Nitric Oxide (NO) Induction Or Exposure May Be a Natural Biofilm Dispersal Method That Can Be Harnessed To Control Biofilm-Related Infections.
Table of Contents
- Introduction
- Endogenously produced NO via inducible nitric oxide synthase (iNOS)
- Endogenously produced NO via endothelial nitric oxide synthase (eNOS)
- UV Light and NO Production in the Skin
- Use of NO donors
- SUNLIGHT – Synergy of Related Compounds
- Theoretical Strategy
- References
Introduction
Diseases associated with biofilms exhibit significant diversity, given that biofilms can develop on various substrates, encompassing both living tissues and abiotic surfaces. Examples of biofilm-related diseases include infections in the respiratory system (rhinosinusitis and CF patients), urinary tract (UTIs), ears (otitis media), heart (endocarditis), oral cavity (plaque, gingivitis, and periodontitis), and wounds. Additionally, biofilms can form on medical devices such as dialysis catheters, contact lenses, and prosthetic implants. The broad spectrum of these conditions poses a considerable challenge in devising effective antibiofilm treatments that can be universally applied across multiple diseases. The complexity arises from the diverse nature of biofilm disruption, dispersal, and formation on different surfaces and tissues, necessitating a novel approach beyond using only antibiotics. Below, I will discuss biofilm, how nitric oxide (NO) relates to biofilm, and my theory on how NO can synergistically be harnessed to disrupt biofilm and assist natural and pharmaceutical antimicrobials to combat bacterial-related diseases.
Biofilm formation represents a complex, multi-stage phenomenon characterized by the synchronized differentiation of cells. After the initial attachment phase, bacteria generate copious extracellular polymeric substances (EPS) consisting of polysaccharides, proteins, extracellular nucleic acids, lipids, and metal ions. These substances irreversibly anchor the cells to the surface, setting the foundation for subsequent stages. As the biofilm matures, intricate three-dimensional structures composed of highly specialized bacteria emerge, contributing to the biofilm environment’s heterogeneous nature and bacterial communities.
In the conclusive phase of biofilm development, known as the dispersal event, a coordinated release of a subpopulation of cells occurs, enabling them to explore and colonize new surfaces. Recent research has pinpointed nitric oxide (NO), a simple gas and pervasive biological signaling molecule, as a crucial mediator in biofilm dispersal, exhibiting conservation across various microbial species (S. aureus, E. coli, P. aeruginosa, B. subtilis, N. gonorrhoeae, Legionella pneumophila, and others). Building upon this discovery, I propose a novel, albeit untested, approach involving NO-inducing compounds and techniques to potentially inhibit, disrupt, and diminish bacterial biofilms via multiple pathways. This speculative strategy aims to leverage the role of nitric oxide in disrupting biofilm integrity, offering a promising avenue for future exploration in the realm of biofilm control.
Endogenously produced NO via inducible nitric oxide synthase (iNOS)
Overview: Hazardous stimuli, including microbial invasion (H. pylori), UVB light exposure, inflammatory cytokines, and biofilm, upregulate the expression of iNOS, and NO produced by the enzyme modulates the resultant inflammatory and immune-system reactions.
NO is produced endogenously during the biofilm life cycle via multiple mechanisms to induce dispersal and trigger the transition to a planktonic lifestyle. In the context of bacteria, the planktonic lifestyle denotes a state where individual bacterial cells are mobile instead of being part of a biofilm or attached to a solid surface. Lack of production or exposure to NO inhibited cell death and dispersal, while endogenous production or exposure to low doses of NO via donor compounds restored the sensitivity of biofilm and dispersed bacteria towards several classes of antimicrobial agents, greatly increasing their efficacy. For example, NOS activity and generation of NO are required to clear infections in the respiratory tract. In cystic fibrosis patients, there is an inability to produce NO in response to pathogen (P. aeruginosa) invasion, which appears to play a major role in establishing chronic infections and compromised NOS activity. As a result, this may be the primary reason for the poor antimicrobial effectiveness in CF patients.
