Name: tools for the real-time assessment of a pseudomonas aeruginosa infection model
Uploaded: 2021-04-06T13:00:00
Description: Watch this Scientific Journal Video about Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model at JoVE.com

S.E.D is supported by start-up funds provided by the Department of Molecular&#160;Medicine, The University of South Florida, as well as a CFF research grant (DARCH19G0) the N.I.H (5R21AI147654 - 02 (PI, Chen)) and the USF Institute on Microbiomes. We thank the Whiteley lab for ongoing collaboration involving data sets related to this manuscript. We thank Dr. Charles Szekeres for facilitating FACS sorting. Figures were created by A.D.G and S.E.D using Biorender.com.

1. Prepare synthetic cystic fibrosis medium (SCFM2)
NOTE: Preparation of SCFM2 comprises three main stages outlined below (Figure 2). For full details and references, see9,10,12.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62420/62420fig02.jpg" /> 
Figure 2...

<ol>
	<li>Ramsay, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+prevalence+of+pulmonary+infection+in+with+fibrosis:+A+longitudinal+analysis.">The changing prevalence of pulmonary infection in with fibrosis: A longitudinal analysis.</a> Journal of Cystic Fibrosis. 16 (1), 70-77 (2017).</li><li>Bessonova, 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=Data+from+the+US+and+UK+cystic+fibrosis+registries+support+disease+modification+by+CFTR+modulation+with+ivacaftor.">Data from the US and UK cystic fibrosis registries support disease modification by CFTR modulation with ivacaftor.</a> Thorax. 73 (8), 731-740 (2018).</li><li>Breuer, O., 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=Changing+prevalence+of+lower+airway+infections+in+young+children+with+cystic+fibrosis.">Changing prevalence of lower airway infections in young children with cystic fibrosis.</a> American Journal of Respiratory and Critical Care Medicine. 200 (5), 590-599 (2019).</li><li>O'Donnell, J. N., Bidell, M. R., Lodise, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+the+treatment+of+patients+with+serious+multidrug-resistant+Pseudomonas+aeruginosa+infections.">Approach to the treatment of patients with serious multidrug-resistant Pseudomonas aeruginosa infections.</a> Pharmacotherapy. 40 (9), 952-969 (2020).</li><li>Bjarnsholt, 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=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends in Microbiology. 21 (9), 466-474 (2013).</li><li>Darch, S. E., 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=Spatial+determinants+of+quorum+signaling+in+a+Pseudomonas+aeruginosa+infection+model.">Spatial determinants of quorum signaling in a Pseudomonas aeruginosa infection model.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (18), 4779-4784 (2018).</li><li>Zhu, K., Chen, S., Sysoeva, T. A., You, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+antibiotic+tolerance+arising+from+antibiotic-triggered+accumulation+of+pyocyanin+in+Pseudomonas+aeruginosa.">Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa.</a> PLoS Biology. 17 (12), 3000573 (2019).</li><li>Ciofu, O., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+resistance+of+Pseudomonas+aeruginosa+biofilms+to+antimicrobial+agents-how+P.+aeruginosa+can+escape+antibiotics.">Tolerance and resistance of Pseudomonas aeruginosa biofilms to antimicrobial agents-how P. aeruginosa can escape antibiotics.</a> Frontiers in Microbiology. 10, 913 (2019).</li><li>Turner, K. H., Wessel, A. K., Palmer, G. C., Murray, J. L., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+genome+of+Pseudomonas+aeruginosa+in+cystic+fibrosis+sputum.">Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (13), 4110-4115 (2015).</li><li>Darch, S. E., 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=Phage+inhibit+pathogen+dissemination+by+targeting+bacterial+migrants+in+a+chronic+infection+model.">Phage inhibit pathogen dissemination by targeting bacterial migrants in a chronic infection model.</a> MBio. 8 (2), 00240 (2017).</li><li>Jorth, 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=Regional+isolation+drives+bacterial+diversification+within+cystic+fibrosis+lungs.">Regional isolation drives bacterial diversification within cystic fibrosis lungs.</a> Cell Host &amp; Microbe. 18 (3), 307-319 (2015).</li><li>Palmer, K. L., Aye, L. M., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+cues+control+Pseudomonas+aeruginosa+multicellular+behavior+in+cystic+fibrosis+sputum.">Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum.</a> Journal of Bacteriology. 189 (22), 8079-8087 (2007).</li><li>Davies, D. G., 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=The+involvement+of+cell-to-cell+signals+in+the+development+of+a+bacterial+biofilm.">The involvement of cell-to-cell signals in the development of a bacterial biofilm.</a> Science. 280 (5361), 295-298 (1998).</li><li>Hartmann, R., 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=Quantitative+image+analysis+of+microbial+communities+with+BiofilmQ.">Quantitative image analysis of microbial communities with BiofilmQ.</a> Nature Microbiology. 6 (2), 151-156 (2021).</li><li>Stacy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+fight-and-flight+responses+enhance+virulence+in+a+polymicrobial+infection.">Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (21), 7819-7824 (2014).</li></ol>

