Wound Bacterial Load Research Articles

Safety and efficacy of a phage cocktail on murine wound infections caused by carbapenem-resistant Klebsiella pneumoniae

ObjectivesCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading pathogen causing difficult-to-treat, healthcare-associated wound infections. Phages are an alternative approach against CRKP. This study established murine wound infection models with a CRKP clinical strain of sequence type 11 and capsular type KL64, which is the dominant type in China, carrying genes encoding KPC-2 and NDM-1 carbapenemases. MethodsA cocktail was made comprising three lytic phages of different viral families against the strain. The phage cocktail restricted bacterial growth for 10 hours in vitro. The efficacy and safety of the phage cocktail in treating a murine wound CRKP infection were then evaluated. Mice were randomly assigned into four groups (16 for each) comprising a phage treatment group, infected with bacteria and 30 minutes later with phages, and three control groups administered with PBS (negative control), bacteria (infection control), or phages (phage control) on the wound. Wound tissues were processed for counting bacterial loads on days 1, 3, and 7 post-infection and examined for histopathological change on days 3 and 7. Two remaining mice in each group were monitored for wound healing until day 14. ResultsCompared with the infection control group, the wound bacterial load in the phage treatment group decreased by 4.95 × 102 CFU/g (> 100-fold; P < 0.05) at day 7 post-treatment, and wounds healed on day 10, as opposed to day 14 in the infection control group. No adverse events associated with phages were observed. ConclusionThe phage cocktail significantly reduced the wound bacterial load and promoted wound healing with good safety.

Bacteriophage therapy as an alternative technique for treatment of multidrug-resistant bacteria causing diabetic foot infection

Diabetic foot ulcer (DFU) represented the most feared diabetic complication that caused the hospitalization of the diabetic patient. DFU was usually characterized with delayed healing as the diabetic neuropathy, angiopathy, and ulcer concomitant infections, among them, are multidrug-resistant (MDR) bacteria that emphasized the clinical importance for developing new therapeutic strategy with safe and effective alternatives for the antibiotics to overcome DFU-MDR bacterial infection. Bacteriophage therapy was considered a novel approach to eradicate the MDR, but its role in the polymicrobial infection of the DFU remains elusive. Thus, the current work was designed to investigate the effect of the topical application of the phage cocktail on the healing of the diabetic wound infected with clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella variicola, Escherichia coli, and Proteus mirabilis. Bacterial isolation was performed from clinical hospitalized and non-hospitalized cases of DFU, identified morphologically, biochemically, molecularly via 16 s rRNA sequencing, and typed for the antibiotic resistance pattern. Moreover, phages were isolated from the aforementioned clinical isolates and identified with electron microscope. Forty-five adult male Sprague–Dawley rats were assigned in 3 groups (15 rats each), namely, the diabetic infected wound group, diabetic infected wound ceftriaxone-treated group, and the diabetic infected wound phage cocktail-treated group. The results revealed that phage cocktail had a superior effect over the ceftriaxone in wound healing parameters (wound size, wound index, wound bacterial load, and mRNA expression); wound healing markers (Cola1a, Fn1, MMP9, PCNA, and TGF-β); inflammatory markers (TNF-α, NF-κβ, IL-1β, IL-8, and MCP-1); anti-inflammatory markers (IL-10 and IL-4); and diabetic wound collagen deposition; and also the histomorphic picture of the diabetic infected wound. Based on the current findings, it could be speculated that phage therapy could be considered a novel antibiotic substitute in the DFU with MDR-polymicrobial infection therapeutic strategies.

Antimicrobial efficacy and toxicity of novel CAMPs against P. aeruginosa infection in a murine skin wound infection model

BackgroundTreatment of P. aeruginosa wound infection is challenging due to its inherent and acquired resistance to many conventional antibiotics. Cationic antimicrobial peptides (CAMPs) with distinct modes of antimicrobial action have been considered as the next-generation therapeutic agents. In the present study, a murine skin surgical wound infection model was used to evaluate the in vivo toxicity and efficacy of two newly designed antimicrobial peptides (CAMP-A and CAMP-B), as chemotherapeutic agents to combat P. aeruginosa infection.ResultsIn the first trial, topical application of CAMPs on the wounds at a dose equivalent to 4 × MIC for 7 consecutive days did not cause any significant changes in the physical activities, hematologic and plasma biochemical parameters, or histology of systemic organs of the treated mice. Daily treatment of infected wounds with CAMP-A and CAMP-B for 5 days at a dose equivalent to 2× MIC resulted in a significant reduction in wound bacterial burden (CAMP-A: 4.3 log10CFU/g of tissue and CAMP-B: 5.8 log10CFU/g of tissue), compared to that of the mock-treated group (8.1 log10CFU/g of tissue). Treatment with CAMPs significantly promoted wound closure and induced epidermal cell proliferation. Topical application of CAMP-A on wounds completely prevented systemic dissemination of P. aeruginosa while CAMP-B blocked systemic infection in 67% of mice and delayed the onset of systemic infection by at least 2 days in the rest of the mice (33%). In a second trial, daily application of CAMP-A at higher doses (5× MIC and 50× MIC) didn’t show any significant toxic effect on mice and the treatments with CAMP-A further reduced wound bacterial burden (5× MIC: 4.5 log10CFU/g of tissue and 50× MIC: 3.8 log10CFU/g of tissue).ConclusionsThe data collectively indicated that CAMPs significantly reduced wound bacterial load, promoted wound healing, and prevented hepatic dissemination. CAMP-A is a promising alternative to commonly used antibiotics to treat P. aeruginosa skin infection.

