We thank the Tolker-Nielsen lab for their generous donation of reporter constructs used to develop the screen. We also thank members of the Ryan laboratory for their helpful comments and critical reading of the manuscript. The work of the authors has been supported in part by grants awarded by the Wellcome Trust (WT100204AIA senior fellowship grant to R.P.R. and 093714/Z/10/Z PhD scholarship to K.N.R.).

Note: Procedures for cloning and transformation of the purified c-di-GMP reporter plasmid are discussed elsewhere12.
1. Generation of GFP Tagged c-di-GMP Reporter P. aeruginosa Strain
<ol>
	<li>Inoculate 5 ml of lysogeny broth (LB) medium with a single colony of P. aeruginosa and incubate overnight at 37 &#176;C with shaking at 200 rpm.</li>
	<li>Transfer 2 ml of the overnight culture into 200 ml of fresh LB medium in a 1 L flask and incubate at 37 &#176;C with shaking at 200 rpm.</li>
	<li>Monitor optical density at 600 nm (OD600) every 30 min by taking a 1 ml sample from the flask and examine using a spectrophotometer (as described previously12).</li>
	<li>When the OD600 reaches between 0.3 - 0.5, place 40 ml of the culture into a 50 ml conical centrifuge tube.</li>
	<li>Pellet the cells by centrifuging at 8,000 rpm for 10 min at 4 &#176;C, discard the supernatant and re-suspend the cells in 40 ml of ice-cold, sterile, 300 mM sucrose solution.</li>
	<li>Pellet the cells a second time by centrifuging as described in step 1.5 and re-suspend in 20 ml of ice-cold, sterile, 300 mM sucrose solution.</li>
	<li>Pellet the cells a final time by centrifuging as described in step 1.5 and re-suspend in 400 &#181;l of ice-cold, sterile, 300 mM sucrose solution. 
	Note: The cell density at this point should be approximately 1 x 1011 colony forming units per milliliter (CFU/ml).</li>
	<li>Chill the cells on ice for 30 min, after which they are ready for electroporation.</li>
	<li>Add 1 &#181;l of a 0.2 &#181;g/&#181;l pCdrA::gfpC plasmid 12 solution to 40 &#181;l of P. aeruginosa electro-competent cells prepared above in a 1.5 ml microcentrifuge tube that has been pre-chilled on ice. Mix the suspension and transfer into a pre-chilled, 2 mm electrode gap, electroporation cuvette. 
	Note: pCdrA::gfpC: pUCP22Not-PcdrA-RBSII-gfp(Mut3)-T0-T1, Ampr Gmr, which gives a fluorescent readout of the intracellular level of c-di-GMP in P. aeruginosa strains, was developed and validated by Rybtke et al.12.</li>
	<li>Remove moisture on the outside of the cuvette with tissue paper and place the cuvette into the sample chamber of the electroporator. Pulse with a voltage of 2.5 kV, capacitance of 25 &#181;F and resistance of 200 &#8486;.</li>
	<li>Remove the cuvette and add 1 ml of LB medium. Then, transfer the cells to a sterile 1.5 ml microcentrifuge tube and incubate for 2 hr at 37 &#176;C with shaking at 200 rpm.</li>
	<li>Spread 10 &#181;l, 50 &#181;l and 100 &#181;l aliquots of the culture onto sterile LB-agar plates supplemented with ampicillin (at a concentration of 100 &#181;g/ml), and incubate the plates at 37 &#176;C overnight.</li>
	<li>Confirm the GFP expression (excitation at 485 nm, emission at 520 nm) by examining the plate under a fluorescence microscope with a standard GFP channel and pick a single colony to inoculate 5 ml LB. Incubate overnight as described in step 1.1. Mix 0.5 ml of the overnight culture with 0.5 ml 50% glycerol in a 2 ml screw top tube and store at -80 &#176;C.</li>