Endogenously produced NO in response to microbial invasion or biofilm is mediated by the enzyme inducible nitric oxide synthase (iNOS), which catalyzes the conversion of arginine into nitric oxide. NOS and iNOS are involved in immunity and autoimmunity. In macrophages and neutrophils, iNOS enzymes are activated by bacterial lipopolysaccharides (LPS), an important outer membrane component of gram-negative bacteria, as well as from the inflammatory cytokines (e.g., Interferon-gamma, IL-1b, and TNF-a). In some cases, impairment of NOS function can lead to infectious disease, inflammation, and exacerbation of autoimmune diseases (colitis, arthritis, and multiple sclerosis…) The cause of NOS malfunction may originate from the host or the bacteria themselves. Note: As a conserved protective mechanism, the bacterium Helicobacter pylori was found to secrete arginase to inhibit NO production in gastric mucosa and potentially aid in its virulence and survival.
Endogenously produced NO via endothelial nitric oxide synthase (eNOS)
Overview: The mechanical force that blood flow exerts on the endothelial cells lining the blood vessels, known as ‘shear stress,’ upregulates the expression of eNOS, and NO produced by the enzyme modulates vascular tone, blood flow dynamics, platelet aggregation, and overall cardiovascular homeostasis (balance).
Endothelial nitric oxide synthase (eNOS) is an enzyme pivotal for synthesizing nitric oxide (NO) within the endothelial cells of blood vessels, responding specifically to mechanical forces known as ‘shear stress.’ Shear stress denotes the mechanical force the blood flow exerts on the endothelial cells lining the blood vessels. Elevated blood flow, such as during physical exertion or alterations in blood pressure, intensifies shear stress, consequently augmenting NO production. This orchestrated increase in nitric oxide level serves as a regulatory mechanism governing vascular tone, blood flow dynamics, platelet aggregation, and overall cardiovascular homeostasis (balance).
UV Light and NO Production in the Skin
Overview: Exposure of the skin to ultraviolet light upregulates the expression of NO in a non-NOS-dependent fashion via a photochemical reaction involving nitrogen-containing compounds.
NO isn’t stored biologically and has a half-life of seconds, but stores of the more stable nitrogen oxides, nitrate (NO−3), nitrite (NO−2), and nitrosothiols (-SNO) in human skin are around ten times as high as those in the circulation. NO can be generated in our epidermal (keratinocytes), microvascular endothelial cells, and melanocytes following exposure to UVB (280-320 nm) and UVA (320-400 nm) wavelengths of sunlight. UV radiation from sunlight induces photochemical reactions, independent of NOS, with stored nitrogen-containing compounds (Nitrates and nitrites), which then could generate NO in keratinocytes as well as produce and release NO to the surrounding smooth muscle cells, causing an immediate dilatory effect. In addition to this, NO or its secondary products can be released directly into the bloodstream to be transported further away to have wide-ranging effects. This process necessitates sufficient quantities of precursor and carrier compounds, including the amino acids arginine and citrulline. Nitrate and nitrite play roles in this process, along with S-nitrosothiols, which are compounds containing a nitric oxide group bound to a sulfur atom. Additionally, nitrosohaemoglobin, a form of hemoglobin in red blood cells, is a carrier of nitric oxide, releasing it in response to various stimuli. This process is distinct from the shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in blood vessels as well as biofilm-induced activation of inducible nitric oxide synthase (iNOS).
Use of NO donors
The most versatile option for delivering NO to infectious biofilm is stimulating endogenous production via NO-donor molecules that can liberate NO in vivo. Release of NO from donors can occur either spontaneously from biofilm cells or surrounding infected tissues, upon activation by enzymatic activity, or through activation under select physical or chemical conditions, e.g., pH, UV light (sunlight) exposure, cardiovascular exercise (HITT).
