0.0.2 - No conflict of interest - general idea(s) presented. Not written or edited or created, reviewed, manipulated with AI.
0.0.3 - Referred within the entire body of writing, B. fragilis is defined specifically as Bacteroides fragilis (subspecies fragilis) nctc 9343 / atcc25285 / or ‘wild-type’ possessing fragilysin (production) / Bft-1,2,3 / categorized as proteolytic/hydrolytic cleavage enzyme (i.e. EC 3.4.24.74) with other secondary virulence factors and possessing mechanisms of action(s) as referred within body of text. Reference is made with etbf only [not ntbf which or other bacteroides members which may impact exotoxigenic B. fragilis (fragilysin producing)].
0.0.4 - Written and expressed for the purpose of option, idea and think-tanking; such is not medical advice, assertion or assumption. The opinions of the author are retained by and of author, only. Such is willed to represent springboard for future research and awaiting cure/resolve of related conditions - such as: atherosclerosis and associated states of dis-ease, dis-order, syn-dromes ranging from cardiovascular (CHF, SA node, fibrotic), clotting, stroke (thrombotic, aneurysm), diabetes, cancer, lung, kidney, (bradykinin, succinate ACE producing, sodium/K+/CL- channel) hypertensive state, CNS/PNS manifestations - as but example(s).
0.0.5 - B. fragilis (exotoxigenic) represents, at very least - a prototypical example - among other possible contributing pathogens (of which it may additionally be involved - as mixed or poly-microbial member).
0.0.6 - Such text is written in good faith and serves as a very dilute, general overview - if not glimpse - into the interplay of some specific, observed and noted associations between B. fragilis and atherosclerosis. Reference is made to particular relevant and curious experiments performed - and resultant outcomes. Such text is written inference and ‘hypo’-thesis - to highlight associations between data - through the lens of exotoxigenic producing Bacteroides fragilis nctc 9343/atcc 25285. The intent is to explore underlying potentially overlooked and dismissed mechanisms of atherosclerosis causality related to said organism, carried and influenced by all humans and transferred with every birth and its unabated continuance (to date) - with every successive generation.
1.0 Introduction
1.1 – Introduction
Hypothesis
Bacteroides fragilis ssp. fragilis is the etiologic pathogen in atherosclerosis and the elusive metabolic “X” link; a maternally and environmentally transmitted nanaerobe that manifests itself as a part of the purported ‘normal’ commensal flora of various mammals. The organism, usually acquired after days 5-7 postgestation perpetuates a pro-inflammatory state within the host as result of chronic and subclinical infection upon disseminating from the intestine early after acquisition and before immunological development and anamnestic response is to such. Dissemination is a continued process throughout the life of the host once the organism has established and especially in the presence of a breach of innate or humoral defenses.
B. fragilis aminopeptidase N-like (CD13-like protease) and fragilysin (Bft-1,2,3) are proposed as the main implicating virulence factors facilitating and resulting in diapedesis and dissemination of the organism via (among other mechanisms) proteolytic cleavage of the many fragile and vulnerable cysteinyl-glycine and cysteinyl-lysine linkages.
Early in evolution, B. fragilis has adaptively developed a very efficacious ability to hydrolyse these peptide linkages as such are quintessential for structure conformation and functionality of a vast array of proteins within the host. Further, cleavage of such linkages provides while fulfilling cysteine (sulfur) requirements for the organism - denatures host proteins which play a pivotal role in innate, humoral, structural and enzymatic abilities orchestrated by the host – as well as detection of the opportunistic commensal.
The outcome of harboring B. fragilis by the host, among the multiple influencing factors which have presented themselves in the vast medical literature as ‘risk factors’ essentially reflects how the organism is cultured within the host organism and ultimately manifests itself relative the loci of establishment, colonization and infection.
Such examples (non-comprehensively) include the availability of specifically rate limiting and influential amino acids like cysteine and histidine, carbohydrates and reduced moieties (such as glucose, fructose, succinate and reduced sugars such as lactose, fucosylated glycans), sterols (cholesterol, estrogens, androgens, corticoids and saponins), trans-saturated fatty acyls (‘trans fats’), sphingolipids, alcohol, concentration gradients of mineral exposes, pharmacologic, chemotherapeutics and recreational agents (nicotine, ephedrine) which are minimal examples of agents that significantly alter the growth rate, survival and virulence of the organism throughout the host’s life cycle.
Other factors such as immunological, coagulatory, oxidative, radiological, the presence of oncologies, hemodialysis, organ transplant as well as genetic dispositions, familial exposures and carriage, contaminated source exposures, nutritional deficit and engrained & propagating behaviors (to name but some) – all influence B. fragilis as well as the host milieu; susceptibility and defense. It is this relational interplay that defines the vague as well as more definitive markers and associations observed between multiple variables, plethora of relations and vast demographic factors – studied from an epidemiological standpoint to wholly derivate some causal constant in relation to atherosclerosis.
Further, it is unlikely that such agent stands in isolation, silently absolved to the many associated conditions, diseases and disorders that coincide with atherosclerosis. Such examples - include (without limitation) and are hypothesized herein to be potentially associated, if not (mediated by the aforementioned virulent mechanisms) influenced by B. fragilis;
schizophrenia, depression, manic depressive illness, Parkinson’s dis-ease and
multiple sclerosis
It is, thus herein hypothesized that current global trends in seemingly disjointed entities such as atherosclerosis, metabolic X syndrome, diabetes, obesity and neurological manifestations such as Alzheimer’s, depression and multiple sclerosis – are commonly associated – if not influenced, mediated and perpetuated by the presence of Bacteroides fragilis NCTC 9343 capable of (among other virulence mechanisms of host evasion) producing aminopeptidase N and Bft-1,2,3.
The continued and current oversight regarding this organism’s involvement in atherosclerosis, diabetes and metabolic X is eerily reminiscent of the relevance of Helicobacter pylori relative gastric and duodenal ulcers, gastric adenocarcinoma and lymphoma – though originally observed in the host in 1906, only to be discovered as causative through the exhaustive efforts of Warren and Marshall 1982, some 76 years later. Interestingly, associations between H. pylori (as well as multiple other organisms and virions) and atherosclerosis have been made without mention of B. fragilis. Upper antral and gastric ulcers, likely represent a mixed H. pylori and exotoxigenic B. fragilis infection. It is curious if the significant H. pylori antibiotic eradication therapy, parallels also the intensity of therapy required to minimize the compounded effects of B. fragilis. It is entirely possible that many acute and chronic host infections (viral, bacterial, protozoal, fungal) potentially represent a constant mixed and/or polymicrobial state involving the presence of B. fragilis.
Perhaps all infective and inflammatory processes are more adaptively defined as multifactorial interactions – so as not to exclude any one variable as improbable. Here’s hoping we take notice regarding B. fragilis organisms.
I) Data scarcity on topic… difficulty in connecting B. fragilis with atherosclerosis…
Bacteroides fragilis (exotoxigenic/fragilysin producing) in general…
Resides in host (mainly) subclinically, in many forms/coats, may degrade its own footprints; a commensal so host generally does not recognize, may intermittently disseminate outside of bowel, testing sample error (anaerobe), homology of genes and proteins to human host, LPS is mild toxic at best, CD13 same as hosts, lives a quiet continuance, major events regarding atherosclerosis - occur early in life brain and nerves are the ultimate object of fixation/aim, only BFT in present at most times in atheroma, host organism is easy for BF cells to move through but BFT remains there, BF cells that are dead simply degrade (all or none effect to host defenses), plaque perpetuated by other members or continuous flow of Bft into blood stream, where a sample is taken/obtained, host cells can phagocytize and carry away! Is LPS and CD13 and BFT all tested (BFT1, and BFT2 and BFT3) are the genes for 9343 tested and/or easily mistaken for another pathogen.
It may be that do to a combination of virulence and host evasion factors and the impact of such by Bacteroides fragilis NCTC 9343 - since its initial impact upon each and every human during fetal development and/or lifelong colonization - subsequent to the reduction of oxygen within the intestine occurring during the first week post gestation - the treatment for atherosclerosis may involve only the eradication of such organisms. Due to its virulence factors, it is difficult to eliminate such organism least of which – eradicate without the necessary lifelong epitopes required for such elimination. In the absence of such, sighting the preceding evolution of Bacteroides fragilis well ahead of the origin of the eukaryotic cell and subsequent mammalian evolution and development, and specifically the early impact and origin of atherosclerosis during the initial phases of human development - it is likely that the latent and reactive impact upon such commensal to alter the progression atherosclerosis - maybe only otherwise influenced by a labor and time intensive approach such as the daily, chronic modification of risk factors and administration of therapeutics which otherwise influence the organism and its affects upon the host.
Unfortunately, in many regards, Bacteroides fragilis is regarded as much a natural human commensal that comprises the human flora, with opportunistic potential as opposed to simply and perhaps more appropriately a ‘commensal pathogen’. In a sense, Bacteroides fragilis NCTC 9343 has perhaps “cried Wolf”, playing a ‘symbiotic card’ far too long - disguised and hidden within the context of its ‘commensal status’. However, beyond a larger host perceived understanding it is likely that the more finite interactions within the host milieu – may be all that truly matters regarding atherosclerosis and certainly regardless of the human hosts interpretation of its understanding and interaction with such organism. Perhaps it is simply time to be realistic.
Perhaps a more probable scenario regarding Bacteroides fragilis NCTC 9343 involves an organism well evolved, developed and much advanced relative the human and other mammalian hosts which harbor it. It is conceivable, that if and when detected inherent virulence factors such as its zwitterionic and varied capsular thickness, responsive extracellular alterations upon host detection, LPS, low toxin endotoxins, and exoproteases (such as that conferred by fragilysin and aminopeptidase N, without including those released during cell lysis), various classes of cellular products and byproducts, substrate adaptability, and oxidative tolerance – to name some – simply enable the organism to evade, neutralize, stifle and avoid host defenses and humoral components.
It is likely then that the chronic carriage of such organism confers upon the host a response unlike that observed relative other organisms regarding signaling, identification, and a successfully neutralized and/or mounted life lasting anamnestic response - but one expected, experienced and witnessed without such. Likely then, atherosclerosis and associated chronic disease states such as non-insulin-dependent diabetes mellitus – more correctly represent and reflect the chronic state of coexisting with such organism in the absence of the host organism to prevent, eliminate, control or decrease such entity within and acting upon its presence. Until demise, the human host is significantly influenced; destined acute and chronic health of the human condition/state - is with much probability - determined by B. fragilis in many important regards. Possibly, that is why all therapies used to modify the biological, biochemical/chemical, physical and stress related outfall of Bacteroides fragilis upon human morbidity and ultimately used to circumvent mortality - are acquired lifelong or as long as the organism remains a non-vaccinated commensal.
Lastly and ironically, the presence of chronic disease states such as atherosclerosis and non-insulin-dependent diabetes mellitus (if not insulin-dependent diabetes mellitus), and Alzheimer's among so many others - not only explain the organism’s direct impact upon the host - while providing an understanding of the discrepancies within the literature and what is assumed or deemed to be of, or culminate from, host origin - but something more can be found.
Regardless of the vast literature and quantitative statistical data collected to date, it becomes apparent that such perturbations within the human existence and in particular, the individuality of one's own lifetime, if and when so impacted – echoes, if not challenges to remind, the contemporary, ego developed and involved human entity - the importance and humble humility and gratitude for an inheritance based upon a magnitude of history that not only dwarfs its own, but more importantly - has afforded an existence too seldom treated as deserved, entitled and owned.
The human has ‘2 stomachs’, limit B. fragilis Bft/fragilysin – you go back to one…
II) Data relating B. fragilis & atherosclerosis - difficult to find studies…
It is possible that a medical and pharmaceutical industry based around acute and chronic diseases and ailments, would perhaps only further contribute to – hiding, if not burying - any etiological relation between Bacteroides fragilis subspecies – such as Bacteroides fragilis NCTC 9343/ ATCC 25285 - to atherosclerosis, diabetes (metabolic X) Alzheimer's, breast cancer, and colon cancer. Such would not only elude the true, hidden, fundamental etiological/causative agent relative such disease processes - but ensure the promotion and perpetuation of chronic disease processes which could be further capitalized upon to secure funding for, as well as directly obtain funding from - the general public - to maintain domains and promote sectors of the medical and pharmaceutical industry for fiscal and financial profit, gain and greed.
