NITRIC OXIDE (NO)
What if you could say NO to biofilm?
The treatment of biofilm infections is a difficult challenge that requires new solutions.
Despite advances in dressing technology and best practice, wound care is in crisis: the number of hard-to-heal wounds is increasing and the implications for the healthcare system, including greater antibiotic usage, are challenging.
To improve the management of hard-to-heal wounds, it is necessary to address the tenacious biofilm
that is present in most of them.1 A range of dressings display antimicrobial efficacy. However, the wound microenvironment is complex and influenced by various factors.
Certain topical agents appear to affect the structure of biofilms to varying degrees.
Nevertheless, there remains a need for solutions capable of eradicating and preventing biofilm reformation, while preserving healthy tissue and fostering an environment conducive to healing.2,3
Nitric oxide, an endogenous antimicrobial agent, has become an attractive candidate in wound therapy protocols.4
Nitric oxide (NO) in wound healing
NO is a natural, broad range antimicrobial produced in response to infection as part of the inflammatory response.5,6
Poorly controlled diabetic patients, for example, display a reduced NO production, which has been linked to impaired wound healing and the development of chronic wounds.4,7
NO’s unique properties have led to its use in innovative medical applications for wound healing, as a topical antimicrobial agent with no demonstrated resistance and combined activity against biofilm, holding the promise of improving wound care and patient outcomes.4,8
The antimicrobial action
NO is a broad-range antimicrobial agent, with multiple modes of action against protein, lipid and nucleic acid microbial components, exerting combined actions to kill bacteria due to its increased ability to penetrate microbial cell wall, inhibited microbial cell replication and disrupt vital microbial processes.9-12
NO‘s antimicrobial action stems from its ability to penetrate inside microbial cells and exert a combination of antibacterial actions working in synergy to kill bacteria.
- NO increases the permeability of microbial cells
As a small, lipophilic molecule, NO freely penetrates inside bacteria, inactivating both and internal cell wall proteins with a dose-dependent breakdown of the lipid bilayer, leading to degradation and increased permeability of microbial cells.6,13-16 - NO inhibits replication
As NO moves into the microbial cell, it can also damage or destroy microbial DNA and inhibit its replication, leading to microbial cell dysfunction and death.13,17,18 - NO disrupts vital microbial processes
NO also disrupts metabolic and respiratory processes by inactivating iron-sulphur clusters, which are essential enzyme co-factors for gene expression, metabolism, and cell respiration.19-21
The journey of NO through the bacterial cells
Stages
NO inactivates extracellular proteins, freely penetrates bacterial cell walls, and inactivates internal cell wall proteins
NO destroys (or damages) microbial DNA
NO inhibits DNA replication
NO inactivates iron-sulphur clusters essential in metabolism, respiration, etc.
The antibiofilm activity
NO‘s antibiofilm action comes from its ability to expose bacteria and inhibit the biofilm’s mechanisms of defence and resistance.6,17-23
- Biofilm matrix breakdown
NO breaks down the protective extracellular polymeric substances that form the biofilm structure, allowing further penetration of NO, other antimicrobials, and host defences to reach the bacteria within.24-26 - Bacteria dispersal
Even at low doses, below bactericidal concentration, NO mimics biofilm signals the bacteria to disperse, exposing them.13 - Impaired bacterial communication
Additionally, NO impairs bacterial communication, reducing both biofilm formation and bacterial virulence to reduce the infection risk, while making the biofilm more susceptible to antimicrobials so removing a barrier to wound healing.27-29
The journey of NO through the biofilm
Stages
NO breaks down biofilm EPS
NO causes biofilm dispersal
NO prevents communication in the biofilm
Antimicrobial action
- Increased permeability of microbial cells
- Replication inhibition
- Microbial processes disruption
Antimicrobial action
- Biofilm matrix breakdown
- Bacteria dispersal
- Impaired bacterial communication
1Murphy C, Atkin L, Swanson T, Tachi M, Tan YK, Vega de Ceniga M, Weir D, Wolcott R. International consensus document. Defying hard-to-heal wounds with an early antibiofilm intervention strategy: wound hygiene. J Wound Care. 2020;29(Suppl 3b):S1–28.
2Cavanagh MH, Burrell RE, Nadworny PL. Evaluating antimicrobial efficacy of new commercially available silver dressings. Int Wound J. 2010;7:394-405.
3Weigelt MA, McNamara SA, Sanchez D, Hirt PA, Kirsner RS. Evidence-Based Review of Antibiofilm Agents for Wound Care. Adv Wound Care (New Rochelle). 2021 Jan;10(1):13-23. doi: 10.1089/wound.2020.1193. Epub 2020 Jun 22. PMID: 32496980; PMCID: PMC7698998.
4Malone-Povolny MJ, Maloney SE, Schoenfisch MH. Nitric Oxide Therapy for Diabetic Wound Healing. Adv Healthc Mater. 2019;8(12):e1801210. doi:10.1002/adhm.201801210.
