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Table of Contents
ORIGINAL ARTICLE
Year : 2018  |  Volume : 22  |  Issue : 2  |  Page : 68-71

Experimental wound ischemia does not promote pseudomonas aeruginosa biofilm formation


1 Departments of Science of Nursing Practice, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan
2 Department of Plastic and Reconstructive Surgery, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan

Date of Web Publication21-Sep-2018

Correspondence Address:
Dr. Emi Kanno
Department of Science of Nursing Practice, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Miyagi
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdds.jdds_33_18

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  Abstract 


Introduction: It has been suggested that wound ischemia is involved in the promotion of bacterial proliferation, which is a detrimental factor in wound healing. Increasing evidence from clinical data suggests that bacteria live within biofilms on nonhealing wounds. Yet, there have been no reports clarifying the contribution of wound ischemia to biofilm formation on nonhealing wounds. Objectives: The present study addresses the question of how ischemia affects biofilm formation at wound sites. Methods: Standardized dorsal ischemic flaps were lifted and sutured on the backs of Sprague–Dawley rats. Partial thickness wounds were created on these flaps, and a suspension of Pseudomonas aeruginosa was applied to each wound. We analyzed wound exudate, histological findings, and biofilm formation. Results: The quantities of exudate from the wounds on the ischemic flaps were greater than those from control wounds. Surprisingly, in ischemic wounds, biofilm formation was diminished and leukocyte infiltration was decreased. Conclusion: Our findings demonstrate for the first time that the ischemic condition may not contribute to the development of biofilm formation on skin wounds through regulating leukocytic responses.

Keywords: Biofilm, ischemic wound, Pseudomonas aeruginosa, rat


How to cite this article:
Kanno E, Tanno H, Yamaguchi K, Sasaki A, Maruyama R, Tachi M. Experimental wound ischemia does not promote pseudomonas aeruginosa biofilm formation. J Dermatol Dermatol Surg 2018;22:68-71

How to cite this URL:
Kanno E, Tanno H, Yamaguchi K, Sasaki A, Maruyama R, Tachi M. Experimental wound ischemia does not promote pseudomonas aeruginosa biofilm formation. J Dermatol Dermatol Surg [serial online] 2018 [cited 2018 Dec 16];22:68-71. Available from: http://www.jddsjournal.org/text.asp?2018/22/2/68/241916




  Introduction Top


The skin wound healing process consists of coagulation, inflammation, proliferation, and remodeling.[1],[2] Certain pathogenic microorganisms that adhere to wounded skin, such as Pseudomonas aeruginosa, can cause persistent infections by forming biofilms.[3],[4] In chronic nonhealing wounds, healing impairment can be caused by various intrinsic factors such as vascular problems and neuropathy as well as various extrinsic factors such as wound infection and excessive pressure to the site.[1] Among these factors, it has been suggested that wound ischemia promotes bacterial proliferation. Increasing evidence from clinical data suggests that bacteria on nonhealing wounds live within biofilms.[4] Thus, it is possible that wound ischemia is involved in the augmentation of biofilm formation. To date, however, there have been no reports about the contribution of the ischemic condition to biofilm formation.

Another possibility is that low inflammatory responses such as leukocyte accumulation might affect biofilm formation in ischemic wounds, because it is not necessary to form a biofilm as a protection from pathogenic microorganisms. In fact, neutrophilic responses have been shown to enhance P. aeruginosa biofilm formation in an in vitro study.[5] While several in vivo models for the study of biofilms have been reported,[3],[6] it is not yet clear how ischemic conditions are related to biofilm formation in vivo.

In the present study, we examined the effect of the ischemic condition on biofilm formation. We also analyzed the infiltration of leukocytes at the wound site after wound creation and P. aeruginosa inoculation. Our findings demonstrate for the first time that ischemic conditions may not contribute to the development of P. aeruginosa biofilm formation.


  Materials And Methods Top


Animals

Male Sprague–Dawley (SD) rats (400–500 g, 10–12 weeks of age) were obtained from Clea Japan, Inc., Tokyo, Japan, and maintained under a 12 h light/12 h dark cycle. Food and water were available ad libitum. Handling of the animals was performed under anesthesia induced by 40 mg/kg of pentobarbital (Nembutal, Dainippon Sumitomo Pharma, Osaka, Japan) and sustained by inhalation anesthesia of isoflurane (Isoflurane, Mairan Pharma, Osaka, Japan). At the end of the experiments, the animals were euthanized by means of a pentobarbital overdose. All experimental protocols described in the present study were approved by the Ethics Review Committee for Animal Experimentation of Tohoku University.

