PcrV immunization enhances survival of burned Pseudomonas aeruginosa-infected mice.

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.

Does the addition of nystatin to 5% mafenide acetate and genitourinary irrigant solutions interfere with their antimicrobial activity? Assessment by two topical antimicrobial test assay systems.

Results previously reported using the Wet Disc Topical Antimicrobial Assay (WDA) suggested that adding nystatin (NY) to a 0.5% mafenide acetate (MA) suspension or genitourinary irrigant (double antibiotic [DAB]) to expand their antimicrobial activity to include Candida sp. antagonized the antibacterial effect of MA but not DAB. We use DAB solution as described by the authors of the previous study, also, but we use a 5% commercially available mafenide acetate solution instead of the in-house prepared 0.5% mafenide acetate suspension that they used. Further, we use both the WDA and the Agar Well Diffusion Topical Antimicrobial Assay (AWDA) to test topical antimicrobials at this institution. In light of the previously reported results, this study 1) examined whether adding nystatin to DAB or the 5% mafenide acetate solution used at this institution caused any interference in the ability of these substances to migrate through the agar matrices and cause zones of growth inhibition in the two test assay systems and 2) compared the assessment of microbial susceptibility (by very precise definition) between the two systems. The addition of nystatin did not interfere with the ability of either DAB or mafenide acetate to migrate through the agar matrices and cause clear zones. However, on the assessment of susceptibility a significantly larger number of organisms were judged susceptible using the AWDA than the WDA. We believe that the disparity is caused by a large difference in agar diffusion kinetics between the two assays. Therefore, we recommend the AWDA rather than the WDA for susceptibility studies.

Antimicrobial mixtures used to store harvested skin: antimicrobial activities tested at refrigerator (4 degrees C) temperatures.

Tissue culture media used by skin and tissue banks to store tissue were tested, with and without antimicrobial mixtures, for their ability to inhibit the growth of bacteria during incubation at refrigerator temperatures. Reductions in bacterial load were found after incubation at 4 degrees C in both the basal tissue culture media with and without the addition of antimicrobial mixtures. The presence of antimicrobials enhanced the degree and rapidity of reduction. Variations occurred among the different formulations, with or without added antimicrobials. No one formulation for either the basal tissue culture medium or antimicrobial mixture was ideal. Methods described in this study can be used to establish optimum basal medium and antimicrobial mixture formulations for general use by skin and tissue banks.

Assessment of the potential for microbial resistance to topical use of multiple antimicrobial agents.

The goal of this study was to reduce the likelihood of the generation and/or persistence of bacterial resistance to some antimicrobial components contained in a topical antimicrobial mixture (neomycin, polymyxin B, mupirocin and ciprofloxacin) for use with cultured skin grafts, by substitution of alternative antimicrobials, specifically fusidic acid for mupirocin and ofloxacin for ciprofloxacin. The alternative agents failed to serve that purpose. However, with the exception of specific genera of bacteria, Proteus sp. and Providencia stuartii, 90% or more of all other bacteria tested were susceptible to the action of one or more of the individual antimicrobial agents contained in the original mixture. This was true when bacteria were highly susceptible to the antimicrobials, generally, or when bacteria resistant to specific antimicrobials such as penicillin-class antibiotics and ciprofloxacin, were tested. These results suggest that the redundancy of antimicrobials contained in this mixture reduces the chance that resistant bacteria generated by the use of this mixture or already present on wounds would persist when the mixture is used clinically.

Antimicrobial mixtures used by tissue banks for harvested skin: comparative in vitro activity.

The activities of antimicrobial combinations from three geographically diverse skin/tissue banks used in the processing of skin/ tissue were compared using bacteria and yeast isolated from burn patients. All formulations showed 90% or more effectiveness against bacteria generally susceptible to antibiotics but were less effective (60-80%) when tested against bacteria resistant to specific antimicrobials. Anti-yeast activity was present when an appropriate antifungal agent was included in the combination. All formulations were stable for at least six weeks. Results of this study raise certain questions about the use of these antimicrobial combinations in contemporary skin/tissue banking and point the way toward areas for future study.

Effect of thermal injury on the adherence of Candida albicans to murine splenic tissue.

In a mouse model of thermal injury, an increase in burn size produced a decrease in the ratio of Candida albicans cells adherent to the marginal zone to those adherent to the white pulp of the spleen, an increase in the number of Candida cells in the circulation and in the kidneys, and an increase in mortality.

Effective management of microbial contamination in cultured skin substitutes after grafting to athymic mice.

