This work was supported by the Italian Cystic Fibrosis Foundation (FFC#22/2017; Associazione &#8220;Gli amici della Ritty&#34; Casnigo and FFC#23/2019; Un respiro in pi&#249; Onlus La Mano tesa Onlus).

Adult zebrafish (Danio rerio) from the AB strain (European Zebrafish Resource Center EZRC) are maintained according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree 4th March 2014, n. 26) on the protection of animals used for scientific purposes. Standard conditions are set in the fish facility with a 14 h of light/10 h dark cycle and tank water temperature at 28&#176; C.
1. Preparation of solutions and tools
<ol>
	<li>Prepare a 50x stock solution and 1x working solutions for E3 embryo medium for growing zebrafish embryos (see Table 1).</li>
	<li>Make pigmentation blocking solution, to block the embryo pigmentation from 24 hours post fertilization (24 hpf) (see Table 1).</li>
	<li>Prepare 25x stock and 1x working Tricane anesthetic solution as described in Table 1. 
	NOTE: To avoid the repeated freezing and thawing of the solution that could damage the stock solution, make aliquots of 2 mL of 25x tricaine and store them at -20 &#176;C.</li>
	<li>Prepare tracks for embryo microinjection by dissolving 1 g of agarose in 100 mL of distilled H2O in a microwavable flask or bottle. Microwave for 1-3 min until the agarose is completely dissolved.</li>
	<li>Position zebrafish microinjection molds (see Table of Materials) turned upside down in a Petri dish (90 mm x 15 mm) and cover the entire surface by pouring the liquid agarose gel with a width of approximately 10 mm.</li>
	<li>Remove the mold once the agarose has solidified. Fill the Petri dish with 1x E3 embryo medium. This can be stored at 4 &#176;C for approximately one week. Before use, replace with the fresh E3 medium containing Tricaine. 
	NOTE: Older molds might develop fungi or bacterial contaminations.</li>
	<li>Use fire polished 10 cm borosilicate capillaries to prepare needles for microinjection and secure them to the micropipette puller by tightening the handles. Set the puller with the following settings: heat 500, velocity 100, time 150, pull 150.</li>
</ol>
2. P. aeruginosa (PAO1) inoculum preparation
<ol>
	<li>Inoculate 1 colony of GFP+ P. aeruginosa strain PAO119 in 5 mL of LB broth (see Table 1) and grow overnight at 37 &#176;C with shaking (200 rpm).</li>
	<li>Dilute the above overnight culture to OD600 = 0.1 in 10 mL of LB broth and grow to OD600 = 0.5 at 37 &#176;C with shaking (200 rpm).</li>
	<li>Centrifuge 2 mL of the culture for 2 min at 4 &#176;C at 16,100 x g and resuspend the cell pellet to OD600 = 1, equivalent to about 1 x 109 cfu/mL, in 1 mL of the physiological solution (see Table 1).</li>
	<li>Dilute the cell suspension to about 5 x 107cfu/mL in physiological solution and store at 4 &#176;C.</li>
</ol>
3. Phage stock preparation
<ol>
	<li>Inoculate 1 colony of P. aeruginosa strain PAO1 in 5 mL of LB broth and incubate overnight at 37 &#176;C with shaking. Dilute the overnight culture to OD600 = 0.01 in 500 mL of LB broth and grow at 37 &#176;C with shaking to OD600 = 0.05, equivalent to about 2.5 x 107 cfu/mL.</li>
	<li>To the diluted culture of P. aeruginosa, add about 1.25 x 107 phages, to get the multiplicity of infection (M.O.I.) of 10-3. Incubate at 37 &#176;C with shaking until the OD600 drops to about 0.1-0.3 (in about 3 to 5 h, depending on the phage used). 
	NOTE: Four phages were selected for this experiment that infect PAO1 strain: Two Podoviridae, GenBank accession numbers vB_PaeP_PYO2, MF490236, and vB_PaeP_DEV, MF490238, and two Myoviridae, vB_PaeM_E215, MF490241, and vB_PaeM_E217, MF490240. Perform the steps below for each phage&#160;individually.</li>
