Ian Alan Holder

Agar well diffusion assay testing of bacterial susceptibility to various antimicrobials in concentrations non-toxic for human cells in culture.

Previously, we showed that microbial susceptibility to antimicrobials in concentrations non-toxic for human cells in culture could be tested using the wet disc topical antimicrobial assay. In this report, wet disc assay and agar well diffusion assay results were compared testing the susceptibility of Ps. aeruginosa isolates from burn patients to concentrations of Polymyxin B non-toxic for cultured cells. Both assays were performed on the same agar plates. No differences in results were observed. Further agar well diffusion assay testing showed that susceptibility/resistance could be demonstrated when testing several antimicrobials in concentrations non-toxic for cultured cells against a variety of bacteria isolated from burn patients. Therefore, the more familiar agar well diffusion as well as the wet disc assay can be used to test microbial susceptibility to these concentrations of antimicrobials.

PcrV Immunization Enhances Survival of Burned Pseudomonas aeruginosa-Infected Mice

ABSTRACT Burned Pseudomonas aeruginosa-infected mice immunized against PcrV, a type III virulence system translocating protein, showed significantly enhanced survival compared to controls. Survival was non-O serotype specific and correlated with a reduced systemic microbial load. Infection with a high-level toxin A-producing strain required supplemental antitoxin treatment to enhance survival.

Cytotoxicity Testing of Topical Antimicrobial Agents on Human Keratinocytes and Fibroblasts for Cultured Skin Grafts

Cultured epidermal skin has become an adjunctive therapy for treatment of major burn injuries, but its effectiveness is greatly limited because of destruction by microbial contamination. To evaluate candidate antimicrobial agents for use with cultured skin, a combined cytotoxicity-antimicrobial assay system was developed for determination of toxicity to cultured human keratinocytes and fibroblasts and for determination of susceptibility or resistance of common burn wound organisms. Candidate agents including chlorhexidine gluconate, polymyxin B, mupirocin, sparfloxacin, or nitrofurazone were tested separately for inhibition of growth of human cells and for inhibitory activity to microorganisms with the wet disk assay. The data showed that (1) chlorhexidine gluconate (0.05%) was uniformly toxic to both cultured human cells and microorganisms; (2) nitrofurazone (0.02%) had dose-dependent toxicity to human cells and limited effectiveness against gram-negative microorganisms; (3) sparfloxacin (30 micrograms/ml) had low toxicity to human cells and retained antimicrobial activity against both gram-positive and gram-negative bacteria; (4) polymyxin B (400 U/ml) was not toxic to human cells and had intermediate effectiveness on gram-negative bacteria; and (5) mupirocin (48 micrograms/ml) had no toxicity to skin cells and had uniform effectiveness against Staphylococcus aureus including methicillin-resistant Staphylococcus aureus. Selection of topical antimicrobial drugs by these assays may improve effectiveness of cultured skin for burns and may be used to control other surgical wound infections.

Assessment of a silver-coated barrier dressing for potential use with skin grafts on excised burns

Acticoat burn dressing is a silver-coated dressing with antimicrobial activity purported to reduce infection from environmental organisms in partial and full-thickness wounds. Acticoat was tested for activity as an antimicrobial treatment and as an antimicrobial barrier dressing in three in vitro assays. It was found that a modified disc assay method gave false negative results but in an assay in which bacteria were inoculated on top of samples of Acticoat, bacterial numbers were reduced, over time, with all microorganisms tested. Acticoat served as a barrier for bacteria, inoculated onto it, from contaminating the surface of an agar plate under the Acticoat. The data show that Acticoat has: antimicrobial capabilities, but to be effective hours of contact between Acticoat and the microorganisms are required; and the capacity to serve as an antimicrobial barrier dressing. These findings support the conclusion that Acticoat has activity to reduce microbial contamination of wounds from environmental sources.

Selection of topical antimicrobial agents for cultured skin for burns by combined assessment of cellular cytotoxicity and antimicrobial activity.

Cultured epidermal skin has become an adjunctive therapy for treatment of major burn injuries, but its effectiveness is greatly limited due to destruction by microbial contamination. To evaluate candidate drugs for use with cultured skin, a combined cytotoxicity-antimicrobial assay system was developed for determination of toxicity to cultured human keratinocytes and fibroblasts, and to common burn wound organisms (20 bacterial and 4 fungal strains). Candidate agents including Hibiclens (n = 3), amikacin, piperacillin, norfloxacin, and nystatin were tested separately and in combination (n = 6 each) for inhibition of growth of human cells and lytic activity on microorganisms in the wet disc assay. The data showed that: (1) Hibiclens was uniformly toxic to both cultured human cells and microorganisms; (2) norfloxacin had dose-dependent toxicity to human cells and broad effectiveness against microorganisms; and (3) norfloxacin (25 μg/mL) plus nystatin (100 U/mL) had low toxicity to human cells and high toxicity to both Gram-positive and Gram-negative bacteria (20 of 20) and fungi (4 of 4). Selection of topical antimicrobial drugs by these assays may improve effectiveness of cultured skin for burns and may be extended to the control of other surgical wound infections. (Plast. Reconstr. Surg. 92: 493, 1993.)

Burn Wounds: Microbiology, Local Host Defenses, and Current Therapy

(1973). Burn Wounds: Microbiology, Local Host Defenses, and Current Therapy. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 4, No. 1, pp. 61-100.

Noncytotoxic combinations of topical antimicrobial agents for use with cultured skin substitutes.