Bacteria

The P. aeruginosa strain PAO1 carrying the gene encoding green fluorescent protein (GFP) plasmid pGREEN was stored at −80°C in a Microbank storage system (Pro-Lab Diagnostics Inc., Richmond Hill, ON, Canada) each containing 25 beads. One of these beads was applied onto a sheep blood agar plate and incubated at 37°C overnight; resulting colonies were cultured in brain heart infusion (BHI) broth (Eiken Chemical Co., Ltd., Tokyo, Japan) at 37°C for 24 h and washed three times in normal saline. After the final suspension was mixed, bacterial counts were performed by measuring absorbance at 600 nm. In every experiment, a quantification culture was performed to confirm the inoculation dose.

Wound creation and tissue collection

The dorsal hairs were shaved to fully expose the skin, which was then disinfected using 70% ethanol. A standardized dorsal ischemic flap was lifted and sutured on the left side of the back of each SD rat [Figure 1]. Deep partial-thickness dermal wounds preserving the cutaneous muscle were created on these flaps and also on the right side of the back of each SD rat using a 6-mm-diameter biopsy punch (Biopsy Punch, Kai Industries Co., Ltd., Gifu, Japan) and scissors under sterile conditions. A suspension (14 μl) of P. aeruginosa PAO1 was applied to the base of each wound at 5 × 107 CFU/wound. Wounds were covered with a polyurethane foam dressing (Tegaderm Transparent Dressing, 3M Health Care, St. Paul, MN), and the whole trunk of each rat was covered with an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) to protect the wound from contamination and mechanical irritation. The day on which the wounds were made was designated as day 0. Tissues were harvested at 1, 3, and 7 days after wound creation by excising the area with an 8-mm-diameter biopsy punch.
Figure 1: A rat model of ischemic wound. A standardized dorsal ischemic flap was lifted and sutured on the back of each Sprague–Dawley rat

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Histological analysis

The wound tissues were collected using an 8-mm-diameter biopsy punch (Biopsy Punch, Kai Industries Co., Ltd., Gifu, Japan) and dissected in the caudocranial direction. Each wound was divided into two equal parts: one part was embedded in paraffin and stained with hematoxylin and eosin according to standard methods; the other part was embedded in optimal cutting temperature (O. C. T). compound (Sakura Finetechnical Co., Tokyo, Japan), quickly frozen and stained with rhodamine-conjugated concanavalin A (50 mg/ml). Specimens were visualized under an Olympus I × 70 fluorescence microscope (Olympus Co., Tokyo, Japan). All measurements were made through the following absorbing filter set: 450–490 nm where fluorescein isothiocyanate-excited GFP was used and 520–550 nm where rhodamine-conjugated concanavalin A (Con A) was used. To map spatial growth patterns of bacteria and GFP within biofilms, GFP reporter gene constructs in P. aeruginosa were used.


  Results Top


Macroscopic histologic findings and bacterial cell attachment

The amounts of exudate from the wound surfaces were greater in ischemic wounds than in control (nonischemic) wounds at 1 day after wound creation [Figure 2]a. The accumulation of inflammatory cells at the wound surface was smaller in ischemic wounds than in control (nonischemic) wounds at 1, 3, and 7 days after wound creation [Figure 2]b and [Figure 2]c. Re-epithelialization was observed in control nonischemic wounds, but not in ischemic wounds [Figure 2]c.
Figure 2: Macroscopic and microscopic appearances of the wound surface. Macroscopic appearances were taken on day 1 after wounding and inoculation of Pseudomonas aeruginosa. Representative histological views of skin wounds on days 1, 3, and 7

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Bacterial counts and biofilm formation

Bacterial cell attachment was indicated by GFP and the materials surrounding each microcolony. Bacteria were positive for rhodamine-conjugated Con A, which binds to alginate.[7] As shown in [Figure 3], there was less attachment of P. aeruginosa (GFP) and materials around the microcolonies (rhodamine) in ischemic wounds than in control wounds.
Figure 3: Biofilm formation on the ischemic wounds. Bacterial cell attachment is indicated by green fluorescent protein. Materials around microcolonies were positive for rhodamine-conjugated concanavalin A and fluorescein isothiocyanate staining. The dotted line indicates the wound surface

