We would like to thank the Laboratory Animal Resource Center (LARC), Indiana University, for their support and the veterinarian care of the animals during the study. This work was partly supported by the National Institutes of Health grants NR015676, NR013898, and DK125835 and the Department of Defense grant W81XWH-11-2-0142. In addition, this work benefited from the following National Institutes of Health awards: GM077185, GM069589, DK076566, AI097511, and NS42617.

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

<ol>
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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=Microscopic+and+physiologic+evidence+for+biofilm-associated+wound+colonization+in+vivo.">Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo.</a> Wound Repair and Regeneration. 16 (1), 23-29 (2008).</li><li>Breuing, K., Kaplan, S., Liu, P., Onderdonk, A. B., Eriksson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+fluid+bacterial+levels+exceed+tissue+bacterial+counts+in+controlled+porcine+partial-thickness+burn+infections.">Wound fluid bacterial levels exceed tissue bacterial counts in controlled porcine partial-thickness burn infections.</a> Plastic and Reconstructive Surgery. 111 (2), 781-788 (2003).</li><li>Nusbaum, A. G., 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=Effective+method+to+remove+wound+bacteria:+Comparison+of+various+debridement+modalities+in+an+in+vivo+porcine+model.">Effective method to remove wound bacteria: Comparison of various debridement modalities in an in vivo porcine model.</a> Journal of Surgical Research. 176 (2), 701-707 (2012).</li><li>Hirsch, 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=Enhanced+susceptibility+to+infections+in+a+diabetic+wound+healing+model.">Enhanced susceptibility to infections in a diabetic wound healing model.</a> BMC Surgery. 8, 5 (2008).</li><li>Roche, E. 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=Increasing+the+presence+of+biofilm+and+healing+delay+in+a+porcine+model+of+MRSA-infected+wounds.">Increasing the presence of biofilm and healing delay in a porcine model of MRSA-infected wounds.</a> Wound Repair and Regeneration. 20 (4), 537-543 (2012).</li><li>Hartoch, R. S., McManus, J. G., Knapp, S., Buettner, M. 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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=Severe+burn+injury+in+a+swine+model+for+clinical+dressing+assessment.">Severe burn injury in a swine model for clinical dressing assessment.</a> Journal of Visualized Experiments. (141), e57942 (2018).</li><li>Sullivan, T. P., Eaglstein, W. H., Davis, S. 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The authors declare no competing interests.

This report provides a detailed protocol for setting up a swine model of chronic wound biofilm infection for experimental studies. Several swine biofilm models have been reported previously22,23,24,25,26, but none of them are swine models involving 8 week long-term studies. Chronic wounds are those that remain open for 4 weeks or more14,27,28. There are no other chronic wound biofilm models reported in the literature. This work addresses the notion of functional wound closure2,7,13,15,17,29. A study conducted in 2014 was the first to report that biofilm-infected wounds may close without the restoration of barrier function7. The measurement of the skin barrier function in the healing wound using transepidermal water loss (TEWL) is reported in this work.
Anatomically and physiologically, the porcine skin, compared to the skin of other small animals, is a closer match to the human skin32,33,34. Both pig and human skin has a thick epidermis33, and the dermal-epidermal thickness ratio ranges from 10:1 to 13:1 in pig, which is comparable to humans34,35. Histologically and biomechanically, the skin of humans and pigs shows similarities in the rete-ridges, subdermal fat, dermal collagen, hair distribution, adnexal structures, and blood vessel size and distribution36,37,38. Functionally, both pigs and humans share similarities in the composition of the lipid, protein, and keratin components of the epidermal layer, as well as comparable immunohistological patterns37,38. The porcine immune system, compared to that of other small animals, shares higher similarities with the human immune system, meaning pigs are an appropriate model for studies on the host interactions that are integral to the complexities of the pathological biofilm in wound infections39. The critical assessment of the pros and cons offered by various animal models has led to the consensus that pigs represent an efficient model for studying wound healing34,38. Additionally, domestic pigs spontaneously develop chronic bacterial infections, as observed in humans10. The burn device used to create the wounds is an advanced and automated burn device that delivers heat energy based on a temperature read out from the targeted skin site22,40. Such an approach improves the rigor and reproducibility of the burn injury. The use of human clinical isolates of bacteria to infect the pig wounds adds value as a pre-clinical model.
Burn injuries are complex and cause several systemic perturbations20,41. Thus, it is important to resuscitate the pig with adequate fluids and prevent hypothermia during anesthesia and recovery. Several factors can interfere with the wound healing, including the post-burn nutrition, fluids, and pain42. Close monitoring of the nutrition and pain assessments is, therefore, of importance. Post-burn pain can be severe and influence the animal&#39;s behavior and diet. Interventions to address behavioral concerns must be actively considered. Regular and continuous pain scoring and management is imperative. A thorough pain assessment sheet with a very detailed pain management plan is included in this protocol. To avoid cross-contamination between the wounds, special attention should be made to apply the first layer of the dressing on each wound separately. Critical care should be taken in handling all the biohazardous materials and when performing the thorough disinfection of the equipment, tools, and entire surgical room. The application of multiple layers of the dressing prevents the pig from exposing the wounds during their effort to rub or scratch the itching back.
The pig in the current model was not compromised by underlying metabolic disorders (e.g., diabetes), and, therefore, the effect being studied was purely the impact of the bacterial biofilm infection on wound healing. However, the model lends itself to the induction of diabetes (using streptozotocin for example) and could be used to study biofilm infection in relation to an underlying metabolic disorder. The other limitation of the model is the controlled infection setting using P. aeruginosa, a bacterium. It is expected that the normal skin micro-flora of the pig may also be growing in the wound and could impact healing. Further analysis using NGS or other advanced techniques to delineate the microbial content of the wound is necessary. The current model could also be applied to mixed infections with differing microbial species (e.g., fungal, viral, etc.). This is an important element, as clinically relevant wounds are likely to be populated by mixed microbes, which may impact wound healing differentially.
There are many potential advantages in this model, including the similarity to the complexity and long-term sequelae of human chronic wounds, the automated and reproducible burn process, and the use of clinically isolated bacterial species. The use of multiple non-invasive imaging modalities represents a powerful approach for collecting useful physiological data characterizing the wound. Finally, the assessment of the functional wound healing via the restoration of skin barrier function based on TEWL is critical. In conclusion, a robust, simple, detailed, and easy-to-use protocol to develop a biofilm-infected severe burn injury using a porcine model system is shown in this work.