In preliminary clinical studies, treatments with nebulized L-arginine in cystic fibrosis patients infected with P. aeruginosa resulted in sustained improvement in lung function associated with significantly increased NOS activity within lung tissues, suggesting that the NO augmentation could potentially reduce bacterial infection.
The ability of NO to induce the dispersal of biofilms offers great promise for developing a novel and efficient therapy for controlling biofilm-related infections and for overcoming biofilm resistance. While exposure to low-dose NO alone appears to be non-toxic to bacteria, the released planktonic cells and cells still residing on surfaces show increased susceptibility to various antibiotics and antimicrobials. Thus, NO-based anti-biofilm strategies could benefit from combined treatments with natural antimicrobials, biofilm disrupters, standard antibiotic therapies, and NO stimulation protocols to assist in clearing infections.
Supplementation with L-Glutamine, a nitrogen-containing amino acid, increases NO in the absence of extracellular arginine. Some studies have demonstrated that glutamine enhances wound healing partly because it increases the concentration of arginine and citrulline, a precursor of arginine. Glutamine thus allows NO production in the absence of extracellular arginine in monocytes and macrophages. Glutamine has also been shown to reduce C-reactive protein (CRP). CRP plays important roles in inflammatory processes and host reactions against infections, including NO release, apoptosis, and IL-6 and tumor necrosis factor-α (TNFα) production. Therefore, CRP levels increase in sick patients and are correlated with the severity of the illness in the patient, thus objectively quantifying the patient’s stress and acuity. Hence, the decline of CRP indicates the reduction of the overall inflammation.
Lastly, circulating nitrate, normally derived both from endogenous NO production and from dietary intake (beet, arugula/rocket, sodium nitrate…), is actively taken up by the salivary glands, excreted in saliva, and reduced to nitrite by commensal bacteria in the oral cavity. By this route, nitrate intake elevates systemic nitrite levels. This nitrate-nitrite-NO pathway represents an alternative and differently regulated system for NO generation that operates in parallel to the classical L-arginine-NOS-NO pathway. One important difference is that, in contrast to the NOSs, all known mechanisms by which nitrite is converted to NO are greatly facilitated during hypoxia and low pH. Dietary Inorganic Nitrate Improves Mitochondrial Efficiency in Humans
SUNLIGHT – Synergy of Related Compounds
Ultraviolet Light – Ultraviolet light not only stimulates NO but other immune-modulating compounds such as Vitamin D and proopiomelanocortin (POMC), which stimulates alpha-melanocyte-stimulating hormone, which increases DOPA melanin, and eumelanin – the most common type of melanin found in humans. Without sunlight exposure, endogenous vitamin D synthesis or melanogenesis does not occur. Supplements and pharmaceuticals are not as effective as nature when it comes to optimizing our body’s defenses against microbial invasion and biofilm, in my opinion.
UVB – UVB upregulates vitamin D levels, which upregulates the expression of cathelicidin antimicrobial peptides (CAMPs). CAMPs, especially LL-37, are a class of small proteins that play a crucial role in the innate immune response against microbial invaders such as bacteria, viruses, and fungi. Combined, vitamin D and CAMPs contribute to maintaining the integrity of our skin barrier, which serves as the first line of defense against microbial invasion. They help prevent pathogens from entering the body through the skin and play a vital role in wound healing and skin homeostasis (balance). Adequate D levels, which means adequate sunlight exposure, are needed for the efficiency of this physiological pathway to occur. Over 50 ng/ml would be the minimum goal I would shoot for, from sunlight, not supplementation. For reference, one multiple sclerosis (MS) study on neonatal vitamin D levels found that individuals whose vitamin D levels were below 20.7 nmol/L had a high risk of MS development, whereas those with above 48.9 nmol/L had the lowest risk. An increase of 25 nmol/L in neonatal vitamin D caused a 30% reduction in MS risk.
