BackgroundThe accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS.ResultsUnder 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48–56 h.ConclusionsThe thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.

Diabetes affects 25.8 million people in the United States, or 8.3% of the population, and these numbers are even higher in developing countries. Diabetic patients are more susceptible to the development of chronic wounds with debilitating bacterial infections than nondiabetics. Previously, we compared the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause biofilm-associated infections in chronic wounds of diabetic and nondiabetic mice (C. Watters, K. DeLeon, U. Trivedi, J. A. Griswold, M. Lyte, K. J. Hampel, M. J. Wargo, and K. P. Rumbaugh, Med. Microbiol. Immunol. 202:131-141, 2013). Unexpectedly, we observed that insulin-treated diabetic mice had significantly more biofilm in their wounds, which correlated with higher antibiotic tolerance. Here, we investigated whether insulin treatment modulates the diabetic immune system to favor P. aeruginosa biofilm formation. Utilizing a murine chronic wound model, we found that DNA protected P. aeruginosa in the wounds of insulin-treated diabetic mice from antibiotic treatment. We also observed increased numbers of neutrophils, reduced numbers of macrophages, and increased cell death in the wounds of diabetic mice on insulin therapy. Taken together, these data suggest that high levels of lysed neutrophils in the wounds of diabetic mice on insulin, combined with fewer macrophages to remove the cellular debris, contribute to increased DNA levels, which enhance P. aeruginosa biofilms.

e Loss of the skin barrier facilitates the colonization of underlying tissues with various bacteria, where they form biofilms that protect them from antibiotics and host responses. Such wounds then become chronically infected. Topical antimicrobials are a major component of chronic wound therapy, yet currently available topical antimicrobials vary in their effectiveness on biofilmforming pathogens. In this study, we evaluated the efficacy of Next Science wound gel technology (NxtSc), a novel topical agent designed to kill planktonic bacteria, penetrate biofilms, and kill the bacteria within. In vitro quantitative analysis, using strains isolated from wounds, showed that NxtSc inhibited biofilm development by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae by inhibiting bacterial growth. The gel formulation NxtSc-G5, when applied to biofilms preformed by these pathogens, reduced the numbers of bacteria present by 7 to 8 log 10 CFU/disc or CFU/g. In vivo, NxtSc-G5 prevented biofilm formation for 72 h when applied at the time of wounding and infection and eliminated biofilm infection when applied 24 h after wounding and infection. Storage of NxtSc-G5 at room temperature for 9 months did not diminish its efficacy. These results establish that NxtSc is efficacious in vitro and in vivo in preventing infection and biofilm development by different wound pathogens when applied immediately and in eliminating biofilm infection already established by these pathogens. This novel antimicrobial agent, which is nontoxic and has a usefully long shelf life, shows promise as an effective agent for the prevention and treatment of biofilm-related infections.