Imposed upon the lesser educated general populous, that at all times, and at any given point within a context relative one’s own contemporary existence - falls within, plus or minus 2 standard deviations of a societally constructed and determined mean - further contributes to the illusion of medical science as intensely complex, scholarly as well as benevolently and altruistically noble. From the vantage of the onlooker, such is a grossly incongruent distortion of reality, equated in self-deluded and contrived perception, a respect and awe otherwise reserved and equivalent that of godliness, least of which magic or mysticism.
In reality, such could not be further from the truth. At best, the understanding and identification of Bacteroides fragilis subspecies fragilis as the etiological agent in atherosclerosis exposes more fundamental and core drives reflective of lesser human behaviors manifest from a lower echelon of human behavior, reasoning, conduct, morality and ethics and perhaps embedded and entranced with unresolved and compensatory mechanisms related to self-worth, self-value and value structure, life meaning, self-esteem, control, and obsession.
It is likely such has come to bare more than the impact of causative agents in chronic illness – surfacing in global crisis – to expose the human in current form – another animal, self- appointed, entitled and inheriting a perceived and deserved global positioning – as ruler of the domain. But ultimately, beyond such embellished dramatics of such humanly contrived cognitions, another organism, an animal among others – stuck on a planet in the middle of a place it can’t understand – simply in search of power, pleasure, transcendence of responsibility and accountability. Trumped only (is the human) by its own righteousness, that orchestrations of such ‘pathetic-isms’, and whose significance and delusions, only matter and have relevance to members of its own mammalian existence – prior to its own forecastable extinction.
2.0 Environment
B. fragilis and atherosclerosis: Environment
2.1 – pH
Examples (non-comprehensive) of why the potential of hydrogen is a suitable & important starting point regarding understanding atherosclerosis…
Environmental pH is a variable determining the rate of growth and colonization of B. Fragilis subspecies fragilis (ATCC 25285). Such not only influences the regions of infection (such as plaque foci) and extensivity of impact but is also an important influence in terms of redox reactions that occur concurrent to and resulting from the interplay of B. fragilis and the associated environment – within and outside of the intestine.
It may be of significance then that the interplay of dietary and pharmacological agents upon B. fragilis over the course of host’s lifetime can present both positive and negative implications regarding the optimal pH alterations that impact B. fragilis. Such may contribute to the organism’s pathogenesis and dissemination from the intestine – ultimately influencing various and multiple systems - simultaneously. As significantly, growth rate can be significantly influenced relative pH especially in relation to carbohydrate, protein and lipid intake. For example, glucose and succinate in combination with optimal pH can have a significant stimulatory effect upon B. fragilis growth (Dalland & Hofstad, 1974).
Such may illuminate the importance of high glycemic dietary intake or disease processes such as diabetes mellitus which can impact such dynamic. Additionally, fibre type and viscosity is associated with the pH present in the intestinal environment and may influence, both positively and negatively, the growth and dissemination of B. fragilis within an organism (Smits et al, 1998) and reflect more accurately other associations between fibre type and atherosclerosis – beyond simply the type of sugar moiety or non-utilizable linkages reducing B. fragilis and promoting competing bacteria flora especially relative preferred reduced sugars such as lactose.
It is of interest to note as illustrated by (Watt & Brown, 1985) that B. fragilis itself is rather robust and can both survive and facilitate ‘striking falls in pH’ in test cultures (i.e., pH 5), perhaps reflective of the observation of lowered pH in atheromas where such ‘nanaerobe’ is potentially associated. For example, plaque sites have been measured at pH values closer to 7 which correlates with the optimal growth pH of B. fragilis. Such conditions may be conducive for B. fragilis survival, especially considering the fact that various antibiotics (if at all effective against B. fragilis - especially as a single chemotherapeutic agent (such as erythromycin) would have further reduced activity as pH falls. Additionally, manipulating pH may circumvent optimal pH required for B. fragilis metallo-beta-lactamase which may further contribute to multi-antibiotic resistance (Wang et al, 1998).
2.2- Oxygen
2.2.1 - Atherosclerosis relative oxygen…
Atherosclerosis can be viewed as the progressive procurement of an anaerobic state within the host that invariably influences circulatory perfusion, and specifically - within the atheroma created. Such stenosis and resultant occlusion can invariably lead to necrosis of cellular processes downstream or proximal to such especially during the precipitation of acute events – such as an acute coronary event and the resultant impact upon myocardial tissue.
2.2.2 - Oxygen therapy…
One of the approaches subsequent to such event is the utility of oxygen therapy to increase hemoglobin and myoglobin O2 concentrations perfusing the anaerobic environment created. It is interesting to note however that the practice of such has recently been called into question given that the prophylaxis of such has proven questionable in terms of actual patient outcome (CO2 production?). Further, given the potential, albeit hypothetical involvement of B. fragilis in atherosclerosis additional factors must be considered when utilizing specific therapeutic methods as analogous and illustrated relative the involvement of B. fragilis in abscess formation (Bieluch & Tally, 1983).
2.2.3 – The effect of oxygen on B. fragilis…
Interestingly, overlooked may be the positive and negative influence of oxygen therapy upon B. fragilis.If oxygen therapy is indeed effective – could such also come from the beneficial influence of such upon B. fragilis – in addition to increasing dissolved O2 for host cell metabolism?
The relative grey area regarding the observed benefit of such practice may be characterized by the fact that B. fragilis, if so incriminated as the etiological agent in atherosclerosis (and diabetes), is an “obligate anaerobe capable of long-term survival in the presence of air”(Smalley et al, 2002) and such adaptive survival is attributable to ‘an elaborate oxidative stress response’ (Smalley et al, 2002) developed and utilized by the organism.
For example, prior to genomic identification, Carlsson et al (1977) demonstrated that “superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis…” and that there existed “distinct forms of SOD in the different species” – in the process, highlighting among other strains B. fragilis NCTC 9343. Later, in 1991, Chen & Gregory identified B. fragilis superoxide dismutase as an iron apoprotein capable of substituting either Fe or Mn – perhaps serving to illustrate the conflicting results relative dietary iron and progression of atherosclerosis.
Compounding such, in 2002, Smalley et al observed that the long-term survival of B. fragilis in the presence of air may be attributable not only to “an elaborate oxidative stress response” but that “during aerobic conditions B. fragilis NrdAB (aerobic-type ribonucleotide reductase) may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery”. It is then probable to suggest that the administration of such therapy, perhaps in the absence of concomitant multiple antibiotics (Poindexter & Washington, 1975) especially given the fact that other facultative species capable of scavenging environmental oxygen may co-infect the atheroma and further reduce the affect of administered dissolved oxygen – and would affect little the ultimate course of progression as opposed to immediate effects upon the host – if indeed the case.
2.2.4 - Mild Bacteriostasis…
Given that anaerobes do not (generally or significantly) multiply in oxygen (Brook, Pediatric Anaerobic infections) – the administration of O2 relative B. fragilis involvement in acute coronary events likely serves at best a mild and temporary bacteriostatic mechanism - resolving inflammatory amplifications directly caused by B. fragilis-host manifestations in acute or chronic illness. However, given the robustness and resistance of the organism – it may be probable that oxygen or hyperbaric oxygen therapy ultimately does little to resolve chronic B. fragilis involvement in atherosclerosis beyond immediate administration (or for a temporarily period thereafter) – such as observed relative mandibular osteomyelitis not eliminating such but resulting in significant improvement (Triplett et al, 1982). As a further example, Tonjum et al (1981) observed that hyperbaric oxygenation had little effect on B. fragilis eradication as the organism continued to be recovered regardless of the length of treatment hyperbaric oxygen was administered to rat livers inoculated with B. fragilis.
It may be, that in order to truly garner the greatest effect of oxygen therapy in atherosclerosis, other variables such as mixed and protective flora at the site of atheroma as well as the physiology mechanics of exposure within the medium (other than hemoglobin and myoglobin saturation) must be kept in mind. It may be probable to shift and further potentiate the benefit of oxygen therapy with improved application of oxygen directly to the atheroma.
The greater the increase in O2 exposure directly applied to atheromas and the arterial bed in general – the more vulnerable B. fragilis – as it’s electron transfer mechanism are highly labile to O2 the hydrogenase activity and the H2-fumarate electron transport system are inhibited and halted by O2 electron transfer (Harris & Reddy, 1977). Its seems then, that improved application of oxygen therapy methods and oxygen delivery in general, especially given the benefits observed with chronic administration of dilatory acetylsalicylic acid and nitric oxide – could further reduce atherosclerosis progression within the host and perhaps help better alleviate symptoms in the immediate and circumvent the probability of such chronically.
2.2.5 - Adverse effects of O2 therapy…
It is possible that oxygen therapy could even worsen such states under various conditions pending particulars such as an advanced stage of progression of the patient administered O2 therapy. That is, therapeutic oxygen administration like that observed regarding antibiotic therapy could result in the liberation of B. fragilis cellular components such as: proteins, endotoxin and antigens which further contribute to an inflammatory state (Rotimi et al, 2000).
It is probable then, liberation of pathogenic particulates could weaken vulnerable atheromas (ie shoulder regions in vulnerable plaques), agglutinate or disseminate coagulatory fragments - further compounding poorly perfused and affected regions of the myocardium, contribute or further amplify an inflammatory state or cause further stenosis in a plaque area or necrosis to sites distal to such – as examples. It may be, the administration of O2 in the absence of B. fragilis would impart the greatest effect by directly supporting the host without inducing oxygen therapy-Bacteroides interactions. Given that B. fragilis is ever present, it is important that such implications be considered – as it is probable that oxygen administration may have varied implications relative its ultimate/net impact upon B. fragilis and consequently the host – during and subsequent acute cardiovascular events.
Alternatively, factoring such in, oxygen may play an under noticed contribution (minimally) affecting and controlling B. fragilis cells that may be present within the host. Such could include plaque foci &/or various other anaerobic regions where such cells may be present such as the general arterial vasculature, metabolic X related plaquing (i.e. beta- amyloid in Alzheimer’s etc) present in the central nervous system and cell viability and dissemination from the intestine - to name some - as B. fragilis cell growth and load is inversely proportional to oxygen tension(Brook, pediatric Anaerobic Infections).
The outcome however, may be overtly, observably and effectively minimal notwithstanding conditions of low dissolved oxygen etc (ie anemia etc) as both organisms - B. fragilis NCTC 9343 contain superoxide dismutase or SOD (Carlsson et al, 1977) – perhaps rendering the impact of such therapy minimal especially pending site of residence such as hypoxic atheromas. It is interesting to note at this point that previous speculation and contention contained within the literature and relative the influence of iron and manganese upon the progression of atherosclerosis outcome can be explained (amongst other ways) here as B. fragilis SOD can bind either Fe or Mn (Chen & Gregory, 1991) - perhaps demonstrating the conflicting data relative Fe and Mn intake relative atherosclerosis (citation).
Relevance of findings related to O2 and reduced O2 states…
Negative atherosclerosis associations: Smoking, exhaust fumes Versus Positive atherosclerosis associations:
2.3- Redox
2.3.1 - Atherosclerosis and Eh…
The redox potential or reduced environment (Eh) observed associated with atherosclerosis is also optimal for the survival and growth of B. fragilis and such represents a second component in addition to the influence of oxygen which strongly influences the growth of B. fragilis.
Anaerobes require -0.2Eh to grow as such influences reaction kinetics. It is probable then that the observed positive oxidation-reduction potential created by the inflammatory response may be directed to moderate, if not minimize and limit the growth and survival of the anaerobe B. fragilis – which prefers a hypoxic and reduced oxidative potential (Walden & Hentges, 1975) and perhaps successfully establishes that observed within the atheroma. It is likely that such is created and established directly by the production of reducing agents by B. fragilis and other mixed mutualistic and noncompetitive commensals within the hypoxic environment of the atheroma.
Dietary foods, functional foods, supplements or otherwise administered elements or chemical compounds which influence Redox (Eh) can influence ( + / - ) atherosclerosis progression as such may invariably affect the growth and dissemination of B. fragilis - perhaps especially critical at early age relative initial progression of atherosclerosis, diabetes and CNS manifestations in the young (ie ADHD, autism etc). More positive (+) eh values can perhaps decrease B. fragilis in intestine and reduce progression of all associated illnesses - especially if such can be manipulated at a site of plaque as alternative form of prophylaxis.
The redox potential of ruminant bacteria is influenced by carbohydrates perhaps representative of the influences of various dietary fibre intakes and influence on atherosclerosis. Additionally, the presence of sulfide and cysteine can influence redox potential and metabolic rate in anaerobes - again, perhaps representative of findings relative inhibitory effect of acetyl-cysteine and CD13 on B. fragilis.