Cultured skin grafts are destroyed more easily than split-thickness skin grafts by common burn wound organisms, including gram-negative and gram-positive bacteria and fungi. To increase the survival and engraftment of cultured skin grafts, formulations of antimicrobial agents were tested for cytotoxicity to cultured human keratinocytes and fibroblasts and for activity against common organisms from burn wounds. On the basis of previous studies, a base formulation containing neomycin (40 micrograms/ml), polymyxin B (700 U/ml), and mupirocin (40 micrograms/ml) was prepared, to which ciprofloxacin (20 micrograms/ml) or norfloxacin (20 micrograms/ml) and amphotericin B (0.25 microgram/ml) or nystatin (100 U/ml) were added. Toxicity to cultured human cells was determined by the growth response of cell cultures (n = 6) to each drug combination over 4 days. Activity against clinical isolates (n = 40) of Staphylococcus aureus, Pseudomonas aeruginosa, other gram-negative bacteria, and Candida spp. was determined by the wet disc assay. Analysis of variance testing showed no significant differences in the growth of keratinocytes or fibroblasts under control or experimental conditions. Medium without antimicrobial agents was not effective against any of the 40 microbial strains tested. The base formulation was effective against all bacterial strains tested but against none of the fungi, while all experimental formulations were effective against all microbial strains tested. These findings suggest that neomycin, mupirocin, and polymyxin B may be combined with a quinolone and an antimycotic agent to provide broad antimicrobial activity for a formulation for topical use with cultured skin on burns. However, the formulations described here are strictly experimental and are not recommended for clinical use without further evaluation.

Circulating levels of tumour necrosis factor, interleukin 6 and proteolytic activity in a murine model of burn and infection

Cytokines and proteinases have both been implicated as mediators in the inflammatory response associated with trauma and sepsis. Using a burned-infected mouse model, it was previously found that mortality is proportional to the amount of proteolytic activity (PA) in the circulation. However, little is known about circulating cytokine levels in hosts that are both burned and infected. With this mouse model, both tumour necrosis factor (TNF) and interleukin 6 (IL-6) were upregulated by a burn and by an infection. Burn plus infection produced an additive effect on each cytokine, but IL-6 levels correlated better with mortality. Treating mice with the proteinase inhibitor aprotinin immediately preburn and infectious challenge significantly decreased IL-6, PA and mortality. This may be a clinically relevant model for studying mediators in burned and/or septic hosts.

Delivery and activity of antimicrobial drugs released from human fibrin sealant.

Engraftment and healing of native or cultured skin grafts depend on adherence, vascularization, and control of microbial contamination in the wound bed. Fibrin sealant is a biocompatible polymer that may be used to promote skin engraftment by serving as a delivery vehicle for antimicrobial drugs. Human fibrin sealant (25 mg/ml) was polymerized with antibacterial agents (mupirocin [32 micrograms/ml], nitrofurazone [0.02% wt/vol], polymyxin B [400 U/ml], or norfloxacin [20 micrograms/ml]) on nitrocellulose (nc) backing and was prepared as 6 mm diameter discs with skin punches. Discs (n = 6) were applied in the Wet Disc Assay to clinical isolates of Staphylococcus aureus (mupirocin, nitrofurazone) or Pseudomonas aeruginosa (polymyxin B, norfloxacin). Controls included drug applied to 6 mm paper discs (25 microliter) and nitrocellulose discs submerged in each drug, blotted, and applied to bacterial cultures on agar in petri dishes. Data were expressed as zone of clearing (mm diameter +/- SEM) after overnight incubation at 35 degrees C. Significant differences (ANOVA and Turkeys test, p < 0.05) were found for each drug released from the disc of fibrin sealant compared with other vehicles. Release from filter paper discs compared with nitrocellulose was significant for nitrofurazone and norfloxacin. Serial transfer of fibrin discs to fresh bacterial cultures after 24 hours showed no zones of clearing. The data show that fibrin sealant releases topical drugs with no inhibition of antimicrobial activity on burn organisms. Greater zones of clearing from fibrin sealant may result from passive fluid retention or from active binding to fibrin followed by protease digestion by burn organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective efficacy of Pseudomonas aeruginosa type-A flagellin in the murine burn wound model of infection

The main goal of this study was to develop a vaccination strategy that would enhance the protective response against the recombinant type A flagellin (r‐fla‐A) of Pseudomonas aeruginosa in the burn wound sepsis model. Inbred mice were immunized with r‐fla‐A with or without alum adjuvant. The vaccinated mice were burned and challenged with P. aeruginosa. To evaluate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. To determine the functional activity of anti r‐fla IgG, opsonophagocytic killing and motility inhibition assay was performed. In vivo administration of r‐fla‐A afforded a remarkable improvement in survival of mice (83.3%) challenged with homologous strain (PAK) in the burn wound infection. The antibodies generated against the r‐fla‐A achieved 25% survival in immunized mice that had been infected with heterologous strain PAO1. Flagellin also induced high level humoral immune response via high titers of serum IgG1 in the burn and challenged mice. Anti r‐fla‐A antibody promoted phagocytosis of the PAK strain, and the number of viable bacterial cells decreased over 53.1%; In contrast, low opsonophagocytic killing activity (17.4%) was observed when the antiserum to r‐fla‐A was treated with the PAO1 strain. The anti r‐fla‐A antisera was able to inhibit the motility of the homologous strains; however, they did not inhibit the heterologous strains. We concluded that active immunization with recombinant type A‐flagellin could protect burn mice against lethal P. aeruginosa challenge via immobilization of the pathogen which promoted the phagocytic activity.