An in vitro biofilm model to examine the effect of antibiotic ointments on biofilms produced by burn wound bacterial isolates.

PURPOSE: Topical treatment of burn wounds is essential as reduced blood supply in the burned tissues restricts the effect of systemic antibiotics. On the burn surface, microorganisms exist within a complex structure termed a biofilm, which enhances bacterial resistance to antimicrobial agents significantly. Since bacteria differ in their ability to develop biofilms, the susceptibility of these biofilms to topically applied antibiotics varies, making it essential to identify which topical antibiotics efficiently disrupt or prevent biofilms produced by these pathogens. Yet, a simple in vitro assay to compare the susceptibility of biofilms produced by burn wound isolates to different topical antibiotics has not been reported. METHODS: Biofilms were developed by inoculating cellulose disks on agar plates with burn wound isolates and incubating for 24h. The biofilms were then covered for 24h with untreated gauze or gauze coated with antibiotic ointment and remaining microorganisms were quantified and visualized microscopically. RESULTS: Mupirocin and triple antibiotic ointments significantly reduced biofilms produced by the Staphylococcus aureus and Pseudomonas aeruginosa burn wound isolates tested, as did gentamicin ointment, with the exception of one P. aeruginosa clinical isolate. CONCLUSIONS: The described assay is a practical and reproducible approach to identify topical antibiotics most effective in eliminating biofilms produced by burn wound isolates.

Serum inhibits P. aeruginosa biofilm formation on plastic surfaces and intravenous catheters.

INTRODUCTION: Biofilm formation on medical devices such as intravenous catheters is a serious manifestation of Pseudomonas aeruginosa infections. Serum has bactericidal activity, a function of multiple serum components. In this study, we determined the effect of serum and serum components on the formation of P. aeruginosa biofilm. MATERIALS AND METHODS: We examined the effect of adult bovine serum (ABS) or bovine serum albumin (BSA) on biofilm development on plastic coverslips. This was done using both static and continuous flow-through culture systems and P. aeruginosa strain PAO1. Biofilms were quantified using crystal violet assays and visualized using confocal scanning laser microscopy and scanning electron microscopy. We examined the effect of ABS on PAO1 swimming and twitching motilities (both contribute to P. aeruginosa biofilm development). We also analyzed the inhibitory effect of adult human serum (AHS) and plasma (AHP) on PAO1 biofilm development on plastic coverslips and intravenous catheters. RESULTS: Compared with M9 minimal medium (M9), 10% ABS-supplemented medium (M9/ABS-10) caused a significant decrease in biofilm development. Coverslips precoated with M9/ABS-10 failed to develop biofilm when placed in M9. In addition to reduced biofilm formation, adding ABS to M9 reduced an already-developed PAO1 biofilm. Compared with M9, M9/ABS-10 enhanced PAO1 twitching motility considerably, but did not affect swimming motility. Similar to ABS, BSA blocked biofilm formation but did not affect PAO1 twitching motility. Both AHS and AHP blocked PAO1 biofilm formation on plastic coverslips and intravenous catheters. CONCLUSIONS: These results suggest that as part of the host innate resistance, serum inhibits P. aeruginosa biofilm formation on plastic surfaces, including intravenous catheters. Two possible scenarios for this inhibition include blocking the direct interaction between P. aeruginosa and the substrates, and the enhancing P. aeruginosa twitching motility.

Organoselenium coating on cellulose inhibits the formation of biofilms by Pseudomonas aeruginosa and Staphylococcus aureus.

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.

Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa.

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.