Name: assessing biofilm dispersal in murine wounds
Uploaded: 2021-08-07T13:00:00
Description: Watch this Scientific Journal Video about Assessing Biofilm Dispersal in Murine Wounds at JoVE.com

This work was supported by grants from the National Institutes of Health (R21 AI137462-01A1), the Ted Nash Long Life Foundation, the Jasper L. and Jack Denton Wilson Foundation and the Department of Defense (DoD MIDRP W0318_19_NM_PP).

This animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center (protocol number 07044). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
1. Preparing bacteria for mouse infections
NOTE: Here we describe infecting mice only with Pseudomonas aeruginosa. However, o...

<ol>
	<li>Rossolini, G. M., Arena, F., Pecile, P., Pollini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+antibiotic+resistance+crisis.">Update on the antibiotic resistance crisis.</a> Current Opinion in Pharmacology. 18, 56-60 (2014).</li><li>Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+Tolerance+in+Biofilms.">Antimicrobial Tolerance in Biofilms.</a> Microbiology Spectrum. 3 (3), (2015).</li><li>Flemming, H. C., 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=Biofilms:+an+emergent+form+of+bacterial+life.">Biofilms: an emergent form of bacterial life.</a> Nature Reviews Microbiology. 14 (9), 563-575 (2016).</li><li>Rumbaugh, K. 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P., Sauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+dispersion.">Biofilm dispersion.</a> Nature Reviews Microbiology. 18 (10), 571-586 (2020).</li><li>Fleming, D., Chahin, L., Rumbaugh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoside+Hydrolases+Degrade+Polymicrobial+Bacterial+Biofilms+in+Wounds.">Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds.</a> Antimicrobial Agents and Chemotherapy. 61 (2), (2017).</li><li>Fleming, D., Rumbaugh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Consequences+of+Biofilm+Dispersal+on+the+Host.">The Consequences of Biofilm Dispersal on the Host.</a> Scientific Reports. 8 (1), 10738 (2018).</li><li>Baker, P., 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=Exopolysaccharide+biosynthetic+glycoside+hydrolases+can+be+utilized+to+disrupt+and+prevent+Pseudomonas+aeruginosa+biofilms.">Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.</a> Science Advances. 2 (5), 1501632 (2016).</li><li>Redman, W. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+treatment+modulates+the+host+immune+system+to+enhance+Pseudomonas+aeruginosa+wound+biofilms.">Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.</a> Infection and Immunity. 82 (1), 92-100 (2014).</li><li>Turner, K. H., Everett, J., Trivedi, U., Rumbaugh, K. P., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+Pseudomonas+aeruginosa+acute+burn+and+chronic+surgical+wound+infection.">Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection.</a> PLOS Genetics. 10 (7), 1004518 (2014).</li><li>Harrison, F., 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=A+1,000-Year-Old+Antimicrobial+Remedy+with+Antistaphylococcal+Activity.">A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity.</a> mBio. 6 (4), 01129 (2015).</li><li>Wolcott, R., 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=Microbiota+is+a+primary+cause+of+pathogenesis+of+chronic+wounds.">Microbiota is a primary cause of pathogenesis of chronic wounds.</a> Journal of Wound Care. 25, 33-43 (2016).