Care must be given to correctly differentiate whether an administered agent is an antioxidant, competitive antagonist or influences ‘focal redox’ as opposed to the broad umbrella or generic wasteland of “antioxidant” which offers little insight in terms of specific mechanism(s) of action & actual influence – while overlooking influence on anaerobic ecology & anaerobic bacterial flora.
The redox potential associated with various elements and compounds may help explain both their positive and negative influences on atherosclerosis when viewed In the context of B. fragilis growth and survival- such as the benefits of copper, gold and silver ions against anaerobes (Berry et al, 1992)(Chin & Neu, 1992) and negative influence relative ions such as lithium and the association of accelerated progression of atherosclerosis in persons consuming higher concentrations during such administration for manic-depressive illness. Such will also allow the understanding of the logic behind various therapies when viewed in reference to periodic table and elements &/or compounds which illustrate mixed results (gray areas) - based on/relative to their reduction potential.
B. fragilis is significantly more resistant to silver ions and several other than other bacteroides members, however all B. fragilis were susceptible to silver sulfadiazine (Riley & Mee, 1982).
Lastly, It is also interesting to note that the redox potential (eh) also influences the ability of B. fragilis to bond to laminin and may reflect the finding that atherosclerosis is associated with reduced environments within the host. Such may also reflect the beneficial utility or administration of compounds to decrease the binding of B. fragilis within the host and influence upon adhesion capacity to extracellular matrix components endothelial lining to name but one.
Further, such studies associating redox potential and binding ability of B. fragilis may illustrate the overlooked effect antioxidants may play relative this component of virulence, especially given the broad (and oftentimes beneficial) influence of antioxidants upon atherosclerosis and its highly associated conditions. Perhaps such represents a glimpse of the potential extent of impact that the zwitterion component of the organism’s polysaccharide A (PSA) coat has upon its host when unaltered by balancing antioxidants, and the capsular polysaccharide (CP) is maintained in its natural polyamphoteric/lyte state. (SEE BF vs TLR2 - Gallorini et al, 2007).
2.4 – Oxidative Fallout
2.4.01 - Free radical theory?
The presence of free radicals contributes to an incomplete picture of atherosclerosis as predominantly culminating from and perpetuated by free radicals – hence, the ‘free radical theory’. Additionally, the presupposition that atherosclerosis is the fulminant expression and elicitation of a pro-inflammatory response would suggest a propensity of factors contributing and not otherwise controlled for. Again, the picture here is both complete and incomplete as such qualitative description usually succumbs to quantitative measures.
It may be that quantification of atherosclerosis without factoring in all possible variables (B. fragilis, due to evasion and limited-to-no literature regarding this area of involvement) only provides for the continuance of producible data and information that nonetheless produces a consensus of thought or paradigm wherein the explainable variable is itself omitted – perpetuating further ambiguation and perplexity.
2.4.02 - The need to explore B. fragilis oxidative relevance
As such, the oxidative stress response by B. fragilis must be explored as relevant. It is important to consider the possibility that the presence of a commensal with the ability to cloak or disguise itself in varied polysaccharide coats is very adaptive within the mammalian host. Compounding such with the ability to (1) respond to and (2) proteolyze host enzymes via exoproteases CD13 and Bft, (3) while neutralizing the host’s oxidative attack and (4) limiting the available cysteine and extracellular glutathione concentrations – could indeed result in a continuous cycle of minimal host detection and persistent amplification within the host chronically – evading and eluding host defenses as well as medical literature.
2.4.03 - B. fragilis: Adaptive abilities (Cloaks of disguise, evasion)
Relative atherosclerosis, such ‘evasive-inflammatory ignition’ serves not only to blunt the response by the host but induces and, perhaps given the pre-dating and development of such organism long before mammalian development, represents a ‘premeditated response’. Such may include ‘procuring up an environment’ within the host in which the opportunistic commensal can adaptively flourish and thrive. Such may be propagated while localized or regionalized within a particular foci of infection or site of colonization (such as the intestine) while simultaneously setting up ‘distal-pseudo intestinal’ satellite sites easily occupied by other commensal rogues thereafter – without acutely destroying the host upon which such depends as observed in atherosclerosis.
B. fragilis possesses many defensive abilities to self-preservate within the human host allowing it to survive an exhibit - a life long resilience. The organism can maintain survival within foci such as atheromas, abscesses and tumors. General mechanisms include the ability to form eight distinct outer coats/capsular layers to evade detection, ability to inhibit immune cells, macrophages, neutrophils/PMNs, cause cellular anergy of T regulatory cell and imbalances within cd4+/cd8+ ratio, form clone-like blebs/OMVs, proteolyze peptides and hormones, utilize various substrates for energy, utilize host glycolytic/TCA/Krebs molecules (fumarate), elicit and skew cytokine/interleukin response - to name some.
The organism is well established in the human host - prior to full maturation and competence of the host’s immune and become recognized/is established as ‘self’ within the host - most likely due to zwitterionic PSA protective shielding. The regular dietary routine and behaviors of the human host - relative providing B. fragilis utilizable substrates (i.e. poor/high risk diet composition), fecal shedding and elimination via exertion/exercise, stasis fermentation (sitting), stress (cortisol, epinephrine etc.) as some examples - allows the continued survival of B. fragilis within the human host as well as pertuation and compounding amplification of the impact (disease progression) of exotoxigenic B. fragilis upon the individual host.
2.4.04 - Intestinal relevance
It may be probable that lessons can be learned and overlaid upon atherosclerosis relative intestinal inflammation – as atheromas predictably and progressively have a predilection to become: hypoxic and separated from general immunological detection (both cell mediated and humoral) in terms of any immediate infiltration and resolution. Similarly, upregulated by the host’s compensatory response is ultimately a set of conditions and variables, the response of which - ultimately supports colonization and infection as B. fragilis is capable of stifling the many measures employed by the host to prevent, eliminate, control or decrease the already illusive ‘self-similar,non-self entity’. Effectively, B. fragilis is able to withstand, tolerate and overcome environmental and host oxidative mechanisms –adaptively surviving and taking such lessons forward - in the atheroma as opposed intestine.
2.4.05 - Atherosclerosis - once begun…
It is possible that once initiated by B. fragilis NCTC 9343, atherosclerosis progression and a pro-inflammatory state is continually perpetuated and influenced by the oxidative fallout produced by (innate, humoral) ‘host- B. fragilis interactions’.
In keeping with such, (1) oxidative and toxic end products have been observed from the anaerobic metabolism of B. fragilis acting upon the host (Kieran et al, 2005). As well, (2) B. fragilis is able to withstand, tolerate and overcome environmental and host oxidative mechanisms which collaterally contribute to such oxidative state. Additionally, (3) the central importance of cysteine in B. fragilis metabolism and growth may result in (a) consumption and (b) reduction of host cysteine levels – while simultaneously resulting in elevated homocysteine and a chronic oxidative state within the host.
Further, (4) it is conceivable that the integral need for cysteine by B. fragilis makes the human host not only an abundant and amiable source but contributes to reduced glutathione levels observed preceding (Biswas et al, 2005) and associated with the progression atherosclerosis via (i) cysteine consumption and (ii) stripping from the intestine as well as (iii) proteolysis via B. fragilis aminopeptidase N at the cysteinyl –glycine bond within the tripeptide.
Coincidently, B. fragilis “lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs” (Rocha et al, 2007). Such may underlie the association of thioredoxin (Trx) in atherosclerotic plaques as observed by Takagi et al (1998) and alterations in Trx system in endothelial progenator cells (Park et al, 2010) – as a blast of the amino acid sequence of B. fragilis Trx (thioredoxin - [Bacteroides fragilis YCH46] NCBI Reference Sequence: YP_097994.1) gives a max. score of 88% similarity with mitochondrial precursor thioredoxin (NCBI Reference Sequence: NP_036605.2). It may be probable that studies linking atherosclerosis - as well as other strong correlating risk factors such as diabetes and hypertension (Ferreira et al, 2010) - to thioredoxin, may in fact be correct while overlooking the contribution of B. fragilis thioredoxin to such observed outcomes.
2.4.06 - Host oxidative promotion
It is conceivable then that host cell induced, responsive oxidation and oxidative bursts (via singlet oxygen, nitric oxide, molecular oxygen, hydrogen peroxide, hypochlorous acid etc – released by host immunological response) negatively impact and progress atherosclerosis within the host. Such cell mediated mechanisms may contribute to a mechanical destruction that adaptively facilitates the survival and nutrient availability for B. fragilis in areas of established buffering and host neutralization – such as the hypoxic and cholesterol esterified environment of the atheroma, cholesterol and protein rich deposits observed in Alzheimer’s disease or the cholesterol and sphingolipid abundant pancreas in diabetes, which consequently itself is not bacteriostatic against B. fragilis.
2.4.07 - Antioxidants
Conversely, antioxidants influencing the optimal reduction potential optimal for the growth and survival of B. fragilis may exert a ‘limited though positive effect’ relative disease pathogenesis (furthered establishment and colonization), reflecting the decreased progression of atherosclerosis (and other disease states B. fragilis may contribute to) as observed within journaled literature upon administration.
The utility of antioxidants may (if titrated, administered and directed appropriately) help buffer and/or compensate for the impact of B. fragilis produced metabolic products and it’s influence upon the host (as well as collateral damage caused by host release of such oxidative mechanisms). Such may underlie the observed beneficial results relative antioxidants upon the atheroma – as antioxidant mechanisms may have a direct influence upon B. fragilis within the atheroma.
2.4.08 - Compounded collateral damage…
The oxidative stress defense of host derived radicals which, if rendered ineffective by B. fragilis - may also further damage host cells and extracellular proteins (compounded collateral damage) and further facilitate the organism’s ability to establish – resulting in chronic infection and inflammation as evidence in atherosclerosis and advanced atheroma progression in particular. Largely, many of the adaptively advantageous or ‘virulent’ mechanisms encoded by B. fragilis ensures its robustness relative host attack and aerotolerance in higher oxygen tension environments.
2.4.09 - Genes carried by…
B. fragilis NCTC 9343 and YCH46 enzymes afford cellular protection via encoded oxidative stress response mechanisms (National Microbial Pathogen Database Resource (NMPDR) -
http://www.nmpdr.org
and help withstand and overcome oxidative attack and oxidative environmental stress. The following represents a non-comprehensive listing of mechanisms utilized by B. fragilis utilized by the host serving as protective oxidative mechanisms and barriers between sites of colonization/colocalization of B. fragilis and somatic cells - such as the intestine and host (i.e. Oxidative burst, oxidative molecules):
Ultimately, such mechanisms may (1) afford B. fragilis survival in (a) the oxidative nanaerobic environment of the atheroma and from (b) the onslaught of immunological attack while (2) contributing to atheroma development, chronic resistance and continued presence of such arterial formations.
Coincidently, the presence of such enzymatic stress response systems may underlie the origin of various measurable surrogate markers of oxidative mechanisms such as the MetO form of HDL observed in atherosclerosis (Panzenbock & Stocker, 2004) possibly resulting from B. fragilis’ bifunction methionine sulfoxide reductase – which among others, present tangible markers for atherosclerosis progression and stage – which may be as indicative of B. fragilis involvement in atherosclerosis as those markers assumed or mistaken to be of host origin.
Such scenario may be especially true if B. fragilis is able to cleave host mechanisms such as the host’s own copper chaperone and extracellular superoxide dismutase at cysteinyl-glycine (CG) and cysteinyl-lysine (CL) linkages (among others) via aminopeptidase N. Such proteolytic mechanisms may also underlie the observation of numerous polymorphisms within the literature on atherosclerosis relative oxidative proteins utilized by the host. Perhaps such simply represents an attempt by the host to circumvent such proteolytic cleavage by B. fragilis - such as superoxide dismutase 3 (SOD3) R231G polymorphism (Grammer et al, 2009)
2.4.11 - Lipid accumulation and peroxidation…
Given the oxidative stress response capacity of B. fragilis, perhaps then, the accumulation of esterified cholesterol within the atheroma may more accurately represent an ‘immunolipid response’ by the host. Supportingly, Saito (1991) observed that the presence of B. fragilis endotoxin can stimulate increased superoxide formation and increase lipid peroxidation.
As such, this may represent an additional and under-explored component regarding the oxidative outfall of the involvement of B. fragilis with atherosclerosis and reason for both the accumulation and resultant lipid peroxidation that occurs. Further, the ingress of lipids within the atheroma, beginning during ‘fatty streak’ formation may represent one of the mechanisms by which the host can ultimately keep B. fragilis cells, Bft and LPS solvated and confined – resulting in accumulating peroxidized lipids from the interaction with B. fragilis and LPS in the process.