Bactericidal activity of 3D-printed hydrogel dressing loaded with gallium maltolate.

Chronic wounds are projected to reach epidemic proportions worldwide because of the aging population and the increasing incidence of diabetes. Despite extensive research, infection remains one of the leading sources of complications in chronic wounds, resulting in improper healing, biofilm formation, and lower extremity amputation. To address the limitations of standard treatments, we have developed a hydrogel wound dressing with self-tuning moisture control that incorporates a novel antimicrobial agent to eliminate and prevent infection. 3D-printing of a hydrogel dressing with dual porosity resulted in a new dressing with greater flexibility, increased water uptake, and more rapid swelling than bulk hydrogel dressings. Additionally, gallium maltolate (GaM) was incorporated into the dressing to investigate the efficacy of this antimicrobial agent. Loading profiles, release kinetics, and the bactericidal activity against Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus) of GaM were investigated in vitro to identify target profiles that supported infection control. Finally, GaM-loaded hydrogel dressings were evaluated in vivo, utilizing a murine splinted-wound model that was inoculated with S. aureus. In comparison to an untreated control, GaM dressings markedly reduced the wound bacterial load without compromising wound closure rates. Overall, this work demonstrates the utility of a 3D-printed hydrogel dressing as an antimicrobial dressing to control infection in chronic wounds.

Polyvinyl alcohol nanofiber formulation of the designer antimicrobial peptide APO sterilizes Acinetobacter baumannii-infected skin wounds in mice.

Native and designer cationic antimicrobial peptides are increasingly acknowledged as host defense molecules rather than true antimicrobials. Due to their ability to activate the innate immune system, these structures are used to treat uninfected and bacterially-infected wounds, including those harboring Acinetobacter baumannii. Previously we documented that when administered intramuscularly or topically in liquid formulations, the proline-rich host defense peptide dimer A3-APO accelerates uninfected wound re-epithelization and eliminates systemic and local A. baumannii, methicillin-resistant Staphylococcus aureus and other pathogen load from infected lesions better than conventional antibiotics. In the current study we sought to produce and characterize a novel delivery system, suitable for immediate and convenient application in non-hospital environments. The APO monomer was incorporated into polyvinyl alcohol nanofibers and the complex was polymerized into a solid patch dressing. Mice were subjected to skin abrasion where the wounds were either left uninfected or were inoculated with a near lethal dose of multidrug resistant A. baumannii strain. Analyzed after 3 days, APO monomer-containing patches improved wound appearance significantly better than polymer patches without antibiotics. When compared to colistin, the APO patches accelerated wound healing, and statistically significantly reduced wound size and wound bacterial load. The in vivo antimicrobial effect was more extensive than after intramuscular administration of the peptide drug, by using only one tenth of the active pharmaceutical ingredient. These data suggest that the APO monomer-impregnated nanofiber dressing can be developed as an economical first-line treatment option to skin injuries in general and battlefield burn and blast injuries in particular.

Autofluorescence imaging device for real-time detection and tracking of pathogenic bacteria in a mouse skin wound model: preclinical feasibility studies.

Bacterial infection significantly impedes wound healing. Clinical diagnosis of wound infections is subjective and suboptimal, in part because bacteria are invisible to the naked eye during clinical examination. Moreover, bacterial infection can be present in asymptomatic patients, leading to missed opportunities for diagnosis and treatment. We developed a prototype handheld autofluorescence (AF) imaging device (Portable Real-time Optical Detection, Identification and Guidance for Intervention - PRODIGI) to noninvasively visualize and measure bacterial load in wounds in real time. We conducted preclinical pilot studies in an established nude mouse skin wound model inoculated with bioluminescent Staphylococcus aureus bacteria. We tested the feasibility of longitudinal AF imaging for in vivo visualization of bacterial load in skin wounds, validated by bioluminescence imaging. We showed that bacteria (S. aureus), occult to standard examination, can be visualized in wounds using PRODIGI. We also detected quantitative changes in wound bacterial load over time based on the antibiotic treatment and the correlation of bacterial AF intensity with bacterial load. AF imaging of wounds offers a safe, noninvasive method for visualizing the presence, location, and extent of bacteria as well as measuring relative changes in bacterial load in wounds in real time.

