Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function

Abstract

In chronic wounds, biofilm infects host tissue for extended periods of time. This work establishes the first chronic preclinical model of wound biofilm infection aimed at addressing the long-term host response. Although biofilm-infected wounds did not show marked differences in wound closure, the repaired skin demonstrated compromised barrier function. This observation is clinically significant, because it leads to the notion that even if a biofilm infected wound is closed, as observed visually, it may be complicated by the presence of failed skin, which is likely to be infected and/or further complicated postclosure. Study of the underlying mechanisms recognized for the first time biofilm-inducible miR-146a and miR-106b in the host skin wound-edge tissue. These miRs silenced ZO-1 and ZO-2 to compromise tight junction function, resulting in leaky skin as measured by transepidermal water loss (TEWL). Intervention strategies aimed at inhibiting biofilm-inducible miRNAs may be productive in restoring the barrier function of host skin.

Keywords: microRNA; mixed-species biofilm; porcine burn wounds; transepidermal water loss (TEWL); wound biofilm.

Figure 1. Establishment of mixed-species biofilm infection in a full thickness porcine burn wound model

Six 2 × 2 sq inch size burn wounds were created with a burning device (Fig S1). Co- infection of the burn wounds was performed day 3 post-burn with P. aeruginosa PAO1 and A. baumannii 19606. A, representative digital photograph of wounds on the day of burn, days 7 and 14 post-infection. Note signs of active infection with localized erythema, yellowish exudates, and friable wound edges on days 7 and 14 post-infection. B, scanning electron microscope (SEM) images of biopsies collected from the wounds on the day of burn and days 7 & 14 post-infection. The images clearly demonstrate a mix of extracellular tissue matrix, fibers, and red blood cells over the surface of burn wounds before bacterial inoculation. Large aggregates of rods attached to the wound surface encased in a layer of extracellular amorphous material was noted on days 7 & 14 post-infection. Upper panel, scale bar = 20 μm, 2500× magnification. Lower panel, magnification of the red dashed boxes in the upper panels. Scale bar = 5 μm, 10000× magnification. C, Gram stained images of inoculated wounds shows presence of bacterial aggregates. Upper panel, representative mosaic image of a day 7 post infection wound. Images collected under 400× magnification using microscope supported with a motorized stage. Scale bar = 200 μm. Es= eschar, Grn= granulation tissue. Lower panels, Zoom of three boxed area in upper panel image showing Gram negative bacilli & coccobacilli over the surface of the burn wound while large Gram negative clumps colonizing the wound tissues (yellow arrows). Scale bar = 50 μm. D, both P. aeruginosa and A. baumannii on the burn wounds were visualized using anti-pseudomonas (green) and anti-acinetobactor (red) antibody and confocal laser scanning (CLSM) microscopy. Merged (red & green) immunofluorescence images of day 7 (post-infection) wound biopsies show heavy colonization of wound tissues with both strains. Mosaic images were collected under 400× magnification using fluorescent microscope supported with a motorized stage. Scale bar = 100 μm. Z-stack images of boxed areas of the lower panel surface of burn wound tissues. Inset, zoom of the boxed area in merged image. The image was created by merging serial scans of thick tissue section (20 μm), viewed under 600× magnification in a confocal laser scanning microscope (CLSM). Dense micro-colonies of P. aeruginosa and A. baumannii with some co-localization in the wound tissues were noted. x/z and y/z planes display the thickness of the microbial clumps within the tissue section. E, representative SEM image of wound biopsies from human chronic pressure ulcers showing that the bacterial colonization and biofilm established in experimental porcine biofilm model is comparable to that of human chronic wounds. left panel, scale bar = 20 μm, 2500× magnification. Right panel, scale bar = 5 μm, 10000× magnification.

