Mast cells are critical for controlling the bacterial burden and the healing of infected wounds.

Skin wound infections are a significant health problem, and antibiotic resistance is on the rise. Mast cells (MCs) have been shown to contribute to host-defense responses in certain bacterial infections, but their role in skin wound superinfection is unknown. We subjected 2 MC-deficient mouse strains to Pseudomonas aeruginosa skin wound infection and found significantly delayed wound closure in infected skin wounds. This delay was associated with impaired bacterial clearance in the absence of MCs. Engraftment of MCs restored both bacterial clearance and wound closure. Bacterial killing was dependent on IL-6 released from MCs, and engraftment with IL-6-deficient MCs failed to control wound infection. Treatment with recombinant IL-6 enhanced bacterial killing and resulted in the control of wound infection and normal wound healing in vivo. Taken together, our results demonstrate a defense mechanism for boosting host innate immune responses, namely effects of MC-derived IL-6 on antimicrobial functions of keratinocytes.

Staphylococcus aureus biofilms release leukocidins to elicit extracellular trap formation and evade neutrophil-mediated killing.

Bacterial biofilms efficiently evade immune defenses, greatly complicating the prognosis of chronic infections. How methicillin-resistant Staphylococcus aureus (MRSA) biofilms evade host immune defenses is largely unknown. This study describes some of the major mechanisms required for S. aureus biofilms to evade the innate immune response and provides evidence of key virulence factors required for survival and persistence of bacteria during chronic infections. Neutrophils are the most abundant white blood cells in circulation, playing crucial roles in the control and elimination of bacterial pathogens. Specifically, here we show that, unlike single-celled populations, S. aureus biofilms rapidly skew neutrophils toward neutrophil extracellular trap (NET) formation through the combined activity of leukocidins Panton-Valentine leukocidin and γ-hemolysin AB. By eliciting this response, S. aureus was able to persist, as the antimicrobial activity of released NETs was ineffective at clearing biofilm bacteria. Indeed, these studies suggest that NETs could inadvertently potentiate biofilm infections. Last, chronic infection in a porcine burn wound model clearly demonstrated that leukocidins are required for "NETosis" and facilitate bacterial survival in vivo.

Analysis and Fine Specificity of the HCMV-Specific Cell-Free and Cell-Associated Antibody-Dependent Cellular Phagocytosis (ADCP) Responses in Lung Transplant Recipients.

Human Cytomegalovirus (HCMV) may cause severe infections in transplant recipients. HCMV-replication can be limited by HCMV-specific antibody responses. The impact of the antibody-dependent cellular phagocytosis (ADCP) on inhibition of HCMV-replication in natural infections has not been clarified. Therefore, we investigated the HCMV-specific ADCP response in a study cohort of lung-transplant recipients (LTRs) with different donor (D) and recipient (R) HCMV-serostatus. Follow-up plasma samples from 39 non/low-viremic and 36 highly viremic (>1000 HCMV copies/mL plasma) LTRs were collected for one (R+ LTRs) or two (D+/R- LTRs) years post-transplantation. The HCMV-specific ADCP responses were assessed by focal expansion assays (FEA) and flow-cytometry. In all LTRs, ADCP responses were detected against HCMV-infected cells and cell-free virions. When measured in fibroblasts as well as with cell-free virus, the HCMV-specific ADPC response was higher in LTRs than in HCMV-seropositive healthy controls. In D+/R- LTRs, a significant ADCP response developed over time after the receipt of an HCMV positive lung, and a level of <19 IE+ cells/focus in the FEA on fibroblasts was associated with further protection from high-level viremia. Taken together, a strong HCMV-specific ADCP response is elicited in transplant recipients, which may contribute to protection from high-level viremia in primary HCMV infection.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1ß secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1ß promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Evaluation of the immune responses following co-administration of PilQ and type b-flagellin from Pseudomonas aeruginosa in the burn mouse model.

