Akhil K. Seth

Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing

A growing body of evidence suggests that in addition to hypoxia, ischemia‐reperfusion injury, and intrinsic host factors, bacterial biofilms represent a fourth major pillar in chronic wound pathogenesis. Given that most studies to date rely on in vitro or observational clinical data, our aim was to develop a novel, quantitative animal model enabling further investigation of the biofilm hypothesis in vivo. Dermal punch wounds were created in New Zealand rabbit ears, and used as uninfected controls, or inoculated with green fluorescent protein‐labeled Staphylococcus aureus to form wounds with bacteria predominantly in the planktonic or biofilm phase. Epifluorescence and scanning electron microscopy revealed that S. aureus rapidly forms mature biofilm in wounds within 24 hours of inoculation, with persistence of biofilm viability over time seen through serial bacterial count measurement and laser scanning confocal imaging at different time points postwounding and inoculation. Inflammatory markers confirmed that the biofilm phenotype creates a characteristic, sustained, low‐grade inflammatory response, and that over time biofilm impairs epithelial migration and granulation tissue in‐growth, as shown histologically. We have established and validated a highly quantitative, reproducible in vivo biofilm model, while providing evidence that the biofilm phenotype specifically contributes to profound cutaneous wound healing impairment. Our model highlights the importance of bacterial biofilms in chronic wound pathogenesis, providing an in vivo platform for further inquiry into the basic biology of bacterial biofilm–host interaction and high‐throughput testing of antibiofilm therapeutics.

Outcomes of tissue expander/implant breast reconstruction in the setting of prereconstruction radiation.

Background: Although the effects of postoperative radiation on tissue expander breast reconstruction are well documented, few data exist regarding the effects of prereconstruction radiation. This study evaluates the outcomes of tissue expander breast reconstruction in women with prereconstruction radiation. Methods: This study retrospectively evaluated two treatments: (1) mastectomy without reconstruction followed by postoperative radiation and delayed reconstruction (10 patients) and (2) failed breast-conserving therapy (lumpectomy plus radiotherapy) necessitating mastectomy and immediate reconstruction (66 patients). Procedures were performed at Northwestern Memorial Hospital between August of 1999 and July of 2008. Average follow-up was 35 months. Results: In both groups, approximately 60 percent of patients successfully completed two stages of reconstruction. Overall complication rates, including major and minor complications, were 70 percent per reconstruction (37 percent first stage, 45 percent second stage) for immediate reconstruction and 50 percent per reconstruction (20 percent first stage, 38 percent second stage) for delayed reconstruction. No differences in complication rates were observed based on age, smoking status, body mass index, or timing between radiation and surgery (p > 0.05). Conclusions: When discussing expander/implant reconstruction with patients who have a history of prior breast radiotherapy, a frank discussion of the risks, benefits, and alternatives should occur. If a 40 percent total explantation or conversion to flap rate is truly understood by the patient, and if immediate autologous breast reconstruction is to be avoided, then the patient may proceed with tissue expander breast reconstruction. For patients who wish to avoid additional scars or more invasive procedures, however, this study demonstrates that they have a 60 percent chance of success. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Utility of acellular dermis-assisted breast reconstruction in the setting of radiation: a comparative analysis.

Background: The role of acellular dermis in immediate prosthetic breast reconstruction remains unclear, particularly within a radiated field. The authors evaluated and compare outcomes following reconstruction with and without acellular dermis, and analyzed patients exposed to radiation therapy. Methods: Retrospective review of 417 consecutive patients (592 breasts) treated from January of 2006 to October of 2008 at one institution was performed. Relevant patient characteristics and follow-up were recorded. Complications were categorized by type and end outcome, including nonoperative, operative, or explantation. Both groups had comparable follow-up (acellular dermis, 23.2 ± 8.9 months; no acellular dermis, 24.4 ± 12.7 months; p = 0.23). Appropriate statistical analyses, including multiple regression, were performed. Results: Acellular dermis patients (n = 199 breasts) had larger body mass indexes (p = 0.0001) and more nipple-sparing mastectomies (p = 0.04) than non–acellular dermis patients (n = 393 breasts). Breasts with acellular dermis had larger intraoperative fill volumes (p < 0.0001) and decreased postoperative expansions (p = 0.02), but no decrease in time to implant exchange. There were no significant differences in complication profiles between acellular dermis and non–acellular dermis breasts, after adjusting for other relevant patient variables on regression analysis. After stratifying patients by exposure to radiation, acellular dermis breasts had a decreased risk of all complications related to radiation as compared with non–acellular dermis breasts. Conclusions: This study suggests that acellular dermis does not adversely affect complication rates following prosthetic breast reconstruction. It may be advantageous, however, in select patients, particularly those undergoing postoperative radiation therapy. Therefore, the choice to use acellular dermis does not compromise outcomes but should be individualized to each patient. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Bacteriophage therapy for Staphylococcus aureus biofilm-infected wounds: a new approach to chronic wound care.

