Abstract:Pseudomonas aeruginosa and Staphylococcus aureus are ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. This study is the first to use laser ablation electrospray ionization mass spectrometry (LAESI-MS) to rapidly study bacteria within a mixed biofilm. Fast, direct, non-invasive LAESI-MS analysis of biofilm could significantly accelerate biofilm studies and provide previously unavailable information on both biofilm composition and the effects of antibiofilm treatment. LA… Show more
“…P. aeruginosa Green-fluorescent protein (GFP) + and S. aureus Red-fluorescent protein (RFP) + were used for these studies. 19 The cells were incubated for 24 h in tryptic soy broth (TSB) medium in the presence of DRGN-1. The next day, the wells were rinsed and the development of a biofilm was compared with a control well (without DRGN-1).…”
Section: Resultsmentioning
confidence: 99%
“…Biofilms of both P. aeruginosa and S. aureus were significantly reduced by DRGN-1. In mixed-culture biofilms grown for 24 h without treatment, P. aeruginosa significantly outgrows S. aureus , 19 thus the appropriate starting ratio was established as a 99:1 ratio S. aureus to P. aeruginosa . Treatment with DRGN-1 had a significant inhibitory effect on the mixed bacterial cultures in contrast with untreated control.Fig.…”
Section: Resultsmentioning
confidence: 99%
“…We have previously characterized P. aeruginosa and S. aureus -mixed biofilms for their heterogeneity and composition; 19 these biofilms were generated and applied to a mouse wound. In our model, we will test whether wound healing in P. aeruginosa and S. aureus biofilm - infected skin wounds is significantly improved following peptide treatment compared to an untreated, infected wound, following the design of other published studies.…”
Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon (Varanus komodoensis), called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus. DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm (Pseudomonas aeruginosa and Staphylococcus aureus)-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial (Pseudomonas aeruginosa & Staphylococcus aureus) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide’s ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was determined to be via anti-biofilm activity and antibacterial activity through membrane permeabilization. This novel peptide may have potential as a future therapeutic for treating infected wounds.
“…P. aeruginosa Green-fluorescent protein (GFP) + and S. aureus Red-fluorescent protein (RFP) + were used for these studies. 19 The cells were incubated for 24 h in tryptic soy broth (TSB) medium in the presence of DRGN-1. The next day, the wells were rinsed and the development of a biofilm was compared with a control well (without DRGN-1).…”
Section: Resultsmentioning
confidence: 99%
“…Biofilms of both P. aeruginosa and S. aureus were significantly reduced by DRGN-1. In mixed-culture biofilms grown for 24 h without treatment, P. aeruginosa significantly outgrows S. aureus , 19 thus the appropriate starting ratio was established as a 99:1 ratio S. aureus to P. aeruginosa . Treatment with DRGN-1 had a significant inhibitory effect on the mixed bacterial cultures in contrast with untreated control.Fig.…”
Section: Resultsmentioning
confidence: 99%
“…We have previously characterized P. aeruginosa and S. aureus -mixed biofilms for their heterogeneity and composition; 19 these biofilms were generated and applied to a mouse wound. In our model, we will test whether wound healing in P. aeruginosa and S. aureus biofilm - infected skin wounds is significantly improved following peptide treatment compared to an untreated, infected wound, following the design of other published studies.…”
Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon (Varanus komodoensis), called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus. DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm (Pseudomonas aeruginosa and Staphylococcus aureus)-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial (Pseudomonas aeruginosa & Staphylococcus aureus) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide’s ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was determined to be via anti-biofilm activity and antibacterial activity through membrane permeabilization. This novel peptide may have potential as a future therapeutic for treating infected wounds.
“…Many advanced versions of the FISH technique have been implemented for different purposes, most commonly; catalyzed reporter deposition-FISH (CARD-FISH) for fluorescent signal enhancement (Lupini et al, 2011), peptide nucleic acid-FISH (PNA-FISH) for better penetration and faster hybridization (Werthén et al, 2010) and CLASI-FISH for expanding the number of distinguishable taxa in complex communities (Valm et al, 2012). In addition, an innovative technique, laser ablation electrospray ionization mass spectrometry (LAESI-MS), has been used for studying the spatial distribution of mixed species biofilms and is showing potential advantages contrasted to CLSM experiments (Dean et al, 2015), as LAESI-MS allows for rapid analysis of unfixed and wet biofilms.…”
Section: Methods To Examine and Link The Spatial Organization To Biolmentioning
Interspecies interactions are essential for the persistence and development of any kind of complex community, and microbial biofilms are no exception. Multispecies biofilms are structured and spatially defined communities that have received much attention due to their omnipresence in natural environments. Species residing in these complex bacterial communities usually interact both intra- and interspecifically. Such interactions are considered to not only be fundamental in shaping overall biomass and the spatial distribution of cells residing in multispecies biofilms, but also to result in coordinated regulation of gene expression in the different species present. These communal interactions often lead to emergent properties in biofilms, such as enhanced tolerance against antibiotics, host immune responses, and other stresses, which have been shown to provide benefits to all biofilm members not only the enabling sub-populations. However, the specific molecular mechanisms of cellular processes affecting spatial organization, and vice versa, are poorly understood and very complex to unravel. Therefore, detailed description of the spatial organization of individual bacterial cells in multispecies communities can be an alternative strategy to reveal the nature of interspecies interactions of constituent species. Closing the gap between visual observation and biological processes may become crucial for resolving biofilm related problems, which is of utmost importance to environmental, industrial, and clinical implications. This review briefly presents the state of the art of studying interspecies interactions and spatial organization of multispecies communities, aiming to support theoretical and practical arguments for further advancement of this field.
“…Dean et al [30] analyzed mixed biofilm of S. aureus and P. aeruginosa by laser ablation electrospray ionization mass spectrometry, and found that S. aureus and P. aeruginosa within the mixed biofilm showed significant colocalization of the cells, rather than self-segregation. Thus, the growth rate of coculture biofilms could be in between the growth rate of monospecies biofilms (that is, greater than P. aeruginosa and less than S. aureus ), according to the findings of the current study.…”
Objective: Pseudomonas aeruginosa and Staphylococcus aureus dual-species biofilm infections are notoriously difficult to manage. This study aimed at investigating the influence of four different culture media on the planktonic growth, adhesion, and biofilm formation of P. aeruginosa and S. aureus. Materials and Methods: We monitored four different culture media including Nutrient Broth, Brain Heart Infusion (BHI) broth, Luria-Bertani broth, and RPMI 1640 medium on the planktonic growth, adhesion, and biofilm formation of P. aeruginosa (ATCC 27853) and S. aureus (ATCC 25923) using MTT assay and scanning electron microscopy (SEM). Results: The most robust growth of the mono- and dual-species cultures was noted in BHI broth. On the contrary, RPMI 1640 medium promoted maximal initial adhesion of both the mono- and dual-species, but BHI broth fostered the maximal biofilm growth. SEM images showed profuse extracellular polysaccharide production in biofilms, particularly in coculture, in BHI medium. Conclusion: Our data demonstrate that BHI broth, relative to the other tested media, is the most conducive for in vitro evaluation of biofilm and planktonic growth kinetics of these two pathogens, both in mono- and coculture.
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