2.5 Nitric Oxide
Nitric oxide is responsible for vasodilation which is associated with the inflammatory response. The ability of the host to dilate the vasculature perfusing host tissues, among other properties, confers the ability to help regulate oxygen flow and provides for an effective immunological response within an area upon antigen detection and presentation.
Given the properties NO affords the host, it is logical and probable to predict that it would be energetically adaptive for B. fragilis to proteolyze proteins or enzymes required for the synthesis of NO. Neutralizing such compound directly or indirectly through selected efficacious sites of cleavage, such as selective cysteine linkages, would negate the abilities conferred to host cells by the production and release of NO – therein promoting its own establishment, colonization and infection.
Supporting, Viera et al have observed that B. fragilis does indeed interfere with iNOS activity in macrophage cells resulting in surface pore formations. It may then be probable to suggest that endothelial inducible nitric oxide synthase perturbations associated with atherosclerosis may itself be a consequence of B. fragilis associated alterations. Potentially, such alterations could be due to the consequential proteolysis of eNOS synthase by Bft &/or aminopeptidase N (CD13) – given the cysteinyl-glycine and cysteinyl-lysine linkages (as examples) such enzyme possesses, especially when viewing such in light of the vulnerability such offers due to expression on outer cell membranes.
In addition to such potential mechanisms, B. fragilis maintains resistance to the actual production of NO or ‘nitrosative stress protection’ conferred by NO protection genes (denitrification and NO detoxification pathway and specifically via DnrN (Nitric oxide-dependent regulator DnrN or NorA) and NnrS. Such mechanism besides enabling the organism to withstand oxidative attack by NO, also provides clues as to why such basal mechanics become altered and require repletion or the addition of NO groups or nitro compounds to restore cellular functionality such as vasotonus – vasodilation relative MI or angina. Further, the encoding of such mechanisms illustrates the evolutionary origins such genes otherwise mediating components of the nitrogen cycle and nitrogen recycling.
It perhaps leads one to speculate whether perturbations in NO synthase in other regions such as that observed in the brain of Alzheimer’s subjects is also directing influenced by B. fragilis within the CNS.
Such may be probable as recent studies identifying the presence of Bacteroides species have been observed in subjects with ADHD and by virtue that such organism disseminates and is easily carried within the circulatory system. Further, the condition of sepsis itself is associated with an overproduction of NO and inducible NOS – perhaps a defensive compensatory mechanism for the host but otherwise overcome by B. fragilis when present.
It may be that the carriage of B. fragilis is both a regular and continued occurrence throughout the lifecycle of the host – perhaps vacillating in precipitation from largely common periods of subclinical infection by members of the general human population as well as those that result in, and present as clinically observable sepsis.
It is perhaps also of significance to note that hyperglycemia (optimal for B. fragilis growth) and the presence of glucosamine (utilized and produced by B. fragilis), represent not only identifiable hallmarks of diabetes and metabolic X syndrome – but is also strongly associated with the progression of atherosclerosis and the inhibition of endothelial nitric oxide synthase (eNOS).
3.0 Nutrition
Relevance and impact of nutrition upon Bacteroides fragilis NCTC 9343 & YCH46
3.1 – Bacteroides fragilis nutrition and relevance in Atherosclerosis
Atherosclerosis is a fulminant condition which obviates the human host as a perfect culturing vehicle for Bacteroides fragilis NCTC 9343 and YCH46 – beyond the context of qualitative literary statements regarding such organism as a simply and wholly an ‘opportunistic commensal’.
Regarding (etbf) B. fragilis NCTC 9343 and YCH46, it may be more insightful, if not adaptive, to state that the human host is in a constant state of inefficient and energetically intensive defense against these organisms (otherwise termed ‘atherogenic’ and ‘pro-inflammatory’ relative atherosclerosis) – at any and every specific moment the organism is harbored. Such may create a more comprehensive picture of the organisms than as simple human commensals, usually referred to in the context as members of the gastrointestinal flora – capable of opportunistic infection.
Perhaps, it is more relevant to suggest that the organisms are ‘proactively opportunistic’ or ‘basally active, somatically-involved’, as opposed to the often associated thought of reactively opportunistic agents of infection. It is conceivable that these organisms are constantly and continually making progressively small, and oftentimes, subclinical strides or ingresses into what – pending time (in terms of acuity or chronicity of the degree of impact) manifests to become overtly obvious, detectable disease pathogenesis.
It is not improbable to suggest that such is the case in atherosclerosis, diabetes, as well as CNS and PNS manifestations of peripheral neuropathy, multiple sclerosis, alzheimer’s disease, ADHD and autism – to name but some. It is likely, however, that such suggestion – least of which one inferring involvement and relevance in so many supposedly non-congruent, disjointed and supposedly mutually exclusive conditions – would be immediately dismissed as provocatively improbable or as irrelevant idealism. Investigation of such matter, however, may be worthy of exploration.
3.1 – Atherosclerosis from the vantage point of B. fragilis …
From the lesser taken vantage point of the organisms B. fragilis NCTC 9343 and YCH46, the mammal - with specific reference to the homo sapien species - which evolved after its own development serves quite adaptively as a specific culturing and cultivating vessel. The homo sapien’s reproductive and gestation processes provide B. fragilis mechanisms of communicability, transference and spreading of its own genetic progeny. Further, the domestication and industrialization of the human organism and its society has proved a demonstrable evolution and invested procurement relative the protection and passage of its progeny as compared to other mammals living in the relative ‘wild’.
Ultimately, for B. fragilis, the host represents a mechanism of hidden, if not layered, anaerobic survival within an aerobic host that walks the earth. The ‘human’ is an abundant nutrient source which presents a varied supply of survival, growth and stimulatory agents through it’s own nutritional requirements and behaviors as well as multiple foci of anaerobic and nanaerobic colonization and means of survival within such erect, terrestrial agent.
Further, if the homo sapien is successfully infiltrated and manipulated by B. fragilis NCTC 9343 and YCH46 – for example, such as that observed within:
(1) anaerobically hypoxic and separate environment of the atheroma,
(2) as observed relative the ineffective response of insulin in non-insulin dependent diabetes mellitus and high serum dextrose availability and compensatory glucose intake optimal for B. fragilis,
(3) increased consumption and substrate abundance in obesity. and
(4) via the fallout of behavioral implications of altered dopaminergic and serotonergic receptors relative states of depression
B. fragilis may indirectly, through its impact upon basal metabolism and host regulatory signalling and subsequent effects - ultimately and adaptively control - the behavioral dispositions, fixations and tendencies of the host. That is, influences by B. fragilis can adaptively reinforce, if not condition - through host behavior and feedback, stimuli and response – the perpetuation of conditions within the host conducive to its own survival, growth and propagation.
Suffice such to the idea that the ultimate purpose of an ‘adaptively virulent organism’, as opposed to simply ‘virulent’, may be the chronic, and if possible – where capable and advantageous, complete control and regulation of the host environment to perpetuate its own continued and optimal bacterial existence. Brought to the extreme, it could be hypothesized that B. fragilis strives (and would) disable and deflect any immunological response, determining and establishing the conditions of the host as simply a ‘larger aqueous niche of its existence’ and controlling and choosing other mutualistic and competitive microbial organisms permissible within the environment maintained under its reign.
However, given the mutual self-interest of B. fragilis to perseverate, examples of aforementioned conditions such as atheroma formation in atherosclerosis allow a slow progression of sparse but strategically located established anaerobic ‘pockets’ or ‘satellites’ which are detrimental in occupation to host system functioning. Further, the establishment of such ‘domains’ omits the need for, or reality of the physiological inability of B. fragilis to take over what would otherwise equate to - the maladaptively futile responsibility and energetically costly functioning of the host systems - by attempting to virulently ingress within the utilized host in the pursuit of dominance and complete control.
It seems probable that the prior evolution of B. fragilis before the homo sapien species (in particular) afforded such organism the understanding, knowledge and wisdom or ‘chemical affinity’ for critical and vulnerable sites of colonization, infection, proteolysis and virulent occupation to ensure and secure its own survival – within, specifically, the homo sapien. Also, such seems to be done so in a logical and adaptive fashion by being dynamically able to move from host to host and forward into the future with the host’s progeny for continued survival.
In an overgeneralized sense, B. fragilis garners the value accrued of its host and, as if transcendent to the disproportionate responsibilities inherent the larger host - relative the energetically expensive and intensive requirements to maintain homeostasis and general integrity – ‘dev-oides’ and detaches itself from integrated somatic host responsibilities.
Viewed from the vantage of bft producing B. fragilis, it may be of consequence - if such is irrefutably correct – that this bacterial organism would have evolved and created, as if donated, the host it’s ‘energetic cellular heart’ or mitochondrion. Perhaps, such prior development along the evolutionary path came both purposefully and at much cost and time, especially given the potential for attack and disintegration of the protypic mitochondrion, was developed to adaptively suit and perpetuate the creation of larger systems.
Such seems logical as the mitochondrion is protected and otherwise suspended within an aqueous environment allowing for the creation of mechanical hosts which would become responsible for its protection and maintenance - and which, invariably, could be used to further secure B. fragilis preservation (and dominion) over time as a commensal living upon such structures. Such is reminiscent if not akin to a Bacteroides initiated ‘frankenstein project’ or human’s creation and reliance upon technological means.
Perhaps the mitochondrion represents an example of a significant investment the earnings of which are being actuated in the present by B. fragilis NCTC 9343 and YCH46 commensalism and resultant outfall relative atherosclerosis and metabolic X brought to bare by influences such as demography, diet, drug resistance, industrialization and overpopulation relative the path trod by the host and brought forward via encoded and expressed bacterial genomics since such time.
The influence of atherosclerosis upon the human condition perhaps even represents a reluctant and inevitable adaptivity of the commensal organism to match that imposed upon the host system by the host itself – as the cessation of viable existence for either organism, which limits propagation of progeny, would compute as illogical and maladaptive – regardless of how well B. fragilis as supposed opportunistic commensal may flourish previous to the host’s demise. It would occur that any insult upon the host that invariably affects B. fragilis is ill intended and maladaptive – even though such may ultimately stem and result from the supposed ‘virulent’ measures willed and imposed upon the host by B.fragilis NCTC 9343 and YCH46, ‘microbially titrating’ it’s necessary survival upon the host in which it is contained and relies.
3.2 – Atherosclerosis, B. fragilis and somatic based culturing media …
Given that various in vitro and in vivo culturing methods inhibit, promote and stimulate the growth of Bacteroides species (1 - Hill, 1978), it may be probable and overlooked that personal lifestyle patterns and behaviors (routines and ritualism’s – i.e. diet, exercise, stress) have an enormous impact upon the organism within the host environment. It is probable that the host’s modifiable behaviors overlayed upon nonmodifiable demographics - directly and indirectly affects B. fragilis NCTC 9343 and YCH 46 - ultimately influencing, and so by, determining the variable rate and degree of atherosclerosis progression.
If such is the case, the vast collection of published epidemiological literature on atherosclerosis may all be connected; the underpinning being the presence and influence of particular subspecies of Bacteroides fragilis – namely, B. fragilis NCTC 9343 and B. fragilis YCH46 – thus far illusive and ultimately contributing to the broad spectrum and unique mosaic of relevant findings to date. Such agent then would represent the eluding variable that accounts for the various and varied published findings representing “A Union of Theories” regarding that forwarded to date within the medical literature relative atherosclerosis.
So too, the agent may represent the proposed “X” variable of ‘metabolic X syndrome’ which strongly correlates with atherosclerosis presence and progression at all ages. It is conceivable then that all data positing associations, positive or negative, between atherosclerosis and the multitude of variables studied and controlled for, overlaid upon diverse genetics, demographics and various hosts ‘unknowns’, may simply reflect and contribute to parts of the a larger picture representing the interplay between studied variables and such outcome - relative and concomitant to the presence of particular subspecies of B. fragilis.
It may be that finite details and interactions within the colonized host, such as that related to: personal dietary regimens, behaviors and habits, non-modifiable variables such as sex (m,f) related tissues and hormones, one’s maternal related carriage, birthing and postpartum exposure sources, coupled with other host specific and/or genetically determined dispositions related to an individual human being – come to bear regarding: gastrointestinal dynamics, B. fragilis load and dissemination, the host organism’s innate and humoral (immunological) responses, conditions of hypoxia – to name but some.