Effective management of pressure ulcers using Hydrofibre technology with silver ions

Pressure ulcers are a common and debilitating form of chronic wounds that impose a considerable burden on individuals and healthcare systems. Colonisation by bacteria complicates the management of pressure ulcers and delays healing. Ionic silver has a well-established role as an antimicrobial agent, providing effective and sustained activity against a broad range of microorganisms, including antibiotic-resistant strains. Innovative silver-containing wound dressings have been designed to assist in the healing of moderate to highly-exuding chronic and acute wounds where there is infection or risk of infection, including ulcers of various causes. This study evaluated the efficacy and contribution to healing of a silver-containing hydrofibre dressing (SCHD; Aquacel ® Ag) in the treatment of bedsores. Twenty patients with bedsore pressure ulcers were treated with SCHD applications for one week. Wound bacterial load change, measured by three different biopsies during therapy, was evaluated for each patient. Wound bioburden decreased by 80% over the treatment period, with 60% of wounds showing no bacterial burden at the end of the study. SCHD was a safe, effective and easy-to-apply treatment for patients with bedsores and eliminated the need for local or systemic antibiotic therapy.

Contrasting mechanisms by which social isolation and restraint impair healing in male mice

Stress modulates vital aspects of immune functioning in both human and non-human animals, including tissue repair. For example, dermal wounds heal more slowly and are associated with prolonged inflammation and increased bacterial load in mice that experience chronic physical restraint. Social stressors also negatively affect healing; however, previous studies suggest that the affected healing mechanisms may be stress model-specific. Here, the effects of either social isolation or physical restraint on dermal wound healing (3.5 mm wounds on the dorsum) were compared in hairless male mice. Social isolation beginning 3 weeks prior to wounding delayed healing comparably to physical restraint (12 h/day for eight days), in spite of marked differences in metabolic and hormonal consequences (i.e. body mass) between the two stress models. Additionally, isolated mice exhibited reductions in wound bacterial load and inflammatory gene expression (interleukin-1beta [IL-1β], monocyte chemoattractant protein [MCP]), whereas restraint significantly increased both of these parameters relative to controls. Experimentally augmenting bacterial concentrations in wounds of isolated mice did not ameliorate healing, whereas this treatment accelerated healing in controls. This work indicates that social isolation and restraint stressors comparably impair healing, but do so through disparate mechanisms and at different phases of healing.

The effects of social isolation on wound healing mechanisms in female mice

Various stressors impair wound healing in humans and rodents. For example, social isolation delays wound closure in rodents, but the healing mechanisms that underlie this delay have yet to be identified. Here, the effects of three weeks of social isolation on hypothalamic–pituitary–adrenal axis responses and healing factors involved in the inflammatory and proliferative phases of wound healing were assessed in adult female hairless mice. Social isolation reduced basal circulating corticosterone concentrations and increased body and thymus weights compared with group-housed controls. Isolation impaired dermal wound closure by up to 30% and reduced initial total wound bacterial load relative to controls. Inflammatory gene expression in the wounds was not affected by the observed differences in wound bacterial load. However, isolation reduced wound gene expression of keratinocyte growth factor and vascular endothelial growth factor, which are involved in keratinocyte proliferation/migration and angiogenesis during the proliferative phase of healing. These data indicate that social isolation induces healing impairments that may be attributed to reductions in growth factors necessary for proper skin cell proliferation and blood vessel growth during healing. This healing impairment occurred in the absence of both high wound bacterial load and elevated circulating glucocorticoids, which have previously been hypothesized to be required for stress-impaired healing in mice.