Considering the increased antibiotic resistance of Pseudomonas aeruginosa, the evaluation of immune response against the antigens of this bacterium seems necessary. In this study, the protective efficacy and immunological properties of P. aeruginosa recombinant PilQ (r-PilQ) and type b-flagellin (FLB) proteins was evaluated in the burn mouse model of infection. The inbred BALB/c mice were immunized with r-PilQ and FLB antigens. To investigate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. After the final immunization, the IL-4, IFN-γ, and IL-17 cytokines level were examined in the spleen of non-challenged mice. Fifty days after lethal challenge, the survival rate and bacterial burden in the skin and other internal organs of experimental mice were assessed. The in vivo administration of r-PilQ, FLB and combined antigen resulted in a significant increase in the survival of mice (66%, 75%, and 83%, respectively) infected by the PAO1 strain of P. aeruginosa in the burn model of infection. Immunization of mice with r-PilQ and FLB mixture induced high titers of IL-4 and IL-17 cytokines compared to control groups (P < 0.05). The high titer of antisera raised against combined antigen was able to inhibit the systemic spread of the PAO1 strain from the site of infection to the internal organs. We concluded that the parallel role of IL-4 and IL-17 is necessary for elimination of the bacteria and promotion of survival in the immunized burn mice.

MC1R gene polymorphisms are associated with dysfunctional immune responses and wound infection after burn injury.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is associated with organ failure and infectious complications after major burn injury. Recent evidence has linked melanocortin signaling to anti-inflammatory and wound-repair functions, with mutations in the melanocortin 1 receptor (MC1R) gene leading to increased inflammatory responses. Our group has previously demonstrated that MC1R gene polymorphisms are associated with postburn hypertrophic scarring. Thus, we hypothesized that MC1R single nucleotide polymorphisms (SNPs) would be associated with increased burn-induced SIRS and increased infectious complications. METHODS: We performed a retrospective cohort study of adults (>18 y of age) admitted to our burn center with >20% total body surface area (TBSA) partial/full thickness burns between 2006 and 2013. We screened for five MC1R SNPs (V60L, V92M, R151C, R163Q, T314T) by polymerase chain reaction from genomic DNA isolated from blood samples. We performed a detailed review of each patient chart to identify age, sex, race, ethnicity, %TBSA burned, burn wound infections (BWIs), and 72-hr intravenous fluid volume, the latter a surrogate for a dysfunctional inflammatory response to injury. Association testing was based on multivariable regression. RESULTS: Of 106 subjects enrolled, 82 had complete data for analysis. Of these, 64 (78%) were male, with a median age of 39 and median burn size of 30% TBSA. A total of 36 (44%) subjects developed BWIs. The median total administered IV crystalloid in first 72h was 24.6 L. In multivariate analysis, the R151C variant allele was a significant independent risk factor for BWI (adjusted prevalence ratio 2.03; 95% CI: 1.21-3.39; P = 0.007), and the V60L variant allele was independently associated with increased resuscitation fluid volume (P = 0.021). CONCLUSIONS: This is the first study to demonstrate a significant association between genetic polymorphisms and a nonfatal burn-induced SIRS complication. Our findings suggest that MC1R polymorphisms contribute to dysfunctional responses to burn injury that may predict infectious and inflammatory complications.

Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro.

Skin quality outcome after skin grafting is adversely affected by wound bed inflammation. Neomycin, gentamicin, and other aminoglycoside antibiotics are known to modulate inflammation, and topical application affords the use of higher doses than are possible to use systemically. Previous data suggest that clinically relevant doses of neomycin, but not gentamicin, may impair angiogenesis, which is critical to the durable survival of skin grafts. The role of gentamicin at ultrahigh doses compared with clinically relevant neomycin doses in regulating inflammatory expression and angiogenesis has been examined. In a porcine skin replacement excisional wound model, continuous exposure to gentamicin increased anti-angiogenic and inflammatory expression at 7 days postgrafting. In in vitro studies, gentamicin also impaired angiogenesis in a human umbilical vein endothelial cell (HUVEC) tube formation model, increased the expression of the anti-angiogenic gene C-X-C motif chemokine 10 (CXCL10) in HUVECs and macrophages, and increased pro-inflammatory cytokine expression of macrophages in a dose-dependent manner. Neomycin exerted similar effects in vitro at clinically relevant doses on HUVEC tube formation and macrophage pro-inflammatory expression. CXCL10 was upregulated in macrophages, but did not exhibit a change in HUVECs with neomycin treatment. Ultrahigh doses of gentamicin and clinically relevant doses of neomycin affect inflammation and angiogenesis in in vivo and in vitro models. These findings suggest that topical administration of aminoglycosides have the potential to adversely influence early skin graft survival.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds.

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.

Immune Modulating Topical S100A8/A9 Inhibits Growth of Pseudomonas aeruginosa and Mitigates Biofilm Infection in Chronic Wounds.