</ol>
2. Preparation of the Starter Culture for Inoculation
<ol>
	<li>Two days before the screen, plate the P. aeruginosa strain (containing the pCdrA::gfpC plasmid) from the -80 &#176;C stock onto an LB-agar plate (supplemented with ampicillin at a concentration of 100 &#181;g/ml) by gently spreading the bacteria over the plate using a sterile inoculation loop. Incubate the plate overnight at 37 &#176;C.</li>
	<li>The evening before the screening, inoculate a single colony of P. aeruginosa from the plate into 10 ml of LB medium (supplemented with ampicillin at a concentration of 100 &#181;g/ml) in a tube and incubate the pre-culture overnight at 37 &#176;C with shaking at 200 rpm.</li>
	<li>On the day of the screen, prepare a subculture from the overnight pre-culture by diluting it first with fresh LB medium to an OD600 of 1.0 and then further diluting with 5% LB to an OD600 of 0.04 (1:25 ratio). 
	Note: The total volume of the inoculated medium depends on the number of plates to be tested. 5% LB is made by diluting 100% LB with phosphate buffered saline (PBS) to the required concentration. Importantly, the media (5% LB) allows constitutive activation of pCdrA::gfpC in P. aeruginosa.</li>
	<li>Add a sterile magnetic stirrer bar into the container and stir the culture at minimal speed on a magnetic stirrer for 30 min at room temperature, allowing the bacteria to acclimatize to the media before they are dispensed into 384-well plates.</li>
</ol>
3. Inoculation and Incubation of the 384-well Plates Containing Small Molecules
Note: Sterility and good aseptic techniques are paramount to the following steps.
<ol>
	<li>To prepare the positive control (100% inhibition), add 20 &#181;l tobramycin sulfate (25 mg/ml) to 10 ml (adequate for up to 12 plates) of the subculture prepared in Step 2.3 and mix gently. Pipette 40 &#181;l of the positive control culture into wells A23 - P23 (Figure 2).</li>
	<li>To prepare the negative control (0% inhibition), add 30 &#181;l dimethyl sulfoxide (DMSO) to 10 ml (adequate for up to 12 plates) of the subculture prepared in Step 2.3 and mix gently. Pipette 40 &#181;l of the negative control culture into wells A24 - P24 (Figure 2).</li>
	<li>For each of the plates stamped with the small molecules, inoculate a volume of 40 &#181;l of the diluted overnight cultures (described in Step 2.3) into the wells A1 - P22 (Figure 2). 
	Note: This can be achieved using a liquid handler robot. Due to the heterogeneous properties of bacterial cultures, it is important to maintain a continuous and moderate agitation to keep bacteria consistently distributed in the media while dispensing. To do this, keep stirring the acclimatized culture from step 2.4 magnetically during dispensing.</li>
	<li>Seal the plates with an air-permeable cover seal and incubate for 6 hr at 37 &#176;C. 
	Note: The air-permeable seal is critical to maintain unchanging conditions across all 384 wells and to circumvent the potential development of oxygen gradients across the plate.</li>
</ol>
4. Measurement of Growth (OD600) and Intracellular c-di-GMP Level (GFP)
<ol>
	<li>Approximately 30 minutes before spectrophotometric measurement, pre-warm the plate reader to 37 &#176;C to avoid condensation.</li>
