Gauze containing designed peptide inhibits bacteria biofilms

Major progress is being reported in developing alternative wound dressings to prevent biofilm-associated infections and overcome difficult-to-treat bacteria.

“These are critical findings as the world faces a growing threat of antibiotic resistance and a decline in the development of traditional antibiotics,” said Valerie Daggett, a University of Washington College of Engineering and UW School of Medicine bioengineering professor who headed the research. 

Biofilms appear when previously free-floating bacteria cluster inside a self-produced slime that guards their colony. Infectious bacteria protected by a biofilm are better able to defend against antimicrobials as well as disease-fighting cells. A bacterial biofilm in a wound can impair healing and require more heavy-duty treatment. 

Wound biofilms in injured or surgical patients are a serious medical concern. They can lead to longer and more costly hospitalizations, the need for painful removal of damaged tissues, and higher post-injury or post-surgery death rates, the researchers noted. 

Wounds are highly prone to bacterial infection. To make matters worse, most infected wounds are colonized with a variety of bacteria. That situation can complicate treatment, as some antibiotics are specific either to gram-negative or gram-positive bacteria. 

In a paper published March 4 in the Journal of Biomedical Materials Research, researchers describe their recent experiments using alpha-sheet peptides to combat biofilm formation by bacteria that commonly infect wounds.  Peptides are short chains of amino acids linked  together, somewhat like proteins, but smaller. 

The alpha-sheet peptides, not found in nature, were computationally designed and chemically synthesized in the Daggett’s laboratory.

Daggett, her graduate student Sarah E. Nick and colleague James D. Bryers, a UW professor of bioengineering, adapted gauze dressings, which functioned as both a biofilm inhibitor and destabilizer as well as a drug delivery vehicle. The major components of this gauze, chitosan and alginate, are antimicrobial but non-toxic to humans. These components were paired with the alpha-sheet peptides in alternating layers. 

In laboratory tests, the layer-by-layer gauze facilitated an initial burst delivery of the alpha-sheet peptides followed by controlled release for 72 hours. When tested against Escherichia coli biofilms, the functionalized gauze reduced colony-forming units of bacteria by 81%, compared to plain gauze, and by 96% when the gauze was accompanied by the antibiotic gentamycin. Similar results were obtained against Staphylococcus aureus with plain gauze and with gauze and the antibiotic vancomycin. 

Daggett and her research team had discovered some years ago that their designed alpha-sheet peptides could disrupt certain toxic molecular structures that appear early during amyloid formation. Amyloids are aggregations of proteins that have undergone structural changes to form fibrils, or slender rods. Abnormal amyloids can amass into fibrous deposits or tangles that have been called out in a variety of troubles, from degeneration of brain cells to damage to pancreatic cells. Amyloid fibrils also play a supporting role in biofilms.

“By targeting amyloid formation with our designed alpha-sheet peptides,” Daggett noted, “we can prevent the formation of the amyloid fibrils that stabilize the biofilms, which subsequently increases the efficacy of existing antibiotics as well as the host immune response.”

In earlier studies, Daggett and her team observed that their alpha-sheet peptides inhibited biofilm formation and increased antibiotic susceptibility in both gram-negative and gram-positive bacteria. Gram negative bacteria are, by their nature, more impervious. The researchers were pleased to see that the effectiveness of the alpha-sheet peptides was not confined to a specific species of bacteria. 

The researchers also found that the alpha sheet peptides did not cause cell death. This finding eliminated the concern that this approach might exert selective pressure on bacteria to push the evolution of more resistant strains. 

“Instead, amyloid inhibition facilitates a shift in the bacterial cells from the biofilm to a free-floating state, rendering them more susceptible to clearance and killing by various mechanisms,” the researchers explained.

Daggett, her graduate student Sarah E. Nick and colleague James D. Bryers, a UW professor of bioengineering, determined that biofilm-associated wound infections would be ideal for evaluating the peptide-based treatment. 

“Treatments such as the alpha-sheet peptides that can target multiple bacterial species might be highly advantageous,” Daggett said. Her research team has demonstrated the efficacy of the alpha peptide sheets in disrupting biofilms in a range of bacteria, including Staphylococcus aureus, Escherichia coli, and Streptococcus mutans. The scientists also mentioned that species like Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptomyces coelicolor and Bacillus subtilis also have amyloids in their biofilm matrix and could therefore be potential targets for this type of treatment testing. 

For their study to improve wound treatment, the researchers adapted gauze dressings, which functioned as both a biofilm inhibitor and destabilizer as well as a drug delivery vehicle. The major components of this gauze, chitosan and alginate, are antimicrobial but non-toxic to humans. These components were paired with the alpha-sheet peptides in alternating layers. 

In laboratory tests, the layer-by-layer gauze facilitated an initial burst delivery of the alpha-sheet peptides followed by controlled release for 72 hours. When tested against Escherichia coli biofilms, the functionalized gauze reduced colony-forming units of bacteria by 81%, compared to plain gauze, and by 96% when the gauze was accompanied by the antibiotic gentamycin. Similar results were obtained against Staphylococcus aureus with plain gauze and with gauze and the antibiotic vancomycin. 

The scientists’ immediate next steps will be to further assess the functionalized gauze in lab dish biofilm models and to perform additional tests of biocompatibility to be sure the dressing is not injurious to host cells or tissues. They are looking to acquire funding to test the layered gauze pads as wound dressings for lab rodents. They will be assessing the shelf-life of the functionalized gauze as well, to accommodate long-term storage requirements for bandages. 

Beyond biofilms in wound infections, the Daggett lab has studied several other amyloid-forming proteins suspected of a role in diseases. These include Alzheimer’s and diabetes. Her team is using the designed alpha-sheet peptides to develop other potential diagnostic and therapeutic agents. For example, her group has developed a blood assay for predicting preclinical to advanced Alzheimer’s disease. This assay has FDA Breakthrough Device status. The National Institutes of Health named it as one of five Promising Medical Findings for 2023. Her lab is also developing a Alzheimer’s therapeutic that is scheduled for human trials in late 2025. 

“The mechanism of action of our compounds is applicable to amyloid proteins of both human and bacterial origin,” Daggett said.

The wound biofilm infection research was supported by grants from the U.S. Army Medical Research Acquisition Activity, Department of Defense Office of the Congressionally Directed Medical Research Program (W81XWH-19-0050, the National Institutes of Health National Institute of Allergy and Infectious Diseases (6R01AI074661), and the NIH National Center for Advancing Translational Sciences through the Clinical and Translational Science Awards Program. Part of the work was conducted at the Washington Nanofabrication Facility, a National Nanotechnology Coordinated Infrastructure at the University of Washington with partial support from the National Science Foundation (NNCI-1542101 and NNCI-2025489.)

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