</li><li>Ibberson, C. B., 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=Co-infecting+microorganisms+dramatically+alter+pathogen+gene+essentiality+during+polymicrobial+infection.">Co-infecting microorganisms dramatically alter pathogen gene essentiality during polymicrobial infection.</a> Nature Microbiology. 2, 17079 (2017).</li><li>Fleming, D., 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=Utilizing+Glycoside+Hydrolases+to+Improve+the+Quantification+and+Visualization+of+Biofilm+Bacteria.">Utilizing Glycoside Hydrolases to Improve the Quantification and Visualization of Biofilm Bacteria.</a> Biofilm. 2, (2020).</li><li>DeLeon, S., 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=Synergistic+interactions+of+Pseudomonas+aeruginosa+and+Staphylococcus+aureus+in+an+in+vitro+wound+model.">Synergistic interactions of Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro wound model.</a> Infection and Immunity. 82 (11), 4718-4728 (2014).</li><li>Darch, S. E., 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=Phage+Inhibit+Pathogen+Dissemination+by+Targeting+Bacterial+Migrants+in+a+Chronic+Infection+Model.">Phage Inhibit Pathogen Dissemination by Targeting Bacterial Migrants in a Chronic Infection Model.</a> mBio. 8 (2), (2017).</li><li>Chua, S. L., 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=Dispersed+cells+represent+a+distinct+stage+in+the+transition+from+bacterial+biofilm+to+planktonic+lifestyles.">Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles.</a> Nature Communications. 5, 4462 (2014).</li><li>Beitelshees, M., Hill, A., Jones, C. H., Pfeifer, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+Variation+during+Biofilm+Formation:+Implications+for+Anti-Biofilm+Therapeutic+Design.">Phenotypic Variation during Biofilm Formation: Implications for Anti-Biofilm Therapeutic Design.</a> Materials (Basel). 11 (7), (2018).</li><li>Sauer, K., 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=Characterization+of+nutrient-induced+dispersion+in+Pseudomonas+aeruginosa+PAO1+biofilm.">Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm.</a> J Bacteriol. 186 (21), 7312-7326 (2004).</li><li>Kuklin, N. A., 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=Real-time+monitoring+of+bacterial+infection+in+vivo:+development+of+bioluminescent+staphylococcal+foreign-body+and+deep-thigh-wound+mouse+infection+models.">Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models.</a> Antimicrobial Agents and Chemotherapy. 47 (9), 2740-2748 (2003).</li><li>Francis, K. 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L., 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=Noninvasive+optical+imaging+method+to+evaluate+postantibiotic+effects+on+biofilm+infection+in+vivo.">Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo.</a> Antimicrobial Agents and Chemotherapy. 48 (6), 2283-2287 (2004).</li><li>Wimpenny, J., Manz, W., Szewzyk, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+biofilms.">Heterogeneity in biofilms.</a> FEMS Microbiology Reviews. 24 (5), 661-671 (2000).</li><li>Stewart, P. S., Franklin, M. 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Here we describe protocols that can be utilized to study the efficacy of biofilm dispersal agents. These protocols can be easily adapted to use with different types of dispersal agents, bacterial species or ex vivo samples, including clinical debridement samples. This protocol also provides a clinically relevant model to collect and study dispersed bacterial cells. The phenotypes of dispersed bacterial cells have been shown to be distinct from those of either planktonic or biofilm cells 5...