This work has introduced methodologies that can be combined to study bacterial aggregate populations in the presence and absence of antibiotic treatment. High-resolution CLSM allows the visualization of changes in aggregate biomass and the structural orientation of aggregates over real time when exposed to antibiotics. In addition, physical and structural features of the biomass that remain after treatment with antibiotics can be quantified, with the goal to correlate these observations with future gene expression studie...

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that establishes chronic infections in immune-compromised individuals. For those with the genetic disease cystic fibrosis&#160;(CF), these infections can span the course of a lifetime. CF causes the buildup of a viscous, nutrient-rich sputum in the airways, which becomes colonized by a variety of microbial pathogens over time. Pa is one of the most prevalent CF pathogens, colonizing the airways in early childhood and establishing difficult-to-treat infections1. Pa remains a significant clinical problem and is considered a leading cause of mortality in those with CF, despite improved therapy regimens in recent years2,3. This persistence phenotype and increasing antibiotic tolerance have earned Pa a place in a group of pathogens identified by both the Centers for Disease Control (CDC) and the World Health Organization (WHO) as research priorities for the development of new therapeutic strategies-the ESKAPE pathogens4.
Like other ESKAPE pathogens, acquired antibiotic resistance is common in Pa, but there are also many intrinsic properties that contribute to Pa antimicrobial tolerance. Among these is the ability of Pa to form aggregates-highly dense clusters of ~10-1,000 cells, which can be observed in multiple infections, including CF patient sputum5,6. Similar to Pa studied in other biofilm systems, Pa aggregates display clinically relevant phenotypes such as increased resistance to antibiotics and activation of cell-cell communication (quorum sensing (QS)). For example, aggregates of Pa have been shown to use QS-regulated behaviors to combat other microbes as well as tolerate antimicrobial treatments such as the production of pyocyanin7. The ability to study such behaviors offers an exciting insight into bacterial ecosystems in an environment similar to the one in which they exist in the human body.
One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Much of what is known about Pa has been discovered using in vitro systems in which cells grow planktonically or in a characteristic surface-attached, &#34;mushroom&#34; architecture that has not been observed in vivo8. While classical biofilm growth models, such as flow cells or solid agar, have yielded extensive and valuable knowledge about bacterial behaviors and mechanisms of antibiotic tolerance, these findings do not always translate in vivo. Many in vitro models have a limited ability to mimic the growth environment of the human infection site, necessitating costly in vivo studies. In turn, many in vivo models lack the flexibility and resolution afforded by in vitro techniques.
Synthetic cystic fibrosis sputum (SCFM2) is designed to provide an environment for Pa growth similar to that experienced during chronic infection in the CF lung. SCFM2 includes nutritional sources identified in expectorated CF sputa in addition to mucin, lipids, and DNA. Pa growth in SCFM2 requires a near identical gene set to that required for growth in actual sputum and supports natural Pa aggregate formation9,10. After inoculation, planktonic cells form aggregates that increase in size through expansion. Individual cells (referred to as migrants) are released from aggregates, migrate to uncolonized areas, and form new aggregates10. This life history can be observed using CLSM and image analysis at the resolution of a single cell. Aggregates of Pa formed in SCFM2 are of similar sizes to those observed in the CF lung10. This model allows the observation of multiple aggregates of varying size in real-time and in three dimensions at the micron scale. Time-lapse microscopy allows the tracking of thousands (~50,000) of aggregates in one experiment. The use of image analysis software allows the quantification of aggregate phenotypes from micrographs, including aggregate volume, surface area, and position in three dimensions to the nearest 0.1 &#956;m, both at the individual aggregate and population levels. Having the ability to group aggregates by phenotype and position allows the differentiation of aggregates at different developmental stages with precision, as well as their response to a changing microenvironment6,11.