Cultured skin substitutes have become therapeutic alternatives for treatment of acute and chronic skin wounds, but all models of these substitutes are avascular and susceptible to microbial destruction during vascularization. To develop a practical management protocol for increased survival of skin substitutes, experimental wounds were contaminated with Pseudomonas aeruginosa and treated with a formulation of noncytotoxic antimicrobial agents (polymyxin B, neomycin, ciprofloxacin, mupirocin, amphotericin B) in a nutrient medium (vehicle). Cultured skin substitutes consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan sponges were grafted to 2 x 2 cm full-thickness wounds on athymic mice that were contaminated with the strain SBI-N of P. aeruginosa at 1 x 10(4), 1 x 10(5), and 1 x 10(6) organisms/wound. Experimental wounds were irrigated with 1 ml/day topical antimicrobial solution for 10 days, and controls received vehicle only. Two, three, and four weeks after grafting, wounds were traced and swabbed for microbial culture and areas were measured with planimetry. At 4 weeks, biopsy samples were scored histochemically for immunoreactivity to HLA antigens. Data analysis by chi-square, analysis of variance, and Tukey's test shows that treatment of contaminated wounds with noncytotoxic topical antimicrobials is associated with an increased area of healed wounds, positive detection of HLA antigens, and negative cultures for P. aeruginosa. These results show that microbial contamination of cultured skin substitutes on full-thickness wounds may be managed effectively during graft vascularization. However, this formulation of antimicrobial agents is not currently approved for human use and is investigational only. Effective management of microbial contamination suggests that clinical efficacy of avascular tissue analogs may be increased by local application of noncytotoxic antimicrobial agents.

Recombinant neutral endopeptidase decreases oedema in the skin of burned guinea-pigs.

Oedema, due to increased vascular leakage postburn, cases significant problems for burn patients. The purpose of this study was to determine if local application of a recombinant neutral endopeptidase (rNEP) would reduce the increased vascular permeability caused by a burn. In a guinea-pig model, a single treatment of rNEP given immediately postburn significantly decreased burn-induced plasma extravasation. This rNEP effect was dependent upon both the dosage of the peptidase and its enzymatic activity. Additional experiments were consistent with the rNEP acting partly by degrading bradykinin, a mediator of increased vascular permeability postburn. These findings suggest that further study of rNEP as a possible treatment for oedema is warranted.

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.

Formulation of 'idealized' topical antimicrobial mixtures for use with cultured skin grafts.

In order to develop antimicrobial mixtures which provide broad-spectrum antimicrobial activity for use with cultured cell autografts, several individual antimicrobial agents, in concentrations non-toxic for cells in culture, were tested against a variety of bacteria and Candida spp. isolated from burn patients. An agar well diffusion topical assay was used. Antimicrobials active against Gram-positive and Gram-negative bacteria and antifungal agents, individually, were uniformly effective against their respective spectra of organisms. Broad-spectrum antibacterials were uniformly effective against Gram-negative bacteria but their activity varied against Gram-positive bacteria. Adding an agent active against Gram-positive bacteria to all broad spectrum antibacterial agents conferred uniform Gram-positive activity to the mixture. One mixture consisting of specific Gram-negative, Gram-positive and broad spectrum antibacterial agents, was uniformly active against all bacteria tested and the addition of antifungal agents extended the activity to cover Candida spp. without interfering with the mixture's overall antibacterial activity. Another mixture showed either additive or antagonistic activities against the battery of microorganisms tested. Thus, these methods can be used to identify mixtures of antimicrobials, in concentrations non-toxic for cells in culture, that have very broad spectra of antimicrobial activity. Such mixtures should be evaluated in patients when cultured skin grafts are used.

Pyoverdin is essential for virulence of Pseudomonas aeruginosa.

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.

Avirulence of a Pseudomonas aeruginosa algC mutant in a burned-mouse model of infection.

The virulence of wild-type Pseudomonas aeruginosa PAO1 and that of a genetically defined algC mutant, PAO1 algC::tet, were compared in a burned-mouse model of infection. Unlike PAO1, PAO1 algC::tet was avirulent, grew less well in the eschar, and did not disseminate to the liver of challenged animals. We have previously shown that the P. aeruginosa algC gene is required for biosynthesis of alginate and lipopolysaccharide (M.J. Coyne, Jr., K.S. Russell, C.L. Coyle, and J.B. Goldberg, J. Bacteriol. 176:3500-3507, 1994). In order to determine whether the alginate or lipopolysaccharide (LPS) defect was responsible for the avirulence of this strain, we constructed a strain with a mutation in an alginate-specific gene, algD. PAO1-algD was virulent in the burned-mouse model, thus implicating the LPS defect in PAO1 algC::tet as the relevant alteration responsible for the avirulence of this strain.

Studies on multiple Pseudomonas aeruginosa isolates from individual burn patients by RFLP, O antigen serotyping and antibiogram analysis.

Multiple isolates of Pseud. aeruginosa from individual burn patients were tested for antibiotic susceptibility-resistance patterns (antibiogram), O serotype lipopolysaccharide and chromosomal DNA restriction fragment length polymorphisms (RFLP) using a PAK pilin gene probe. Some patients were colonized by isolates identical by all three analytical procedures whereas other patients were found where multiple isolates were identical on the basis of serotype and antibiogram analysis, but different on the basis of RFLP analysis. Examples were found where multiple isolates from an individual patient appeared to be identical on the basis of serotyping and RFLP data, but different on the basis of antibiogram. Strains refractory to O serotyping could be characterized by RFLP type. These results indicate that RFLP analysis provides a valuable addition to routine serotyping and antibiogram studies on Pseud. aeruginosa isolates and that significant numbers of burn patients become co-colonized/co-infected with phenotypically diverse strains of this organism.