	<li>Gently remove the air-permeable cover seal before reading. Measure the optical density at a wavelength of 600 nm with a setting of 10 flashes per well and a settling time of 0.2 sec.</li>
	<li>Prior to measuring fluorescence, make an automatic gain and focal adjustment based on well A24 (negative control) and set the gain target value at 75% (Figure 2).
	<ol>
		<li>From the plate reader control software, click on start measurement and click on the &#39;&#39;Focus and Gain Adjustment/Plate IDs&#39;&#39; tab.</li>
		<li>Select &#39;&#39;Focus Adjustment&#39;&#39; and &#39;&#39;Channel A&#39;&#39; on the right on the window.</li>
		<li>Select &#39;&#39;Gain Adjustment&#39;&#39; and &#39;&#39;Selected well&#39;&#39;, and type in &#39;&#39;75&#39;&#39; as the &#39;&#39;Target value&#39;&#39;. Finally, pick well A24 in the plate layout before clicking on &#39;&#39;Start Adjustment&#39;&#39;. Once the adjustment is carried out, click on &#39;&#39;Start measurement&#39;&#39;.</li>
		<li>Measure the fluorescence from the GFP reporter at an excitation/emission maxima of 485/520 nm with a setting of 10 flashes per well and a settling time of 0.2 sec.</li>
	</ol>
	</li>
	<li>Collect data from all plates examined. Note: Data readings are automatically saved as default by the plate reader control software.
	<ol>
		<li>View readings by clicking on the &#34;Mars&#34; icon within the control software to open the statistical analysis package.</li>
		<li>Retrieve data by &#34;double clicking&#34; on the plate number of interest to access the data for analysis.</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>Evaluate the uniformity and reproducibility of the assay using robust z&#39; analysis, which is the most common quality metrics reported for high-throughput screens. Calculate robust z&#39; using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/54115/54115eq1.jpg" /> 
	Where MAD (Median Absolute Deviation) is an estimate of standard deviation, p is the positive control (100% inhibition) and n is the negative control (0% inhibition) for both OD600 and GFP values. Note: An assay with a robust z&#39; value (calculated for both OD600 and GFP raw data) greater than or equal to 0.5 is considered as an excellent assay.</li>
	<li>Calculate% inhibition of growth using the formula: 
	<img alt="Equation 2" src="/files/ftp_upload/54115/54115eq2.jpg" /> 
	Where XiOD600 is the iterated OD600 value of each sample well. %&#160;InhibitionOD600 &#62; 0, growth inhibitor; %&#160;InhibitionOD600 &#60; 0, growth promoter.</li>
	<li>Assess the %&#160;inhibition of the intracellular level of c-di-GMP using the formula (the change in arbitrary fluorescence intensity units are corrected for the change in cell density): 
	<img alt="Equation 3" src="/files/ftp_upload/54115/54115eq3.jpg" /> <img alt="Equation 4" src="/files/ftp_upload/54115/54115eq4.jpg" /> 
	Where XiGFP is the iterated GFP value of each sample well. %&#160;InhibitionGFP &#62; 0, c-di-GMP inhibitor; %&#160;InhibitionGFP &#60; 0, c-di-GMP promoter.</li>
	<li>Set a threshold for hit detection at 3 MAD from the median (median &#177;3 MAD), calculated from the sample well read-outs of each plate, assuming a normal distribution of data-points. Note: However, a &#177;50% inhibition cut-off can be selected for more reproducible and accurate dose response assays if required.</li>
</ol>