5Edmonds ME, Bodansky HJ, Boulton AJM, Chadwick PJ, Dang CN, D‘Costa R, Johnston A, Kennon B, Leese G, Rajbhandari SM, Serena TE, Young MJ, Stewart JE, Tucker AT, Carter MJ. Multicenter, randomized controlled, observer-blinded study of a nitric oxide generating treatment in foot ulcers of patients with diabetes-ProNOx1 study. Wound Repair Regen. 2018 Mar;26(2):228-237. doi: 10.1111/wrr.12630. Epub 2018 Jul 17. PMID: 29617058.
6Fang FC. Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest. 1997;99:2818-2825.
7Ahmed R, Augustine R, Chaudhry M, et al. Nitric oxide-releasing biomaterials for promoting wound healing in impaired diabetic wounds: State of the art and recent trends. Biomed Pharmacother. 2022;149:112707. doi:10.1016/j.biopha.2022.112707.
8Seabra, A.B. (2016). Antibiotic Resistance. || Can Nitric Oxide Overcome Bacterial Resistance to Antibiotics?. , (), 187-204. doi:10.1016/B978-0-12-803642-6.00009-5.
9Schairer DO, Chouake JS, Nosanchuk JD, Friedman AJ. The potential of nitric oxide releasing therapies as antimicrobial agents. Virulence. 2012 May 1;3(3):271-279. doi: 10.4161/viru.20328. Epub 2012 May 1. PMID: 22546899; PMCID: PMC3442839.
10Waite RD, Stewart JE, Stephen AS, Allaker RP. Activity of a nitric oxide-generating wound treatment system against wound pathogen biofilms. Int J Antimicrob Agents. 2018 Sep;52(3):338-343. doi: 10.1016/j.ijantimicag.2018.04.009. Epub 2018 Apr 14. PMID: 29665443.
11Barraud N, Storey MV, Moore ZP, Webb JS, Rice SA, Kjelleberg S. Nitric oxide-mediated dispersal in single-and multi-species biofilms of clinically and industrially relevant microorganisms. Microb Biotechnol. 2009;2:370-378.
12Barraud N, Kelso MJ, Rice SA, Kjelleberg S. Nitric oxide: a key mediator of biofilm dispersal with applications in infectious diseases. Curr Pharm Des. 2015;21:31-42.
13Rong et al. Nitric oxide-releasing polymeric materials for antimicrobial applications: A review. Antioxidants 2019;8(11).
14Carpenter & Schoenfisch. Nitric oxide release: Part II. Therapeutic applications. Chem Soc Rev. 2012;41(10):3742.
15Wiegand et al. Antimicrobial effects of nitric oxide in murine models of Klebsiella pneumonia. Redox Biology. 2021;39(Dec 2020):101826.
16Dupree & Schoenfisch. Morphological analysis of the antimicrobial action of nitric oxide on Gram-negative pathogens using atomic force microscopy. Acta Biomat. 2009;5(5):1405-1415.
17Lepoivre et al. Inactivation of ribonucleotide reductase by nitric oxide. Biochem Biophys Res Com. 1991;179(1):442-448.
18Torrents. Ribonucleotide reductases: Essential enzymes for bacterial life. Front Cell Inf Micro. 2014;4(Apr):1-9.
19Fitzpatrick & Kim. Synthetic Modeling Chemistry of Iron-Sulfur Clusters in Nitric Oxide Signaling. Acc Chem Res. 2015;48(8):2453-2461.
20Radi. Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of Functional Effects. Acc Chem Res. 2013;46(2):550-559.
21Vanin. Physico-chemistry of dinitrosyl iron complexes as a determinant of their biological activity. Int J Mol Sci. 2021;22(19).
22Möller & Denicola. Diffusion of nitric oxide and oxygen in lipoproteins and membranes studied by pyrene fluorescence quenching. Free Radical Biology and Medicine. 2018;128:137-143.
23Hall, et al. Mode of Nitric Oxide Delivery Affects Antibacterial Action. ACS Biomat Sci Eng. 2020;6(1):433-441.
24Yu. Molecular Insights into Extracellular Polymeric Substances in Activated Sludge. Envir Sci Tech. 2020;54(13):7742-7750.
25Vu, et al. Bacterial extracellular polysaccharides involved in biofilm formation. Molecules. 2009;14(7):2535-2554.
26Chislett et al. Structural changes in model compounds of sludge extracellular polymeric substances caused by exposure to free nitrous acid. Water Res. 2021;188:116553.
27Heckler & Boon. Insights Into Nitric Oxide Modulated Quorum Sensing Pathways. Frontiers in Microbiology. 2019;10(Sept):1-8.
28Sharma, et al. Bacterial Virulence Factors: Secreted for Survival. Indian J Micro. 2017;57(1):1-10
29Vestby, et al. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics. 2020;9(2).
Big losses can start from small misses
In a disease with many challenging variables to control, small misses can be devastating. Improving our understanding of diabetic foot ulcers will enhance prevention and management strategies for DFUs.
The invisible barrier to healing
One of the main reasons why wounds are so difficult to heal is related to the presence of biofilms, which affects all phases of wound healing and is considered an independent factor in delaying normal wound healing.