	<li>Incubate the lysate with 1 mg/mL of DNase and RNase for 30 min at 37 &#176;C. Centrifuge at 5,000 x g for 30 min at 4 &#176;C and carefully recover the supernatant (SN). Filter the SN with a filter having 0.8 &#956;m pore diameter.</li>
	<li>To the SN, add 58 g/L of NaCl and 105 g/L of PEG6000. Dissolve with stirring at RT 20 min and then keep it overnight at 4 &#176;C. Centrifuge at 20, 000 x g for 30 min at 4 &#176;C for phage precipitation. Remove the SN and carefully dissolve the phage pellet in 15 mL of TN buffer (see Table 1).</li>
	<li>Purify the phages with CsCl density gradient as described in steps below.
	<ol>
		<li>In a polyallomer ultracentrifuge tubes for SW41 rotor, prepare a CsCl step density gradient by stratifying 2 mL of the four CsCl solutions described in Table 1. &#160;Add 3.5 mL of phage suspension in each of the 4 tubes. 
		NOTE: CsCl is toxic, adopt proper safety procedures to handle and discard it.</li>
		<li>Introduce the tubes into the rotor and ensure that the tubes are balanced (the weight difference between the two facing tubes must be &#8804; 0.01g).</li>
		<li>Centrifuge for 2 h at 4 &#176;C and 100, 000 x g (25,000 rpm for SW41 rotor). After centrifugation, slowly remove the tubes and carefully immobilize them on a support. Using a syringe with 19 G needle, draw up the white/opalescent band that is located usually between the d=1.5 and the d=1.4 density region.</li>
		<li>Transfer the suspensions from the syringe into new polyallomer tubes for SW60 rotor and use the d=1.4 solution to precisely balance the tubes.</li>
		<li>Centrifuge the suspension at 4 &#176;C and 150,0000 x g (38,000 rpm for the SW60 rotor) for at least 16 h.</li>
		<li>Collect the visible band as above (see step 3.3.3) and transfer them into dialysis tubes with 6,000 Da cut-off. Dialyze the obtained visible band 2x for 20 min against 500 mL of water and overnight against 500 mL of TN buffer. Filter with a 0.22 &#181;m pore filter and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Phage cocktail preparation
<ol>
	<li>Make a mix of four phages that infect the strain PAO1, previously isolated and characterized8. Before mixing, estimate the titer of each phage stock by plaque assay20.</li>
	<li>Assemble the phage cocktail, with an overall titer of 5 &#215; 108 pfu/mL, by equally mixing four phage preparations at the same pfu/mL immediately before each experiment, to ensure accurate phage titers.&#160;</li>
</ol>
5. Collection and preparation of zebrafish embryos for cftr morpholinos microinjection
NOTE: Collect 1-2 cell embryos from wild type zebrafish for cftr morpholinos (cftr-MOs) microinjection.
<ol>
	<li>Two days before the bacterial microinjection experiment, set up breeding pairs and collect embryos as described previously21. Adult zebrafish (Danio rerio) of the AB strain were purchased from the European Zebrafish Resource Center, EZRC.</li>
	<li>The day after, collect the embryos immediately after fertilization with a plastic pipette or using a fine-mesh strainer. Place the collected embryos in a Petri dish containing E3 embryo medium and carefully remove the debris with a plastic pipette to avoid contamination that might damage the embryo development.</li>
	<li>Prepare 5 &#181;L of morpholino injection solution by diluting the two morpholino stock solutions (1 mM) in sterile water to a final concentration of 0.25 pmol/embryo ATG-MO + 0.25 pmol/embryo splice-MO. To trace the embryo injection, add 0.5 &#181;L of phenol red to the morpholino injection solution mix.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the morpholino mix solution using a 20 &#956;L micropipette with a fine gel loading tip. Secure the needle to the micromanipulator connected to a stand and position it under a stereomicroscope. 
