Antimicrobial activity of silver-containing dressings on wound microorganisms using an in vitro biofilm model.

Antimicrobial dressings such as those containing silver are now being used widely to control wound bioburden, and tests to demonstrate their efficacy predominantly involve in vitro models using free-living or planktonic bacteria. In this present study a wide range of antibiotic-sensitive and resistant bacteria were tested in their quasi-sessile state using a standard agar assay and a second method used a poloxamer gel (true biofilm state - poloxamer encourages microorganisms to exhibit a more clinically relevant biofilm phenotype) technique. The antimicrobial activity of two silver dressings, a silver-containing Hydrofiber (SCH) dressing and a nanocrystalline silver-containing dressing (NCS), were evaluated on a variety of microorganisms, using a zone-of-inhibition (ZOI) test. When grown on agar (presenting a quasi-sessile state of each organism), the antibiotic-susceptible microorganisms were generally more susceptible to the SCH dressing compared with the NCS. ZOIs associated with SCH dressing ranged between 5.7 and 17.5 mm; those for the NCS against the same group of organisms ranged between 1.9 and 8.6 mm. When grown on poloxamer gel, (presenting the biofilm state of each organism) the same group of microorganisms were less susceptible to both dressings. The SCH dressing was most effective against strains of Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus (ZOI range: 2.6-6 mm); the NCS was most effective against strains of Klebsiella pneumoniae, Enterococcus faecalis and Escherichia coli (i.e. ZOI range: 1-2.8 mm). Similarly to the antibiotic-susceptible microorganisms, nine of ten antibiotic-resistant bacterial strains when grown on agar were more susceptible to the SCH dressing compared with the NCS. Although the microorganisms tested were universally less susceptible to the silver dressings when in their biofilm state, in the majority of cases, the SCH dressing demonstrated greater biofilm-inhibiting activity than the NCS.

Evaluating antibiotics for use in medicine using a poloxamer biofilm model.

BACKGROUND: Wound infections, due to biofilms, are a constant problem because of their recalcitrant nature towards antibiotics. Appropriate antibiotic selection for the treatment of these biofilm infections is important. The traditional in vitro disc diffusion method for antibiotic selection uses bacterial cultures grown on agar plates. However, the form of bacterial growth on agar is not representative of how bacteria grow in wounds and other tissue sites as here bacteria grow naturally in a biofilm. The aim of this research was to test a more appropriate method for testing antimicrobial efficacy on biofilms and compare with the standard methods used for antibiotic sensitivity testing. METHODS: Outer Membrane Protein analysis was performed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Acinetobacter juni when grown on Mueller Hinton agar ('quasi-biofilm state') and 30% Poloxamer hydrogel ('true- biofilm state). Susceptibility to antibiotics on 28 clinical isolates was determined using the modified Kirby Bauer disc diffusion method, on agar and 30% Poloxamer. RESULTS: Similar outer membrane proteins [OMPs] were identified in bacteria grown in a biofilm state and on a 30% poloxamer hydrogel, which were very different to the OMPs identified in bacteria grown on Mueller-Hinton agar and broth. There was a significant difference between the means of the clearance zones around the antibiotic discs on standard agar and poloxamer gels [P < 0.05]. The zones of clearance were generally smaller for poloxamer-grown bacteria than those grown on standard agar. Diffusion distances of various antibiotics through agar and 30% poloxamer showed no significant difference [P > 0.05]. CONCLUSION: The findings of this experiment suggest that poloxamer gel could be used as an appropriate medium on which to conduct biofilm antibiotic susceptibility tests as it enables bacteria to be grown in a state representative of the infected surface from which the culture was taken.

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium.

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.

A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion.

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.