The application of SCFM2 to study Pa aggregates in low volume and high-throughput assays make it a flexible, cost-effective model. As a defined medium, SCFM2 offers uniformity and reproducibility across multiple platforms, providing a nutritionally and physically relevant method to study Pa aggregates in vitro9. Applications include its use in combination with CLSM to observe spatial organization and antibiotic tolerance at high resolution (as described in this methods paper). The ability to perform experiments that provide real-time, micron-scale data allows the study of intra-species and inter-species interactions as they may occur in vivo. For example, SCFM2 has previously been used to study the spatial dynamics of cell-cell communication in aggregate populations via a network of systems utilized by Pa to regulate multiple genes that contribute to virulence and pathogenesis6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62420/62420fig01.jpg" /> 
Figure 1: Graphical depiction of the main experimental steps. (A) SCFM2 is inoculated with Pa cells and allowed to form&#160;aggregates in a glass-bottomed culture dish. (B) Aggregates are transferred to the confocal microscope, and antibiotic is added. Depicted are three technical replicates (chambers 1-3) and a control well (4) of inoculated SCFM2 without antibiotic treatment. Aggregates are imaged using CLSM over the course of 18 h. (C) After the initial 18-h imaging, aggregates are treated with propidium iodide to visualize dead cells and imaged using CLSM (D) Aggregates with desired phenotype are separated from SCFM2 using FACS. Abbreviations: SCFM2 = synthetic cystic fibrosis sputum medium; Pa = Pseudomonas aeruginosa; CLSM = confocal laser scanning microscopy; FACS = fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/62420/62420fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, the utility of SCFM2 to study the impact of antibiotic treatment on Pa aggregates in real time is demonstrated, followed by the use of a cell-sorting approach to isolate populations of aggregates with distinct phenotypes for downstream analysis (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino acids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alanine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>56-41-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Arginine HCl</td>
			<td>MP</td>
			<td>1119-34-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Asparagine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>56-84-8</td>
			<td>Prepared in 0.5 M NaOH</td>
		</tr>
		<tr>
			<td>Cystine HCl</td>
			<td>Alfa Aesar</td>
			<td>L06328</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamic acid HCl</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>138-15-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine HCl H2O</td>
			<td>Alfa Aesar</td>
			<td>A17627</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>73-32-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Alfa Aesar</td>
			<td>A12311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine HCl</td>
			<td>Alfa Aesar</td>
			<td>J62099</td>
			<td></td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>63-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ornithine HCl</td>
			<td>Alfa Aesar</td>
			<td>A12111</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>63-91-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Alfa Aesar</td>
			<td>A10199</td>
			<td></td>
		</tr>
		<tr>
			<td>Serine</td>
			<td>Alfa Aesar</td>
			<td>A11179</td>
			<td></td>
		</tr>
		<tr>
			<td>Threonine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>72-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>73-22-3</td>
			<td>Prepared in 0.2 M NaOH</td>
		</tr>
		<tr>
			<td>Tyrosine</td>
			<td>Alfa Aesar</td>
			<td>A11141</td>
			<td>Prepared in 1.0 M NaOH</td>
		</tr>
		<tr>
			<td>Valine</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>72-18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Alfa Aesar</td>
			<td>J6194903</td>
			<td></td>
		</tr>
		<tr>