Further, such imposed upon the specific context of a particular human’s exact demographics, may determine at any moment – the shifting flux and milieu of exposures to which B. fragilis subspecies contends, resultantly influencing the gastrointestinal and serological opportunistic dynamics of such organism. In short, the summation of such interplay and variables relative B. fragilis may inevitably impact the atherosclerotic progression within a specific colonized human host – the individual specificity of disease particulars representing a ‘fingerprint of the regions influenced’ by B. fragilis to date.
What can be proposed, if not inferred from such hypothesis regarding the relation between B. fragilis ssp. and atherosclerosis, is that reductions and advancements in disease progression observed and imposed through epidemiological and experimental studies, respectively, and associated with modifiable risk factors may be attributable to the antagonistic and agonistic impact of such variables upon the microbiological ecology of B. fragilis.
Specifically, putative modifiable risk factors for atherosclerosis may have specific an inhibitory or stimulatory affect upon factors affecting B. fragilis metabolism, redox, virulence factors etc – which are conducive to acute and/or chronic patterns of colonization and infection, dissemination, by-product and toxic release, tissue infiltration and manifesting in the meriade of associated and measurable infectious and inflammatory markers.
Also, it is probable that some serological markers of atherosclerosis may not only be representative of the direct involvement with B. fragilis (such are CRP , HSP) but actually produced by the organism (B. fragilis origin) in addition to, compounding and screened by that responsively and reflexively produced by the host – but not discerned biochemically from that of the host; akin to a solution of isomers.
3.3 – Atherosclerosis, B. fragilis and somatic based culturing media
As illustrated by (Tamimi et al, 1960), it is clear that B. fragilis is capable of growing amidst various substrates and preferentially with the added addition of specific essential and rate limiting nutrients including, but not limited to, cysteine, cystine, glucose, sodium phosphate, pantothenate and inositol. As well, it was observed that reducing agents in particular such as thioglycolate and specific mediums such as rabbit serum solutions resulted in increased total grow and rate of B. fragilis.
Given the hypothetical causative role of B. fragilis in atherosclerosis, it is observed that (1) reducing agents (nitrates, nitrites etc) as well as (2) sulphur sources such as sulphates, sulphites and cysteine are relatively high in the homo sapien diet - especially from sources such as food preservatives and food additives.
Additionally, though the amount of dietary cysteine is relatively low, the human host contains ample supply built within structural and functional proteins and perhaps accessible upon proteolysis by B. fragilis – specifically given the affinity of the organism’s aminopeptidase N for cysteinyl-glycyl bonds and fragilysin for cysteinyl-lysyl bonds, respectively. Interestingly, aberrations in host levels has been observed to coincide in atherosclerosis, diabetes and CNS manifestations – usually restored upon dietary administration with beneficial impact subsequent to the host consumption of such.
3.4 – Atherosclerosis, B. fragilis and Mediterranean Diet…
It seems unlikely that B. fragilis would thrive upon the conditions of a Mediterranean Diet or similarly antagonistic and selectively limiting diets maintained by various sectors of the global population where, amidst other factors, reducing agents and those essential and optimally required for enhanced or stimulated growth of the organism are limited - such as within the Inuit and Yuit population. Though such dietary patterns can be assessed and give relevance to compounding variables, highlighting probable constants within such diets which influence atherosclerosis - omitted may be the involvement with B. fragilis ssp – which, in the absence of such factoring may create confusion and unnecessary energies expended pursuing such.
As a case in point, Brown et al (2007) reviewed literature relative the “protective effects of the Mediterranean diet against atherosclerosis in humans” to illuminate whether the suggested implicating dietary consumption of monounsaturated fatty acids (MUFA) was responsible for such observations, as opposed to the association of dietary saturated fatty acids in other diets. However, using animal modelling to directly measure the outcome of dietary administration of MUFAs - no such association could be observed. (“MUFA-enriched diets are not atheroprotective when compared with SFA-enriched diets”). Further, such findings would otherwise “refute the idea the MUFAs per se are atheroprotective”.
In this particular case, and as an example, non factoring of an agent such as B. fragilis could not only illustrate such outcome – but explain such. That is, given the minimally low production and concentration of unsaturated fats as structural entities by B. fragilis it is probable that such administration further reduces its growth potential due to hydrogen bonding (or ‘trapping’) further reducing the amount of hydrogen available for reductive metabolism along the organism’s fumarate reductase pathway.
Given the predominant synthesis and utilization of saturated fatty acyls as a component of the organism’s cellular structure – it would be probable to infer that such would increase growth rate, while negating any effect upon it’s fumarate reductase pathway - as no hydrogen bonding or hydrogen trapping would occur (due to saturated SP3 carbon bonds contained by saturated fatty acyls), limiting hydrogen available for reduction.
Unfortunately, as the title itself specifies, such results may not be reciprocated in “Animal models” if such strains of ‘adaptively virulent organisms’ are not: (1) present or (2) active - within the model utilized to establish or replicate such findings. Conversely, no such atheroprotective association may be found relative MUFAs (in the absence of B. fragilis), meriting such observational finding the same degree of statistical significance as an observed lack of atheroprogression otherwise caused by SFAs; both results contrary to what is generally found in humans which constantly and ubiquitously harbor B. fragilis NCTC 9343 and YCH46.
Further, as this publication eludes (and respectfully utilizing such professional work as an illustrating example), relative it’s tabular “summary of strength on lifestyle factors and risk of developing cardiovascular disease” (Brown et al, table 10, pg) – again, provided is an illustration, where in the absence of B. fragilis ssp. – a very different result is observed.
Without factoring B. fragilis as an implicating variable, it becomes otherwise difficult to establish, decipher and discern what has to date been assessed and ranked as a set of dietary risk factors related to atherosclerosis - with what may very simply represent - a collection of correlates relating the strength and influence of various nutrients to inhibit or antagonize the growth of B. fragilis (to varying degrees) when present within the human host.
3.5 – Atherosclerosis, B. fragilis media and dietary fat and SCFAs…
Compounding the above, studies on past short chain fatty acid utilization and production make an interesting case for the implication of B. fragilis involvement in atherosclerosis. In 1975, Mayhew et al, observed that “incubation time and composition of the medium are important determinants in short chain fatty acid production by B. fragilis”.
Given that succinate is implicated in the progression of atherosclerosis (aka ‘succinate hypothesis’) it may possible that such not only becomes elevated due to shifting dietary patterns, but that either stable glucose levels or a total decrease or elimination of B. fragilis may be important in reducing the production and implications of SCFAs.
Further, Wu et al (2011) have related dietary fat of animal origin to increases in Bacteroides spp. And perhaps gives an element of credence to relations between selectivity of dietary fat consumption relative atherosclerosis. Also, Zimmer et al (2011) observed that total counts of Bacteroides spp. have been observed in vegan faecal samples versus control samples – lending support to the relevance of dietary fat and B. fragilis involvement.
As B. fragilis NCTC 9343 and YCH46 are but two of a large number of human commensals occupying the gastrointestinal tract (GI), strict and specific dietary modification(s) and adherence (with limited variation), particularly aimed at restricting the organisms are likely required to show the greatest or most overt effects pursuant any interceding and influential dietary measure utilized and studied.
Perhaps otherwise, any such marked association illustrating a direct or indirect ‘cause and effect’, that might be otherwise overtly obvious, too soon - becomes lost - so as to be masked or screened against the backdrop of competing flora and the dynamism of the host metabolism. Developed experiments and studies, not otherwise controlling for such, may then result in contradictory, limited or inconclusive results – furthering ambiguity and illusiveness of B. fragilis continued involvement in the progress and processes of atherosclerosis.
3.6 - Atherosclerosis, B. fragilis media and sugar precursors …
Illustrated tangentially to the studies by Mayhew et al (1975) regarding B. fragilis and SCFAs - it can be observed graphically as one might assume that steady decreases in medium glucose resulted in abrupt decreases B. fragilis colony forming units (CFUs). Though this is an expected observation such may have implications regarding the relevance of high and low glycemic diets, and the organisms hypothetical involvement in diabetes – and the strong statistical relation of such to atherosclerosis and progression. Further, it also would confirm the voluminous literature regarding the relation of dietary fibre types to the progress of atherosclerosis and influence upon diabetes and metabolic X.
As such, this study outlines the relevance here regarding the importance of dietary glucose and utilizable SCFA restrictions as well as the selective incorporation of fibre sources in the human diet that particularly influence B. fragilis NCTC 9343 and YCH46.
4.0 Energetics:
Fumarate reductase pathway
4.1 - Bacteroides fragilis & the Fumarate Reduction Pathway
An overlooked aspect in atherosclerosis is the specific kinetics of the commensal Bacteroides fragilis energy production. This organism's electron transport system contains many similarities and yet many important differences from that of the host. A specific aspect, it's fumarate reductase pathway utilized by backwards fragilis for energy production. A brief review of the metabolism of Bacteroides fragilis along the FRP allows one to piece together the metabolism of accurate fragilis the host - relative atherosclerosis.
It has been noted by Brian and Mahoney that the aconitase of B. Fragilis is “most similar to aconitases of the mitochondrial group”. What may be more fundamental than a simple understanding of the origin of mitochondrial Krebs cycle components however is a broader understanding of the interplay of B. fragilis in atherosclerosis.
Within the human host glucose is invariably converted to pyruvate via glycolysis under anaerobic conditions. However b. Fragilis has carbon dioxide dependent metabolism. As such, when the organism is grown in the presence of glucose under an atmosphere of 100% carbon dioxide growth rate is slow and cell yield low (reference: pathway succinate and propriety formation impact rates fragilis).
The major fermentation products formed under these conditions are fumarate and lactate (= Warburg). However when hemin is included in the same medium growth is both faster and cell yield greater. The major fermentation products are acetate, propionate and succinate. As stated by it is obvious that the evolution of the carbon dioxide dependent metabolism of Bacteroides fragilis has adaptive utility allowing it to compete and survive not only in the presence of competing microorganisms but with the host itself for nutrients.
An example, may include the relation of obesity with atherosclerosis and implications of gastric bypass upon human gut microbiota. Succinic acid production is not only a byproduct of the fumarate reduction pathway but is also strongly associated with atherosclerosis and obesity. Of interest, succinic acid can be efficiently produced by Bacteroides fragilis and has been harnessed and optimized in scale up bioreactors (Isar at al).
Interestingly, the impact of Bacteroides fragilis an originating source of succinate production altering host metabolism relative obesity in atherosclerosis is currently and chronically overlooked. Such an association may be both relevant and of significance as fumarate reduction by NADH has been demonstrated in cell extracts of B fragilis (Macy et al.), and in light of the fact that Bacteroides fragilis 25285 is capable of catalyzing fumarate reduction by hydrogen. Interestingly, hydrogen therapy (<4% administration) and consumption of hydrogen infused drinks have been associated with a significant reduction in BMI. Administration of hydrogen rich water and inhaled hydrogen enhance performance and endurance as well as prevents atherosclerosis (Ohsawa 2008).
It is also of significance to note that germ-free animals require ~30% more calories to maintain body weight (Gilmore & Ferretti, 2003). B. fragilis - a member of the human flora - may play a significant contribution to weight gain and such may be corrected by eliminating BFT producing B. fragilis in germ-free models. This also is similar to the ~28% reduction in weight observed by Aysan et al (2011) upon administration of low dose oral boric acid - which significantly reduces colonic bacterial load (including B. fragilis).
4.2 - Fumarate reductase pathway and proteases
What may also be of consequence regarding Bacteroides fragilis metabolism, is that enzymes that carry out similar reactions within the host are structurally and therefore significantly different regardless of the degree of similarity. Using the fumarate reduction pathway as a case in point, is conceivable that Bacteroides fragilis may scavenge and utilize host fumarate for its own metabolism while simultaneously denaturing host enzymes through the up regulation of proteases such as ‘aminopeptidase N (CD13) - like’ in the catalytic ability conferred by Bacteroides fragilis toxin (Bft) - especially as vulnerable cysteinyl-glycine and cysteinyl –leucine linkages, respectively.
Such may not only afford Bacteroides fragilis an adaptive mechanism by which to exploit host substrates, but could conceivably provide a chronic mechanism which helps perpetuate its own long-term survival, while controlling (if not regulating) aspects of altered host metabolism. Further, if otherwise antagonistic host enzymes such as fumarase which would theoretically oppose Bacteroides fragilis growth can be proteolytically hydrolyzed, such may represent a mechanism which maintains a chronic or steady-state necessary for the organism's establishment, survival and perpetuation within various locations such as the atheromas observed in atherosclerosis.
