acute+diarrhoea+in+Calcutta,+India.">Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India.</a> Journal of Medical Microbiology. 50 (8), 667-674 (2001).</li></ol>

The authors have nothing to disclose.

Since Salmonella spp./Shigella spp. are often associated with food poisoning and fecal-oral transmission of acute gastroenteritis28,29 and the routine method is either labor-intensive or time-consuming7, we describe a high-throughput platform for the Salmonella spp./Shigella spp. screening, using real-time PCR combined with guided culture.
There are several steps that need con...

Diarrhea is still a common health issue with a high incidence rate globally1,2. Though the mortality is relatively low, some patients show various symptoms for weeks (such as loose and watery stools, an urgency to go to the bathroom), which make the socioeconomic impact very high3,4. More seriously, some patients may even develop irritable bowel syndrome if left untreated5. There are various kinds of bacteria, viruses and parasites that can cause diarrhea6. Salmonella spp./Shigella spp. are among the most common bacteria for the transmission of acute gastroenteritis7,8,9,10,11. Therefore, many counties have issued laws or regulations for regular Salmonella spp./Shigella spp. screening among people who would handle food and water. For example, the Chinese government have issued laws for obligatory Salmonella spp./Shigella spp. screening once a year.
The gold standard method for Salmonella spp./Shigella spp. detection is bacteria culture. Through bacteria culture and successive biochemical identification and serological characterization, we can identify the species of bacteria, which could facilitate disease outbreak management and antimicrobial profiling to aid the treatment of patients12. It could also help trace the source of infection during the Salmonella spp./Shigella spp. outbreak13. However, this method is labor-intensive (requiring manual operation) and time-consuming (taking several days), especially for the testing of large numbers of samples7. Moreover, viable but non-culturable (VBNC) Salmonella spp./Shigella spp.may exist in some stool samples14. In view of these drawbacks, many laboratories have tried to develop new techniques for the detection of Salmonella spp./Shigella spp.15,16,17,18,19,20,21,22,23,24,25. All these methods use the nuclear acid amplification test (NAAT), among which the polymerase chain reaction (PCR) is the most common. One major limitation of these NAAT based methods is that dead bacteria, even bacterial debris containing incomplete genomic DNA, could show positive results26, which could largely influence the accurate diagnosis of disease. Blanco et al. showed that molecular assay is highly sensitive, not only to viable Salmonella in cultures, but also to partial genomes and dead or unviable bacteria from past infections or contamination26. Therefore, new technology should be developed.
Here, we described a novel method that combines the NAAT based method and culturing. As shown in Figure 1, this new method applies real-time PCR screening first and then positive samples are sent for bacteria culture and identification.

<table><tbody><tr><td>Tris</td><td>Sigma</td><td>10708976001</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>798681</td><td></td></tr><tr><td>NP40</td><td>Sigma</td><td>11332473001</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Takara</td><td>9012</td><td></td></tr><tr><td>PrimeSTAR HS (Premix)</td><td>Takara</td><td>R040Q</td><td></td></tr><tr><td>Nutrient Broth</td><td>LandBridge</td><td>CM106</td><td></td></tr><tr><td>Nutrient agar</td><td>LandBridge</td><td>CM107</td><td></td></tr><tr><td>Selenite Cystine medium</td><td>LandBridge</td><td>CM225</td><td></td></tr><tr><td>XLD</td><td>LandBridge</td><td>CM219</td><td></td></tr><tr><td>MAC&amp;nbsp;</td><td>LandBridge</td><td>CM908</td><td></td></tr><tr><td>&lt;em&gt;Salmonella&lt;/em&gt; chromogenic agar</td><td>CHROMagar</td><td>SA130</td><td></td></tr><tr><td>&lt;em&gt;Salmonella&lt;/em&gt; diagnostic serum</td><td>Tianrun</td><td>SAL60</td><td></td></tr><tr><td>&lt;em&gt;Shigella&lt;/em&gt; diagnostic serum</td><td>Tianrun</td><td>SHI54</td><td></td></tr><tr><td>anal swab (collecting tube plus)</td><td>Huachenyang</td><td></td><td></td></tr><tr><td>slide</td><td>Mingsheng</td><td>7102</td><td></td></tr><tr><td>micro-loop</td><td>Weierkang</td><td>W511</td><td></td></tr><tr><td>incubator</td><td>Jinghong</td><td>DNP-9082</td><td></td></tr><tr><td>autoclave</td><td>AUL</td><td>SS-325</td><td></td></tr><tr><td>dry bath</td><td>Jinghong</td><td>KB-20</td><td></td></tr><tr><td>automated microbial identification system</td><td>bioM&amp;eacute;rieux</td><td>VITEK2</td><td>other equivalent system could be used</td></tr><tr><td>fluorescent real-time PCR machine</td><td>ThermoFisher</td><td>ABI7500</td><td>other equivalent machine could be used</td></tr></tbody></table>

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.

The protocol was applied for the screening of Salmonella spp./Shigella spp. in anal stool samples from people who would handle food and water.
In the real-time PCR step, as shown in Figure 5A, there was a successful amplification in HEX channel, which meant that the mixed sample was positive for Salmonella spp. Then a further real-time PCR was conducted on...

Watch this Scientific Journal Video about A High-throughput Platform for the Screening of Salmonella spp./Shigella spp. at JoVE.com

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.

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 ...

a-high-throughput-platform-for-screening-salmonella-sppshigella

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Zhuhai International Travel Healthcare Center. Deze methode biedt een hoog doorvoerplatform voor de screening van Salmonella en Shigella. Het belangrijkste voordeel van deze techniek is herhalen, specifiek, gevoelig en hoge doorvoer. Deze techniek combineert NAAT snelle methode en cultuur waarin real-time PCR screening werd toegepast eerste, en vervolgens positieve degenen werden verzonden voor bacteriën cultuur en identificatie.Hoewel deze methode zich richt op Salmonella en Shigella screening, kan het ook worden toegepast op and...

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical &amp; Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

Environment

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Bioengineering

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

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.

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.

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

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

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

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