Biofilm infection complicates burn and chronic wounds and causes chronicity1,2,3,4,5. In microbiology, biofilm mechanisms are primarily studied, with a focus on the microbes1,6. The lessons learned from these studies are of paramount importance from a biological science standpoint but may not necessarily be applicable to clinically relevant pathogenic biofilms6,7,8. Clinically relevant biofilm structural aggregates should include microbial as well as host factors8,9,10. Such a microenvironment allows for the inclusion of host-microbe iterative interactions, which are critical to developing a clinically relevant biofilm7,8. In such a process, the participation of immune cells and blood-borne factors is critical11,12. The host-microbe interactions underlying clinical pathogenic biofilms, as seen in chronic wounds, occur over a long period of time. Thus, any experimental approach aimed at developing a translationally relevant model of biofilm infection must account for these factors. So, we sought to develop a clinically reproducible swine chronic biofilm infection model.
While human studies clearly represent the best approach to studying healing outcomes, often they are not best suited to addressing the underlying mechanisms and new mechanistic paradigms. Ethical concerns limit the use of study designs requiring the collection of multiple biopsies from a chronic wound at different time points. It is, therefore, critical to have a well-established and reproducible animal model to enable invasive studies for the thorough examination of biofilm fate7,13. The selection of an animal model depends on several factors, including scientific/translational relevance and logistics. The porcine system is widely acknowledged to be the most translationally valuable experimental model to study human skin wounds7. Thus, this work reports an established swine model of biofilm-infected full-thickness burn injury. This work is based on several original publications reported in the literature2,7,13,14,15,16,17. In this study, a clinical isolate of multidrug-resistant&#160;Pseudomonas aeruginosa (PA01) was chosen to infect the wound. P. aeruginosa is a common cause of wound infections2,18,19,20. It is a Gram-negative bacterium that can be difficult to treat due to its resistance to some antibiotics11,19,21. None of the swine biofilm models reported so far involved 8 week long-term studies22,23,24,25,26. Chronic wounds are those that remain open for 4 weeks or more14,27,28. There are no other chronic wound biofilm models reported in the literature. This work addresses the notion of functional wound closure2,7,13,15,17,29.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sedation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Zoetis</td>
			<td>10004027</td>
			<td>100mg/ml</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>106-111</td>
			<td>100mg/ml</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Pivetal</td>
			<td>04606-6750-02</td>
			<td>100mg/ml Anased</td>
		</tr>
		<tr>
			<td>3ml syringe w/ 20g needle</td>
			<td>Covidien-Monoject</td>
			<td>8881513033</td>
			<td></td>
		</tr>
		<tr>
			<td>Winged infusion set 21g</td>
			<td>Jorgensen Labs</td>
			<td>J0454B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal</td>
			<td>21295097</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Church and Dwight Co. Inc</td>
			<td>70506572</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine Solution</td>
			<td>First Priority Inc.</td>
			<td>179925722</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Isopropyl Alcohol</td>
			<td>Uline</td>
			<td>S-17474</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Saline Solution</td>
			<td>ICU Medical</td>
			<td>&#160;RL-7282</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven gauze</td>
			<td>Pivetal</td>
			<td>21295051</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper tape</td>
			<td>McKesson</td>
			<td>455531</td>
			<td></td>
		</tr>
		<tr>
			<td>2&#34; Elastic tape</td>
			<td>Pivetal</td>
			<td>21300869</td>
			<td></td>
		</tr>
		<tr>
			<td>18-22g Intravenous Angiocath</td>
			<td>SurVet</td>
			<td>(01)14806017512306</td>
			<td></td>
		</tr>
		<tr>
			<td>Spay hook</td>
			<td>Jorgensen Labs</td>
			<td>J0112A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile lube</td>
			<td>McKesson</td>
			<td>16-8942</td>
			<td></td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Jorgensen Labs</td>