As fumarate hydratase has a dual role in repairing DNA strand breaks in the nucleus (Yogev et al, 2010), and using such as a case in point, it becomes conceivable that a competitive interaction between host and commensal energy requirements - could not only perpetuate its continued existence by altering host homeostatic mechanisms, but also resultin an amplification of such dis-balance by limiting the host's ability to respond to such agent as well as the host's ability to repair itself within a foci of anaerobic occupation (i.e. plaque).
What is interesting about factoring in an alternative pathway used by an opportunistic commensal such as Bacteroides fragilis relative atherosclerosis, is that such may help explain the involvement of various compounds which decrease atherosclerotic progression such as particular therapeutics, dietary and herbal supplements - in light of their direct and/or indirect impact upon the fumarate reduction pathway. Such As well, factoring in such variable may afford further insight into existing, as well as the development of novel, approaches to control or decrease atherosclerosis progression.
Further, by factoring in Bacteroides fragilis as a variable findings such as the relation of red meat and cooked meat consumption to atherosclerosis and diabetes may be viewed in terms of the relevance and role that increased hemin, ammonia as well as preservatives and additives may play relative to such organism.
As an example, and with reference to the fumarate reductase pathway, it is probable that nitrate containing food preservatives may provide nitrates which serve as a proton acceptor (reducing agent) for B fragilis subspecies fragilis along such pathway. As observed by Shen at al (2010), ingestion of cook meats altered human gut and fecal microbiota samples which was postulated with an increased risk of intestinal and systemic diseases associated with a high intake of particular meats and those processed origin. Such may have relevance regarding the specific cultivation Bacic & Smith (2008), perpetuation and virulence relative Bacteroides species within the host - thus influencing atherosclerosis in non-insulin-dependent diabetes mellitus.
As well, Migta and Nishimura (2006), successfully purified and characterized a chloride and nitrate stimulated enzyme which appeared to be involved in an increase in free amino acids during postmortem storage of meats. Such, can analogously represent a similar influence of enzymes possessed by Bacteroides fragilis such as aminopeptidase N-like and aminopeptidase D.
Interestingly, 16 years earlier, Hung at al (1988) purified and characterized also a chloride stimulated cellobiosidase from Bacteroides succinogenes. Though both represent two classes of enzymes with different actions, such may be important regarding the importance of association of bacterial and specifically Bacteroides enzymes integrating nutrient products consumed by the host which may also promote commensal overgrowth.
5.0 Carbohydrates
Substrate Utilization
Linking Bacteroides fragilis with atherosclerosis & diabetes
5.0.1 – Carbohydrates (Point form)
· B. fragilis – Embden-Meyerof pathway influenced by host diet
· B. fragilis - Relevance and implications in atherosclerosis & diabetes
· B. fragilis - Monosaccharide preference & utilization
· B. fragilis - Glucose & glycogen utilization
· B. fragilis - Catalase link in diabetes
· B. fragilis - Lactose (galactose) & Insulin Secretagogue
· B. fragilis - Galactose utilization (LPS & Capsule)
· B. fragilis - Glucosamine & Proteoglycans
· B. fragilis - Glucose, CO2 and the runner’s conundrum
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5.1 – Dietary carbohydrates and the Embden-Meyerof Pathway
Dietary carbohydrate consumption patterns have been the subject of voluminous published literature related to atherosclerosis and metabolic X syndrome as such is strongly associated with non-insulin-dependent diabetes mellitus and obesity. However, studies analyzing and comparing dietary intake upon Bacteroides fragilis NCTC 9343 is comparatively scant to nonexistent - with the exception of in vitro and past, in vivo, studies conducted upon animal models undertaken to explore such mechanisms and more fully understand the organism.
Given the potential impact and significance of Bacteroides fragilis upon atherosclerosis and particularly - the potential relevance of the Embden-Meyerhof pathway of glycolysis used by B. fragilis for the catabolism and fermentation of glucose (Macy et al, 1978), as an underexplored aspect in host diabetes -further exploration regarding the specific impact of such within the human host is past due.
A case in point, illustrating the importance of diet not only upon the host but colonizing flora as well, Frantz and McCallum (1980), while studying the composition morphology of Bacteroides fragilis within various medium, noted alterations in cellular carbohydrate composition during different phases of growth - reporting that such is indicative of the highly complex metabolic capability of the organism. During the study, changes in media carbohydrate incorporation and retention were observed to range in value - fluctuating with growth time and comprising between 10 to 20% of the organism’s dry cellular weight.
Though the gut microbiota in general has been implicated as potentially contributing to atherosclerosis and NIDDM, Bacteroides fragilis is as intriguing as it is unique in its diversity of monosaccharide substrate utilization and conversion to glucose-6-phosphate or fructose-6-phosphate along the Embden-Meyerhof (EM) pathway – especially when compared to other gut organisms (MJ Hill, Role of Gut Bacteria in Human Toxicology and Pharmacology).
Further, as a commensal, the array of various sugar moieties, and predominantly those that preferentially enhance and stimulate Bacteroides fragilis growth such as glucose and galactose (i.e. lactose), maltose, fructose (i.e. sucrose, fruits) as well as mannose and xylose (Blatch & Woods, 1993) – are regularly, if not abundantly, consumed in the host Homo sapiens diet - and are easily accessible, utilized and converted as per the organisms needs.
Such may be of consequence given that it was observed by Kritchevsky et al, (1980) who successfully initiated atherosclerosis by diet alone in baboons, that a ranking for the development of aortic sudanophilia (observed plaque formation) in 40% carbohydrate, 0.1% cholesterol supplemented diets relative sugars followed the order: lactose>starch>glucose>fructose and sucrose (similar). This may not be surprising given that Macfarlane et al (1990), regarding Bacteroides ovatus, which is also highly adapted for intestinal growth - confirmed polysaccharide utilization as “partly regulated at the level of enzyme synthesis”.
If such is similar within Bacteroides fragilis, then utilization of simple sugars is preferrable and an abundance of primary sugars would likely contribute to progressed growth and unabated – contribute to the process and progression of atherosclerosis. Further, and perhaps most interestingly, Blatch & Woods (1993), during their characterization of Bacteroides fragilis fructanase noted the same order relative inulinase and carbon source ranking.
Beyond such, however, it should additionally be mentioned that Bacteroides fragilis is also able to utilize other important monosaccharide components such as: D-ribose, L-fucose, D-galacturonate, D-glucuronate and is involved in deoxyribose and deoxynucleoside catabolism – factors which may have relevance regarding atherosclerosis.
It should be noted also, that Bacteroides fragilis has an elaborate myriad of mechanisms to utilize and convert an extensive grouping of carbohydrates. As an example, and - to stress the importance of the interplay with the human host - those related to generating energy and incorporating the involvement of various host similar components through its central carbohydrate metabolism include: methylglyoxal metabolism, pyruvate:ferredoxin oxidoreductase, pyruvate metabolism I anaplerotic reactions (PEP), pyruvate metabolism II: acetyl-CoA acetogenesis from pyruvate, pyruvate alanine serine interconversions, glycolysis and gluconeogenesis, Entner-Doudoroff pathway, TCA Cycle, pentose phosphate pathway and glycolate, glyoxylate interconversions (and synthesis).
Further, Bacteroides fragilis is involved the biosynthesis and utilization of Di and oligosaccharides such as sucrose metabolism and utilization as well as mechanisms for the uptake and utilization of lactose and galactose - the consumption, all of which, is high in western diets and is particularly associated with atherosclerosis progression and advanced development as well as positively and strongly correlated with associated states such as non-insulin dependent diabetes mellitus and obesity. In addition to such, B. fragilis has the capacity for trehalose biosynthesis, uptake and utilization, as well as capacity for melibiose, maltose and maltodextrin utilization.
Other mechanisms, which may have relevance regarding atherosclerosis and biomarker formation (as well as other disease states and cancers such as malignancies of the breast and colon) include: propionyl-CoA to Succinyl-CoA, acetoin and butanediol metabolism, butanol biosynthesis, and mixed acid fermentation (obesity).
Additionally, the organism’s one-carbon metabolism by tetrahydropterin (homocysteine/folate implications relative atherosclerosis), lactose and lactate utilization and fermentation (perhaps relevant in breast cancer),those related to amino sugars such as chitin and N-acetylglucosamine utilization (arthritis), inositol and hexitol catabolism (related to CNS manifestations) and glycogen polysaccharide metabolism – which may have primary and fundamental relevance regarding both atherosclerosis and non-insulin dependent diabetes mellitus.
Bacteroides: Dietary Monosaccharides
Taken on a more general scale, it is perceivable that Bacteroides fragilis may be directly and indirectly impacted by the substrates available through individual diet and dietary behavior. For example, many polysaccharides observed as regularly and preferably utilized and degraded by B. fragilis, and constitute the human diet can, perhaps barring a strict daily dietary review, intake and regiment – regularly, and even inadvertently, as a result of food chemistry constitution and overlooked dietary supplementation, become consumed providing exposure to vast substrates not otherwise absorbed by the duodenum and present within the upper intestine.
Such may, not comprehensively, include common dietary substrates such as starch, cellulose (plant), chondroitin sulphate (&/or supplemented), arabinogalactan (food stabilizer), xylan (algae), inulin (fibre), dextrin (fibre), raffinose (digestive aide), pectin (gelling sugar), guar gum (additives) as well as that compounding and of host derivation such as mucins (mucosal defense) and heparin (anticoagulant) (Cato & Johnson, 1976).
It is also worthy of note, that Bacteroides fragilis (NCTC 9343/ATCC 25285) has been observed as inhibited or slow of grow relative sugar moieties such as: arabinose which has shown strong antioxidant properties (Quan et al, 2011 – PMID 21535815), dulcitol, erythritol a strong antioxidant and sweetener (den Hartog, 2010 - PMID), glycerol, inositol, mannitol, melezitose, rhamnose, salicin (ASA relevance), sorbitol, and trehalose. Other sugars with mixed results include amygdalin (purported cancer reductions), as well as those more pH dependent: cellobiose, esculin, mannose (Cato & Johnson, 1976).
Bacteroides: Glucose & Glycogen utilization
As mentioned, B. fragilis is capable of polysaccharide metabolism –which, indeed may be of relevance regarding diabetes. As observed by Linder et al (1979), 27 strains of Bacteroides fragilis were capable of intracellular, cytosolic, granular polysaccharide formation from glucose; dependent on environmental pH, cellular growth phase and glucose concentrations – that upon characterization was revealed as glycogen.
Further, Franz & McCallum (1979) noted that “B. fragilis is capable of rapid rates of growth in vitro” and more importantly “by using glucose as the sole energy source”. It is both probable and likely that particular sugars such as glucose - beyond a mere natural assumption - plays not only a particularly intrinsic role in its metabolism but may be involved in a dynamic interplay with the host – to adaptively increase and secure chronic supply of such substrates within the environment of the human host.
Additionally, it was observed by Franz & McCallum (1979) that “greater amounts of glucose were utilized during the early phase growth of Bacteroides fragilis”, and that “the rapid growth rate of B. fragilis does not correspond to an economical utilization of glucose as an energy substrate”. It is tempting then to consider the possibility that a state of chronically increased glucose (dextrose) as observed in diabetes, if so induced may supply and fulfill such need – within the host environment, especially considering the relative proximity of the pancreas to the upper intestine regarding the infective and colonization potential of B. fragilis.
It is conceivable then that the findings of Frantz and McCallum (1979 ) may both be overlooked, as well as lend credence to the fundamental possibility of such interplay regarding Bacteroides fragilis which was observed to demonstrate “a dynamic and rapid growth capability in glucose enriched medium”.
Further, it was suggested that ‘under more conducive conditions it may be possible to obtain more rapid rates of growth’ then that tested in vitro, and that given similar nutrients and conditions – “rapid growth rates would likely be possible within the gut or diseased tissue”. Perhaps, such findings require reconsideration, review and further exploration.
Compounding such, Frantz and McCallum (1979), concluded that “B. fragilis is capable of rapid rates of growth in vitro by using glucose as the sole energy source”. It is therefore possible that this inherent ability of substrate utilization and conversion, may have far reaching effects regarding diabetes and atherosclerosis progression and account for the significance of diet upon the progression of both disease states.
Lastly, Allison et al (1984), when studying alternative biosynthesis pathways for Bacteroides ruminicola and Bacteroides fragilis noted that “carbon from [U-14C] glucose was used for the synthesis of leucine and other cellular amino acids – again, re-stating the importance of glucose utilization as of a centralized importance within Bacteroides.
