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% l...

<ol>
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P., Van Herendael, B., Van Schaeren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screening+algorithm+for+diagnosing+bacterial+gastroenteritis+by+real-time+PCR+in+combination+with+guided+culture.">A screening algorithm for diagnosing bacterial gastroenteritis by real-time PCR in combination with guided culture.</a> Diagnostic Microbiology and Infectious Disease. 85 (2), 255-259 (2016).</li><li>Liu, J., 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=Use+of+quantitative+molecular+diagnostic+methods+to+identify+causes+of+diarrhoea+in+children:+a+reanalysis+of+the+GEMS+case-control+study.">Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.</a> Lancet. 388 (10051), 1291-1301 (2016).</li><li>Wang, S. 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=Surveillance+of+shigellosis+by+real-time+PCR+suggests+underestimation+of+shigellosis+prevalence+by+culture-based+methods+in+a+population+of+rural+China.">Surveillance of shigellosis by real-time PCR suggests underestimation of shigellosis prevalence by culture-based methods in a population of rural China.</a> Journal of Infection. 61 (6), 471-475 (2010).</li><li>Wikswo, M. E., Hall, A. 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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 border="0" cellpadding="0" cellspacing="0" style="width:732px;" width="732">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tris</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">10708976001</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">EDTA</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">798681</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">NP40</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">11332473001</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:249px;">ddH2O</td>
			<td style="width:112px;">Takara</td>
			<td style="width:119px;">9012</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PrimeSTAR HS (Premix)</td>
			<td style="width:112px;">Takara</td>
			<td style="width:119px;">R040Q</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nutrient Broth</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM106</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nutrient agar</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM107</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Selenite Cystine medium</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM225</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">XLD</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM219</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">MAC&#160;</td>
			<td style="width:112px;">LandBridge</td>
			<td style="width:119px;">CM908</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Salmonella chromogenic agar</td>
			<td style="width:112px;">CHROMagar</td>
			<td style="width:119px;">SA130</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Salmonella diagnostic serum</td>
			<td style="width:112px;">Tianrun</td>
			<td style="width:119px;">SAL60</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Shigella diagnostic serum</td>
			<td style="width:112px;">Tianrun</td>
			<td style="width:119px;">SHI54</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">anal swab (collecting tube plus)</td>
			<td style="width:112px;">Huachenyang</td>
			<td style="width:119px;"></td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">slide</td>
			<td style="width:112px;">Mingsheng</td>
			<td style="width:119px;">7102</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">micro-loop</td>
			<td style="width:112px;">Weierkang</td>
			<td style="width:119px;">W511</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">incubator</td>
			<td style="width:112px;">Jinghong</td>
			<td style="width:119px;">DNP-9082</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">autoclave</td>
			<td style="width:112px;">AUL</td>
			<td style="width:119px;">SS-325</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">dry bath</td>
			<td style="width:112px;">Jinghong</td>
			<td style="width:119px;">KB-20</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">automated microbial identification system</td>
			<td style="width:112px;">bioM&#233;rieux</td>
			<td style="width:119px;">VITEK2</td>
			<td style="width:252px;">other equivalent system could be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">fluorescent real-time PCR machine</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:119px;">ABI7500</td>
			<td style="width:252px;">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 ...