	NOTE: It is not necessary that the solution reaches the tip of the needle as the pressure of the microinjector pump will push it.</li>
	<li>Clip off the tip of the needle by cutting the needle tip with fine sterile tweezers. Measure the diameter of the drop using a scale bar in the ocular of the microscope. Alternatively, create a drop of mineral oil onto a micrometer slide and set the microinjection volume by injecting the solution into the oil-drop to evaluate the droplet size. Use a drop with a 156 &#956;m diameter to inject a volume of 2 nL.</li>
	<li>Set the microinjector by adjusting the compensation pressure to 15 hPa, injection time to 0.5 s, and injection pressure between 300 and 600 hPa to obtain the correct injection volume depending on the needle used.</li>
	<li>With a plastic pipette arrange the embryos from step 5.2 onto a glass positioned in a 96 mm diameter Petri dish. 
	NOTE: Too much E3 medium will prevent the proper penetration of the microinjection needle into the chorion of the embryos.</li>
	<li>Penetrate the chorion and then the yolk with the needle to inject 2 nL into the embryo as described previously22.</li>
	<li>Place injected embryos into a Petri dish with E3 medium and put them in an incubator at 28 &#176;C to let them develop until the day after.</li>
</ol>
6. Microinjection of zebrafish embryos with bacteria and phage cocktail
NOTE: To perform a systemic infection, the embryo must have blood circulation that usually starts after 26 hpf.
<ol>
	<li>After 26 hpf (for zebrafish developmental stages refer to Kimmel21) dechorionate embryos with pronase solution prepared by dissolving pronase powder in E3 medium at a concentration of 2 mg/mL. Gently pipette the embryos to break the chorion. Discard the pronase/E3 medium and rinse the dish several times with fresh E3 to remove all pronase.</li>
	<li>Anesthetize zebrafish embryos in E3 medium containing Tricaine approximately 5 min prior to injections.</li>
	<li>Pipette the anesthesized embryos into the track prepared in step 1. Use a fine gel loading tip to line the embryos in the lateral position.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the P. aeruginosa preparation as described in part 5.</li>
	<li>Insert the needle dorsally to the starting point of the duct of Cuvier where it starts spreading over the yolk sac. Inject a volume of 1-3 nL and ensure that the volume expands directly within the duct and enters into the circulation.</li>
	<li>Approximately after every 50 injected-embryo, inject a drop of the bacteria into a 1.5 mL centrifuge tube with 100 &#956;L of sterile PBS to check if the injection volume remains the same. Plate the inoculum on LB agar (see Table 1) at 37 &#176;C overnight to assess the CFU.</li>
	<li>Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 &#176;C. &#160;</li>
	<li>At two selected time points: 30 min or 3 h after the bacterial injection, take out one of the Petri dishes with bacterial-injected embryos from the incubator and align them in the track as described in 6.3&#160;for the phage cocktail injection.</li>
	<li>Load a microinjection needle with approximately 5 &#956;L of the phage cocktail (prepared in step 4) with a fine gel loading tip and fix it to the microinjector (see step 5).</li>
	<li>Inject the phage cocktail in the duct of Cuvier of the embryos previously injected with bacteria.</li>
	<li>Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 &#176;C.</li>
</ol>
7. Evaluation of the bacterial burden of embryos injected with PAO1 and phages
<ol>
	<li>At 8 hpi, pipette 15 anesthetized embryos from the Petri dish to a 1.5 mL centrifuge tube.</li>
