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Abstract Background Chronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms. Results The helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low μg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities. Conclusions The NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections.

Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS) (Type B) was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC) in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA) were found to be less sensitive in vitro to Az compared to the wild-type. Az treatment prolonged the survival of Galleria (G.) mellonella infected with Francisella. Conclusion These studies demonstrate that Type A Francisella strains, as well as F. novicida and F. philomiragia, are sensitive to Az in vitro. Francisella LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular Francisella, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of Francisella, especially in macrophages. Az treatment prolonged survival an in vivo model of Francisella-infection.