			<td>Day-of Stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 * 2H2O</td>
			<td>Fisher Chemical</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-glucose)</td>
			<td>Fisher Chemical</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Fisher (Avanti Polar Lipids)</td>
			<td>4235-95-4</td>
			<td>shake 15-20 min at 37 &#176;C to evaporate chloroform</td>
		</tr>
		<tr>
			<td>FeSO4 * 7H2O</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>7782-63-0</td>
			<td>this stock equals 1 mg/mL, MUST make fresh</td>
		</tr>
		<tr>
			<td>L-lactic acid</td>
			<td>Alfa Aesar</td>
			<td>L13242</td>
			<td>pH stock to 7 with NaOH</td>
		</tr>
		<tr>
			<td>MgCl2 * 6H2O</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylglucosamine</td>
			<td>&#160;TCI</td>
			<td>A0092</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepared solids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine mucin</td>
			<td>Sigma</td>
			<td>M1778-100G</td>
			<td>UV-sterilize</td>
		</tr>
		<tr>
			<td>Salmon sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Alfa Aesar</td>
			<td>J66764MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Alfa Aesar</td>
			<td>A13975</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Alfa Aesar</td>
			<td>J64189</td>
			<td>add solid directly to buffered base</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>7757-79-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Alfa Aesar</td>
			<td>A12914</td>
			<td>add solid directly to buffered base</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271-500</td>
			<td>add solid directly to buffered base</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>RPI</td>
			<td>S23100-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>RPI</td>
			<td>S23120-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Acr<img alt="Equation 1" src="/files/ftp_upload/62420/62420eq1v10.png" />s Organics</td>
			<td>12125-02-9</td>
			<td>add solid directly to buffered base</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (15 mL)</td>
			<td>Olympus plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (50 mL)</td>
			<td>Olympus plastics</td>
			<td>28-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tubes w/air flow cap</td>
			<td>Olympus plastics</td>
			<td>21-129</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm four chamber glass-bottom dish</td>
			<td>CellVis</td>
			<td>NC0600518</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani (LB) broth</td>
			<td>Genessee Scientific</td>
			<td>11-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Fisher Bioreagents</td>
			<td>BP2944100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips (p200)</td>
			<td>Olympus plastics</td>
			<td>23-150RL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips (p1000)</td>
			<td>Olympus plastics</td>
			<td>23-165RL</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (5 mL)</td>
			<td>Olympus plastics</td>
			<td>12-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (25 mL)</td>
			<td>Olympus plastics</td>
			<td>12-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (50 mL)</td>
			<td>Olympus plastics</td>
			<td>12-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water (RNAse/DNAse free); nanopure water</td>
			<td>Genessee Scientific</td>
			<td>18-194</td>
			<td>Nanopure water used for preparation of solutions in Table 1</td>
		</tr>
		<tr>
			<td>Syringes (10&#160; mL)</td>
			<td>BD</td>
			<td>794412</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (50 mL)</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 mm PES syringe filter</td>
			<td>Olympus plastics</td>
			<td>25-244</td>
			<td></td>
		</tr>
		<tr>
			<td>PS cuvette semi-mico</td>
			<td>Olympus plastics</td>
			<td>91-408</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biorender</td>
			<td></td>
			<td></td>
			<td>To prepare the figures</td>
		</tr>
		<tr>
			<td>FacsDiva6.1.3</td>
			<td>Becton Dickinson, San Jose, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>version 9.6</td>
		</tr>
		<tr>
			<td>Zen Black</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FacsAriallu</td>
			<td>Becton Dickinson, San Jose, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 880 confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tools for the real-time assessment of a pseudomonas aeruginosa infection model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