<ol>
	<li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recover+the+lost+art+of+drug+discovery.">Recover the lost art of drug discovery.</a> Nature. 485, 439-440 (2012).</li><li>Caly, D. L., Bellini, D., Walsh, M. A., Dow, J. M., Ryan, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Cyclic+di-GMP+Signaling:+A+Strategy+to+Control+Biofilm+Formation?.">Targeting Cyclic di-GMP Signaling: A Strategy to Control Biofilm Formation?.</a> Curr Pharm Design. 21, 12-24 (2015).</li><li>Hengge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+c-di-GMP+signaling+in+bacteria.">Principles of c-di-GMP signaling in bacteria.</a> Nat Rev Microbiol. 7, 263-273 (2009).</li><li>Ryan, R. P., Tolker-Nielsen, T., Dow, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+the+PilZ+don't+work:+effectors+for+cyclic+di-GMP+action+in+bacteria.">When the PilZ don't work: effectors for cyclic di-GMP action in bacteria.</a> Trend Microbiol. 20, 235-242 (2012).</li><li>Romling, U., Galperin, M. Y., Gomelsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-GMP:+the+First+25+Years+of+a+Universal+Bacterial+Second+Messenger.">Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger.</a> Microbiol Mol Biol Rev. 77, 1-52 (2013).</li><li> Boyd, C. D., GA, O. '. T. o. o. l. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+Messenger+Regulation+of+Biofilm+Formation:+Breakthroughs+in+Understanding+c-di-GMP+Effector+Systems.">Second Messenger Regulation of Biofilm Formation: Breakthroughs in Understanding c-di-GMP Effector Systems.</a> Ann Rev Cell Dev Biol. 28, 439-462 (2012).</li><li>Ryan, R. P., 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=HD-GYP+domain+proteins+regulate+biofilm+formation+and+virulence+in+Pseudomonas+aeruginosa.">HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa.</a> Environ Microbiol. 11, 1126-1136 (2009).</li><li>Christensen, L. D., 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=Clearance+of+Pseudomonas+aeruginosa+Foreign-Body+Biofilm+Infections+through+Reduction+of+the+Cyclic+Di-GMP+Level+in+the+Bacteria.">Clearance of Pseudomonas aeruginosa Foreign-Body Biofilm Infections through Reduction of the Cyclic Di-GMP Level in the Bacteria.</a> Infection and Immunity. 81, 2705-2713 (2013).</li><li>Chua, S. L., 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=Dispersed+cells+represent+a+distinct+stage+in+the+transition+from+bacterial+biofilm+to+planktonic+lifestyles.">Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles.</a> Nat Comm. 5, (2014).</li><li>Sambanthamoorthy, K., 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=Identification+of+Small+Molecules+That+Antagonize+Diguanylate+Cyclase+Enzymes+To+Inhibit+Biofilm+Formation.">Identification of Small Molecules That Antagonize Diguanylate Cyclase Enzymes To Inhibit Biofilm Formation.</a> Antimicrob Agents Chemother. 56, 5202-5211 (2012).</li><li>Sambanthamoorthy, K., 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=Identification+of+small+molecules+inhibiting+diguanylate+cyclases+to+control+bacterial+biofilm+development.">Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development.</a> Biofouling. 30, 17-28 (2014).</li><li>Rybtke, M. T., 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=Fluorescence-Based+Reporter+for+Gauging+Cyclic+Di-GMP+Levels+in+Pseudomonas+aeruginosa.">Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa.</a> App Environ Microbiol. 78, 5060-5069 (2012).</li></ol>