The rise of antibiotic resistance worldwide is leading to a lack of antibiotic options to treat a variety of bacterial infections1. In addition to antibiotic resistance, bacteria can gain antibiotic tolerance by adopting a biofilm-associated lifestyle2. A biofilm is a community of microorganisms that are protected by a matrix of polysaccharides, extracellular DNA, lipids, and proteins3, collectively called the extracellular polymeric substance (EPS). As the antibiotic resistance crisis continues, new strategies that prolong the use of, or potentiate the efficacy of, antibiotics are sorely needed. Anti-biofilm agents are one promising solution4.
Amongst the different anti-biofilm strategies that have been proposed, the utilization of dispersal agents, which target different components of the biofilm EPS, are at the forefront of therapeutic development5. Glycoside hydrolases (GH) are one such class of dispersal agent. GH are a large class of enzymes that catalyze the cleavage of different bonds within the polysaccharides that provide structural integrity to the EPS. Our group, as well as others, have shown that GH can effectively degrade biofilms, induce dispersal and improve antibiotic efficacy for a number of different bacterial species, both in vitro and in vivo6,7,8,9,10,11.
With a growing interest in biofilm dispersal,it is important to develop effective methods that assess dispersal efficacy. Here, we present a detailed protocol for the treatment of biofilm-associated wound infections with a dispersal agent in mice,&#160;and the assessment of dispersal efficacy, in vivo and ex vivo. The overall goal is to provide effective methods that can be used with preclinical models to measure biofilm dispersal effectively and efficiently.
A murine surgical excision infection model was used in these studies to establish a biofilm-associated infection. We have used this model for over 15 years and published our observations extensively7,9,12,13,14,15,16,17,18,19,20,21. In general, this is a non-lethal infection model where bacteria remain localized to the wound bed and are biofilm-associated (bacteria seen in aggregates surrounded by EPS), setting up a chronic infection that lasts up to 3 weeks. However, if mice are immunocompromised (with Type 1 diabetes for example), they can become more susceptible to developing a fatal systemic infection in this model.
In this report, we provide protocols for assessing the dispersal of bacteria from a wound, both in vivo and ex vivo. Both protocols can be used to examine the efficacy of a dispersal agent and have their own strengths and weaknesses. For example, assessing dispersal in vivo can provide important, real-time information about the spread of bacteria to other parts of the body after dispersal, and how the host responds. On the other hand, assessing dispersal ex vivo may be more desirable for screening multiple agents, doses, or formulations, as the tissue can be divided into multiple sections that can be tested separately, thus reducing the number of mice required. When assessing multiple agents, we typically measure dispersal first in vitro as previously described 6,9,22. We then test the most effective ex vivo and reserve in vivo testing for a limited number of very promising agents.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Fisher</td>
			<td>14823434</td>
			<td>Use to complete serial dilutions of samples</td>
		</tr>
		<tr>
			<td>25G 58 in needle</td>
			<td>Fisher</td>
			<td>14823434</td>
			<td>Attaches to 1 mL syringe</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium Salt</td>
			<td>Fisher</td>
			<td>BP1760-5</td>
			<td>Make a 50 mg/ mL stock solution and add 100 &#181;L to 10 mL of LB broth for both overnight and subculture</td>
		</tr>
		<tr>
			<td>Amylase</td>
			<td>MP Biomedicals</td>
			<td>2100447</td>
			<td>Make a 5% w/v solution, vortex- other dispersal agents can be used</td>
		</tr>
		<tr>
			<td>Buprenorphine SR-LAB 5 mL (1 mg/mL)</td>
