Name: phage therapy application to counteract pseudomonas aeruginosa infection in cystic fibrosis zebrafish embryos
Uploaded: 2020-05-12T14:45:00
Description: Watch this Scientific Journal Video about Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos at JoVE.com

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
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	<li>Prepare a 50x stock...

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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 height="20" style="height:20px;width:275px;">Bacto Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:108px;">214010</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CsCl</td>
			<td>Sigma-Aldrich</td>
			<td>289329</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s phospate buffered saline PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Femtojet Micromanipulator</td>
			<td>Eppendorf</td>
			<td>5247</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fleming/brown P-97</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LE-Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>11685660001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low Melting Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 9012-36-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl Blue</td>
			<td>Sigma-Aldrich</td>
			<td>28983-56-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microinjection needles</td>
			<td>Harvard apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Phenylthiourea &#62;=98%</td>
			<td>Aldrich-P7629</td>
			<td>103-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oligo Morpholino</td>
			<td>Gene Tools</td>
			<td></td>
			<td>designed by the researcher</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG6000</td>
			<td>Calbiochem</td>
			<td>528877</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phenol Red Solution</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 143-74-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pronase</td>
			<td>Sigma-Aldrich</td>
			<td>9036-06-0</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Z-MOLDS Microinjection</td>
			<td>Word Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