The authors have nothing to disclose.

In order to improve the treatment of bacterial infections, it is clear that a better understanding of bacterial behavior at the molecular regulatory level is required. The procedure described here will be beneficial for microbiologists, biochemists and clinicians who want to uncover small molecules that have the potential to manipulate or interfere with cellular concentrations of c-di-GMP in bacteria. The method utilizes a recently developed GFP bioreporter to monitor cellular levels of c-di-GMP in P. aeruginosa12. This bioreporter has been validated and shown importantly not to affect the growth of the strain used when cultured in 5% LB in PBS. The use of a whole-cell screen to identify small molecules that alters c-di-GMP levels in vivo overcomes the major difficulties of target-based drug discovery in terms of molecule penetration through the bacterial membranes, particularly through those of Gram-negative bacteria. Importantly, the assay appears very robust as a robust z&#39; value consistently above 0.5 was observed in all screens to date. Screening using this protocol will reveal a number of small molecules that inhibit and/or promote intracellular c-di-GMP levels in P. aeruginosa. Moreover, this assay also has the potential to identify bactericidal or bacteriostatic compounds resulting in a decrease in the OD600 read-out.
Although not discussed in the protocol section, there are several important considerations for the preparation of the experiment. It is vital to bear in mind that the GFP bioreporter is based on a plasmid. Although the reporter plasmid is known to be very stable in P. aeruginosa, it can be lost after continuous re-plating, hence the need to use freshly plated strains from a -80 &#176;C glycerol stock, and checking fluorescence expression is critical. It is also very valuable to maintain uniform growth conditions throughout the screen since any fluctuations in these conditions could have knock-on effects on the screen. This would include making certain that media and antibiotics are premade in batch and used throughout the course of the screen. Bacterial cultures not growing (based on OD600 read-outs) in a uniform fashion is a common issue for most high-throughput screens of this nature. This can be due to edge effects or dispensing a non-homogeneous bacterial culture into the plates. For the former, making sure a gas-permeable seal is used during incubation is vital. While for the latter, priming the liquid handler tubing with a volume of the culture at least three times the dead volume of the tubing itself is recommended. It is crucial to keep the magnetic stirrer at minimal speed during dispensing. While monitoring and storage of the 384-well plates for consistent growth over the course of the experiments is paramount, a situation to avoid is the stacking of plates during incubation as it can cause unwanted oxygen gradients leading to a skew in the growth pattern. It is also important to ensure that the DMSO vehicle used as a negative control is not negatively impacting growth of the strain of interest. Many of these issues associated with growth can be avoided by performing a mock screen with the bacterial strain of interest prior to screening. Data interpretation also merits consideration given that c-di-GMP inhibition results are calculated by the change in arbitrary fluorescence intensity units that must be corrected for the change in cell density. With this in mind different mathematical formulas to assess the data output from these experiments should also be considered.&#160;For example, a compound could appear as a c-di-GMP inhibitor but actually be a growth inhibitor or vice versa, requiring prudent interpretation of identified hits.
There are several drawbacks and limitations that must be considered during the development and performance of this high-throughput cell-based screen. For example, the bio-reporter functions on an indirect measure of cellular c-di-GMP levels using a fluorescent probe whose detection properties could potentially be affected by small molecules used in a screen. A tangential issue is the fact that the cell-based assay gives no information regarding the mechanism behind changes in the levels of intracellular c-di-GMP. Therefore, important observations from experiments must be confirmed using a high-performance liquid chromatography-mass spectrometry approach, which is considered the &#34;gold-standard&#34; method for measuring intracellular c-di-GMP fluctuations in bacteria. Moreover, our procedure can only provide information regarding the behavior of bacteria grown in vitro, as bacterial cells grown in the context of the host (in vivo) quickly modify their activities due to this environment. Furthermore, the process of using a GFP bioreporter means the screen does not take into account the physiological status of the bacterial cells. However, the protocol could be adapted to microscopic monitoring of individual wells during the course of the screen.
Even with these considerations and limitations, this high-throughput assay is still a robust screen for small molecules capable of interfering with intracellular c-di-GMP levels. Since many microbial species including many bacterial pathogens have been studied in order to characterize the modulation of intracellular c-di-GMP levels, our protocol could be applied to diverse bacterial species and expanded to the study of more complex multispecies bacteria models. The assay can be easily optimized for other bacterial species by changing the growth conditions used, or can be adapted to read other outputs by using different bioreporters. Different incubation times can be applied depending on the growth phase of interest. However, when scaling up or increasing through-put, it is important to keep in mind that the bacteria will still be growing during plate preparations and read-out measurements. Therefore it is recommended to screen a maximum of 15 plates at a time using this protocol. Moreover, using plates with more than 384 wells may not allow uniform growth, requiring further optimization. When scaling down the number of plates, it may be more appropriate to manually inoculate using an electronic pipette instead of a liquid handling robot. It is clear that this protocol could be used to investigate small molecules that disperse biofilms, given that anti-biofilm compounds interfere with c-di-GMP signaling. The protocol could also be redeveloped to understand various aspects of bacterial physiology. Given that c-di-GMP signaling is prevalent in most bacteria, by studying the physiologies of these organisms using this protocol we can identify different chemical stimuli to determine whether or not their working mechanisms require c-di-GMP signaling.
In summary, the robustness and versatility of the approach presented in this protocol will aid the identification of chemical modulators of c-di-GMP signaling in many biological systems.