			<td>J0449S</td>
			<td></td>
		</tr>
		<tr>
			<td>Roll gauze</td>
			<td>Pivetal</td>
			<td>21295032</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube (7-9mm)</td>
			<td>Covidien</td>
			<td>86112</td>
			<td>Shiley Hi-Lo Oral Nasal Tracheal Tube Cuffed</td>
		</tr>
		<tr>
			<td>15gtt/ml IV administration set</td>
			<td>ICU Medical</td>
			<td>12672-28</td>
			<td></td>
		</tr>
		<tr>
			<td>LRS 1000ml bag</td>
			<td>ICU Medical</td>
			<td>07953-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Three Quarter Drape Sheet</td>
			<td>McKesson</td>
			<td>16-i80-12110G</td>
			<td></td>
		</tr>
		<tr>
			<td>Analgesia</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>RX Generics</td>
			<td>42023-0179-05</td>
			<td>0.3mg/ml</td>
		</tr>
		<tr>
			<td>Fentanyl Transdermal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>21294548</td>
			<td>Pivetal</td>
			<td>50mg/ml Levafen</td>
		</tr>
		<tr>
			<td>Bandaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent film dressing 26x30</td>
			<td>Genadyne Biotechnologies</td>
			<td>A4-S00F5</td>
			<td></td>
		</tr>
		<tr>
			<td>Film dressing 4 x 4-3/4 Frame Style</td>
			<td>McKesson</td>
			<td>886408</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetrap</td>
			<td>3M</td>
			<td>1410BK BULK</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic tape 4&#34;</td>
			<td>Pivetal</td>
			<td>21300931</td>
			<td></td>
		</tr>
		<tr>
			<td>Kerlix Roll Gauze</td>
			<td>Cardinal Health</td>
			<td>3324</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Canon EOS 80D</td>
			<td>Canon</td>
			<td>1263C004</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedlight 600EX II-RT</td>
			<td>Canon</td>
			<td>1177C002</td>
			<td></td>
		</tr>
		<tr>
			<td>EFS 17-55mm Ultrasonic</td>
			<td>Canon</td>
			<td>1242B002</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Logiq E9</td>
			<td>GE</td>
			<td>5197104-2</td>
			<td></td>
		</tr>
		<tr>
			<td>ML6-15 Probe</td>
			<td>GE</td>
			<td>5199103</td>
			<td></td>
		</tr>
		<tr>
			<td>PeriCamPSI</td>
			<td>Perimed</td>
			<td>90-00070</td>
			<td></td>
		</tr>
		<tr>
			<td>DermaLab</td>
			<td>Cortex Technologies Inc</td>
			<td>4608D78</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy/Tissue Collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6mm punch biopsy</td>
			<td>Integra Lifesciences</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr>
			<td>bupivicaine 0.5%</td>
			<td>Auromedics Pharma</td>
			<td>55150017030</td>
			<td></td>
		</tr>
		<tr>
			<td>Size 10 Disposable Scalpel</td>
			<td>McKesson</td>
			<td>16-63810</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Pivetal</td>
			<td>21294806</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tooth thumb tissue forceps</td>
			<td>Aesculap</td>
			<td>BD512R</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-adherent Dressing</td>
			<td>Covidien</td>
			<td>2132</td>
			<td>Telfa</td>
		</tr>
		<tr>
			<td>50ml Conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf/microcentrifuge tube</td>
			<td>Fisherbrand</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non Woven Gauze 4x4</td>
			<td>Pivetal</td>
			<td>21295051</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low salt LB agar</td>
			<td>Invitrogen</td>
			<td>22700-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Low salt LB broth</td>
			<td>Fisher scientific</td>
			<td>BP1427-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate</td>
			<td>Falcon</td>
			<td>REF-351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyprophyline round bottom tubes (14 ml)</td>
			<td>Falcon</td>
			<td>REF-352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas Agar Base (Dehydrated)</td>
			<td>Thermo Scientific</td>
			<td>OXCM0559B</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar, powder (Lennox L agar)</td>
			<td>Thermo Fisher Scientific (Life Technologies)</td>
			<td>22700025</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco&#8482; DPBS, calcium, magnesium</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthanasia</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18-22g Intravenous Angiocath</td>
			<td>SurVet</td>
			<td>(01)14806017512306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatal Plus</td>
			<td>Vortech Pharmaceuticals</td>
			<td>9373</td>
			<td></td>
		</tr>
	</tbody>
</table>

