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

Summary

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

Abstract

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.

Introduction

Phage therapy, the use of the natural enemies of bacteria to fight bacterial infections, is garnering renewed interest as bacterial resistance to antibiotics becomes widespread^1,2. 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 antibiotics^2,3. Advantages of antibiotic therapy are that bacteriophages multiply at the infection....

Protocol

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 4^th 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° C.

1. Preparation of solutions and tools

1. Prepare a 50x stock solution and 1x working solutions for E3 embryo medium for growing zebrafish embryos (see Table 1).
2. Make pigmentation bloc....

Results

Results and figures presented here are referred to CF embryos generated through the injection of cftr morpholinos as described previously¹⁰ 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.  .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	BD	214010
Calcium chloride	Sigma-Aldrich	10043-52-4
CsCl	Sigma-Aldrich	289329
Dulbecco's phospate buffered saline PBS	Sigma-Aldrich	D8537
Ethyl 3-aminobenzoate methanesulfonate	Sigma-Aldrich	886-86-2	common name tricaine
Femtojet Micromanipulator	Eppendorf	5247
Fleming/brown P-97	Sutter Instrument Company	P-97
LE-Agarose	Sigma-Aldrich	11685660001
Low Melting Agarose	Sigma-Aldrich	CAS 9012-36-6
Magnesium sulfate	Sigma-Aldrich	7487-88-9
Methyl Blue	Sigma-Aldrich	28983-56-4
Microinjection needles	Harvard apparatus
N-Phenylthiourea >=98%	Aldrich-P7629	103-85-5
Oligo Morpholino	Gene Tools		designed by the researcher
PEG6000	Calbiochem	528877
Phenol Red Solution	Sigma-Aldrich	CAS 143-74-B
Potassium chloride	Sigma-Aldrich	7447-40-7
Pronase	Sigma-Aldrich	9036-06-0
Sodium chloride ACS reagent, ≥99.0%	Sigma-Aldrich	S9888
Stereomicroscope	Leica	S9I
Tris HCl	Sigma-Aldrich	T5941
Triton X	Sigma-Aldrich	T9284
Tryptone	Oxoid	LP0042B
Yeast extract	Oxoid	LP0021B
Z-MOLDS Microinjection	Word Precision Instruments