Background: Bacterial biofilms, which are critical mediators of chronic wounds, remain difficult to treat with traditional methods. Bacteriophage therapy against biofilm has not been rigorously studied in vivo. The authors evaluate the efficacy of a species-specific bacteriophage against Staphylococcus aureus biofilm–infected wounds using a validated, quantitative, rabbit ear model. Methods: Six-millimeter dermal punch wounds in New Zealand rabbit ears were inoculated with wild-type or mutant, biofilm-deficient S. aureus. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Wounds were left untreated, or treated every other day with topical S. aureus–specific bacteriophage, sharp débridement, or both. Histologic wound healing and viable bacterial count measurements, and scanning electron microscopy were performed following harvest. Results: Wild-type S. aureus biofilm wounds demonstrated no differences in healing or viable bacteria following bacteriophage application or sharp débridement alone. However, the combination of both treatments significantly improved all measured wound healing parameters (p < 0.05) and reduced bacteria counts (p = 0.03), which was confirmed by scanning electron microscopy. Bacteriophage treatment of biofilm-deficient S. aureus mutant wounds alone also resulted in similar trends for both endpoints (p < 0.05). Conclusions: Bacteriophages can be an effective topical therapy against S. aureus biofilm–infected wounds in the setting of a deficient (mutant) or disrupted (débridement) biofilm structure. Combination treatment aimed at disturbing the extracellular biofilm matrix, allowing for increased penetration of species-specific bacteriophages, represents a new and potentially effective approach to chronic wound care. These results establish principles for biofilm therapy that may be applied to several different clinical and surgical problems.

In vivo modeling of biofilm-infected wounds: a review.

Chronic wounds continue to represent a difficult and complex problem for both patients and healthcare providers. Bacterial biofilms represent a critical component of nonhealing wounds, utilizing several different mechanisms to inhibit innate inflammatory pathways and resist traditional therapeutics. Although in vitro biofilm systems have been well described and studied, understanding the intricacies of wound biofilm pathology requires appropriate in vivo models to understand the interactions between bacteria and host. In an effort to clarify the available literature, this review describes and critically evaluates all of the in vivo wound biofilm models currently published to-date, including model advantages and clinical applicability. We will also address the need for continued therapeutic development and testing using these currently available in vivo models.

Comparative Analysis of Single-Species and Polybacterial Wound Biofilms Using a Quantitative, In Vivo, Rabbit Ear Model

Introduction The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model. Methods Six-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10∧6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1β, TNF-α) expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM). For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections. Results Bacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001). SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01), and increased expression of IL-1β and TNF-α (p<0.05). In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01) with decreased host cytokine expression (p<0.01), despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds. Conclusions This study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The increased virulence of polybacterial biofilm appears dependent on the combined pathogenicity of each species, verified using a mutant strain. These data suggest that individual bacterial species can interact synergistically within a single biofilm structure.

Human Acellular Dermis versus Submuscular Tissue Expander Breast Reconstruction: A Multivariate Analysis of Short-Term Complications

