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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+well+are+we+translating+biofilm+research+from+bench-side+to+bedside.">How well are we translating biofilm research from bench-side to bedside.</a> Biofilm. 2 (100028), (2020).</li><li>Rumbaugh, K. 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. K., Welch, G. S., Rumbaugh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Efficacy+of+Glycoside+Hydrolases+to+Disperse+Biofilms.">Differential Efficacy of Glycoside Hydrolases to Disperse Biofilms.</a> Frontiers in Cellular and Infection Microbiology. 10, 379 (2020).</li><li>Zhu, 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=Glycoside+hydrolase+DisH+from+Desulfovibrio+vulgaris+degrades+the+N-acetylgalactosamine+component+of+diverse+biofilms.">Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms.</a> Environmental Microbiology. 20 (6), 2026-2037 (2018).</li><li>Fell, C. F., Rumbaugh, K. 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> Antibacterial Drug Discovery to Combat MDR. , 527-546 (2019).</li><li>Dalton, T., 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=An+in+vivo+polymicrobial+biofilm+wound+infection+model+to+study+interspecies+interactions.">An in vivo polymicrobial biofilm wound infection model to study interspecies interactions.</a> PLoS One. 6 (11), 27317 (2011).</li><li>Wolcott, R. 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=Biofilm+maturity+studies+indicate+sharp+debridement+opens+a+time-+dependent+therapeutic+window.">Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window.</a> Journal of Wound Care. 19 (8), 320-328 (2010).</li><li>Korgaonkar, A., 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=Community+surveillance+enhances+Pseudomonas+aeruginosa+virulence+during+polymicrobial+infection.">Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (3), 1059-1064 (2013).</li><li>Watters, 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=Pseudomonas+aeruginosa+biofilms+perturb+wound+resolution+and+antibiotic+tolerance+in+diabetic+mice.">Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.</a> Medical Microbiology and Immunology. 202 (2), 131-141 (2013).</li><li>Watters, C., Everett, J. A., Haley, C., Clinton, A., Rumbaugh, K. 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. 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=Visualizing+pneumococcal+infections+in+the+lungs+of+live+mice+using+bioluminescent+Streptococcus+pneumoniae+transformed+with+a+novel+gram-positive+lux+transposon.">Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.</a> Infection and Immunity. 69 (5), 3350-3358 (2001).</li><li>Kadurugamuwa, J. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+heterogeneity+in+biofilms.">Physiological heterogeneity in biofilms.</a> Nature Reviews Microbiology. 6 (3), 199-210 (2008).</li><li>Tipton, C. 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=Chronic+wound+microbiome+colonization+on+mouse+model+following+cryogenic+preservation.">Chronic wound microbiome colonization on mouse model following cryogenic preservation.</a> PLoS One. 14 (8), 0221656 (2019).</li></ol>

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 ...

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Immunology and Infection

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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 ...

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 ...

In vitro Biofilm Formation in an 8-well Chamber Slide

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. ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

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

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. ...

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

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. ...

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, ...