			<td>ZooPharm</td>
			<td>RX216118</td>
			<td>Use as pain mainagement- may use other options</td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>MP Biomedicals</td>
			<td>2150583</td>
			<td>Add 5% w/v to the 5% w/v amylase solution, vortex, activate at 37 &#176;C for 30 min- other dispersal agents can be used</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Walmart</td>
			<td>287746</td>
			<td>Use a small amount to massage into the hair follicles on the back of the animal and allot 10 min to remove hair</td>
		</tr>
		<tr>
			<td>Dressing Forceps, Serrated Tips</td>
			<td>Fisher</td>
			<td>12-460-536</td>
			<td>Can use other forms of forceps</td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks baffled 125 mL</td>
			<td>Fisher</td>
			<td>101406</td>
			<td>Use to grow overnights and sub-cultures of bacteria</td>
		</tr>
		<tr>
			<td>FastPrep-24 Benchtop Homogenizer</td>
			<td>MP Biomedicals</td>
			<td>6VFV9</td>
			<td>Use 5 m/s for 60 s two times to homogenize tissue</td>
		</tr>
		<tr>
			<td>Fatal Plus</td>
			<td>Vortech Pharmaceuticals</td>
			<td>0298-9373-68</td>
			<td>Inject 0.2 mL intraperitaneal for each mouse</td>
		</tr>
		<tr>
			<td>Homogenizing tubes (Bead Tube 2 mL 2.4 mm Metal)</td>
			<td>Fisher</td>
			<td>15340151</td>
			<td>Used to homogenize samples for plating</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Diamond Back Drugs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride/xylazine hydrochloride solution C-IIIN</td>
			<td>Sigma Aldrich</td>
			<td>K4138</td>
			<td>Use as anasethia- other options can also be utilized to gain a surgical field of anasethia</td>
		</tr>
		<tr>
			<td>LB broth, Miller</td>
			<td>Fisher</td>
			<td>BP1426-2</td>
			<td>Add 25 g/L and autoclave</td>
		</tr>
		<tr>
			<td>Lidocaine 2% Injectable</td>
			<td>Diamond Back Drugs</td>
			<td>2468</td>
			<td>Inject 0.05 mL through the side of the marked wound bed area so it is deposited in the center of the mark. Allot 10 min prior to cutting</td>
		</tr>
		<tr>
			<td>Meropenem</td>
			<td>Sigma Aldrich</td>
			<td>PHR1772-500MG</td>
			<td>Make 5 mg/mL to add to the GH solution to apply topically and a 15 mg/mL solution to inject intraperitaneal 4 h prior and 6 h post-treatment</td>
		</tr>
		<tr>
			<td>Non-sterile cotton gauze sponges</td>
			<td>Fisher</td>
			<td>13-761-52</td>
			<td>Use to remove the depilatory cream</td>
		</tr>
		<tr>
			<td>PAO1 pQF50-lux bacterial strain</td>
			<td>Ref [13]</td>
			<td>N/A</td>
			<td>PAO1 pgF50-lux was used as the P. aeruginosa strain of interest in this paper&#39;s representative results</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher</td>
			<td>PHR1772-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline 10x</td>
			<td>Fisher</td>
			<td>BP3991</td>
			<td>Dilute 10x to 1x prior to use</td>
		</tr>
		<tr>
			<td>Polyurethane dressing</td>
			<td>Mckesson</td>
			<td>66024007</td>
			<td>Cut the rounded edge off and cut the remaining square into 4 equal sections</td>
		</tr>
		<tr>
			<td>Pseudomonas isolation agar</td>
			<td>VWR</td>
			<td>90004-394</td>
			<td>Add 20 mL/L of glycerol and 45 g/mL to water, autoclave, and pour 20 mL into petri dishes</td>
		</tr>
		<tr>
			<td>Refresh P.M.</td>
			<td>Walmart</td>
			<td></td>
			<td>Use on eyes to reduce dryness during procedure.</td>
		</tr>
		<tr>
			<td>Sterile Alcohol Prep Pads</td>
			<td>Fisher</td>
			<td>22-363-750</td>
			<td>Use to clean the skin immediately prior to wounding to disinfect the area</td>
		</tr>
		<tr>
			<td>Straight Delicate Scissors</td>
			<td>Fisher</td>
			<td>89515</td>
			<td>Can also use curved scissors</td>
		</tr>
		<tr>
			<td>Swiss Webster mice</td>
			<td>Charles River</td>
			<td>551NCISWWEB</td>
			<td>Other mice strains can be used</td>
		</tr>
		<tr>
			<td>Syring Slip Tip 1 mL</td>
			<td>Fisher</td>
			<td>14823434</td>
			<td>Used to administer drugs and enzyme treatment</td>
		</tr>
	</tbody>
</table>