This work details methods to observe Pa aggregates at a high resolution and in an environment similar to that of chronic infection of the CF lung9,10,12. SCFM2 provides an in vitro system that promotes natural aggregation of Pa cells in sizes similar to those observed during actual infection10. The adaptability of SCFM2 as a defined medium can be leveraged to approach many resea...

Watch this Scientific Journal Video about Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model at JoVE.com

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Our protocol provides a way for us to study antibiotic-resistant bacteria in a nutritional and physical environment similar to how they likely exist in vivo. One of the greatest advantages of this technique is the ability to capture high-resolution images and phenotypic data of bacteria in infection-relevant population sizes. That's aggregates of approximately 10 to 1, 000 cells.Demonstrating the procedure will be Alexa Gannon, a graduate research assistant from my laboratory. To begin, sterilize the mucin-containing medium under ultraviolet light for four hours. Transfer the UV-treated mucin into sterile 1.7 milliliter tubes under sterile conditions and store the tubes at minus 20 degrees Celsius.After preparing the buffered base, add the stock solution of mucin into the buffered base containing DNA as described in the text manuscript. On the evening before the experiment, inoculate five milliliters of LB broth with several colonies of Pseudomonas aeruginosa PAO1 pMRP91 from an antibiotic containing LB agar plate and culture the bacteria overnight at 37 degrees Celsius with agitation at 250 revolutions per minute. The next morning, at least two hours before starting the experiment, turn on the confocal laser scanning microscope and open the incubation module in the associated imaging software.Dilute 500 microliters of the culture in five milliliters of fresh LB broth and incubate the bacterial suspension at 37 degrees Celsius and 250 revolutions per minute for 60 to 90 minutes. When the bacteria have entered the log phase, pellet the bacterial cells by centrifugation and wash the cells three times with filter sterilized PBS. After the last wash, resuspend the pellet in one milliliter of PBS.Measure the absorbance of the bacterial cell suspension at 600 nanometers to determine the culture volume required for an optical density of 0.05 in five milliliters of synthetic cystic fibrosis sputum medium two. Inoculate the calculated volume of bacterial cell suspension in the medium and vortex gently before adding one milliliter of cells to each chamber of a four-chambered optic culture dish, then incubate the bacteria for four hours under static conditions at 37 degrees Celsius. To image the Pseudomonas aeruginosa aggregates by confocal laser scanning microscope, at the end of the incubation, designate three wells of the culture plate as antibiotic treatment replicates and one well as the no treatment control and place the plate on the heated stage in the microscope.Select a 63X oil immersion objective and open the locate tab to identify the bacterial aggregates in the brightfield. Define an area for imaging within each well and use the positions module to store the area position. Set the excitation wavelength to 488 nanometers and the emission wavelength to 509 nanometers.In the acquisition module, select the Z-stack option to acquire the images and use the line averaging module to reduce the background fluorescence in the GFP channel within the total volume of the Z-stack images. Set the time series option to capture 60 slices in each well at 15-minute intervals for 18 hours and use the definite focus strategy to store a focal plane for each position that will be re-imaged at each time point throughout the experiment. 4-1/2 hours after setting up the imaging experiment, image each position to determine the aggregate biomass within each of the four wells before the addition of the antibiotic.After six hours, gently add antibiotic at two times below the minimum inhibitory concentration to the middle of each well just blow the air liquid interface and click start experiment to begin the post-antibiotic treatment imaging. To isolate the live cells, at the end of the 18-hour imaging period, label the bacteria in each well with the appropriate volume of propidium iodide according to the manufacturer's recommendations. At the end of the incubation, use an insulated container to transfer the plate to the flow cytometer and set the cytometer to detect GFP and propidium iodide using a 70 micron nozzle, then run one milliliter aliquots of each culture supernatant at the lowest flow rate to sort the viable GFP-positive and non-viable propidium iodide negative Pseudomonas aeruginosa aggregates.To quantify the aggregate dynamics, load the images into an appropriate image analysis software program and create histograms of the counts produced for the untreated control and antibiotic-treated cultures in the GFP channel to allow quantification of the background fluorescence. To ensure that the detected GFP voxels correlate to the bacterial biomass, define a GFP-positive voxel as greater than or equal to 1.5 times the GFP background count value. Produce the ISO surfaces for all of the remaining voxels and to detect individual aggregates, enable the split objects option and define the aggregates as objects.Use the vantage module to calculate the volume XYZ and sum of GFP voxels for each individual object. Export this data to an external statistical platform. Filter the exported data by size to define objects with volumes greater than or equal to five cubic micrometers.Remove any objects less than 0.5 cubic micrometers and use the vantage module to calculate the distance of each object in relation to other objects within each image. Alternatively, the distance can be calculated using the formula, then use the sum and average calculations to determine the total biomass in the average aggregate volume. Although multiple viable bacterial aggregates remain after four hours of antibiotic application, the total biomass of the aggregate populations within the antibiotic-treated cultures is significantly reduced compared to that of untreated cultures.Time series microscopy can be used to determine the spatial patterns between sensitive propidium iodide positive and tolerant GFP-positive aggregates after antibiotic treatment. By calculating how aggregates of different sizes contribute to the overall population, patterns of how aggregates respond to antibiotic treatment in relation to their size, shape, and position can be identified. After antibiotic treatment, viable and non-viable aggregates can be successfully separated according to their fluorescent signal expression.We hope this protocol inspires other researchers to study their organism of choice at a similar resolution. Our goal is to find common biological threads in these complex communities regardless of the infection site.

Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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 ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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 ...

Pseudomonas aeruginosa Induced Lung Injury Model

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. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...