swine model of biofilm infection and invisible wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

A standardized burn device was used to create full-thickness burn wounds at 150 &#176;C for 1 min, which resulted in a homogenous deep burn with a uniform margin of erythema and inflammation (Figure 3 and Figure 7). Each pig received eight full-thickness burn wounds on their back, as depicted in Figure 3C. 
The non-invasive real-time assessment of the burn wounds by B-mode high-resolution ultrasound to confirm the wound depth and progression of wound healing over time showed the destruction of all skin layers up to the subcutaneous fat (Figure 4). Laser speckle imaging (LSI) was used for further characterization of the wound perfusion (Figure 4A).
The burn wounds showed a thick pyogenic membrane on the wound surface by day 7 post-inoculation, thus confirming the infection and the establishment of the burn wound biofilm (Figure 7A). Digital planimetry showed an increased wound area at day 3 post-inoculation with PAO1 due to the inflammatory response at the wound site and margins (Figure 7A,B). Although the wound area started to shrink by day 14 post-inoculation, incomplete healing to approximately 25% of the original wound size was observed at day 56, indicating the chronicity of the wounds (Figure 7B). Wound chronicity and impaired wound healing were further confirmed by the TEWL, which showed high transepidermal water loss. The TEWL results reflected the loss of skin barrier function compared to normal skin at all measured the timepoints, thus indicating functional impairment of the burn wound healing (Figure 7B). This was also confirmed by the suppression of the tight junctional proteins ZO-1 and 213 and the impairment of the restoration of skin barrier function, as reflected in the high TEWL values seen at day 35 (mid) and day 56 (late) despite the visual wound closure (Figure 7I).
The burn depth was further validated by H&#38;E staining, which showed distortion and necrosis of all the histological skin layers, as shown in Figure 7C. The established biofilm of PA01 was further validated at day 7 post-inoculation by CFU (Figure 7E,F), SEM imaging (Figure 7G), and immunofluorescence staining (Figure 7H).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65301/65301fig01.jpg" /> 
Figure 1: Setup for the procedure. (A) Surgical table preparation. (B) Ear vein cannulation for IV fluids and drug administration. (C)&#160;Thermal blanket covering to protect the pig from hypothermia during the procedure. (D) Burner and timer setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65301/65301fig02.jpg" /> 
Figure 2: Surgical site sterilization and marking. (A) Hair clipping and sterilization. (B) Marking of the burn site using a sterile eight-wound standard template (each wound is 2 in x 2 in). (C)&#160;Final marking using a sterile skin marker. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65301/65301fig03.jpg" /> 
Figure 3: Burn wound induction. (A,B) Standardized burner with a pressure gauge and automated controller unit (2 in x 2 in) applied to the pre-marked wound site. (C)&#160;The whole back showing the eight full-thickness burn wounds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65301/65301fig04.jpg" /> 
Figure 4: Noninvasive burn wound imaging and assessment. (A) Laser speckle imaging (LSI) with proper orientation of the laser beam indicator to the center of the wound is shown in the left-side image; the right-side image shows the LSI device and the real-time skin vascular perfusion map. (B) Transepidermal water loss (TEWL) probe application to the wound site at five different spots (four wound corners and the center demonstrated in lower-right corner image) is shown in the left-side image; the right-side image is a representative real-time captured screen of the TEWL measurement. (C) Harmonic ultrasound scanning of the burn wound using a high-resolution 16 MHz ultrasound probe is shown on the left side; the right-side image shows the ultrasound device and the real-time screen recording. (D) Structural (B-mode images, grayscale ultrasound) and biomechanical (elastography, color ultrasound) images of the burn wound site at the inoculation day and day 7 post-inoculation. The wound depth is indicated by the yellow dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65301/65301fig05.jpg" /> 
