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Protocol

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Immunology and Infection

Assessing Biofilm Dispersal in Murine Wounds

Published: August 7th, 2021

DOI:

10.3791/62136

1Department of Surgery, Texas Tech University Health Sciences Center, 2Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, 3TTUHSC Surgery Burn Center of Research Excellence, Texas Tech University Health Sciences Center
* These authors contributed equally

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 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.

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. ....

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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.......

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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

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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.......

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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).

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

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