assessing biofilm dispersal in murine wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

In this experiment, 8-10 week old female Swiss Webster mice were infected with 104 CFU of PAO1 carrying the luminescence plasmid pQF50-lux. As described above, an infection was allowed to establish for 48 h prior to administering 3 x 30 min treatments of either PBS (vehicle control) or 10% GH (treatment) to digest the biofilm EPS. Mice were imaged pre-treatment, directly after treatment (0 h) and at 10 h and 20 h post-treatment. Figure 2A and Supplemental Figure 1 </strong...

Watch this Scientific Journal Video about Assessing Biofilm Dispersal in Murine Wounds at JoVE.com

Assessing Biofilm Dispersal in Murine Wounds

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

Microbiologists and infectious disease physicians are starting to realize how important biofilm-associated infections are in chronic disease. The NIH has even said that over 80%of chronic infections are due to bacterial biofilms. So this has urged on the emphasis on addressing biofilm, and developing therapeutics that address the biofilm problem.And a lotta those therapeutics are focused on dispersing bacterial cells that are in biofilms out of the biofilm so that they can be more readily killed. But there really aren't a lot of protocols in the literature currently that address how to determine the efficacy of such a dispersal agent. So we feel like this is a good protocol to use.It is one type of bacterial infection that's biofilm-associated. And we also think it can be optimized pretty easily to study many different types of bacterial species, and how they form biofilms and disperse, as well as different dispersal agents. And we think it's important to deliver this in a video format, because we have seen that there are nuances to especially the delivery of the agent that we have found work optimally in mice.To begin, assess the plane of anesthesia by toe pinch, and by monitoring the respiratory rate and effort. Shave the dorsal surface and apply a depilatory cream to the shaved region for 10 minutes, or as per product's instructions. Gently remove the depilatory cream.And ensure the skin is dry. Using an alcohol-proof marker, draw a circle with a diameter of 1.5 centimeters towards the posterior region of the back. For pain management, administer 0.05 milliliters of lidocaine subcutaneously into the area to be excised.And 0.02 milliliters of buprenorphine subcutaneously in the scruff of the neck. Wait 10 minutes to ensure pain management is reached. Before excision, disinfect the dorsal surface using an alcohol swab.Maintain a sterile surgical field throughout the procedure, and use autoclaved instruments and sterile gloves to limit contamination. Administer a full thickness excisional skin wound to the level of the panniculus muscle, with surgical scissors. After excision, immediately cover the wound with a semipermeable polyurethane dressing.Carefully separate the backing from the dressing using two fine-tipped forceps, if applicable. Inject approximately 10 to the fourth CFU of bacteria, and 100 microliters PBS under the dressing and onto the wound bed to establish an infection. Note, wounds can also be infected with multiple species of bacteria and/or fungi simultaneously, or by placing preformed biofilms or even debridement tissue extracted from patients onto the wound bed.After infection, place mice in cages on top of paper towels to prevent the mice from asphyxiating on the bedding material. Place the cages on heating pads and monitor the mice until they have regained their righting reflex. Remove the paper towel at this point.Allow wound infections to establish for 48 hours. Inspect the dressing daily for tears in areas of nonadherence and replace it if needed. Place mice under isoflurane anesthesia at 3%and one liter per minute of oxygen, while administering treatments.Inject 200 microliters of enzyme solution or PBS control onto the wound by lifting the skin slightly anterior of the wound with forceps, carefully piercing the lifted skin with the syringe, and slowly injecting the solution into the area between the wound bed and the dressing. To ensure that the entire wound bed is covered by solution, gently raise the dressing with forceps, pulling it away from the wound bed, while slowly injecting the solution. After treatment, place the mice back in cages for 30 minutes.After 30 minutes of exposure to the treatment, reanesthetize the mice with isoflurane and aspirate the remaining solution with a syringe, puncturing in the same area as before. Note, this aspirated solution contains dispersed bacterial cells, which may be saved for various downstream analyses. Administer the second and third irrigation treatments as was done for the first using a new syringe each time.If a luminescent strain of bacteria is utilized to initiate infection, an in-vivo imaging system can be used to visualize dispersal from the wound bed. For analysis, open the images in the IVIS software and select the area to be analyzed with the tool palette. To account for background, place a circle on the background of an image, and then placed the same sized circle on top of the wound bed for each mouse.From the tool palette, select Measure ROIs and upload the table of measurements to a spreadsheet. Subtract the background circle reading from each of the wound bed measurements to calculate luminescence for each sample. After humane euthanasia, harvest the wound bed tissue by first gently removing the dressing with sterile forceps, and cutting around the perimeter of the wound bed with sterile surgical scissors.Use forceps to gently lift one end of the wound bed, then use scissors to cut the tissue sample away from the muscle layer. Place the tissue sample in a pre-weighed two-milliliter homogenization tube containing one milliliter of PBS. Weigh the tube again after inserting the sample.To harvest the spleen, place the mouse in a supine anatomical position and secure by pinning the extremities to a dissection surface. Soak the area to be dissected with 70%ethanol to help prevent contamination. Make