Bacteroides: Catalase link in Diabetes
Curiously, a link between diabetes and Bacteroides fragilis may be observed by reviewing enzyme catalase production relative fermentable carbohydrate and nutritional status as well as the influence of oxidative stress and Bacteroides fragilis. Gregory et al (1977), for example, observed a dramatic and immediate cessation of catalase production by glucose and eight other fermentable carbohydrates including: lactose, fructose, raffinose, starch, mannose, sucrose, galactose and maltose - utilized by Bacteroides fragilis strains (i.e NCTC 9343).
Two decades later, Rocha & Smith (1997) noted that in the absence of oxygen, the expression of the katB catalase gene in Bacteroides fragilis (638) was strongly repressed by glucose. Additionally, it was observed that glucose induced katB mRNA repression “was abolished upon oxygen exposure, and the levels of mRNA increased dramatically… regardless of carbon sources used”.
The relevance of such can be noted in light of the association of serum catalase in type 2 diabetes (NIDDM) as well as atherosclerosis and alzheimer’s disease. Serum catalase has been observed as lower in subjects with type 2 diabetes than nondiabetic subjects though no associated genotype, mutation, or polymorphism has been observed – and has been interpreted as possibly suggestive of down regulation of host catalase production (Goth, 2008).
It is however, entirely probable that higher circulating catalase in nondiabetic subjects may represent the influence of higher basal concentrations resulting also from total Bacteroides species contributions relative chronically controlled and therefore lower circulating blood glucose levels - and, in the presence of higher dissolved oxygen content.
The situation may be somewhat different however in type II diabetic subjects, who may, for example, hypothetically represent a state of ‘unabated infection and extraintestinal dissemination’, beyond normal colonization by Bacteroides fragilis NCTC 9343 and YCH46. Given that anemia, especially subclinical anemia, is a common presentation and state associated with type 2 diabetes (Merlin et al, 2003) - lower dissolved oxygen levels may contribute to a lower required upregulation of oxidative mechanisms utilized by B. fragilis (Sund et al, 2008) – as would be energetically efficient and adaptive.
Given that higher circulating blood sugar levels is the primary manifestation of insulin insensitivity observed in NIDDM, it is likely that such would result in a measurable reduction of catalase as observed in B. fragilis (Gregory et al, 1977) – and otherwise observed and misinterpreted as departure from the otherwise ‘baseline norm’ for catalase (Goth, 2008).
Further, the reduction in catalase as a result of higher circulating glucose (and likely consumed fermentable carbohydrates within the intestine) upon katB mRNA repression in Bacteroides fragilis (Rocha & Smith, 1997), as well as the rapid growth rates and influence upon B. fragilis metabolism subsequent to increased sugars (Frantz and McCallum, 1979). This may ultimately reflect the higher hydrogen peroxide levels chronically observed in NIDDM and atherosclerosis (Yang et al, 2004), especially relative the endogenous host response to B. fragilis (Rocha & Smith, 1999).
Bacteroides flourishes under reduced redox conditions. Its preference and utility for reduced sugars such as lactose, is then no surprise. The organism is capable of both galactose and lactose uptake and utilization amongst a host of other monosaccharides, disaccharides and oligosaccharides – as per substrate availability or endogenous synthesis.
Bacteroides fragilis (such as NCTC 9343, YCH46 & 638) contains the LacZ gene which encodes for lactose galactohydrolase (beta-galactosidase) capable of catalysing the reversible reaction between lactose and H2O and alpha-D-glucose and D-galactose. Additionally carried are the genes GalM (aldose 1-epimerase), GalK (galactokinase) and GalE (UDP-glucose 4-epimerase) required for galactose degradation.
Interestingly, oral galactose is considered a “potent insulin secretagogue” and was observed to affect secreted and circulating insulin levels (Ercan, 1993). In fact, it was observed that the insulin response to galactose in type 2 diabetic (NIDDM) subjects is additive to that following glucose administration – however, only a modest increase in plasma glucose concentration was observed as a result of galactose ingestion and the co-administration of both resulted in decreased plasma galactose concentration.
It may be therefore possible that the observed increase in insulin levels without an otherwise assumed - simultaneous impact or rise in plasma glucose may be due to increases in galactose (and lactose) uptake and utilization by Bacteroides fragilis NCTC 9343 (Cato & Johnson, 1976).. Such is analogous to the preferential use of lactose in media utilized to isolate and optimize growth for the presumptive identification of B. fragilis (Ushijima, 1983).
Similarly, galactose would likely optimize growth and increase total B. fragilis load within the host – upon its consumption or administration. Further the impact of galactose in the synthesis of its Lipopolysaccharide cannot be overlooked, especially relative its upon the pancreas subsequent to such increased production – likely connecting further the impact of B. fragilis upon the host in type 2 diabetes (in addition to other mechanisms such as cysteine proteases (fragilysin and aminopeptidase N - to name some).
If B. fragilis NCTC 9343 & YCH46 are the causative agents acutely and severely impacting pancreatic insulin production in insulin dependent diabetes mellitus and chronically in type 2 diabetes (NIDDM), then it is foreseeable that dietary substrates which increase cellular growth, rate and total load would likely further perpetuate and adversely affect the host – resulting in progressive stages and advanced disease states observed over time.
In diets, providing increased lactose – containing both galactose and glucose – to intestinal B. fragilis cells within the intestine and areas of its dissemination - especially in the absence of competing gut flora members capable of utilizing such and maintaining a balanced B. fragilis composition and control of strains NCTC 9343 and YCH46 - such would likely reflect the higher incidence and prevalence of diabetes and atherosclerosis within such groups, and help explain demographic associations observed.
Such may account, for example, in the increased burden of cardiovascular disease observed in India where the interplay of higher cholesterol and lactose consumption (milk, milk based products) within the diet in association with a noted absence of small intestine lactase – would theoretically result in “significant amounts of lactose reaching the colon”. Similarly, such results and influences may be observed with regard to the regular use of agents like lactulose (used as a stool bulking agent) which provide galactose within the intestine for use by B. fragilis NCTC 9343 and YCH46.
Additionally, it is likely that decreases in both the incidence and prevalence of atherosclerosis and diabetes would then be expected in the presence of a compensatory mechanism to counter the dietary impact of lactose upon B. fragilis NCTC 9343 and YCH46 - such as that afforded through lactose competition by lactobacillus or as simply associated with a significantly decreased consumption of such galactose and reduced dietary sugars in general.
It is also of note, that the presence of endogenous enzymes of (endo)-beta-galactosidase (LacZ; EC 3.2.1.23), lactose galactohydrolase (EC 3.2.1.108) by Bacteroides fragilis (Scudder et al, 1983) serves not only to utilize and interconvert lactose and galactose but is fundamental to the production of LPS – wherein galactose moieties can bind ceramide.
It is plausible then that the relation with dietary sugars and circulating serum insulin may be viewed, if not reflect – in type 2 diabetes (NIDDM) - the degree to which dietary sugars such as glucose and galactose influence: (1) Bacteroides fragilis growth and (2) production of virulence factors – and in this example – the rate or total degree of LPS synthesis afforded by the administration of galactose a substrate with dual purposes substituting for energy needs or LPS generation.
If correct, such may provide insight into discrepancies and particular dietary influences observed upon both diabetes and atherosclerosis – hypothetically assumed herein to be caused by B. fragilis NCTC 9343 and YCH 46. Further, it becomes apparent that even the strictest dietary regimens, adjusting for cholesterol, trans and saturated fatty acids - may hypothetically still contribute to a continued and progressed disease state – as lactose is contained within many food products. As a case in point, lactose (galactose) is contained in, but not limited to, a diverse range of dietary food stuffs such as:
Common high-lactose (galactose) foods, can include:
Food examples, which may contain lactose (galactose) in smaller quantities:
bread, various baked goods, milk chocolate, salad dressings, various sauces, breakfast cereals, cereal bars, instant potatoes, instant soup noodle and rice mixes, luncheon meats, candies, various snacks, mixes for pancakes biscuits and cookies, margarine, organ meats (i.e. liver), sugar beats, peas and lima beans.
Ingredients derived from milk that contain lactose (galactose):
Thus it is obvious, the many and diverse foods/foodstuffs containing - but one - dietary monosaccharide which is capable of dramatically influencing Bacteroides fragilis NCTC 9343 and YCH46. This is by no means an anomaly, as using galactose as a representative index, foods varied in composition but containing nutrients utilizable to B. fragilis are commonly found in contemporary society.
Again, such may serve not only as abundant dietary staples to the human host, but amortized over the life of the human host may accumulate compounded impact through the ingestion of chronic substrates, vacillating daily in inconsistent patterns of consumption on a nutrient level. It is therefore probable that nutrients are regularly, continually ‘leached’ or percolate’ from the human diet from - conception until demise - and constantly contribute to changes in B. fragilis growth, rate and load – relative cells and cellular byproducts, within the intestine and areas of B. fragilis dissemination and influence.
Hints of Atherosclerosis and Diabetes: Galactose based LPS & Capsule
The enzyme – endo-beta-galactosidase – as referred, contributes both to the energy production as well as the incorporation of galactose and ceramides into capsular polysaccharides and lipopolysaccharides produced by Bacteroides strains and in particular B. fragilis NCTC 9343 (Scudder et, 1983), (Kasper et al, 1984). Specifically, Scudder et al (1983) observed that particular strains of B. fragilis were able to hydrolyze various oligosaccharides via endo-beta-galactosidase – yielding free glucose – providing an index of enzymatic hydrolytic potential relative the type of oligopolysaccharide used.
As such, and while circumstantial supporting relevance to the significant impact dietary fibres play in noninsulin dependent diabetes and atherosclerosis relative Bacteroides strains, it was observed that endo-beta-galactosidase retained a high percentage of hydrolysis between Galβ1→4Glc linkages of lacto-N-neotetraose, lacto-N-tetraose as well as the form of lacto-N-tetraose fucosylated at the 3 Glc (glucose) position via an alpha 1,4 bond and lacto-N-tetraose fucosylated (via an alpha 1,2 bond) at the first galactose position.
It was observed however, that the percentage of hydroxylation decreased in lacto-N-tetraose (compared to lacto-N-neotetraose) due to Galβ1→3Glc. Further, successive decreases in hydroxylation were observed upon fucosylation. α1,4 fucosylation at the 3glucose in Galβ1→3Glc decreased hydrolysis due to endo-β-galactosidase and a further reduction was observed due to the position α1,2 fucosylation of the first galactose within lacto-N-tetraose. A further reduction in hydrolysis was observed due to the presence of fucose on both monosaccharides.
Bacteroides: Glucosamine, galactosamine and proteoglycan receptors
In diets supplemented with the crustacean derived dietary fiber chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) units, a reported decrease in blood lipids and body weight has been observed.
Initially such may present as remarkable in observation. However, when considered in relation to the potential impact of Bacteroides fragilis upon the host a clearer picture presents.
Chitosan has been observed as a bacteriostatic and bactericidal agent within the intestine. And though such properties may influence all residing members of the intestinal flora, it's potential impact upon Bacteroides fragilis may be unique.
That is, chitosan, the second most abundant fiber after cellulose, would likely have also provided a potential energy substrate prior to the evolution of the mammalian species. As such, the polyglucosamine structure could theoretically be cleaved to provide free D-glucosamine and N-acetyl-D-glucosamine molecules – for bacteria energy and structural needs as well as a binding ligand for niche creation.Overlaid upon findings relative the use of glucosamine by Bacteroides fragilis - merit to such statement may be found.
As an example, glucosamine is utilized in the formation of capsular polysaccharide (PSA) and lipopolysaccharide (LPS) by Bacteroides fragilis (Kasper et al, 1984), (Pragani et al, 2010). Further, Chen et al (2002) observed that “Bacteroides fragilis could utilize N-acetylchitooligosaccharides (under particular conditions) more efficiently for growth than glucose”.
Also , regarding the potential relation between B. fragilis and atherosclerosis, such may provide additional insight into the observations made by Tannock et al (2006) that; (1) glucosamine accelerates early atherosclerosis progression and (2) that long-term glucosamine consumption increases cholesterol and triglyceride concentrations in male mice, but does not effectively progress further atherosclerosis.
Perhaps then it is plausible, that timing (host immunological, floral/gastrointestinal and vascular development) and forms of glucosamine administered - vary in influence upon B. fragilis. That is, during early host development increased glucosamine may contribute to the establishment, growth and virulence of Bacteroides fragilis. However, once established dietary forms of glucosamine may mainly replete and enable the host to repair in the presence of the pathogen.
