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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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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	<tbody>
		<tr height="20">
			<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 ...

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