Pseudomonas aeruginosa biofilm maintains and perturbs local host defense, hindering timely wound healing. Previously, we showed that P. aeruginosa suppressed S100A8/A9 of the murine innate host defense. We assessed the potential antimicrobial effect of S100A8/A9 on biofilm-infected wounds in a murine model and P. aeruginosa growth in vitro. Seventy-six mice, inflicted with a full-thickness burn wound were challenged subcutaneously (s.c.) by 106 colony-forming units (CFUs) of P. aeruginosa biofilm. Mice were subsequently randomized into two treatment groups, one group receiving recombinant murine S100A8/A9 and a group of vehicle controls (phosphate-buffered saline, PBS) all treated with s.c. injections daily for up to five days. Wounds were analyzed for quantitative bacteriology and contents of key inflammatory markers. Count of blood polymorphonuclear leukocytes was included. S100A8/A9-treatment ameliorated wound infection, as evaluated by quantitative bacteriology (p ≤ 0.05). In vitro, growth of P. aeruginosa was inhibited dose-dependently by S100A8/A9 in concentrations from 5 to 40 µg/mL, as determined by optical density-measurement (OD-measurement) and quantitative bacteriology. Treatment slightly augmented key inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), but dampened interferon-γ (IFN-γ) levels and blood polymorphonuclear count. In conclusion, topical S100A8/A9 displays remarkable novel immune stimulatory and anti-infective properties in vivo and in vitro. Importantly, treatment by S100A8/A9 provides local infection control. Implications for a role as adjunctive treatment in healing of chronic biofilm-infected wounds are discussed.

[DYNAMICS OF OCCURRENCE AND REGRESSION OF NEUTROPHIL EXTRACELLULAR TRAPS IN THE EXPERIMENTAL MODEL OF THE WOUND INFECTIOUS-INFLAMMATORY PROCESS].

The dynamics of the appearance and regression of neutrophilic extracellular traps (NETs) in the experimental model of the wound infectious-inflammatory process that was caused by infection of open wounds by microbes surrounding environment, was studied. It was established that on the second day from the start of the experiment a significant number of mature neutrophilic granulocytes show the ability to form NEТs. The number of NETs was reached its maximum by the 2th and 4th days from the beginning of the experiment, that was associated with maximum occurrence of symptoms of inflammation in the wound. The regression of the wound inflammatory process was associated with decreasing of the number of NETs to 7th day and the complete disappearance of NETs to 14th day of the experiment, which coincided with the healing of wounds.

Modelling the immune system response to epithelial wound infections.

In this paper, a cell-colony based formalism for the healing of superficial wounds is presented. The paper incorporates the migration, proliferation, and death of constituent cells, in the context of wound healing. The present study considers wound healing under ischemic conditions where a bacterial infection develops, which impairs the motility of the constituent cells. In this work, the performance of the immune response system is incorporated in the sense that migrating leukocyte are modelled which engulf the infectious pathogens. The model is based on both deterministic and stochastic principles. Simulation results are discussed in a biological context.

Cellular and molecular mechanisms of repair in acute and chronic wound healing.

A considerable understanding of the fundamental cellular and molecular mechanisms underpinning healthy acute wound healing has been gleaned from studying various animal models, and we are now unravelling the mechanisms that lead to chronic wounds and pathological healing including fibrosis. A small cut will normally heal in days through tight orchestration of cell migration and appropriate levels of inflammation, innervation and angiogenesis. Major surgeries may take several weeks to heal and leave behind a noticeable scar. At the extreme end, chronic wounds - defined as a barrier defect that has not healed in 3 months - have become a major therapeutic challenge throughout the Western world and will only increase as our populations advance in age, and with the increasing incidence of diabetes, obesity and vascular disorders. Here we describe the clinical problems and how, through better dialogue between basic researchers and clinicians, we may extend our current knowledge to enable the development of novel potential therapeutic treatments.

A Twenty-Three-Year-Old Man with a Chronic Anterior Chest Wall Wound, Eosinophilia, and Elevated IgE-Job's Syndrome.

