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.

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

Research

JoVE Journal

Immunology and Infection

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 surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

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

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

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

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 ...

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 these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

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

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

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