Background Acellular dermal matrix (ADM) allografts and their putative benefits have been increasingly described in prosthesis based breast reconstruction. There have been a myriad of analyses outlining ADM complication profiles, but few large-scale, multi-institutional studies exploring these outcomes. In this study, complication rates of acellular dermis-assisted tissue expander breast reconstruction were compared with traditional submuscular methods by evaluation of the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) registry. Methods Patients who underwent immediate tissue expander breast reconstruction from 2006-2010 were identified using surgical procedure codes. Two hundred forty tracked variables from over 250 participating sites were extracted for patients undergoing acellular dermis-assisted versus submuscular tissue expander reconstruction. Thirty-day postoperative outcomes and captured risk factors for complications were compared between the two groups. Results A total of 9,159 patients underwent tissue expander breast reconstruction; 1,717 using acellular dermis and 7,442 with submuscular expander placement. Total complications and reconstruction related complications were similar in both cohorts (5.5% vs. 5.3%, P=0.68 and 4.7% vs. 4.3%, P=0.39, respectively). Multivariate logistic regression revealed body mass index and smoking as independent risk factors for reconstructive complications in both cohorts (P<0.01). Conclusions The NSQIP database provides large-scale, multi-institutional, independent outcomes for acellular dermis and submuscular breast reconstruction. Both thirty-day complication profiles and risk factors for post operative morbidity are similar between these two reconstructive approaches.

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm-containing chronic wounds.

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm‐impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll‐like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild‐type mice. However, diabetic biofilm‐containing wounds had significantly less TLR 2, TLR 4, interleukin‐1β, and tumor necrosis factor‐α expression than wild‐type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log‐fold greater bacterial burden and significant delay of wound epithelization for biofilm‐impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.

Long-term outcomes following fat grafting in prosthetic breast reconstruction: A comparative analysis

Background: Autologous fat grafting has become a common technique for optimizing aesthetic outcomes following breast reconstruction. Its long-term oncologic implications, however, remain unclear. The authors evaluated long-term outcomes following tissue expander breast reconstruction with and without fat grafting. Methods: A retrospective review was performed of consecutive patients undergoing mastectomy with immediate tissue expander reconstruction from April of 1998 to August of 2008. Demographic, operative, oncologic, and postoperative factors were recorded, including the use of fat grafting. Mean follow-up was 42.1 ± 28.8 and 43.6 ± 27.2 months for non–fat-grafting and fat-grafting patients, respectively (p = 0.63), including 24.8 ± 5.9 months after the first fat-grafting procedure. Fishers exact test, t test, and regression analysis were used for statistics. Results: A review of 886 patients (n = 1202 breasts) revealed no significant differences in demographics, operative characteristics, tumor staging, or radiation therapy exposure between fat-grafting (n = 90 breasts) and non–fat-grafting (n = 1112 breasts) patients. Ninety-nine fat-grafting procedures were performed an average of 18.3 months after reconstruction, with one complication (fat necrosis). Grafting did not affect local tumor recurrence or survival when compared with non–fat-grafted breasts. Complication following reconstruction, including a poor cosmetic result, was an independent predictor of undergoing subsequent fat grafting (p < 0.0001). Conclusions: The analysis suggests that fat grafting after breast reconstruction does not adversely affect local tumor recurrence or survival on long-term follow-up. Autologous fat grafting can be used as an aesthetic adjunct to prosthetic reconstruction with minimal complications. These results also indicate the need for multi-institutional, prospective studies to definitively establish its oncologic safety. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Quantitative Comparison and Analysis of Species-Specific Wound Biofilm Virulence Using an In Vivo, Rabbit-Ear Model

BACKGROUND Although bacterial biofilm is recognized as an important contributor to chronic wound pathogenesis, differences in biofilm virulence between species have never been studied in vivo. STUDY DESIGN Dermal punch wounds in New Zealand white rabbit ears were inoculated with Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa, or left uninfected as controls. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Virulence was assessed by measurement of histologic wound healing and host inflammatory mediators. Scanning electron microscopy (SEM) and bacterial counts verified biofilm viability. Extracellular polymeric substance (EPS)-deficient P aeruginosa was used for comparison. RESULTS SEM confirmed the presence of wound biofilm for each species. P aeruginosa biofilm-infected wounds showed significantly more healing impairment than uninfected, K pneumoniae, and S aureus (p < 0.05), while also triggering the largest host inflammatory response (p < 0.05). Extracellular polymeric substance-deficient P aeruginosa demonstrated a reduced impact on the same quantitative endpoints relative to its wild-type strain (p < 0.05). CONCLUSIONS Our novel analysis demonstrates that individual bacterial species possess distinct levels of biofilm virulence. Biofilm EPS may represent an integral part of their distinct pathogenicity. Rigorous examination of species-dependent differences in biofilm virulence is critical to developing specific therapeutics, while lending insight to the interactions within clinically relevant, polybacterial biofilms.