Chronic wounds contain complex polymicrobial communities of sessile organisms that have been underappreciated because of limitations of standard culture techniques. The aim of this work is to combine recently developed next-generation investigative techniques to comprehensively describe the microbial characteristics of chronic wounds. Tissue samples were obtained from 15 patients with chronic wounds presenting to the Johns Hopkins Wound Center. Standard bacteriological cultures demonstrated an average of 3 common bacterial species in wound samples. By contrast, high-throughput pyrosequencing revealed increased bacterial diversity with an average of 17 genera in each wound. Data from microbial community profiling of chronic wounds was compared to published sequenced analyses of bacteria from normal skin. Increased proportions of anaerobes, Gram-negative rods and Gram-positive cocci were found in chronic wounds. In addition, chronic wounds had significantly lower populations of Propionibacterium compared to normal skin. Using epifluorescence microscopy, wound bacteria were visualized in highly organized thick confluent biofilms or as scattered individual bacterial cells. Fluorescent in-situ hybridization allowed for the visualization of Staphylococcus aureus cells in a wound sample. Quorum sensing molecules were measured by bioassay to evaluate signaling patterns amongst bacteria in the wounds. A range of autoinducer-2 activities were detected in the wound samples. Collectively, these data provide new insights into the identity, organization, and behavior of bacteria in chronic wounds. Such information may provide important clues to effective future strategies in wound healing.

Biofilms confer protection from adverse environmental conditions and can be reservoirs for pathogenic organisms and sources of disease outbreaks, especially in medical devices. The goal of this research was to evaluate the anti-biofilm activities of silver nanoparticles (AgNPs) against several microorganisms of clinical interest. The antimicrobial activity of AgNPs was tested within biofilms generated under static conditions and also under high fluid shears conditions using a bioreactor. A 4-log reduction in the number of colony-forming units of Pseudomonas aeruginosa was recorded under turbulent fluid conditions in the CDC reactor on exposure to 100 mg ml(-1) of AgNPs. The antibacterial activity of AgNPs on various microbial strains grown on polycarbonate membranes is reported. In conclusion, AgNPs effectively prevent the formation of biofilms and kill bacteria in established biofilms, which suggests that AgNPs could be used for prevention and treatment of biofilm-related infections. Further research and development are necessary to translate this technology into therapeutic and preventive strategies.

Objective-To determine whether or not there are any significant differences in the effects of wound dressings on bacterial bioburden.Method-A selection of non-occlusive, non-adhesive dressings was tested for their effect on bacterial bioburden. The dressings selected included two dressings with antimicrobial properties (one containing silver and one containing PHMB), a cotton-based dressing enclosed in a perforated sleeve of poly(ethylene terephthalate), a carboxymethyl cellulose-based dressing, a fibre-free alginate dressing, and a 12-ply 100% cotton gauze. Using the colony-drip flow reactor (DFR) model, a meticillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa biofilm was grown underneath a dressing sample. Biofilm growth was examined via plate counts, fluorescent microscopy and scanning electron microscopy.Results-The dressings containing antimicrobial agents had the greatest effect on bacterial load. In the MRSA experiments, both antimicrobial dressings produced lower bacteria counts than the other dressings (p≤0.001), while in the P. aeruginosa experiments, only the silver-containing sample had fewer bacteria (p≤0.0001). However, neither antimicrobial dressing was able to completely eradicate the bacteria when testing with either microorganism. Conclusion-The results presented herein illustrate that bacteria can grow unchallenged within the dressing environment and that an antimicrobial dressing can limit this bacterial growth. Keywordsantimicrobial dressings; biofilms; plate counts; fluorescent microscopy; scanning electron microscopy Wounds are an ideal environment for bacterial colonisation and biofilm formation. The wound bed provides both a surface on which to grow and an ample supply of nutrients. Recent studies have shown that bacteria inoculated into wounds form biofilms. 1,2 Another study found that as many as 60% of chronic wound specimens contained a biofilm. 3 Current research indicates that biofilms are present in chronic wounds. [3][4][5][6] Furthermore, recent studies suggest that biofilms should be targeted when developing and testing therapies for these wounds. [7][8][9][10][11] A literature review of culture data from 62 published studies dating from 1969 to 1997 revealed that the predominant wound isolate in both chronic and acute wounds was Staphylococcus aureus (reported in 63% of the studies), followed by coliforms (45%), Bacteroides spp. (39%), Peptostreptococcus spp. (36%), Pseudomonas aeruginosa (29%), Enterococcus spp. (26%) and Streptococcus pyogenes (13%).

Aims The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip-flow reactor, and a three-species biofilm model was established using methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and C. perfringens in the colony-drip-flow reactor model. Plate counts revealed that MRSA, P. aeruginosa, and C. perfringens grew to 7.39±0.45, 10.22±0.22, and 7.13±0.77 log CFU per membrane, respectively. The three-species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity™ AMD and Acticoat™, compared to sterile gauze controls. Microbial growth on Curity™ AMD and gauze were not significantly different, for any species, whereas Acticoat™ was found to significantly reduce growth for all three species. Conclusions Using the Colony-DFR, a three-species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of Study The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.

Aims:  To develop an in vitro model (Colony/drip‐flow reactor – C/DFR) for the growth and analysis of methicillin‐resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results:  Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 μm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions:  The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study:  The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.