Figure 2. Resistance of mixed species biofilm to antimicrobial and debridement

A-C, antimicrobial Acticoat 7™ effectively kills bacteria in planktonic phase while ineffective against wound biofilm. A, planktonic cultures of P. aeruginosa and A. baumannii were incubated in presence or absence of Acticoat 7™ for 24h followed by determination of bacterial CFU. The treatment was highly effective in killing the bacteria. nd, not detectable. Data are mean ±SD (n=4), *, p<0.05. B, images of wounds covered with Tegaderm™ or Acticoat 7™ .C, Microbiology analysis of porcine burn wound were covered with Tegaderm™ or Acticoat 7™ on days 7 or 14 post-inoculation. Acticoat 7™ was ineffective in attenuating any bacterial load from wounds suggesting that the bacteria in biofilm were resistant to anti-microbial treatment. Data are mean ± SD, n=3. D-H, debridement was performed using a 0.12 inch Weck Blade by removing necrotic and infected tissues until bleeding healthy tissue is exposed. D, digital images of burn wound pre debridement (pre-D), immediate post-debridement (post-D, 0h) or 48h (post-D, 48h) after debridement. E, microbiology analysis reveals a significant decrease in bacterial burden after debridement. The bacterial burden for P. aeruginosa was restored to almost to the initial level 48h post-D suggesting debridement alone does not eradicate biofilm. Data are mean ± SD. * p<0.05 (n=3); F, laser doppler imaging was performed to demonstrate that there was no blood flow (blue color) in pre debrided burn wounds while the healthy well perfused (red) tissue is exposed immediately after debridement. G, bar graph represents quantitation of the laser Doppler blood flow imaging. H, Immuno-histochemical staining of P. aeruginosa (green) and A. baumannii (red) burn wounds following debridement demonstrate that the bacterial biofilm (micro colonies) are restored in deeper layers of the wounds on days 14 post-debridement. White dashed line represents the line of debridement. Scale bar = 100μM.

Figure 3. Abundant expression of biofilm specific genes in biofilm tissue elements captured using LCM

A, frozen serial sections (10 μm) from biofilm infected wound tissue were stained with anti-PAO1 (green) antibody. The next serial sections were stained with hematoxylin to visualize corresponding biofilm affected area. Images are representative (left) anti-PAO1 stained image; (middle) anti-PAO1 stained tissue marked to delineate the biofilm affected area; and (right) the area of biofilm was cut/captured using laser capture microdissection (LCM) from corresponding hematoxylin stained section. B, to collect planktonic bacteria from wounds, a sterile double open-ended plastic tube was placed on a wound followed by washing once with 4ml of PBS and collected. The wash suspension was collected and spun down to retrieve bacterial pellets. This procedure results in removal of loosely adhered or planktonic form of bacteria. C, LCM captured tissue was used for quantification of mRNA levels of biofilm specific mRNA using real-time PCR and normalized against 16S rRNA expression. 16S rRNA level have been presented to demonstrate comparable levels in both plankonic and captured biofilm groups. Data are mean ±SD (n=4); * p<0.05 compared to planktonic.

Figure 4. Biofilms infection compromised re-establishment of skin barrier function

A, wound closure in spontaneous infection (blue line, control) or induced infection (red line) wounds. Data have been presented as percentage of the initial wound area. Data are mean ± SD (n=4). B, representative digital images from spontaneous infection or induced infection wounds burn wounds. C, trans epidermal water loss (TEWL) analysis from spontaneous infection and induced infection burn wounds. Inset presents the DermaLab TEWL Probe used for the measurement of the trans-epidermal water loss from the wounds. TEWL was expressed in g/m²/h. Data are mean ±SD (n=4); * p<0.05 compared to spontaneous infection.

Figure 5. Silencing of tight junction proteins by biofilm infection

Wound biopsies were collected at specified time-points after inoculation from spontaneous infected (spont, no inoculation, control) or induced infected (induced), inoculated with A. baumannii and P. aeruginosa. A, representative mosaic (scale bar=200 μm) and corresponding zoomed (scale bar=50 μm) images of ZO-1 and ZO-2 stained sections on days 35 & 56 post inoculation demonstrating reduced expression of the proteins following induced infection. OCT embedded frozen sections (10 μm) and stained using anti-ZO-1 (green) or anti-ZO-2 (green). The sections were counterstained using DAPI. Bar graphs present quantitation of ZO-1 and ZO-2 signal intensity. Data are presented as mean ± SD (n=3), * p<0.05 compared to spontaneous. B, expression of miR-146a and miR-106b in wound biopsies collected on days 7-35 post inoculation from spontaneous or induced burn wounds. Data presented as mean ± SD (n=3), * p<0.05 compared to spontaneous infection.

