Name: generation of in-frame gene deletion mutants in pseudomonas aeruginosa and testing for virulence attenuation in a simple mouse model of infection
Uploaded: 2020-01-08T14:00:00
Description: Watch this Scientific Journal Video about Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection at JoVE.com

This work was supported by the National Institutes of Health (NIH) grants R44GM113545 and P20GM103434.

Before beginning animal experiments, the protocol to be used must be approved by the Institutional Animal Care and Use Committee (IACUC). Approval for the protocol described was obtained through the IACUC at Marshall University (Huntington, WV, USA).
1. Plasmid Design
<ol>
	<li>To generate a genetic deletion using the pEX100T-NotI plasmid, clone the regions of DNA flanking the desired deletion sequence and insert into the NotI restriction site of the plasmid. The plasmid insert should contai...

<ol>
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N., 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=Production+of+Biopharmaceuticals+in+E.+coli:+Current+Scenario+and+Future+Perspectives.">Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives.</a> Journal of Microbiology and Biotechnology. 25 (7), 953-962 (2015).</li><li>Marisch, K., Bayer, K., Cserjan-Puschmann, M., Luchner, M., Striedner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+three+industrial+Escherichia+coli+strains+in+fed-batch+cultivations+during+high-level+SOD+protein+production.">Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.</a> Microbial Cell Factories. 12, 58 (2013).</li><li>Ferrer-Miralles, N., Domingo-Espin, J., Corchero, J. L., Vazquez, E., Villaverde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+factories+for+recombinant+pharmaceuticals.">Microbial factories for recombinant pharmaceuticals.</a> Microbial Cell Factories. 8, 17 (2009).</li><li>Horton, R. M., Cai, Z. L., Ho, S. N., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+splicing+by+overlap+extension:+tailor-made+genes+using+the+polymerase+chain+reaction.">Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.</a> Biotechniques. 8 (5), 528-535 (1990).</li><li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+an+origin-containing+derivative+of+plasmid+RK2+dependent+on+a+plasmid+function+provided+in+trans.">Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (4), 1648-1652 (1979).</li><li>Choi, K. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nature Protocols. 1 (1), 153-161 (2006).</li><li>Choi, K. H., Kumar, A., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+10-min+method+for+preparation+of+highly+electrocompetent+Pseudomonas+aeruginosa+cells:+application+for+DNA+fragment+transfer+between+chromosomes+and+plasmid+transformation.">A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation.</a> Journal of Microbiological Methods. 64 (3), 391-397 (2006).</li><li>Liang, R., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scarless+and+sequential+gene+modification+in+Pseudomonas+using+PCR+product+flanked+by+short+homology+regions.">Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions.</a> BMC Microbiology. 10, 209 (2010).</li><li>Martinez-Garcia, E., de Lorenzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+multiple+genomic+deletions+in+Gram-negative+bacteria:+analysis+of+the+multi-resistant+antibiotic+profile+of+Pseudomonas+putida+KT2440.">Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440.</a> Environmental Microbiology. 13 (10), 2702-2716 (2011).</li><li>Song, C. W., Lee, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+one-step+inactivation+of+single+or+multiple+genes+in+Escherichia+coli.">Rapid one-step inactivation of single or multiple genes in Escherichia coli.</a> Biotechnology Journal. 8 (7), 776-784 (2013).</li><li>Yan, M. Y., 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=CRISPR-Cas12a-Assisted+Recombineering+in+Bacteria.">CRISPR-Cas12a-Assisted Recombineering in Bacteria.</a> Applied and Environmental Microbiology. 83 (17), (2017).</li><li>Prakash, O., Nimonkar, Y., Shouche, Y. 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The pEX100T-Not1 plasmid is an efficient mediator of sequential genomic deletions that are marker-free and in-frame. When engineering bacterial strains for attenuated virulence, deletion of entire gene sequences rather than generating point mutations decreases the likelihood of reversion to a virulent phenotype. Additionally, each pathogenicity gene deletion attenuates the pathogen further, reinforcing the stability of the attenuation.
This method can also be used to generate genomic modificat...