a small incision perpendicular to the midline with surgical scissors in the dermis of the lower abdomen.Make sure to pull the skin up and away from the internal organs. Continue the incision along the midline until the sternum is reached. Once the peritoneal cavity is exposed and the internal organs are visible, separate the spleen from the connective tissue and place in a separate pre-weighed homogenization tube.Weigh the tube again after inserting the spleen. Homogenize the samples at five meters per second, for 60 seconds, two times. To enumerate CFU, serially dilute and spot plate the homogenized samples.Serially dilute by pipetting 100 microliters of the homogenized sample into 900 microliters of PBS and a 1.5 milliliter centrifuge tube. Vortex before continuing the dilution by pipetting 100 microliters of the diluted sample into a separate tube containing 900 microliters of PBS. Repeat these steps until six dilutions are achieved.Pipette 10 microliters of each dilution onto the indicated area on an agar plate as shown. If more precise CFU quantitation is required, spread 100 microliters of each dilution of the sample across separate agar plates. Wound and infect mice with approximately 10 to the fourth CFU of bacteria, as described in the in vivo protocol.Allow the infection to establish for 48 hours, then extract the wound bed in a similar fashion to the in-vivo protocol. Begin by carefully removing the dressing. Cutting around the perimeter of the wound.And separating from the muscle layer. The wound bed sample can be divided into multiple sections to increase the number of samples. Then place the divided samples into separate pre-weighed empty homogenization tubes.Weigh the tube again and calculate the weight of the sample by subtracting the weight of the tube without the sample from the weight of the tube with the sample. Add one milliliter of PBS to the homogenization tube containing the sample, and gently invert the tube a few times to rinse. Remove the PBS wash and discard.Add one milliliter of GH treatment, or PBS as a negative control, and incubate for two hours at 37 degrees Celsius, with shaking at 80 RPM. Remove the bile from dispersal solution and place in a separate 1.5 milliliter centrifuge tube. This contains the dispersed cells.Wash the remaining tissue with one milliliter of PBS, as done previously, and discard the PBS wash. Add one milliliter of PBS to the remaining tissue and homogenize at five meters per second, for 60 seconds. Serially dilute both the solution of dispersed cells and the homogenized tissue sample by pipetting 100 microliters of the sample into 900 microliters of PBS in a 1.5 milliliter centrifuge tube.Vortex the diluted sample and continue the dilution five more times for a total of six dilutions. Spot plate the sample by pipetting 10 microliters of each dilution onto the indicated area on a selective agar plate. Count the CFU and calculate percent dispersal by dividing the dispersed CFU by the total CFU, and multiplying by 100.In this experiment, eight to 10 week old female Swiss Webster mice were infected with 10 to the fourth CFU of PA01 carrying the luminescence plasmid PQF50-lux. The infection was allowed to establish for 48 hours prior to administrating three 30-minute treatments of either PBS as a vehicle control, or 10%GH as an experimental treatment to digest the biofilm EPS. Shown in section A, mice were imaged pre-treatment, directly after treatment, and at 10 and 20 hours post-treatment.Using IVIS, an established infection can be visualized within the wound bed generating a bright bioluminescent signal. The dispersal of bacteria out of the wound bed can be visualized immediately after the GH treatment, but not after the PBS treatment. Bacterial dissemination into the organs can also be seen by placing the mouse on its side for imaging, as shown in the lower panels.In section B, images from both the PBS and GH-treated mice demonstrated an increase in luminescent signal at 20 hours post-treatment, compared to pre-treatment. However, it is still important to quantitate CFU due to the detection threshold of a luminescence in IVIS. At 20 hours post-treatment, the mice were euthanized and the wound beds and spleens were collected to enumerate CFU per gram of tissue.Section C shows the bacterial load in the wound bed, while section D shows the bacterial load in the spleens. These data suggest disseminated spread of the dispersed bacteria, since bacteria was only detected in the spleens of the mice treated with the EPS-targeting GH solution. Although these protocols are very useful, there are some limitations.First, it should be noted that there is a detection limitation with the in-vivo imaging system, or IVIS. A decrease in luminescent signal can be seen when the bacteria enter stationary phase. Therefore it is important to collect tissue of interest and measure the CFU present, as there can be a disconnect between the luminescence and CFU enumerated.However, IVIS is useful for visualizing the efficacy of the treatment throughout a study, without having to actually euthanize the animal. Another limitation to take into consideration is the requirement of close monitoring of the bandages. The bandages are required to reduce contractile healing as well as keep the wound sterile from other bacteria.Bandages will need to be replaced often, which can lead to accidental debridement of the wound and opportunity for contamination. These protocols describe novel methods to study bile from dispersal, and the evaluation of therapeutic bile from dispersal agents. These models allow for the development of complex polymicrobial biofilm-associated infections that mimic clinically relevant human infections.Our hope is that these protocols will be utilized and refined to further the development of anti-biofilm dispersal agents for therapeutic use.

assessing-biofilm-dispersal-in-murine-wounds

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 ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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. ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...