The rapid development of bacterial resistance against clinically important antibiotics is one of the major concerns currently facing health professionals worldwide. This failure of traditional antibiotics has driven new searches for chemical matter that can interfere with bacterial processes involved in virulence and disease progression1. One such regulatory system that utilizes the intracellular second messenger cyclic di-GMP (c-di-GMP) has recently become a target with promising validity2-4. It has been established that this global second messenger signal molecule regulates many functions including antibiotic resistance, adhesion, biofilm formation and disease2-4.
It is now understood that the cellular level of c-di-GMP in the bacterial cell is controlled by synthesis and degradation whereby two molecules of GTP are used to synthesize c-di-GMP by GGDEF domain-containing diguanylate cyclases (DGCs) whereas c-di-GMP degradation is catalyzed by phosphodiesterases (PDEs) that have either an EAL or an HD-GYP domain (reviewed in (3, 5)). Proteins containing these domains often contain other signaling domains, suggesting that their activity in c-di-GMP turnover is regulated either directly or indirectly by environmental or cellular cues3,5. Consequently, c-di-GMP signaling functions to link the sensing of diverse environmental cues to modifications in bacterial phenotype. c-di-GMP exerts its regulatory effect in bacteria at the level of transcription, post-transcription and post-translation by various mechanisms4.
A major influence of c-di-GMP in many bacterial cells is in the determination of bacterial &#39;lifestyle&#39; and, in particular, in the control of transitions between motile planktonic cells and sessile cells attached to surfaces or organized in the multicellular structures of biofilms3,5. In general, high cellular levels of c-di-GMP are associated with biofilm formation and sessility, while low cellular levels encourage motility and virulence factor synthesis in many bacterial pathogens3,5. Thus, a more detailed knowledge of the workings of c-di-GMP signaling could afford strategies for inhibition of biofilm formation and virulence in bacterial pathogens. This is a daunting task given that most bacterial genomes encode numerous proteins with GGDEF, EAL and/or HD-GYP domains (for example P. aeruginosa has over 40 proteins) and multiple effectors6,7.
However, even with this complexity, recent evidence suggests that strategies manipulating c-di-GMP signaling may be developed to either prevent antibiotic resistant infections developing or make them susceptible to the immune system or efficient treatment by co-administration of classic antibiotics2. In line with this, it has been experimentally demonstrated that artificial decrease of intracellular c-di-GMP in in vitro-grown P. aeruginosa leads to decreased biofilm formation and increased susceptibility to antimicrobials, while P. aeruginosa-developed biofilms on silicone implants, located in the peritoneal cavity of mice, can be dispersed in a similar fashion8-11.
Here, we describe a high-throughput, fluorescence-based reporter assay to screen small molecules that can potentially modulate the cellular c-di-GMP levels in P. aeruginosa (Figure 1). The assay is based on measuring c-di-GMP cellular levels using a previously developed GFP reporter whose expression is transcriptionally linked to the c-di-GMP-responsive cdrA promoter12. This protocol describes the methodology for expression of the reporter construct in the P. aeruginosa strain of interest, compound plate preparation, culture inoculation into 384-well plates, growth conditions, as well as details regarding data collection, management and analysis (Figure 1). Overall, this protocol will aid researchers to potentially identify novel compounds targeting c-di-GMP signaling in bacteria, and for use in research aiming at understanding the biology of P. aeruginosa.