	<li>Replace the anesthetic solution with 300 &#956;L of 1% Triton X-100 in PBS (PBSTritonX) and homogenize the embryos by passing them at least 15x through an insulin syringe with a sterile 27-G needle.</li>
	<li>Prepare serial dilutions of homogenates in sterile PBS by transferring 100 &#956;L of the diluted homogenate into 900 &#956;L of sterile PBS.</li>
	<li>To select for the naturally amp-resistant PAO1 strain, plate 100 &#956;L of the dilutions on LB agar added with ampicillin (100 &#956;g/mL), and incubate at 37 &#176;C over-night.</li>
	<li>The day after, count the number of colonies, multiply by the dilution factor to determine the total number of CFU, and divide by the number of embryos homogenized to obtain the average number of CFU/infected embryo.</li>
</ol>
8. Evaluation of the lethality of embryos injected with PAO1 and phages
<ol>
	<li>To evaluate the lethality of the PAO1 infection, score the injected embryos at 20 hpi under a stereomicroscope and count for the dead embryos (white/not transparent).</li>
	<li>Calculate the half-maximal lethal concentration 50 (LD50) dose of PAO1 that determines the death of 50% of injected embryos at 20 hpf.</li>
</ol>
9. Embryo preparation for stereomicroscope time-lapse imaging of GFP+ PAO1 infection
<ol>
	<li>Preparate the mold for live-embryo imaging by dissolving 1.5% low melting agarose in 100 mL of E3 solution.</li>
	<li>Add 1% Tricaine (see Table 1) to anesthetize the embryos.</li>
	<li>At 4 hpi transfer one embryo with a plastic pipette in a glass bottom dish and add the warm low-melting-point agarose solution.</li>
	<li>Position the glass bottom dish under a stereomicroscope and with a tip position the embryo in the desired orientation. Use a plastic pipette to carefully add a drop of agarose on the embryo.</li>
	<li>Let the agarose cool for 5-10 min and gently fill the glass bottom dish with E3 containing the anesthetic solution to keep the embryo moist.</li>
	<li>Place the glass bottom dish with the embryo under the fluorescent stereomicroscope with a fluorescent filter (channel 488 nm) for GFP+ bacteria. Do not remove the Petri dish until further acquisitions with the same parameters at 9, 14 and 18 hpi.</li>
</ol>
10. Expression analyses of pro-inflammatory cytokines
<ol>
	<li>At 20 hpi anesthetize the embryos with tricaine 1x solution and transfer them from the Petri dish to a 1.5 mL centrifuge tube.</li>
	<li>Under a fume hood remove the anesthetic solution and replace with 200 &#956;L of guanidium hydrochloride reagent.</li>
	<li>Homogenize embryos by pipetting them repetitively through a 200 &#956;L pipette first and then at least 15x with an insulin syringe with a sterile 27 G needle.</li>
	<li>Extract total RNA from homogenized zebrafish embryos using a commercially available guanidium hydrochloride reagents as per the manufacturer&#8217;s instructions.</li>
	<li>Treat 1 &#956;g of each sample of RNA with 2 &#956;L of 1U/&#956;L of DNase I &#160;(RNase-free), following the manufacturer&#8217;s instructions.</li>
	<li>Use 1 &#956;g of DNase I treated RNA for reverse-transcription reaction (RT) using commercially available kit (see Table of Materials), in a total volume of 20 &#956;L according to manufacturer&#8217;s instructions.</li>
	<li>Perform RT qPCR reaction in a total volume of 10 &#956;L containing 1x SYBR Green mastermix using 1.5 &#956;L of a 1:6 dilution of RT reaction. For normalization purposes, use the primers for the house-keeping genes rpl8 and for pro-inflammatory cytokines, use primers for TNF-&#945;&#160;and IL-1&#946;&#160;(see Table 2). qPCR protocol was: cycle 1 step 1: 95 &#176;C, 2 min, repeat once; cycle 2: step 1 95 &#176;C, 10 s, step 2 55 &#176;C, 30 s, repeat 40x; cycle 3: step 1 72 &#176;C, 15 min.</li>
</ol>