91. Sex modulates social isolation-induced wound healing impairments in mice

Many factors (e.g. sex, stress) modulate wound healing and thereby alter the risk of developing bacterial infections or chronic, non-healing wounds. Previous research indicates that sex influences dermal wound healing; females heal quicker than males, likely due to differences in testosterone. Stressors (e.g., social isolation) can also impair wound healing. In combination, sex modulates the effects of stressors on other types of immune processes. However, the mechanisms underlying isolation stress-impaired healing, and how sex may modulate these mechanisms, remain unexplored. Here, we hypothesized that social isolation inhibits healing more in female than in male mice. Male and female SKH-1 mice were single- or group-housed (5/cage) for 3 weeks prior to wounding. Two 3.5 mm dorsal skin wounds were then placed on each mouse and wound closure was assessed daily for 5 days. Wound tissue samples were collected 1, 3, or 5 days after wounding and used to quantify gene expression of factors associated with wound healing and wound bacterial load. Isolation delayed wound closure in both sexes, but was more pronounced in males. Isolation also reduced wound bacterial burden in both sexes, which persisted in males only and corresponded with decreased wound inflammatory gene expression (IL-1beta, monocyte chemoattractant protein-1). Isolation also decreased alpha-smooth muscle actin (involved in wound contraction) in males, but not females, which may have contributed to their lasting healing delays.

Infection and Colonization in Epidermolysis Bullosa

In all forms of epidermolysis bullosa (EB), skin fragility may result in bacterial colonization or infection, particularly in the more severe forms where wounds may be multiple and long-standing. A balance exists between a wound's bacterial load and the host defenses, such that there is a spectrum from simple contamination, through colonization, critical colonization, to overt infection. The increased bioburden in critically colonized or infected wounds impairs healing and therefore recognition of these situations, and appropriate measures to promote a healing environment, are fundamental to the care of EB wounds.

Interleukin-8 production by polymorphonuclear leukocytes from patients with chronic infected leg ulcers treated with Lactobacillus plantarum

Bacterial infection impairs the healing process, promoting the chronicity of inflammation and wounds. Because antibiotics fail to eradicate bacteria, especially in biofilm form, new therapeutic modalities may be required. In the present study, the effectiveness of bacteriotherapy with Lactobacillus plantarum on infected chronic venous ulcers was investigated and its effects on interleukin (IL)-8 production by cells from the ulcer bed and neutrophils isolated from peripheral blood that were previously challenged in vitro with Pseudomonas aeruginosa and L. plantarum were studied. Topical application of L. plantarum culture to lesions (25–60 cm2) of 14 diabetic and 20 non-diabetic patients induced debridement, granulation tissue formation and total healing after 30 days in 43% diabetics and in 50% non-diabetics. No significant differences between the groups were observed. The cells from ulcer beds collected after treatment with L. plantarum for 10 days showed a decrease in the percentage of polymorphonuclear, apoptotic and necrotic cells and an enhancement of IL-8 production. IL-8 production by isolated neutrophils from these patients was compared with that in diabetics without ulcers, as well as normal subjects under basal conditions, and after infection of polymorphonuclear cells with P. aeruginosa preincubated either with or without L. plantarum. The basal values in diabetic and ulcer patients were higher than normal (p <0.001) and were increased by P. aeruginosa infection in normal, diabetics (p <0.001) and non-diabetics with ulcers (p <0.01). Preincubation with L. plantarum decreased IL-8 production in patients with ulcers non-diabetic and diabetic (p <0.001). Lactobacillus plantarum treatment reduced wound bacterial load, neutrophils, apoptotic and necrotic cells, modified IL-8 production and induced wound healing.

Evaluation of a bi-layer wound dressing for burn care: II. In vitro and in vivo bactericidal properties

We have recently designed a medicated bi-layer wound dressing to address the key requirements for treating external, contaminated war wounds. This study assessed the in vitro and in vivo bactericidal efficacies of the DRDC hydrogel/polyurethane wound dressing. Chloramphenicol- and chlorhexidine-loaded DRDC dressings produced significantly larger zones of inhibition against Pseudomonas aeruginosa than the other medicated dressings for 4 d. Chlorhexidine-loaded Allevyn ® and Hydrasorb ® remained bactericidal for 48 h only. Chloramphenicol-loaded Hydrasorb ® and Allevyn ® remained bactericidal for 1 and 3 d, respectively. Ps. aeruginosa and Staphylococcus epidermidis counts in wounds treated with chlorhexidine- and chloramphenicol-loaded DRDC dressings for 24 h were 1–3-log lower than those of control wounds. While Ps. aeruginosa counts in the wounds on day 4 were comparable following daily changes of either antiseptic-loaded dressings, chlorhexidine showed a 75% greater bactericidal efficacy against Staph. epidermidis than chloramphenicol. Though increasing the frequency of dressing changes led to a greater reduction in the wound bacterial load, the contamination levels of all antiseptic-treated wounds remained below 10 5 CFU/g of wound. Cerium nitrate-loaded dressings did not exert any bactericidal effect, irrespective of the experimental conditions. These data show that the DRDC dressing is effective in delivering medications, such as an antimicrobial agent, to the wound bed.