Vitamin D – Vitamin D is a main player in our defense against microbial invasion and biofilm formation. Adequate vitamin D (UVB) is responsible for the upregulation of DEFENSINS, which, Like CAMPs, are antimicrobial peptides that can neutralize microbes by disrupting their cell membranes; NOD-LIKE RECEPTORS (NLRs), which are a group of intracellular sensors that detect microbial components and initiate immune responses; TOLL-LIKE RECEPTORS (TLRs), which are another group of receptors that recognize microbial patterns and activate immune responses; INTERFERONS, which are signaling proteins that play a role in antiviral defenses and modulate immune responses against various pathogens, including bacteria and fungi; PHAGOCYTE and AUTOPHGAY REGULATION, which engulf and digest microbes, and target intracellular pathogens and dysfunctional components for degradation, respectively; and inflammatory CYTOKINES, which are another type of signaling molecules that regulate immune responses. This can influence the inflammatory environment and the body’s ability to combat microbial infections and biofilm formation (NO is also generated during biofilm formation).
Theoretical Strategy
The signaling role of NO in regulating biofilm dispersal across microbial species offers an unprecedented opportunity to develop novel treatments to induce biofilm dispersal. Optimizing NO physiology via exercise, diet, and sunlight is an evolutionary cocktail that is missing from our modern life and may be contributing to the rise in resistant and chronic bacterial and yeast infections. Going old-school may be just what the doctor ordered to improve treatments for chronic infections.
The data above supports the idea that by utilizing a comprehensive, natural approach, combining donors and activators, NO could be more efficiently and abundantly produced. First, one would ingest daily natural activators and donors to support NO synthesis. These include, but are not limited to, L-glutamine, L-arginine, L-citrulline, arugula, and beetroot. Next, consistently increase blood flow (sheer force), upregulating the expression of eNOS. This is done via aerobic exercise and, more importantly, anaerobic exercise, thus capitalizing on its hypoxic and acidic nature. Lastly would be regular ultraviolet (sunlight) exposure, which not only benefits our mood and the efficiency of our immune system but also has a strong upregulatory effect on NO production. This alone may not be sufficient in all cases, but in those cases that require antibiotic therapy, applying the above strategy will most doubtlessly add rocket fuel to any pharmaceutical intervention.
The future of chronic biofilm management is looking brighter and more promising than ever.
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News:
Erectile Dysfunction Drugs May Protect Against Alzheimer’s – My commentary: Both Cialis and Viagra are phosphodiesterase type 5 (PDE5) inhibitors. PDE5 normally breaks down cyclic guanosine monophosphate (cGMP), which is a secondary messenger involved in the nitric oxide pathway. By inhibiting PDE5, Viagra and Cialis increase the levels of cGMP, leading to enhanced vasodilation (dilation of blood vessels) and improved blood flow. While the primary therapeutic effect of Viagra (sildenafil) and Cialis (tadalafil) is targeted at the erectile tissue of the penis, these medications can have vasodilatory effects throughout the body. This can result in increased blood flow to various peripheral tissues, including fingers, toes, brain, and other organs. NO precursors and NO stimulation are needed for NO synthesis, but the effects can be short-lived. Viagra and Cialis assist by increasing cGMP, thus synergistically supporting increased vasodilation. This synergistic effect allows for more oxygen to tissues. I believe this is why these medications can confer protection against neurodegenerative disease.
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Regarding Viagra : I had dark blue gangrene below the ankles in 2021. Instead of undergoing amputation, I stayed in a bed for 5 months. Long enough to allow some healing and learning how to walk again…(Long story).
Winters became very tough and painful. The tip of my toes was still rock hard. In 2024, 3 days after starting viagra 50 mg per day, the oxygen starved blue blood stuck, started to ooze out of my toes (no cut, no pain). The previous hard tip toes, became soft and the cold so far is not painful. Waiting for January to tell how much it had improved.
I wish I was given viagra back when I was in bed, letting my feet recover on their own. You can email me for more details.
Amin,
Interesting story. The power of nitric oxide at work.
Dr. E