Figure 5: Wound dressing and bandaging. (A) Application of the transparent film dressing for each wound separately. (B)&#160;All the dorsal inoculated burn wounds are covered with the first layer of dressing. (C)&#160;A larger transparent film dressing is placed over the entire wound area.(D)&#160;Application of the second layer of gauze and a loose layer of stretchy elastic bandage around the entire trunk of the pig to absorb any fluid exudate that comes from the wounds. (E)&#160;Covering of the entire wound area with a final layer of 4 in adhesive dressing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65301/65301fig06.jpg" /> 
Figure 6: Bacterial inoculation. (A) Setup for the Pseudomonas aeruginosa (PA01) inoculation at day 3 post-burn. (B)&#160;Topical application of the inoculum with a pipette using a 500 &#181;L volume for each wound. (C)&#160;The inoculum is dispersed across the wound surface evenly using a sterile disposable spreader. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65301/65301fig07.jpg" /> 
Figure 7: Wound healing progress and biofilm confirmation. (A) Representative images of the wound closure over the timeline of the study. Scale bar = 1 cm. (B) Quantitation of the wound area and TEWL measurements over the timeline of the study (n = 6). The data are represented as mean &#177; SD. N.S. refers to the TEWL value of normal skin. (C)&#160;Schematic diagram showing different wound biopsy sites. D. H&#38;E staining with its corresponding Masson&#39;s trichrome staining showing distortion and necrosis of all the skin layers at day 3 post-burn and day 7 post-inoculation. Scale bar = 500 &#181;m. (E) Representative digital images of non-selective agar (Luria-Bertani agar) and selective agar (Pseudomonas Isolation Agar) with bacterial colonies grown from porcine wound bed tissue. The selective medium enables the accurate counting of the PA01 colonies only. (F) A sample colony forming unit (CFU) calculation from the colony counts taken from processed day 7 post-inoculation wound biopsies is shown. (G) Representative scanning electron microscopy (SEM) images of the inoculated burn wounds at day 7 post-inoculation showing the established PA01 biofilm, with a zoomed-in image on the right side. Scale bar = 1 &#181;m. The red arrowheads point to extracellular polymeric substances (EPS). (H) P. aeruginosa on the burn wounds were visualized using anti-Pseudomonas (green) antibody; the immunofluorescence images of the day 7 post-inoculation wound biopsies show heavy colonization of the wound tissues by P. aeruginosa. Scale bar = 100 &#181;m.&#160;(I) Representative mosaic (scale bar = 200 &#181;m) and corresponding zoomed-in (scale bar = 50 &#181;m) images of ZO-1- and ZO-2-stained sections on day 35 and day 56 post-inoculation, demonstrating reduced expression of the proteins following the induced infection. The OCT-embedded frozen sections (10 &#181;m) were stained using anti-ZO-1 (green) or anti-ZO-2 (green). The sections were counterstained using DAPI. The bar graphs present the quantitation of the ZO-1 and ZO-2 signal intensity. The data are presented as mean &#177; SD (n = 3); * p &#60; 0.05 compared to spontaneous ones. Mann-Whitney or Kruskal-Wallis one-way analysis of variance tests were performed to test the significance. Figure 7H,I has been modified from Roy et al.13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65301/65301fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Host-Microbe Interactions in Wound Biofilm Formation at JoVE.com

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

swine-model-of-biofilm-infection-and-invisible-wounds

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.7K Views.  Indiana University School of Medicine. Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Video: Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

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. ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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 ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

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. ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Swine Model of Biofilm Infection and Invisible Wounds

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Chapters in this video

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Microtiter Dish Biofilm Formation Assay

In vitro Biofilm Formation in an 8-well Chamber Slide

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

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

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

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

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

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model