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause life-threatening diseases in humans, especially in the immunocompromised. The pathogenicity of P. aeruginosa is due to the expression of many virulence factors, including proteases and lipopolysaccharide, as well as its ability to form a protective biofilm1. Because of its ability to produce virulence factors and cause disease in humans, using P. aeruginosa to make commercial products presents safety concerns. Nonpathogenic strains of E. coli have traditionally been used to bioengineer medical and commercial products for human use. However, some products are difficult for E. coli to make, and many are packaged in inclusion bodies, making extraction laborious. Engineered bacterial strains with the ability to make and secrete specific products is highly desirable, as secretion would likely increase yield and ease purification processes. Thus, nonpathogenic strains of other species of bacteria (e.g., species that utilize more secretion pathways) may provide useful alternatives to E. coli. We recently reported the development of a strain of P. aeruginosa, PGN5, in which the pathogenicity and toxicity of the organism is highly attenuated2. Importantly, this strain still produces large quantities of the polysaccharide alginate, a commercially interesting component of the P. aeruginosa biofilm.
The PGN5 strain was generated using a two-step allelic exchange procedure with the pEX100T-NotI plasmid to sequentially delete five genes (toxA, plcH, phzM, wapR, aroA) known to contribute to the pathogenicity of the organism. pEX100T-NotI was generated by changing the SmaI to a NotI restriction enzyme recognition site within the multiple cloning site of the plasmid pEX100T, which was developed in Herbert Schweizer&#39;s lab3,4. The recognition site for the restriction enzyme NotI is a rarer DNA sequence compared to SmaI and less likely to be present in sequences being cloned, thus it is more convenient for cloning purposes. The plasmid carries genes that allow for selection, including the bla gene, which encodes &#223;-lactamase and confers resistance to carbenicillin, and the B. subtilissacB gene, which confers sensitivity to sucrose (Figure 1A). The plasmid also carries an origin of replication (ori) compatible with E. coli, and an origin of transfer (oriT) that allows for plasmid transfer from E. coli to Pseudomonas species via conjugation. However, the plasmid lacks an origin of replication compatible with Pseudomonas, and thus cannot replicate within Pseudomonas species (i.e., it is a Pseudomonas suicide vector). These characteristics make pEX100T-NotI ideal for targeting genetic deletions from the Pseudomonas chromosome. Plasmid cloning steps are carried out using E. coli and the resultant plasmid is transferred to Pseudomonas by transformation or conjugation. Then, through homologous recombination events and selective steps, the targeted in-frame deletion is generated, marker-free. This method of sequentially deleting genomic regions from the chromosome of P. aeruginosa could be used to generate highly attenuated Pseudomonas strains, like PGN5, or to design strains for other specific uses (e.g., strains deficient in endonucleases for plasmid propagation or strains deficient in proteases for production of proteins of interest).
The overall virulence of strains of bacteria is affected by growth conditions and phases, during which mutations occur frequently. Therefore, measuring the safety of genetically-engineered strains can be challenging. To evaluate bacterial isolates for systemic virulence, we adapted a previously published protocol of infection by intraperitoneal injection of C57BL/6 mice5. We modified this procedure to use frozen bacterial stocks for injection, which allowed for precise dosing and easy validation of the strains used. In this model, the E. coli strain BL21, which has been FDA-approved for production of biopharmaceuticals, was used as a control safety standard for determining the relative pathogenesis of the strain6,7,8. The main advantage to using this method is that it is reproducible and minimizes sources of variation, as infecting strains are validated for bacterial cell number, phenotype, and genetic markers both before and after infection. With these controlled steps, the number of animals required is reduced. In this model, P. aeruginosa strains that result in C57BL/6 murine mortality rates equal to or less than E. coli BL21 when injected intraperitoneally may be considered attenuated. This simple mouse model of infection may also be used to assess the attenuated pathogenicity of genetically engineered strains from other species using the FDA-approved E. coli strain as the reference. Steps 1-7 detail the generation of sequential genomic deletions in P. aeruginosa (Figure 1) and steps 8-12 detail the use of a mouse model to test the pathogenicity of P. aeruginosa strains.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL tubes with flat caps</td>