<ol>
	<li>Cisek, A. A., D&#261;browska, I., Gregorczyk, K. P., Wy&#380;ewski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Therapy+in+Bacterial+Infections+Treatment:+One+Hundred+Years+After+the+Discovery+of+Bacteriophages.">Phage Therapy in Bacterial Infections Treatment: One Hundred Years After the Discovery of Bacteriophages.</a> Current Microbiology. 74 (2), 277-283 (2017).</li><li>Trend, S., Fonceca, A. M., Ditcham, W. G., Kicic, A., Cf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+phage+therapy+in+cystic+fibrosis:+Essential+human-bacterial-phage+interactions+and+delivery+considerations+for+use+in+Pseudomonas+aeruginosa-infected+airways.">The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways.</a> Journal of Cystic Fibrosis. 16 (6), 663-667 (2017).</li><li>Pacios, 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=Strategies+to+combat+multidrug-resistant+and+persistent+infectious+diseases.">Strategies to combat multidrug-resistant and persistent infectious diseases.</a> Antibiotics. 9 (2), 65 (2020).</li><li>Dubos, R. J., Straus, J. H., Pierce, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiplication+of+bacteriophage+in+vivo+and+its+protective+effect+against+an+experimental+infection+with+shigella+dysenteriae.">The multiplication of bacteriophage in vivo and its protective effect against an experimental infection with shigella dysenteriae.</a> Journal of Experimental Medicine. 78 (3), 161-168 (1943).</li><li>Marza, J. A. S., Soothill, J. S., Boydell, P., Collyns, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplication+of+therapeutically+administered+bacteriophages+in+Pseudomonas+aeruginosa+infected+patients.">Multiplication of therapeutically administered bacteriophages in Pseudomonas aeruginosa infected patients.</a> Burns. 32 (5), 644-656 (2006).</li><li>Heo, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+efficacy+of+phages+against+Pseudomonas+aeruginosa+infections+in+mice+and+Drosophila+melanogaster.">Antibacterial efficacy of phages against Pseudomonas aeruginosa infections in mice and Drosophila melanogaster.</a> Antimicrobial Agents and Chemotherapy. , 01646 (2009).</li><li>McVay, C. S., Vel&#225;squez, M., Fralick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+therapy+of+Pseudomonas+aeruginosa+infection+in+a+mouse+burn+wound+model.">Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model.</a> Antimicrobial Agents and Chemotherapy. , 01028 (2007).</li><li>Forti, F., 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=Design+of+a+broad-range+bacteriophage+cocktail+that+reduces+Pseudomonas+aeruginosa+biofilms+and+treats+acute+infections+in+two+animal+models.">Design of a broad-range bacteriophage cocktail that reduces Pseudomonas aeruginosa biofilms and treats acute infections in two animal models.</a> Antimicrobial Agents and Chemotherapy. , 02573 (2018).</li><li>Jault, 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=Efficacy+and+tolerability+of+a+cocktail+of+bacteriophages+to+treat+burn+wounds+infected+by+Pseudomonas+aeruginosa+(PhagoBurn):+a+randomised,+controlled,+double-blind+phase+1/2+trial.">Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial.</a> Lancet Infectious Diseases. 19 (1), 35-45 (2019).</li><li>Phennicie, R. T., Sullivan, M. J., Singer, J. T., Yoder, J. A., Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+resistance+to+Pseudomonas+aeruginosa+infection+in+zebrafish+is+mediated+by+the+cystic+fibrosis+transmembrane+conductance+regulator.">Specific resistance to Pseudomonas aeruginosa infection in zebrafish is mediated by the cystic fibrosis transmembrane conductance regulator.</a> Infections and Immunity. 78, 4542-4550 (2010).</li><li>Clatworthy, A. 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=Pseudomonas+aeruginosa+infection+of+zebrafish+involves+both+host+and+pathogen+determinants.">Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants.</a> Infections and Immunity. 77, 1293-1303 (2009).</li><li>Bernut, 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=CFTR+Protects+against+Mycobacterium+abscessus+Infection+by+Fine-Tuning+Host+Oxidative+Defenses.">CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses.</a> Cell Reports. 26 (7), 1828-1840 (2019).</li><li>Semler, D. D., Goudie, A. D., Finlay, W. H., Dennis, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosol+phage+therapy+efficacy+in+Burkholderia+cepacia+complex+respiratory+infections.">Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.</a> Antimicrobial Agents and Chemotherapy. , 02388 (2014).</li><li>Benard, E. 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=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments. (61), e3781 (2012).</li><li>Doring, G., Gulbins, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+and+innate+immunity:+how+chloride+channel+mutations+provoke+lung+disease.">Cystic fibrosis and innate immunity: how chloride channel mutations provoke lung disease.</a> Cellular Microbiology. 11, 208-216 (2009).</li><li>Navis, A., Bagnat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+cftr+function+leads+to+pancreatic+destruction+in+larval+zebrafish.">Loss of cftr function leads to pancreatic destruction in larval zebrafish.</a> Developmental Biology. 399, 237-248 (2015).</li><li>Navis, A., Marjoram, L., Bagnat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cftr+controls+lumen+expansion+and+function+of+Kupffer's+vesicle+in+zebrafish.">Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish.</a> Development. 140, 1703-1712 (2013).</li><li>Balloy, V., 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=Normal+and+cystic+fibrosis+human+bronchial+epithelial+cells+infected+with+Pseudomonas+aeruginosa+exhibit+distinct+gene+activation+patterns.">Normal and cystic fibrosis human bronchial epithelial cells infected with Pseudomonas aeruginosa exhibit distinct gene activation patterns.</a> PLoS One. 10, 0140979 (2015).</li><li>Cafora, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+therapy+against+Pseudomonas+aeruginosa+infections+in+a+cystic+fibrosis+zebrafish+model.">Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.</a> Science Reports. 9, 1527 (2019).</li><li>Hershey, A. D., Kalmanson, G. M., Bronfenbrenner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+methods+in+the+study+of+the+phage-antiphage+reaction.">Quantitative methods in the study of the phage-antiphage reaction.</a> Journal of Immunology. 46, 267-279 (1943).</li><li>Kimmel, C., Ballard, W., Kimmel, S., Ullmann, B., Schilling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), e1115 (2009).</li><li>Traver, 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=The+Zebrafish+as+a+Model+Organism+to+Study+Development+of+the+Immune+System.">The Zebrafish as a Model Organism to Study Development of the Immune System.</a> Advances in Immunology. 81, 253-330 (2003).</li></ol>