<table border="0" cellpadding="0" cellspacing="0" width="622">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Lysogeny Broth (Lennox)</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">L3022-1KG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Sucrose</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">S0389-1KG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Lysogeny Broth with Agar (Lennox)</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">L2897-1KG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Ampicillin sodium</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">A0166-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Glycerol</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">G5516-1L</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Phosphate Buffered Saline, pH 7.4</td>
			<td style="width:208px;">Life technologies</td>
			<td style="width:167px;">10010-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Tobramycin sulfate</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">T1783-100MG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">276855-250ML</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Genesys 10S UV-Vis spectrophotometer</td>
			<td style="width:208px;">Thermoscientific</td>
			<td style="width:167px;">840-208100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">2 mm gap electroporator cuvette</td>
			<td style="width:208px;">Bio-Rad</td>
			<td style="width:167px;">1652092</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">BioRad GenePulser XCell electroporator</td>
			<td style="width:208px;">Bio-Rad</td>
			<td style="width:167px;">1652662</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Leica Fluorescent Stereomicroscope</td>
			<td style="width:208px;">Leica Microsystems</td>
			<td style="width:167px;">MZ16FA</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">BRAND magnetic stirring bar, PTFE, cylindrical with pivot ring (sterilise by autoclaving before use)</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">Z328952-10EA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">384-well, white-walled, clear-bottom plates</td>
			<td style="width:208px;">Greiner</td>
			<td style="width:167px;">781098</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Multidrop Combi reagent dispenser</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td style="width:167px;">5840300</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Multidrop Combi tubing</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td style="width:167px;">24073290</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">VIAFLO II 16-channel electronic pipette</td>
			<td style="width:208px;">Integra Biosciences</td>
			<td style="width:167px;">4642</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">100 mL sterile disposable reagent reservoirs</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:167px;">12399175</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">AeraSeal air-permeable membranes</td>
			<td style="width:208px;">Sigma Aldrich</td>
			<td style="width:167px;">MKBQ1886</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Pherastar plate reader (Software version: 4.00 R4, Firmware version: 1.13)</td>
			<td style="width:208px;">BMG Labtech</td>
			<td style="width:167px;">Pherastar FS</td>
		</tr>
	</tbody>
</table>

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.