			<td>ThermoScientific</td>
			<td>AB-0620</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD</td>
			<td>22-253-260</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>1.5 mL disposable polystyrene cuvette</td>
			<td>Fisher Scientific</td>
			<td>14955127</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL Cryogenic Vials</td>
			<td>Corning</td>
			<td>430659</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>BD</td>
			<td>14-821-13B</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Accu block Digital Dry Bath</td>
			<td>Labnet</td>
			<td>NC0205808</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge 5804R</td>
			<td>Eppendorf</td>
			<td>04-987-372</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Benchtop Microcentrifuge</td>
			<td>Sorvall</td>
			<td>75-003-287</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cabinet Incubator</td>
			<td>VWR</td>
			<td>1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt</td>
			<td>Fisher Scientific</td>
			<td>BP2648250</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Test Tube, Polystyrene</td>
			<td>Fisher Scientific</td>
			<td>14-956-6D</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Diposable Inoculation Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-597</td>
			<td></td>
		</tr>
		<tr>
			<td>Dneasy UltraClean Microbial Kit (50)</td>
			<td>Qiagen</td>
			<td>12224-50</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr>
			<td>E.Z.N.A. Cycle Pure Kit (50)</td>
			<td>Omega bio-tek</td>
			<td>D6493-01</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr>
			<td>EcoRI-HF, restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R3101L</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation Cuvettes</td>
			<td>Bulldog Bio</td>
			<td>NC0492929</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>FastLink II DNA Ligation Kit</td>
			<td>Epicentre Technologies</td>
			<td>LK6201H</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP918-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq G2 Colorless Master Mix</td>
			<td>Promega</td>
			<td>M7833</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Isothesia Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina XRMS Series III In Vivo Imaging System</td>
			<td>Perkins and Elmer</td>
			<td>CLS136340</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin monosulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>LE agarose</td>
			<td>Genemate</td>
			<td>3120-500</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Luria Broth</td>
			<td>Difco</td>
			<td>240230</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>MicroPulser Electroporator</td>
			<td>BioRad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Noble agar, ultrapure</td>
			<td>Affymetris/USB</td>
			<td>AAJ10907A1</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>NotI-HF, restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R3189</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot TOP10 Electrocomp E. coli</td>
			<td>Invitrogen</td>
			<td>C404052</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline powder</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td><a href="https://www.graphpad.com/scientific-software/prism/" target="_blank">https://www.graphpad.com/scientific-software/prism/</a></td>
		</tr>
		<tr>
			<td>Pseudomonas isolation agar</td>
			<td>Difco</td>
			<td>292710</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pseudomonas isolation broth</td>
			<td>Alpha Biosciences</td>
			<td>P16-115</td>
			<td>Custom made batch</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 4080</td>
			<td>shake at 200 rpm</td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td>A24811</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk</td>
			<td>Difco</td>
			<td>DF0032-17-3</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Small Plates (100 O.D. x 10 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec Plus Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2525</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothpicks, round</td>
			<td>Diamond</td>
			<td></td>
			<td>Any brand of toothpicks, autoclaved</td>
		</tr>
		<tr>
			<td>TOPO TA Cloning Kit, for seqeuncing</td>
			<td>Invitrogen</td>
			<td>45-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>XAF-8 Anesthesia System Filters</td>
			<td>Perkins and Elmer</td>
			<td>118999</td>
			<td></td>
		</tr>
		<tr>
			<td>XGI 8 Gas Anesthesia System</td>
			<td>Caliper Life Sciences/Xenogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