The authors have nothing to disclose.

In this manuscript, we described the protocol to perform P. aeruginosa (PAO1) infection in zebrafish embryos and how to apply phage therapy with a cocktail of phages previously identified as able to infect PAO1 to resolve it. The use of bacteriophages as an alternative to antibiotic treatments has been of increasing interest since the last few years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections, which constitute a serious issue for public health. Of course, the scope of ...

Phage therapy, the use of the natural enemies of bacteria to fight bacterial infections, is garnering renewed interest as bacterial resistance to antibiotics becomes widespread1,2. This therapy, used for decades in Eastern Europe, could be considered a complementary treatment to antibiotics in curing lung infections in patients with CF and a possible therapeutic alternative for patients infected with bacteria that are resistant to all the currently in use antibiotics2,3. Advantages of antibiotic therapy are that bacteriophages multiply at the infection site, whereas antibiotics are metabolized and eliminated from the body4,5. Indeed, the administration of cocktails of virulent phages isolated in different laboratories has proven to be effective in treating Pseudomonas aeruginosa infections in animal models as different as insects and mammals6,7,8. Phage therapy was also shown to be able to reduce the bacterial burden in burn wounds infected with P. aeruginosa and Escherichia coli in a randomized clinical trial9.
Zebrafish (Danio rerio) has recently emerged as a valuable model to study infections with several pathogens, including P. aeruginosa10,11, Mycobacterium abscessus and Burkolderia cepacia12,13. By microinjecting bacteria directly into the embryo blood circulation14 it is easy to establish a systemic infection that is counteracted by the zebrafish innate immune system, which is evolutionary conserved with neutrophils and macrophage generation similar to the human counterpart. Moreover, during the first month of life, zebrafish embryos lack the adaptive immune response, making them ideal models for studying the innate immunity, which is the critical defense mechanism in human lung infections15. Zebrafish recently emerged as a powerful genetic model system to better understand the CF onset and to develop new pharmacological treatments10,16,17. The CF zebrafish model of cftr knock-down generated with morpholino injection in zebrafish presented a dampened respiratory burst response and reduced neutrophil migration10, while the cftr knock-out leads to impaired internal organ position and the destruction of the exocrine pancreas, a phenotype that mirrors human disease16,17. Of greatest interest was the finding that the P. aeruginosa bacterial burden was significantly higher in cftr-loss-of-function embryos than in controls at 8 hours post-infection (hpi), which parallels the results obtained with mice and human bronchial epithelial cells2,18.
In this work, we demonstrate that phage therapy is effective against P. aeruginosa infections in zebrafish embryos.

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			<td>10043-52-4</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
			<td>289329</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s phospate buffered saline PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
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		</tr>
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			<td height="20" style="height:20px;">Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td>common name tricaine</td>
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			<td height="20" style="height:20px;">Low Melting Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 9012-36-6</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
			<td>28983-56-4</td>
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			<td height="20" style="height:20px;">Microinjection needles</td>
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			<td>103-85-5</td>
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			<td height="20" style="height:20px;">Oligo Morpholino</td>
			<td>Gene Tools</td>
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			<td height="20" style="height:20px;">PEG6000</td>
			<td>Calbiochem</td>
			<td>528877</td>
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			<td height="20" style="height:20px;">Phenol Red Solution</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 143-74-B</td>
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			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
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			<td height="20" style="height:20px;">Pronase</td>
			<td>Sigma-Aldrich</td>
			<td>9036-06-0</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride ACS reagent, &#8805;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica</td>
			<td>S9I</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042B</td>
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			<td height="20" style="height:20px;">Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021B</td>
			<td></td>
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			<td height="20" style="height:20px;">Z-MOLDS Microinjection</td>
			<td>Word Precision Instruments</td>
			<td></td>
			<td></td>
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phage therapy application to counteract pseudomonas aeruginosa infection in cystic fibrosis zebrafish embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Results and figures presented here are referred to CF embryos generated through the injection of cftr morpholinos as described previously10&#160;and in step 5. To validate the CF phenotype, the impaired position of internal organs such as heart, liver, and pancreas as previously described17 (Figure 1) were considered. Similar results were obtained in case of the WT embryos as reported in our previous publication19</su...

Watch this Scientific Journal Video about Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos at JoVE.com

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

phage-therapy-application-to-counteract-pseudomonas-aeruginosa

Research

JoVE Journal

Immunology and Infection

6.4K Views.  Università degli Studi di Milano. Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Video: Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

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 ...

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 leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

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

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

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

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

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

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 fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

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

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of 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

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 ...

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. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

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 tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

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

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium 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 ...

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 ...