The approach described here allows a single researcher to efficiently and successfully screen an average of 3,500 compounds within a 48-hour period. An overview of the high-throughput screening protocol is illustrated as a flowchart in Figure 1. For the current article, we screened a rule-of-five compliant compound library for potential c-di-GMP modulating compounds at a final concentration of 600 &#181;M. However, there are many commercially available drug-like and non-drug like compound sets that could be used in the assay. These sets can be bacteria-focused compounds or random pooled compounds but each library would have predicated physicochemical properties and characteristics assigned such as the set screened here, which had polar functional groups and multiple stereogenic centers. In this protocol, bacteria are inoculated into the plate along with compounds, an antibiotic positive control and a DMSO vehicle control, according to the layout shown in Figure 2. Figure 3 depicts the results of the primary screening exemplified by a single library plate. Representative OD600 and GFP raw data are shown in Figure 3A and 3B, respectively. A color gradient heat map, with hot (red) to cool (green) colors indicating low to high OD600/GFP values, has been applied to the well values. The heat maps were generated in a spreadsheet software by applying a 3-Color scale conditional formatting spanning from the lowest OD600/GFP read-out up to the highest OD600/GFP read-out. Low OD600 and GFP values relative to the DMSO negative control correspond to an inhibition in growth and c-di-GMP levels respectively, while high values relative to the DMSO negative control correspond to a promotion in growth and c-di-GMP levels respectively. The robust z&#39; values for OD600 and GFP are calculated according to the formula provided in the protocol (5.1). As they are both above 0.5 (0.694 and 0.761 respectively), the assay quality was deemed robust and the data was further analyzed. The % inhibition of growth (OD600) and intracellular c-di-GMP level (GFP) are calculated by the formulae provided in the protocol (5.2, 5.3). Representative data are shown in Figure 4A and 4B respectively. A color gradient heat map, with hot (red) to cool (green) colors indicating low to high % inhibition, has been applied to the well values. A scatter plot of the % inhibition(OD600/GFP) from individual wells based on the primary screen is plotted in Figure 5A and 5B for OD600 and GFP data respectively. A &#177;50% cut-off (indicated with dotted lines) is selected for hit detection. Potential hits based on this cut-off are indicated with red dots. Two types of compounds of interest can be discerned from the assay. Hits identified in Figure 5A are compounds that inhibit bacterial growth, while hits identified in Figure 5B are compounds that potentially possess the ability to modulate intracellular levels of c-di-GMP. Two compounds have been identified as representative hits for this assay. These are compound A, a potential growth inhibitor and compound B, a potential c-di-GMP inhibitor. Compound A corresponds to well F8 in Figures 3 and 4 and had a 72.5% inhibition in growth. There was an expected corresponding inhibition in cyclic di-GMP levels. Compound B corresponds to well K3 in Figures 3 and 4 and had a 61% inhibition in cyclic di-GMP levels with no change in growth. Select hit compounds were further tested by a 10-point dose-response assay with a top concentration of 2 mM and two-fold dilution series. IC50 values are calculated based on the dose-response curves (Figure 6A and B).
<img alt="Figure 1" src="/files/ftp_upload/54115/54115fig1.jpg" /> 
Figure 1. Overview: High-throughput Setup for Screening Small Molecules for Their Potential to Modulate Cellular Levels of c-di-GMP in P. aeruginosa. This overview shows a step-by-step representation of the protocol used in this assay. A bacterial colony of P. aeruginosa is grown overnight in an LB starter culture. Using a reagent dispenser, the cells are inoculated into 384-well plates containing the selected compounds. The plates are incubated for 6 hours, after which the OD600 and GFP values are measured. Using these read-outs, compounds that affect the cellular levels of c-di-GMP can be identified. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54115/54115fig2.jpg" /> 
Figure 2. A 384-well Plate Map Used for the Small Molecule Screening Assay. The compounds from the library (light blue) are aliquoted into wells A1 - P22. The &#34;positive control&#34; (red) consisting of a final concentration of 50 &#181;g/ml tobramycin sulfate is added to wells A23 - P23 and the &#34;negative control&#34; (green) containing a final concentration of 1% DMSO is added to wells A24 - P24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54115/54115fig3.jpg" /> 
Figure 3. Representative Results for One 384-well Screening Plate. Representative raw data is for OD600 (A) and GFP (B). A color gradient heat map, with hot (Red: OD600&#160;= 0.65 or GFP = 250,000) to cool (Green: OD600&#160;= 0.10 or GFP = 40,000) colors indicating low to high values, has been applied to the well values. Wells that have lower OD600/GFP readings relative to the DMSO negative control will tend to be in red while wells that have higher OD600/GFP readings relative to the DMSO control will tend to be in green. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54115/54115fig4.jpg" /> 
Figure 4. Representative Results for Percentage Inhibition Calculated for a 384-well Plate. The analyzed % inhibition data is represented for both of the OD600 (A) and GFP (B) read-outs. A color gradient heat map, with hot (Red: OD600&#160;= -150% or GFP = -20%) to cool (Green: OD600&#160;= 100% or GFP = 100%) colors indicating low to high inhibition values, has been applied to the well values. Small molecules, which potentially inhibit growth and intracellular c-di-GMP will tend to be in green while small molecules which potentially promote growth and intracellular c-di-GMP levels will tend to be in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54115/54115fig5.jpg" /> 
Figure 5. Representative Scatter Plots for % Inhibition(OD600/GFP) Obtained From Each Tested Small Molecule. Each small molecule is represented by a dot and the % inhibition distribution for each of the OD600 (A) and GFP (B) read-outs is shown. A &#177;50% cut-off is selected for hit identification. Potential hits are highlighted in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54115/54115fig6.jpg" /> 
Figure 6. Representative Results From a Dose - Response Assay. Two compounds (potential growth inhibitor A and potential c-di-GMP inhibitor B) identified from previous screens are further tested in a 10 point dose-response assay with a top concentration of 2 mM and two-fold dilution series. Fitting a four-parameter logistic function to the data yielded IC50 values of 158 &#181;M and 193 &#181;M respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54115/54115fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa at JoVE.com

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.

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. ...

establishment-high-throughput-setup-for-screening-small-molecules

Research

JoVE Journal

Immunology and Infection

8.3K Views.  University of Dundee. 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.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

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 ...

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 ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

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. ...