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  Discussion Top


The major findings in the present study are as follows: (1) the amount of wound exudate was greater in ischemic wounds than in control wounds; (2) the accumulation of leukocytes was diminished and re-epithelialization was decreased in ischemic wounds compared with control wounds; and (3) bacterial attachment and biofilm formation were diminished in ischemic wounds compared with control wounds. These results suggest that ischemic conditions may not contribute to the development of biofilm formation on skin wounds.

The process of wound healing is affected by various factors at the wound site, including infection, necrotic tissue, ischemia, pressure, and other factors underlying systemic diseases. For many years, ischemic conditions have been considered to contribute to impairment of wound healing.[8] It is also well known that certain pathogenic microorganisms that adhere to wounded skin, such as P. aeruginosa, can cause persistent infections by forming biofilms.[3],[4] To date, however, the interaction of ischemic conditions with biofilm formation has not been investigated. Contrary to expectations, the current study revealed that ischemic conditions in our experimental model did not contribute to biofilm formation. Taken together with previous findings, our results suggest that P. aeruginosa biofilms are not more likely to develop in ischemic wounds on skin flaps.

The accumulation of neutrophils was smaller in ischemic wounds than in control wounds. In addition, biofilm formation was diminished in ischemic wounds. In a previous in vitro study, Jesaitis et al.[9] reported that neutrophils accumulated on P. aeruginosa biofilms were phagocytically engorged, partially degranulated, immobilized, and rounded.[9] Moreover, Walker et al.[5] demonstrated that human neutrophils can serve to enhance the initial development of P. aeruginosa biofilms.[5] Considered alongside these previous results, our findings suggest that neutrophils may be involved in promoting biofilm formation at wound sites.


  Conclusion Top


The present study constitutes the first demonstration that wound ischemia in our experimental model does not promote the development of biofilm formation by P. aeruginosa. This finding may lead to the development of novel therapeutic strategies for the prevention of biofilm formation on skin wounds. In the present study, the precise nature of the relationship between biofilm formation and neutrophils remains to be determined. Further investigation is necessary to address this issue.

Acknowledgments

The authors would like to thank Dr. Naomasa Gotoh, Kyoto Pharmaceutical University, for his kind gift of P. aeruginosa PAO1 carrying the gene encoding GFP.

Financial support and sponsorship

This work was supported in part by a Grant-in-aid for Scientific Research (C) (16K11909) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;366:1736-43.  Back to cited text no. 1
    
2.
Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585-601.  Back to cited text no. 2
    
3.
James GA, Swogger E, Wolcott R, Pulcini Ed, Secor P, Sestrich J, et al. Biofilms in chronic wounds. Wound Repair Regen 2008;16:37-44.  Back to cited text no. 3
    
4.
Schultz G, Bjarnsholt T, James GA, Leaper DJ, McBain AJ, Malone M, et al. Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds. Wound Repair Regen 2017;25:744-57.  Back to cited text no. 4
    
5.
Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, et al. Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils. Infect Immun 2005;73:3693-701.  Back to cited text no. 5
    
6.
Gurjala AN, Geringer MR, Seth AK, Hong SJ, Smeltzer MS, Galiano RD, et al. Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing. Wound Repair Regen 2011;19:400-10.  Back to cited text no. 6
    
7.
Kanno E, Toriyabe S, Zhang L, Imai Y, Tachi M. Biofilm formation on rat skin wounds by Pseudomonas aeruginosa carrying the green fluorescent protein gene. Exp Dermatol 2010;19:154-6.  Back to cited text no. 7
    
8.
Mustoe TA, O'Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies: A unifying hypothesis. Plast Reconstr Surg 2006;117:35S-41S.  Back to cited text no. 8
    
9.
Jesaitis AJ, Franklin MJ, Berglund D, Sasaki M, Lord CI, Bleazard JB, et al. Compromised host defense on Pseudomonas aeruginosa biofilms: Characterization of neutrophil and biofilm interactions. J Immunol 2003;171:4329-39.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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