Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria–protein interactions in vivo and the accompanying alterations in phenotype, virulence factor production, and ultimately antibiotic tolerance. We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus (MRSA or MSSA, respectively) and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein–bacteria interactions characteristic of physiological fluids.
Background: Ethyl chloride spray is used frequently in the outpatient setting as a local anesthetic for injections and aspirations with varying consensus about the sterility of the spray. We hypothesize that ethyl chloride spray remains sterile and would show no bacterial growth during routine clinical use. Methods: Thirteen ethyl chloride bottles were collected for testing. Two unopened bottles were used as controls. Eleven unopened bottles were placed in orthopedic clinics and recollected after varying duration of use. The final volume and duration of use were recorded. Each bottle was sprayed in a separate test tube and allowed to evaporate. Trypticase soy broth was added to each tube and incubated for 48 hours. Control test tubes with broth alone were prepared and incubated under the same conditions. Cultures were evaluated at 24 and 48 hours. Results: The mean duration of ethyl chloride bottle use prior to culturing was 26 days. The average volume used per day was 1.9 mL. Each ethyl chloride bottle had an initial volume of 103.5 mL. Using the average daily volume usage, an extrapolated lifespan of each bottle was estimated at 7.7 weeks. None of the samples showed bacterial or fungal growth at 24 or 48 hours. Conclusion: Ethyl chloride bottles used in the clinical settings showed no bacterial or fungal contamination through their shelf life and routine use. The duration and amount of use did not affect sterility. Although the antimicrobial activity of ethyl chloride spray on skin is debated, ethyl chloride itself remains sterile through clinical use.
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