This protocol facilitates the preparation and administration of a viral and phage cocktail against pseudomonas aeruginosa in zebrafish embryos. The main advantage of this technique is that it allows an in vivo assessment of the phage efficacy in counteracting the bacterial infection. The phage cocktail is efficient in both wild type and cystic fibrosis zebrafish models.Opening the possibility of applying the phage therapy to bacterial infections in cystic fibrosis patients. Phage therapy can also be applied to counteract specific bacterial infections in other model systems. To prepare a phage stock for the experiment.Add 1.25 times 10 to the seventh phages to an OD 600 of 0.05 P.aeruginosa culture to a multiplicity of infection of one times 10 to the negative three and incubate the culture for three to four hours with shaking until the OD 600 drops to 0.1 to 0.3. At the end of the incubation incubate the lysate with one microgram per milliliter of DNAse and RNAse for 30 minutes at 37 degrees Celsius. And pellet the cells by centrifugation.At the end of the centrifugation filter the supernatant through a 0.8 micro meter pore diameter and add 58 grams per liter of sodium chloride and 105 grams per liter of PEG 6000. Incubate the solution at four degrees Celsius overnight. The next morning, precipitate the phage by centrifugation.At the end of the centrofugation remove the supernatant and carefully dissolve the phage pellet in 15 milliliters of Tris sodium chloride buffer. To purify the phages by cesium chloride density gradient, first stratify two milliliters of four different cesium chloride solutions in polyallomer ultracentrifuge tubes for an SW 41 Rotor and add 3.5 milliliters of the phage suspension to each tube. Cesium chloride is toxic.Always adopt proper safety procedures when handling and discarding this compound. When all of the tubes have been loaded, place the tubes into the rotor taking care that the tubes are balanced. And centrifuge the samples for two hours at 100, 000 times g and four degrees Celsius.At the end of the centrifugation, use a syringe with a 19 gauge needle to aspirate the white layer between the D equals 1.5 and the D equals 1.4 density regions. And transfer the suspensions into new SW 60 size polyallomer tubes. Centrifuge the suspensions for at least 16 hours at 150, 000 times g and four degrees Celsius.And transfer the visible bands into dialysis tubes with a 6, 000 Dalton cutoff. Then dialyze the collected samples two times against 500 milliliters of water for 20 minutes per dialysis and overnight against 500 milliliters of Tris sodium chloride buffer. The next morning, filter the resulting phage stock with a 0.22 micro meter pore filter and store the stock at four degrees Celsius.On the day of the micro injection dilute two one micromolar morpholinos stock solutions in sterile water to a final concentration of 0.25 picomolar per embryo to obtain a five microliter morpholino injection solution. And add 0.5 microliters of phenol red to each solution. Next use a 20 microliter micropipette with a fine gel loading tip to load a micro injection needle with the entire volume of morpholino mixed solution and secure the needle to a micro manipulator connected to a stand under a stereo microscope.Then with one or two cells, zebra fish embryos arranged on a glass slide placed within a 96 millimeter diameter Petri dish, penetrate the chorion and the yolk with the micro injection needle tip and inject two nanoliters of the morphlino mixture into the embryo. At 26 hours post fertilization, load a micro injection needle with approximately five microliters of P.aeruginosa inoculum and insert the needle dorsally to the starting point of the duct of Cuvier at which the duct starts spreading over the yolk sac. Inject one to three nanoliters of the PAO1 inoculum into the embryo making sure that the volume expands directly within the duct and enters into the circulation.After the injection transfer the embryo into one of two new Petri dishes containing fresh E3 medium supplemented with phenylthiourea for a 30 minute or three hour incubation at 28 degrees Celsius. Next, load a micro injection needle with approximately five microliters of the phage cocktail and fix the needle to the micro injector. Then inject one to three nanoliters of phage cocktail into the duct of Cuvier of each embryo previously injected with bacteria and place the embryos into one of two new Petri dishes containing fresh E3 medium supplemented with phenylthiourea at 28 degrees Celsius.At four hours post-infection, use a plastic pipette to transfer an anesthetized embryo into a glass bottom dish. And fill the dish with warm low melting point agarose solution. When the agarose is cold, gently fill the dish with E3 medium supplemented with anesthetic solution.Place the dish under the stereo microscope and use a pipette tip to position the embryo in the desired orientation. Then place the dish under a fluorescent stereo microscope with a fluorescent filter for GFP positive bacteria for up to 18 hours post-infection and image the embryo for the progression of GFP expression at nine, 14 and 18 hours post-infection. To determine the bacterial burden at eight hours post-infection, transfer 15 anesthetized micro-injected embryos into a 1.5 milliliter centrifuge tube and replace the anesthetic solution with 300 microliters of 1%Triton X-100 in PBS.Pass the needles through the sterile 27 gauge needle of an insulin syringe at least 15 times to homogenize the embryos. And prepare serial dilutions of the homogenate by transferring 100 microliters of the resulting mixture into 900 microliters of sterile PBS per dilution. To select for the naturally ampicillin resistant P.aeruginosa strain, plate 10 microliters of the dilutions onto LB agar supplemented with ampicillin and incubate them overnight at 37 degrees Celsius.The next day count the number of colonies. To allow calculation of the total number of colony forming units and the average number of colony forming units per infected embryo. To evaluate the lethality of the P.aeruginosa infection score the injected embryos at 20 hours post-infection under a stereo microscope by counting the number of dead white opaque embryos.Then calculate the half maximal lethal concentration 50 dose of P.aeruginosa that resulted in the death of 50%of the injected embryos at 20 hours post-infection. To validate the cystic fibrosis phenotype the impaired position of internal organs such as the heart liver and pancreas can be evaluated. The bacterial burden is reduced by phage therapy in cystic fibrosis embryos infected with P.Aeruginosa.In 48 hours post fertilization cystic fibrosis embryos infected with GFP positive P.Aeroginosa bacteria a 30 colony forming units per embryo dose demonstrates a 50%lethality at 20 hours post-infection. Phage therapy is equally effective at 20 hours post-infection when delivered 30 minutes or seven hours after bacterial injection. Here, a cystic fibrosis and GFP positive P.Aeruginosa injected embryo at four, nine, 14 and 18 hours post-infection is shown.In contrast the cystic fibrosis plus P.Aeruginosa plus phage injected embryo demonstrates a reduced fluorescence due to the phage action against the bacteria. The inflammatory response generated by P.aeruginosa infection in cystic fibrosis embryos is significantly increased as revealed by the expression of the pro-inflammatory cytokines, TNF alpha and interleukin 1 beta following P.aeruginosa injection compared to control injected zebrafish. The expression of both inflammatory cytokines is reduced in phage cocktail co-injected animals however.The phages must be carefully purified to remove any contaminating endotoxin that may be retained during the preparation. The phage preparations can be analyzed by electron microscopy to assess the virion morphology. It would be interesting to test whether the infection in human patients by bacteria other than pseudomonas aeruginosa can be cured with phage therapy in the cystic fibrosis zebrafish model.

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 ...

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 ...

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. ...

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

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 ...

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 ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...