A 23-year-old man presented with a chronic anterior chest wall wound. Previously he had a split thickness skin graft in the area in where the wound bed had become infected, developing a thick purulent drainage. The infected skin graft was excised. Histologic examination of the failed graft revealed skin with surface ulceration, focal abscess formation, deep penetrating acute and chronic inflammation with numerous eosinophils, and granulomatous changes demonstrating a foreign body-type reaction to fungal hyphae (highlighted by periodic acid-Schiff staining, Figures 1A-E). The patient's past medical history included scoliosis, acute lower back pain, right shoulder degenerative joint disease, atopic dermatitis, lymphadenitis, rhonchi, insomnia, depression, and a long history of recurrent infections, particularly cutaneous staphylococcal and candida albicans infections, often accompanied by a purulent drainage. Review of the patient's laboratory studies revealed chronically elevated alkaline phosphatase, with highly elevated serum IgE (2,922 IU/ml) and eosinophilia (925/µL3) since childhood. Other lab studies were unremarkable, except for episodic elevations of the white blood cell count. The patient's family history was largely unremarkable and the patient's parents and siblings had no histories of unusual infections.

A novel treatment in X-linked agammaglobulinaemia - hyperbaric oxygen therapy in refractory chronic wounds.

Chronic wounds are a rare complication of X-linked agammaglobulinaemia (XLA). Fastidious organisms such as helicobacter bills have been reported in XLA with chronic wounds but sterile chronic wounds also occur. Hyperbaric Oxygen Therapy has been used in chronic wounds but has not previously been reported in primary antibody deficiencies. We present a case of a chronic wound in a patient with XLA refractory to antimicrobial therapy that made a remarkable recovery following Hyperbaric Oxygen Therapy.

Contrasting host immuno-inflammatory responses to bacterial challenge within venous and diabetic ulcers.

Within chronic wounds, the relationship between the clinical diagnosis of infection and bacterial/immuno-inflammatory responses is imprecise. This study prospectively examined the interrelationship between clinical, microbiological, and proinflammatory biomarker levels between chronic venous leg ulcers (CVLUs) and diabetic foot ulcers (DFUs). Wound swabs and fluids were collected from CVLUs (n = 18) and DFUs (n = 15) and diagnosed clinically as noninfected or infected; and qualitative/quantitative microbiology was performed. CVLU and DFU fluids were also analyzed for cytokine, growth factor, receptor, proteinase/proteinase inhibitor; and oxidative stress biomarker (protein carbonyl, malondialdehyde, and antioxidant capacity) levels. While no correlations existed between clinical diagnosis, microbiology, or biomarker profiles, increasing bacterial bioburden (≥10(7) colony-forming unit/mL) was associated with significant alterations in cytokine, growth factor, and receptor levels. These responses contrasted between ulcer type, with elevated and decreased cytokine, growth factor, and receptor levels in CVLUs and DFUs with increasing bioburden, respectively. Despite proteinase biomarkers exhibiting few differences between CVLUs and DFUs, significant elevations in antioxidant capacities correlated with increased bioburden in CVLU fluids, but not in DFUs. Furthermore, oxidative stress biomarker levels were significantly elevated in all DFU fluids compared with CVLUs. This study provides further insight into the contrasting disease-specific host responses to bacterial challenge within infected CVLUs and DFUs.

Prostaglandin E2 induces oncostatin M expression in human chronic wound macrophages through Axl receptor tyrosine kinase pathway.

Monocytes and macrophages (m) are plastic cells whose functions are governed by microenvironmental cues. Wound fluid bathing the wound tissue reflects the wound microenvironment. Current literature on wound inflammation is primarily based on the study of blood monocyte-derived macrophages, cells that have never been exposed to the wound microenvironment. We sought to compare pair-matched monocyte-derived macrophages with m isolated from chronic wounds of patients. Oncostatin M (OSM) was differentially overexpressed in pair-matched wound m. Both PGE2 and its metabolite 13,14-dihydro-15-keto-PGE2 (PGE-M) were abundant in wound fluid and induced OSM in wound-site m. Consistently, induction of OSM mRNA was observed in m isolated from PGE2-enriched polyvinyl alcohol sponges implanted in murine wounds. Treatment of human THP-1 cell-derived m with PGE2 or PGE-M caused dose-dependent induction of OSM. Characterization of the signal transduction pathways demonstrated the involvement of EP4 receptor and cAMP signaling. In human m, PGE2 phosphorylated Axl, a receptor tyrosine kinase (RTK). Axl phosphorylation was also induced by a cAMP analogue demonstrating interplay between the cAMP and RTK pathways. PGE2-dependent Axl phosphorylation led to AP-1 transactivation, which is directly implicated in inducible expression of OSM. Treatment of human m or mice excisional wounds with recombinant OSM resulted in an anti-inflammatory response as manifested by attenuated expression of endotoxin-induced TNF-α and IL-1ß. OSM treatment also improved wound closure during the early inflammatory phase of healing. In summary, this work recognizes PGE2 in the wound fluid as a potent inducer of m OSM, a cytokine with an anti-inflammatory role in cutaneous wound healing.

