The development of an infected acute wound porcine model

D. Boone, MD; E. Braitman, MD; J. Afthinos, MD; C. Gendics, RN; E. Sordillo, MD, PhD; G. Todd, MD; J. Lantis, MD; St. Luke’s-Roosevelt Hospital Center, New York, NY

Introduction: To assess potential antimicrobial and pro-growth agents available for wound healing, we found it necessary to develop a medium size animal model for infected wounds. While there are many animal models for infection, most of them include a wound inhibiting therapy as an adjunct, which can alter the potential effects of tested therapies. In addition, most models leave no delay between inoculation and treatment, which makes them sub-optimal for assessing treatment of critical colonization.

Methods: A total of 12 animals (50-kg pigs) had four 6-cm in diameter full-thickness wounds created. The reservoir of the wound was filled with a mixture of Pseudomonas aeruginosa, coagulase negative Staphylococcus, and Bacteroides fragilis in a ratio of 2:2:1 at a concentration of 10⁷ cfu/mL. Standard gauze was then placed in the wound and covered with a bio-occlusive dressing. Quantitative biopsy cultures and semi-quantitative swab cultures were obtained at 72 hours; histological biopsies and digital images were also obtained at this point in time. At that point, therapeutic device trials were initiated.

Results: One animal suffered a post-operative fatality of unknown etiology. One animal was euthanized secondary to an unrecognized medical comorbidity. Of the 40 remaining wounds, all became clinically infected, with significant bacterial counts and histological markers of infection. Two of the animals (8 wounds) required systemic antibiotics prior to their first biopsy due to evidence of systemic infection, including fever and tachycardia in addition to grossly infected wounds; the wounds on these two animals were the only ones that failed to achieve the critical bacterial count of 10⁵ cfu/g of tissue.

Conclusions: We were able to create an effective model for invasive bacterial infection without systematically impairing the host. However, the classic teaching that systemic antibiotics do not significantly affect the local wound bacterial burden may not be true, based upon the findings from the two animals treated with systemic antibiotics.

Diagnosing chronic wound infection: comparison of routine cultures, quantitative microbiology, and real time PCR

Yelena Frankel, MD, MPH; Johan Melendez, MS; Lance Price, PhD; Jonathan Zenilman, MD; Gerald Lazarus, MD; Johns Hopkins University, Baltimore, MD

Background: Wounds account for > $15 billion in medical costs in the US. Diagnosing wound infection versus colonization in chronic wounds remains a major clinical challenge. Microbiology labs frequently report qualitative results (scant/moderate/heavy growth) without linkage to clinical outcome and often use a threshold of 10⁵ organisms/g. Reliable quantification of bacterial loads and pathogen speciation are critical to correlating changes in microbial population with healing rates.

Objectives: We evaluated biopsies from chronic wounds and compared quantitative and qualitative results. We determined microbiology by semiquantitative cultures in a clinical lab and quantitative cultures in a research lab. Species detection was compared between quantitative microbiology and real time polymerase chain reaction (RT-PCR).

Methods: 17 patients presenting to an outpatient teaching hospital wound center with suspected chronic wound infection were enrolled. Tissue was obtained from the leading wound edge using a 3-mm curette.

Results: By quant. microbiology 16/17 specimens were infected at > 10⁵ cfu/g. Nine and 7 of the bacterial organisms present were at 10⁷ cfu/g and 10⁸ cfu/g, respectively. In contrast, only 25% of the semi quant. cultures showed “heavy” growth. RT-PCR results were comparable to quantitative culture results. Most common pathogens included MRSA in 46% of the patients, Streptococcus group B in 22%, and Pseudomonas aeruginosa in 17%. Minimum tissue needed for reproducible measurements was 20 mg, easily obtained using a 3-mm curette and topical anesthesia, with little loss of tissue or discomfort for the patient.

Conclusion: Chronic wounds are highly complex ecosystems. Quantitative culture is more reproducible and reliable for determining bacterial load compared to routine semi quantitative cultures. Modern molecular methods are promising for characterizing wound bacterial populations. PCR analysis provides results within 24 hours and can detect difficult to culture organisms, including bacteria residing in biofilms, anaerobes, and microaerophilic organisms, providing insight into inflammation and healing.

Generation of transgenic mice that inducibly express beta ig-h3 in the epidermis

T. Kwak, MD; T. Yufit; J. Butmarc; S-J Kim; V. Falanga; Roger Williams Medical Center, Providence, RI

Increasing evidence suggests persistent phenotypic alterations in fibroblasts from non-healing human chronic wounds, which may result in faulty extracellular matrix deposition and keratinocyte migration. We have previously indicated that these cells are characterized by morphological changes, low proliferative potential, and unresponsiveness to TGF-beta 1. Recent work from our laboratory has shown a decreased expression of beta ig-h3 in chronic wounds and their fibroblasts compared to acute wounds. Beta ig-h3 is a transforming growth factor beta (TGF-beta) induced extracellular protein involved in a number of functions essential to cell adhesion, proliferation, and tumor invasion, but little is known of the specific role of beta ig-h3 in cutaneous wound healing. To establish a viable transgenic model for studying functions of beta ig-h3 in wound healing, we have generated transgenic mice, which allow focal induction of the beta ig-h3 transgene in the epidermis at different expression levels. Target transgenic mice that have a human beta ig-h3 gene fused with GAL4 binding sites upstream of the TATA promoter were generated. The transactivator mice were used that targeted the expression of GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAL4-DNA binding domain, with a human keratin 14 promoter. The target mice (TATA.beta ig-h3) and the transactivator mice (K14.GLp65) were mated to generate bigenic mice, and beta ig-h3 transgene expression was induced by topical application of RU 486 (mifepristone, a progesterone receptor antagonist). These inducible beta ig-h3 transgenic mice will be a novel method for better understanding of the role of beta ig-h3 in the pathogenesis of cutaneous wound healing.

Acknowledgement: NIH sponsored Center of Biomedical Research Excellence (COBRE) P20RR018757 and NIDDK 067836.

The Young Investigators Award is presented for outstanding achievement in clinical and laboratory research by a young investigator

To qualify, participants must be the first author of, and therefore, made significant contributions to the work; submit clinical or laboratory research that has not been previously published; be under the age of 40 -or- have an appointment at a rank of assistant professor or lower (fellows/residents included) to an academic institution (4 years or fewer) -or- be in the process of obtaining a terminal degree (MD/PhD); attend the SAWC/WHS and present the work.