As shown in Figure 2, the targeted genomic deletion can be confirmed using colony PCR with specific primers that amplify the region of interest. Colonies that carry a genomic deletion will yield a shorter PCR band size in comparison to wild-type colonies. A PCR-screen of 10-12 colonies is usually sufficient to detect at least one colony that carries the targeted deletion. If no deletions are detected after multiple rounds of screens, repeat the procedure begi...

Watch this Scientific Journal Video about Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection at JoVE.com

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.

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 ...

This protocol is significant for creating and determining mind-body desired mutations in the genome of Pseudomonas aeruginosa, as well as the testing the effect of mutations on virulence reduction in a reproducible mouse model. The key advantage of this technique is a chrome validation and a reproducibility of mouse infection model. Bacteria are constantly working, continually changing.To slow down this process, we use a frozen stops, bag them, hang them on multiple rungs on modification before testing them in the mouse model. Someone unfamiliar with these methods will most likely struggle with the development and selection of possible crossover recombinant to test. Furthermore, the handling of the mice for infection will be difficult for someone unfamiliar with animal work.This method can be applied to testing other pathogens and their mutants, as well as virulence infect in mice. Compared to current models, procedures with reproducibility plus clone validation before and after infection can benefit other investigators in the field. Visual demonstration of this protocol provides insight to extensive procedures that may be difficult to understand or comprehend through reading alone.Start by growing single crossover, recombinant colonies in Pseudomonas Isolation Broth, or PIB. Inoculate and streak 10 microliters of each culture onto pre-warmed PIA plates, supplemented with 10%sucrose. Then incubate the plates overnight at 37 degrees Celsius.On the next day, remove the plates from the incubator and inspect them for growth. The sucrose resistant colony should be double crossover recombinants. Use sterile toothpicks to patch at least 20 colonies onto pre-warmed plates of PIA, PIA supplemented with 10%sucrose, and PIA supplemented with carbenicillin.Incubate the plates overnight and examine them for growth on the next day. True double crossover recombinants will be carbenicillin-sensitive and sucrose-resistant. Screen 10 to 20 colonies for deletion, using colony PCR.Pick up the growth of a suspected double crossover recombinant with a sterile toothpick and suspend it in 50 microliters of PBS. Boil the suspension at 100 degrees Celsius for 10 minutes. Centrifuge it for three minutes at 13, 000 times G and place it on ice.Then, perform PCR according to manuscript directions. Once PCR is finished, perform Agarose gel electrophoresis on the products. Smaller amplification products indicate colonies in which the region of interest has been deleted.On the morning of injections, thaw the cryovials of bacterial cells at four degrees Celsius for three to four hours. Keep the vials on ice after thawing and inject the mice within two hours. Transfer the contents of each cryovial to a new two milliliter tube and centrifuge it at 4, 500 times G for 10 minutes.Discard the supernatant and re-suspend the cell pellet in one milliliter of PBS. Repeat the centrifugation and re-suspend the cells in PBS to a final concentration of 2.5 times 10 to the ninth colony forming units per milliliter. Take three samples from the final suspension of each strain to validate concentration, genotype, and phenotype.For each strain, aliquot 1.5 milliliters of the cell suspension into a two milliliter tube and prepare the PBS for control injections. Gather the mice and materials needed for injections in a sterile animal surgical room and wipe all surfaces with sanitizing wipes prior to starting. Wear two pairs of latex gloves to limit the risk of puncture, if bitten, as well as a lab coat, safety glasses, and face mask.Remove the mouse from the cage and weigh it, marking the tail with permanent marker for post-injection tracking. Open a new one milliliter syringe with a 27 gauge needle and draw up 200 microliters of sterile PBS. Grab the mouse behind its ears, using the thumb and forefinger, and pinch to create a skin fold at the nape of the neck.Then, secure the tail into the palm using the pinky to hold the mouse flat and immobile. Insert the needle at a 30 degree angle into the peritoneal cavity to the left or right of the midline. Slightly lift the needle to ensure that it was not inserted into organs.Then, slowly inject the PBS and withdraw the needle. A bolus at the injection site is typical. Place the needle in the designated sharps disposal container and move the mouse to a separate cage.Repeat the procedure with the next mouse and after all mice from one cage are injected, move them back to their original cage. After injecting the control group, inject the test groups using the same procedure. Once all injections are complete, return the mice to the housing room and clean the work area with sanitizing wipes.To image the animals, prepare the imaging system by setting the camera parameters and heating the stage. Set the oxygen flow to 1.5 liters per minute and isoflurane to 3.5%and move the mouse to the anesthetic chamber, then to the temperature stabilized stage, following anesthesia. Position the mouse on its back with arms outstretched and fit a nose cone for administration of 2.5%isoflurane during imaging.Close the door and take bioluminescent images and x-rays of the mouse. When imaging is complete, return the mouse to its cage and monitor it. It should regain consciousness within three to five minutes.The targeted genomic deletions were confirmed by colony PCR with specific primers that amplified the region of interest. Colonies with the genomic deletion yield a shorter PCR band compared to wild type colonies. Intraperitoneal injection of the attenuated strain of P aeruginosa, PGN five, resulted in 0%mortality, equivalent to mortality observed with E.coli BL 21.Injection of the parent strain, however, was fatal to 80%of the mice. Infection progression was tracked using bioluminescence-marked parent and attenuated strains. The attenuated strain remained localized at the injection site until bioluminescence faded, which likely coincided with clearance of infection.The method presented only examined the mortality produced by the strain. More in depth immunological and toxicological aspects could be utilized to determine the dynamics of infection and the end effect the infection has that does not result in death. The most important thing in the procedure is the extensive validation done, between various steps from initial identification to animal testing.Missing certain validation steps could potentially lead to wrong result, as the strains tested may undergo mutation and selection, or become contaminated. With the development of these two techniques, we think this will allow our researchers in the field of infection immunology to more effectively investigate how powerful package interaction leads to the extreme phenotype, sepsis, and mortality. Different nutrients'impact on variants can be compared through size dosing of bacteria.

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Research

JoVE Journal

Immunology and Infection

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 ...

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. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

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 ...