Neutrophil survival and c-kit(+)-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.

Polymorphonuclear neutrophils (PMNs) are critical for the formation, maintenance, and resolution of bacterial abscesses. However, the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound, combined with real-time imaging of genetically tagged PMNs, we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound, where their lifespan was markedly extended. A continuous rise in wound PMN number, which was not accounted for by trafficking from the bone marrow or by prolonged survival, was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound, where they proliferated and formed mature PMNs. Furthermore, by blocking their recruitment with an antibody to c-kit, which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival, we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound, but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells.

Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin graft donor sites.

Cold atmospheric plasma has already been shown to decrease the bacterial load in chronic wounds. However, until now it is not yet known if plasma treatment can also improve wound healing. We aimed to assess the impact of cold atmospheric argon plasma on the process of donor site healing. Forty patients with skin graft donor sites on the upper leg were enrolled in our study. The wound sites were divided into two equally sized areas that were randomly assigned to receive either plasma treatment or placebo (argon gas) for 2 minutes. Donor site healing was evaluated independently by two blinded dermatologists, who compared the wound areas with regard to reepithelialization, blood crusts, fibrin layers, and wound surroundings. From the second treatment day onwards, donor site wound areas treated with plasma (n = 34) showed significantly improved healing compared with placebo-treated areas (day 1, p = 0.25; day 2, p = 0.011; day 3, p < 0.001; day 4, p < 0.001; day 5, p = 0.004; day 6, p = 0.008; day 7, p = 0.031). Positive effects were observed in terms of improved reepithelialization and fewer fibrin layers and blood crusts, whereas wound surroundings were always normal, independent of the type of treatment. Wound infection did not occur in any of the patients, and no relevant side effects were observed. Both types of treatment were well tolerated. The mechanisms contributing to these clinically observed effects should be further investigated.

Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model.

Chronic wounds are presumed to persist in the inflammatory state, preventing healing. Emerging evidence indicates a clinical impact of bacterial biofilms in soft tissues, including Pseudomonas aeruginosa (PA) biofilms. To further investigate this, we developed a chronic PA biofilm wound infection model in C3H/HeN and BALB/c mice. The chronic wound was established by an injection of seaweed alginate-embedded P. aeruginosa PAO1 beneath a third-degree thermal lesion providing full thickness skin necrosis, as in human chronic wounds. Cultures revealed growth of PA, and both alginate with or without PAO1 generated a polymorphonuclear-dominated inflammation early after infection. However, both at days 4 and 7, there were a more acute polymorphonuclear-dominated and higher degree of inflammation in the PAO1 containing group (p < 0.05). Furthermore, PNA-FISH and supplemented DAPI staining showed bacteria organized in clusters, resembling biofilms, and inflammation located adjacent to the PA. The chronic wound infection showed a higher number of PAO1 in the BALB/c mice at day 4 after infection as compared to C3H/HeN mice (p < 0.006). In addition, a higher concentration of interleukin-1beta in the chronic wounds of BALB/c mice was observed at day 7 (p < 0.02), despite a similar number of bacteria in the two mouse strains. The present study succeeded in establishing a chronic PA biofilm infection in mice. The results showed an aggravating impact of local inflammation induced by PA biofilms. In conclusion, our findings indicate that improved infection control of chronic wounds reduces the inflammatory response and may improve healing.

Novel models for bacterial colonization and infection of full-thickness wounds in rats.

An animal model is needed to study the pathophysiology of wound infections; however, an animal model that is reproducible and clinically relevant has not previously been available. In addition, an animal model of wound colonization generated in a manner similar to the wound infection model would be useful. Here, we describe new animal models of the wound infection continuum for the characterization of essential host-pathogen relationships. We determined the conditions needed to establish rat models of stable wound colonization and infection, without the use of disturbing factors (e.g., foreign bodies or induction of diabetes mellitus). We found that the age of the rats, bacterial inoculum size, and wound location were important elements in generating reproducible, obvious, spreading wound infections. We inoculated approximately 6-month-old rats with 2.06 × 10(9) or 4.12 × 10(9) colony-forming units of Pseudomonas aeruginosa to generate the wound colonization and wound infection models, respectively. Wounds were made 2 cm cranial to the greater trochanter. These clinically relevant and highly reproducible animal models can be used to investigate the mechanisms of wound infection and monitor the effect of therapeutic agents in vivo.