Figure 6. Biofilm inducible miRNA silence ZO-1 and ZO-2

Human keratinocyte (HaCaT) cells were infected with a static biofilm infection as described in methods. A, Western blot of ZO-1 and ZO-2 expression following 12h of infection as compared to non-infected cells (control). Data are mean ± SD (n=3), * p<0.05 compared to control. B, expression of miR-146a and miR-106b in keratinocytes following 12h of P.aeruginosa PAO1 (Pa), A. baumannii 19606 (Ab) mixed (both Pa and Ab) infection compared to non-infected control. Data represented as mean ± SD (n=4), * p<0.05 compared to control, † p<0.05 compared to A. baumannii and ‡ p<0.05 compared P.aeruginosa. C, expression of miR-146a and miR-106b in keratinocytes following 12h of poor-biofilm forming P.aeruginosa Δpsl PAO1 (Pa), wild type biofilm forming P.aeruginosa PAO1 (Pa) infection compared to non infected control. Data represented as mean ± SD (n=4), * p<0.05 compared to control. D, expression of miR-146a and miR-106b in keratinocytes following 12h of incubation with planktonic bacterial media and biofilm bacterial medium compared to non infected control media. Data represented as mean ± SD (n=4), * p<0.05 compared to control media and † p<0.05 compared to planktonic control media. E, human keratinocytes were transfected with miR-146a a miR-106b mimics for 48h followed by Immunocytochemistry. Images of HaCaT stained with anti- ZO-1 or anti-ZO-2 (green) and counter stained with DAPI (nuclear, blue). The quantification of ZO-1 and ZO-2 has been provided in Fig S4. Scale bar =10 μm. F, expression of ZO-1 and ZO-2 expression in HaCaT cells transfected with either miRIDIAN hsa-miR-146a or hsa-miR-106b mimic or control-mimic for 48h. Data represented as mean ± SD (n=4), * p<0.05 compared to control miRNA mimic. G, expression of ZO-1 and ZO-2 in presence of anti-miR to miR-146a and miR-106b inhibitors. Data represented as mean ± SD (n=4), * p<0.05 compared to control anti-miR. H, to test if ZO-1 or ZO-2 are direct targets of miR-146a or miR-106b keratinocytes were transfected with pmiR Target-ZO1-3′-UTR or pmiR Target-ZO2-3′-UTR firefly luciferase expression constructs and co-transfected with pRL-TK Renilla luciferase expression construct along with either miR-146a, miR106b or control mimics. I, expression of ZO-1 and ZO-2 mRNA in presence of miR-146a and miR-106b mimics. Data represent mean ± SD (n=3). *, p<0.05 compared to control transfected cells.

Figure 7. miRNA 146a and miR106b disrupts tight junction proteins in epidermis and increases trans epidermal water loss (TEWL) in skin

MiR-146a mimic, miR-106b mimic and non-targeting miRNA mimic (C. elegans miR-67 as negative control) were mixed in a cream and applied on a marked area on the dorsal skin of mice for 10 days. TEWL measurements were taken daily using Dermalab Series Skinlab Combo. The animals were sacrificed at day 11 and the skin where the mimic was applied was harvested for miRNA and immunohistochemistry (IHC) analysis. A, TEWL analysis of mice skin following delivery of miR-146a and miR-106b or control mimics. Data are mean ± SD (n=3), * p<0.05 compared to mice applied control miRNA mimic. B, quantitative Real Time PCR of skin where miR-146a and miR-106b mimic was applied. Data are mean ± SD (n=3), *p<0.05 compared to mice applied with control miRNA mimic. C, IHC images of mouse skin delivered with miR-146a and miR-106b mimic stained with tight Junction proteins ZO-1 and ZO-2 (green) and counterstained with DAPI (blue). The quantification of ZO-1 and ZO-2 has been provided in Fig S5. D, summary illustration depicting acute phase induction of biofilm-inducible miRs followed by silencing of ZO-1 & ZO-2 resulting in longer-term compromise of skin barrier function.