Oxygen, Dysox, and Biofilms

Majid Ali, M.D. 

Biofilms are microbial tribes abiding by Darwinian imperatives—adapting to dynamic changes in the oxygen order and availability of their growth requirements in their microecologic conditions. Oral dysoxygenosis is the state in which oxygen homeostasis is regionally disrupted by local conditions in the mouth. Mercury from dental amalgams appears to be one of the most, if not the most, potent disrupters of oxygen metabolism in the oral cavity. Other such disrupters are thioethers and other microbial toxins. Those factors also alter the local conditions that either inhibit or foster microbial growth, so facilitating biofilm formation. Such dynamics seem to play crucial roles in the pathogenesis of systemic disorders rooted in the oral cavity.

In the classical sense, Costerton described biofilms as complex, heterogeneous bacterial colonies embedded in a protective slimy, polymeric polysaccharide matrix that they themselves produce.7-9 Such microbial communities are well known for their resistance to various antibiotics and are increasingly recognized as the roots of many chronic and indolent infections.10,11 Disruptions of local tissue architecture — fibroproliferative or occlusive lesions, for instance — facilitate biofilm formations and so impede healing with resolution. Antibiotic resistance of biofilms has been attributed to differentiated, structured groups of cells with communal characteristics that are distinct from those of the parent cells. Some Candida albicans biofilms examined were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of such biofilms were made up of tangled masses of hyphae with openings between germ tubes about 10 to 50 mM across. Such biofilm architecture markedly alters effective diffusion coefficients of various substances, such as chlorhexidine digluconate. Such changes in diffusion rates are of considerable consequence in terms of diminished fungicidal activities of various agents employed to eradicate infections caused by Candida and other mycotic species.

Biofilms also appear to play central roles in initiating, amplifying, and perpetuating anomalous and unremitting inflammatory responses — locally in the oral cavity and other locations as well as systemically in tissues far removed from the sites of biofilm formation. Such phenomena also set the stage for chronic and indolent lesions—infectious as well as noninfectious — again, not only locally in the dental, periodontal, and sinusoidal tissues, but also systemically in tissues distant from the oral and sinusoidal cavities.

The biofilm studies of direct relevance to the subject of oral dysoxygenosis, of course, are those that directly examine the oxygen dynamics and effects of reactive oxygen species on the biofilm formation and how the characteristics of biofilms, in turn, affect oxygen metabolism. In essence, we are interested in how dysfunctional oxygen metabolism may lead to biofilm formation and how biofilms increase the degree of local dysoxygenosis. In the context of dental amalgams, such considerations lead to the larger question of the impact of the “mercury-dysoxygenosis -biofilm dynamics” on the health/dis-ease/disease continuum in the oral cavity as well as in systemic tissues.

Oxygen mediates several of the bioelectric phenomena involving biofilms. It seems safe to predict that it will eventually be proven to influence all types of bioelectric effects in biofilms. In view of the enormous functional diversity of oxygen, I expect that oxygen among the products of electrolysis—protons, hydrogen, reactive oxygen species, hydroxyl ions and heat—would exert important effects on the system, though some might think reactive oxygen species might be more important. In the case of some species of Pseudomonas aeruginosa, in the biofilms at least, the role of oxygen in moderating the biofilm dynamics has been documented with experimental work. Electrolytically generated oxygen accounts for a major part of the electrical enhancement of efficacy of tobramycin against P. aeruginosa biofilm. The addition of sodium thiosulfate—an agent that rapidly neutralizes reactive oxygen intermediates—does not abolish electrical enhancement of microbial killing by the antibiotic. Interestingly, the increase in microbial killing with bubbling of gaseous oxygen through the chamber alone—without electric current—was far less than that with electrolysis.

Complex multispecies biofilms develop in the oral cavity under diverse conditions. In certain cases, pathogens in such films exist largely when supported by other nonpathogenic species in the biofilm, and vice versa. For example, Porphyromonas gingivalis and Streptococcus gordonii exist in oral biofilms only in the presence of each other. In this relationship, P. gingivalis adheres to S. gordonii by interacting with a specific region of the streptococcal SspB polypeptide, designated BAR.12 P. gingivalis in oral biofilm is of interest since it appears to play some role in the pathogenesis of atherosclerosis.

Of direct relevance to the mercury-related oxygenative dysfunction (MROD) model presented here are the altered redox dynamics in biofilms. The protective role of catalase and biofilm resistance to hydrogen peroxide has been investigated in Pseudomonas aeruginosa by comparing the susceptibility to hydrogen peroxide of wild-type strain PAO1 and the katA and katB catalase mutants.13-15 One-hour wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells in that film were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking H2O2-inducible catalase katB, exhibited a pattern of H2O2 resistance similar to that of wild-type strain, whereas plankton katA mutant cultures, possessing no detectable catalase activity, were hypersensitive to a single dose of 50 mM H2O2. Biofilms were capable of a rapid response to oxidant stress, as revealed by significant katB catalase induction detected by increased catalase activity bands in nondenaturing polyacrylamide gels.

Mercury, Biofilms, and Oxidative Coagulopathy

In the oral cavity, mercury-induced molecular and cellular injury, biofilm formation, osteonecrosis, and regional oxidative coagulopathy form a spectrum, the various aspects of which can be separated only in a theoretical sense. From the clinical standpoint, it is essential that that spectrum be seen as a continuum and that all involved factors be addressed integratively.

Biofilms pose special hazards to the structural and functional integrity of tissues in their vicinity. A very common histopathologic feature of all infectious lesions, as well as non-infectious inflammatory processes, is a prominent endothelial response designated as endothelial hyperplasia. Such endothelial responses are more pronounced in the vicinity of biofilms. Endothelial lining of arteries and capillaries is very responsive to changes in the concentration and partial pressure of oxygen circulating within those vessels. A host of genes and proteins—most notably belonging to the hypoxia-inducible factor (HIF) cascades—are involved in endothelial responses.16,17 This subject is presented at length in the chapter entitled “Dysoxygenosis.” Alterations in the pathophysiology of nitric oxide are equally important in endothelial dynamics. That subject is discussed in Nature’s Preoccupation With Complementarity and Contrariety, the first volume of this textbook.18

Biofilm-triggered and/or enhanced endothelial responses directly induce oxidative coagulopathy, since such responses are oxidative in nature.19 Microbial populations of biofilms also exaggerate the degree of oxidative coagulopathy initiated and perpetuated by other etiologic factors. Beyond inflicting endothelial injury, biofilms also facilitate invasion of the subendothelial stroma by their microbial residents. Collagen in the stroma is thought to provide a favorable microenvironment to microbial species spreading from biofilms, including the availability of receptors for some bacterial metabolites which serve as anchors for microbes. Not unexpectedly, those changes also put in jeopardy the structural and functional integrity of the bone in proximity to biofilms. The osseous tissue devitalized or rendered ischemic by periodontal infections and root canal procedures is especially vulnerable to necrosis. Those observations led some investigators to propose that a large number of cases of myocardial infarction and cerebrovascular accidents are related to a hypercoagulable state produced by oral biofilms.

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