14 research outputs found

    Inflammatory and Antimicrobial Responses to Methicillin-Resistant <i>Staphylococcus aureus</i> in an <i>In Vitro</i> Wound Infection Model

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    <div><p>Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an <i>in vitro</i> thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human ╬▓-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.</p> </div

    Morphology, basement membrane protein collagen type IV and keratin expression in control and thermal wounded HSEs.

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    <p>Shown are cross-sections of the paraffin embedded control and thermally wounded HSEs that were stained with either (<b>a</b>-<b>b</b>) hematoxylin and eosin (H&E), or immunolabeled for (<b>c</b>-<b>d</b>) collagen type IV, (e-f) K16, or (<b>g</b>-<b>h</b>) K17. Control HSEs (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>) and HSEs 48 hours after thermal wounding (<b>b</b>,<b>d</b>,<b>f</b>,<b>h</b>). Arrows indicate dead epidermis and fibroblasts in the dermis. * indicates re-epithelialization. Pictures are representative for 3 NHK donors. Scale bars = 50 ╬╝m.</p

    Course of MRSA growth on control and thermal wounded HSEs.

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    <p>The number of (<b>a</b>) detachable MRSA and (<b>b</b>) of tightly adherent MRSA to HSEs were assessed by the colony forming unit (CFU) assay (mean CFU/ml). Control (open circles) and thermal wounded (closed circles) HSEs were exposed for one hour to 1x10<sup>5</sup> CFU MRSA, next the non-adherent bacteria were removed and at 24 and 48 hours thereafter. Results show the mean ┬▒ SEM. (<b>c</b>) Hematoxylin staining of thermal wounded HSEs exposed to MRSA for 48 hours. Arrows indicate bacterial biofilm and (*) indicates re-epithelialization. N=3. Scale bar = 50 ╬╝m.</p

    Antimicrobial protein expression by HSEs after thermal wounding and MRSA exposure.

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    <p>Expression of (<b>a</b>) hBD-2 mRNA, (<b>b</b>) hBD-3 mRNA, (<b>c</b>) RNase-7 mRNA, (<b>d</b>) hBD-2 protein and (<b>e</b>) hBD-3 protein by control, thermal wounded and/or MRSA colonized HSEs. *P <0.05. Bars are represented as the median. (<b>f</b>) hBD-2 and hBD-3 expression in normal human skin, control HSEs, thermal wounded HSEs, MRSA colonized HSEs, and thermal wounded and MRSA colonized HSEs. N=7-9. Scale bars = 50 ┬Ám.</p

    TLR2 and TLR3 expression by thermal wounded and/or MRSA exposed HSEs.

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    <p>The expression of TLR2 was assessed by quantitative PCR. (<b>a</b>) TLR2 mRNA expression in normalized fold change and (<b>b</b>) TLR3 by thermal wounded and/or MRSA exposed HSEs after 24 hours as compared to control HSEs. Horizontal lines represent the median. *P<0.05, as compared to control HSEs. (<b>c</b>) TLR2 protein expression in cross-sections of control HSEs. (<b>d</b>) Thermal wounded HSEs. N=6-8. * indicates re-epithelialization. Scale bars = 50 ╬╝m.</p

    Eradication of MRSA from thermal wounded HSEs by mupirocin.

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    <p>(<b>a</b>) The number of detachable (white bars) and adherent bacteria (black bars) before and after mupirocin treatment were determined using the CFU assay. Results are expressed as mean CFU per HSE ┬▒ SEM. The dotted line represents the lower limit of detection. *P<0.05, as compared to untreated HSEs. (<b>b</b>) PAS/Alcian blue stained cross-section of mupirocin-exposed thermal wounded HSE that was infected by MRSA. * indicates re-epithelialization. N=3. Scale bar = 50 ╬╝m.</p