This method provide a high throughput platform for the screening of Salmonella and Shigella. The main advantage of this technique is repeat, specific, sensitive, and high throughput. This technique combine NAAT fast method and culture in which real-time PCR screening was applied first, and then positive ones was sent for bacteria culture and identification.Though this method focuses on Salmonella and Shigella screening, it can also be applied to other pathogens such as Vibrios, Staphylococcus, E.Coli, and et cetera. Visual demonstration of this method is critical as the selection of the correct colony is difficult due to background flora in samples. To begin, add three milliliters of nutrient broth to each sample.Then, incubate the samples at 36 degrees Celsius for six hours. After this, centrifuge the pre-enrichment culture at 800 gs for two minutes. Then, transfer the supernatant to a new tube, and centrifuge it again for five minutes at 12, 000 gs.After discarding the supernatant, add 100 microliters of DNA extraction solution to the pellet. Then, vortex the tube for one minute. Next, heat the sample at 1, 000 degrees Celsius in the dry bath.After repeating the centrifugation, collect the supernatant into a new tube. First, create the reaction mixture and set the cycling program according to the text protocol. Then, perform real-time PCR on a fluorescent real-time PCR machine.First, add 100 microliters of the pre-enrichment culture to five milliliters of selenite crystal medium. Then, incubate the culture at 36 degrees Celsius for 18 to 24 hours. Next, collect one loop of the culture and spread it onto a plate.Then, incubate the plate at 36 degrees Celsius for 18 to 24 hours. After incubation, select suspicious colonies and transfer them to a plate. Incubate the plate at 36 degrees Celsius for 18 to 24 hours.Then, identify the suspicious colony using an automated microbial identification system. To begin O-antigen characterization, add one drop of the O-antigen polyvalent sera to a clean slide. Transfer one loop of the colony to the slide and grind it in the sera.If the bacteria look like flowing sand, repeat the previous treatment with O-antigen monovalent sera until the antigen is characterized. To begin H-antigen characterization, add one drop of H-antigen polyvalent sera to a clean slide. Then, collect one loopful of the colony and grid it in the sera.Use H-antigen monovalent sera to repeat treatment until the specific H-antigen is characterized. To separate the culture, collect one loop of the pre-enrichment culture and spread it onto a plate. Then, incubate the place at 36 degrees Celsius for 18 to 24 hours.After incubation, collect a suspicious looking colony onto a plate. Incubate the plate at 36 degrees Celsius for 17 to 24 hours. Then, subject the colony to biochemical identification using an automated microbial identification system.Next, apply one drop of Shigella species polyvalent sera to a clean slide. Then, use a micro-loop to collect some of the bacteria and grind it into the sera. Finally, if the sera resemble flowing sand, use Shigella species monovalent sera to further characterize the bacteria.Using this protocol, stool samples from patients were screened for Salmonella and Shigella species. Using real-time PCR, there was successful amplification in the HEX channel, indicating that the sample was positive for Salmonella species. Further real-time PCR indicated that sample two was positive for Salmonella and was chosen for guided Salmonella species culture.In the guided culture of Salmonella species, the pink and purple colonies were plated separately and subjected to biochemical identification. The results indicated that the colonies from sample two were Salmonella paratyphi B.Using real-time PCR, there was successful amplification in the FAM channel, indicating that the sample was positive for Shigella species. Further real-time PCR indicated that sample 10 was positive for Shigella and was chosen for guided culture.In the guided culture of Shigella species, the pink-red and colorless colonies were plated separately and subjected to biochemical identification. The results indicated that sample 10 contained Shigella sonnei phase II bacteria. While attempting this procedure, it's important to remember the limitation of the method due to sequence variation.Following this procedure, other methods, like direct culture, can be performed in order to avoid false negative real-time PCR results. This technique paves the way for researchers in the field of Salmonella and Shigella detection, as the new protocol could increase the positive rate by twofolds and decrease workload and TAT significantly. Don't forget that working with Salmonella and Shigella can be extremely dangerous, and the personal protective equipment, PPE, should be always be taken while performing this procedure.

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JoVE Journal

Immunology and Infection

8.7K Views.  Zhuhai International Travel Healthcare Center. This method provide a high throughput platform for the screening of Salmonella and Shigella. The main advantage of this technique is repeat, specific, sensitive, and high throughput. This technique combine NAAT fast method and culture in which real-time PCR screening was applied first, and then positive ones was sent for bacteria culture and identification.Though this method focuses on Salmonella and Shigella screening, it can also be applied to other pathogens such as Vibrios, Staphyl...

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

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...