Further, it has been shown that Bacteroides fragilis NCTC 9343 attaches to mucosal surface glucosamine and galactosamine units via a lectin binding receptor with a specific avidity for such (Rogemond & Guinet, 1986). It was also observed during such study, that preincubation of cellular lectin receptors or binding ligands with D-glucosamine and D-galactosamine inhibited B. fragilis NCTC 9343 adherence.
Such may be analogous, if not representative of the attenuation of attachment to host mucosal layers subsequent the dietary administration of such molecules – as free sugars or linear polysaccharides such as glucosamine (and chondroitin) or chitosan, respectively.
It is possible then, that lectin binding interference within the intestine, and perhaps circulatory system, and conferred by glucosamine ingestion - may reduce attachment of the organism. Additionally, glucosamine, N-acetyl-D-glucosamine, chitin, chitosan and N-acetylgalactosamine, galactosamine, chondroitin (among others) can variably inhibit theenzyme beta-N-acetylhexosaminidase (EC 3.2.1.52) possessed by Bacteroides fragilis NCTC 9343 (NCBI: YP_210645.1), used to obtain N-acetylhexosamines from the environment - antagonizing its function.
However, it may well be, that the effect of chitosan may have a very variable influence upon mechanisms of B. fragilis-host attachment, which again would perhaps be separate and different from the ultimate utilization of polysaccharides (such as chitosan) by B. fragilis. It would be plausible that the administration of chitosan, may result in cleavage of the molecule by Bacteroide fragilis for substrate utilization which increases its growth, but influences attachment due to liberation of glucosamine units.
Such may echo then the observed increase in early atherosclerosis as well as the increase in blood lipids upon chronic consumption of glucosamine (Tannock et al, 2006). Further, such would be directly in line with observations made by Mrazek et al (2010) of “a raised level of fecal Bacteroides in response to chitosan intake…found in all samples” during long-term chitosan supplementation. Consequently, it may be possible that other Bacteroides members such as B. thetaiotaomicron possessing beta-N-acetyl glucosaminidase (GenBank: AAL39074.1) may additionally contribute to substrate liberation increasing further Bacteroides load during chitosan administration.
Finally, perhaps a similar model and series of mechanisms regarding the impact of chondroitin (N-acetylgalactosamine and glucuronic acid) administration upon B. fragilis lectin attachment and extracellular chondroitin sulfatase (Rudek & Haque, 1976) is relevant. That is, B. fragilis is capable of utilizing the amino sugar N-acetylgalactosamine - found within chondroitin – but liberation of such is normally directed to minimize the possible influence of liberated monosaccharides upon lectin (glycosaminoglycan) binding between itself and a host surface.
If so, such may similarly accounts for findings relating “chondroitin sulfate proteoglycan and coronary atherosclerosis in the youth” (Yang et al, 1996) and positive findings relative atherosclerosis associated with supplemental chondroitin administration (Martínez-Calatrava MJ, 2010).
Further, findings regarding the use of glycosaminoglycan antibody-based immunotherapy as a potential tool to prevent atherosclerosis (Soto et al, 2012) – may support the statement that agents capable of reducing B. fragilis NCTC 9343 and YCH 46 lectin binding (intestinal and/or extra-intestinal) may potentially reduce atherosclerosis progression even in the presence of increasing numbers of the organism within the host. As stated by (Rogemond & Guinet, 1986), regarding the finding of glucosamine and galactosamine receptors in seven subspecies of B. fragilis:
“…the presence of a lectinlike adhesion among B. fragilis group organisms may help to elucidate the roles of these bacteria in the colonization of intestinal mucosal surfaces and in the predominance of infections with B. fragilis alone as well as in synergy with other bacteria. These lectin-like adhesions may have the potential for the specific control and prevention of these infections…”
Glucose, CO2 and the runner’s conundrum…
Within the literature amassed regarding atherosclerosis and noninsulin dependent diabetes mellitus - associations have been raised regarding various human activities and endeavors relative the incidence of acute cardiovascular events (ACEs). One such example, involving the chronic intake of higher carbohydrate diets associated with ACEs - involves that of the long distance (marathon) runner. However, when viewed through the lens of Bacteroides fragilis – a potential explanation may become more apparent.
It is well known that runners require increased carbohydrate to restore liver and muscle glycogen. As well, periods of anemia and subclinical anemia may surface over the life course of a long distance runner attributable to physical exertion and compounded by factors such as reduced intake of hematopoietic nutrients ranging from vitamin D, cobalamin, pyridoxine, cobalamin, folate, ascorbic acid, iron to a low total protein intake.
It has been also observed that such may occur as the result of foot strike resulting erythrocyte lysis, intestinal bleeding, helminth infections (i.e. giardia, cryptosporidium) and increased intestinal transit resulting in reduced B12 absorption.Interestingly though, even in the absence of such risk factors - atherosclerosis and manifestations of such, have been associated with the activity of long-distance running.
However, overlooked in many cases is the rise in serum carbon dioxide levels (CO2) which can stand well above that observed in individuals undertaking activities of lesser intensity and duration, especially when performed repetitively without allowance for adequate recovery. When higher dissolved CO2 combined with a chronically high carbohydrate intake and other variables such as the circulatory and intestinal availability of hemin produced during erythrocyte lysis (and required by B. fragilis) in the presence of reduced O2 – such may be advantageous to, and promote further colonization and virulence of Bacteroides fragilis.
Observations made by Macy et al (1978), for example, on glucose fermentation by Bacteroides fragilis illustrated thatC02 not only promotes an anaerobic environment for cultures studied ‘in vitro’, but was observed as “necessary for growth because it is needed for the formation of C4 acids” – during glucose fermentation. Similar findings were confirmed by Frantz & McCallum (1979) who observed that though the rate of growth was “consistently reproducible, reflecting a high capability for energy production by B. fragilis from glucose” (Frantz & McCallum, 1979).
It was also noted that “the exclusion of vigorous gassing with anaerobic grade CO2 reduced growth rate values…suggesting a stimulation of growth of B. fragilis by carbon dioxide (during glucose fermentation) agreeing with a possible role for carbon dioxide in energy production via condensation with phosphoenolpyruvate”.
It may be probable then to suggest that in certain scenarios, high carbohydrate consumption, perhaps especially high glycemic food nutrients, in the presence of higher dissolved blood values of CO2, may, in some cases negate the anticipated, if not expected health protection inherent to physical activity - beyond otherwise shear weight loss, increased circulatory perfusion and developed endurance. That is, particular activates undertaken and maintained may ultimately increase the impact of Bacteroides fragilis upon the host over time – by optimizing the conditions required for its growth, colonization, dissemination and infectivity.
Perhaps such presents an example of a scenario which literally and increasingly ‘ferments’ with time and more precisely one the host cannot, and preferably should not ‘run away from’ – if and when higher CO2 values are observed – given the chronic presence of Bacteroides.
Hints of Atherosclerosis and Diabetes: Mucin
In the absence of dietary fibre to continually maintain regular digestive base by shedding and bulk removal of Bacteroides fragilis from within the intestine…
5.0 CHOLESTEROL
(‘chole’-sterol vs. i.e. stigma-stero-l, stero-ne, stero-id…)
B. fragilis & Sterols structures
5.1 - Cholesterol:
A fundamental connection between Bacteroides fragilis and atherosclerosis
In no small way can the importance of cholesterol levels within the host be dismissed with regard to it's impact upon, and larger relation, with atherosclerosis. The importance of such interaction may be more adaptively, logically, and specifically attributed, however, to a more fundamental dynamic – the primary impact of cholesterol upon Bacteroides fragilis NCTC 9343 and YCH46, which manifests such disease state.
What if, for example, serum cholesterol levels such as low serum, high density lipoproteins and high serum levels of low-density lipoproteins essentially reflect or equilibrate the host's response to the impact of Bacteroides fragilis and its products such as the protease aminopeptidase N and fragilysin (i.e. especially BFT-1) –upon the host?
As a human commensal, the continued trends associated with serum cholesterol may represent the chronic presence and ‘disease process’ of Bacteroides fragilis within the host - with specific variations in cholesterol and serum lipid profiles representing the acuity of impact caused by the organism upon the host and anyone period of time.
Also, given the stimulatory effect of cholesterol as a substrate upon the metabolism and growth of Bacteroides fragilis, it is conceivable that Bacteroides fragilis represents - a fundamental intermediate, if not link - between dietary sources and behavior and the ultimate progression or alternatively - prevention, control or decrease of atherosclerosis.
If such is probable, then a simple overgeneralized algebraic relation may be in order regarding cholesterol, Bacteroides fragilis and atherosclerosis . Atherosclerosis is associated with cholesterol such that cholesterol substrate availability for B. fragilis utilization and observed serum cholesterol profiles are associated with viable B fragilis cells as well as B fragilis products and influence. A simpler equation, which overgeneralizes the impact of cholesterol upon atherosclerosis may be:
5.2 - Exploring dietary cholesterol, B. fragilis and atherosclerosis
Possibly one of the most expanded and explored fields relative to the understanding of atherosclerosis is the relationship between disease progression variation in dietary patterns, and particularly the relationship between cholesterol and atherosclerosis. If cholesterol intakes can profoundly influence the progression advancement of atherosclerotic plaques, yet little-to-no literature exists relating the increase in serum cholesterol and Bacteroides fragilis as an important virulent member of the intestinal flora.
Those few studies published date and designed to study the effects of other related phenomena then that alluded, however, provide significant insight into the possible progression of atherosclerosis when viewed outside of the scope of their originally designed and contextualized meaning.
Studies in relation to metronidazole,such as that conducted by von Bergmann et al (1985) for example, have revealed the influence of particular antibiotics on serum lipids. Specifically, metronidazole resulted in abrupt decrease in cholesterol - measurable in days, as opposed to months - with a degree of outcome generally unattainable by synthetic cholesterol-lowering agents, niacin and least of which, dietary restrictions.
Such findings have been subsequently and unintentionally supported, through observations made regarding the use of antibiotics, such as metronidazole, ampicillin and clindamycin upon the biotransformation activity of the intestinal flora (Midtvedt et al, 1986), directly by studying the effects of metronidazole upon serum lipids (Ducobu & Fontaine, 1986), use of metronidazole to treat biliary tract infections caused by anaerobes (Fu, 1989) and while analyzing the conversion of cholesterol to coprostanol by intestinal bacteria within human subjects (Midtvedt et al, 1990) - to name just some.
These studies, not only highlight the importance of the anaerobic flora upon serum cholesterol and lipid profiles, but hint at a more fundamental relationship between cholesterol and anaerobic metabolism.
5.1a - Cholesterol is not the Root cause of Atherosclerosis, B. fragilis is Root Cause, Fragilysin (BFT/Bft-1,2,3) is Direct cause, initiates & propagates CVD outcome(s)
DIRECT CAUSE:
Fragilysin (Bft-1,2,3) - EC 3.4.24.74 (BFT binding is cholesterol sensitive) i.e. PMID: 16926433
INDIRECT CAUSE:
Cholesterol: is an INDIRECT contributing cause, with slow onset. i.e. (1) Bft binding - PMID: 16926433, i.e. (2) B. fragilis growth - PMID: 2995802, i.e. (3) B. fragilis + cholesterol = atherosclerosis - PMID: 290725
while studying the relationship between fecal mutagens, Bacteroides fragilis and dietary dimethylhydrazine in rats, observed that fecal levels of Bacteroides fragilis were significantly increased by the inclusion of cholesterol in the diets administered. As stated within the study results:
· “the cholesterol added to the diet had the most pronounced effect for each sampling period examined and for the overall experiment. Cholesterol induced a marked proliferation of B fragilis group organisms compared to all other dietary factors. The effect predominated even as all values decline toward the end of the experiment.”
Similarly, it may be both relevant and probable to suggest that strains of Bacteroides fragilis such as NCTC 9343 and YCH46 would be analogously, and perhaps significantly, increased by the inclusion and consumption of high dietary cholesterol sources within the human host.
As such, if Bacteroides fragilis NCTC 9343 and YCH46 are involved as aetiological and progressive agents in atherosclerosis, high dietary consumption of cholesterol or increased synthesis of cholesterol within the human host should invariably contribute to atheroma progression and advanced stages of arterial plaquing - as similarly observed, subsequent to such dietary consumption.
Further, if dietary and enterohepatic cholesterol concentrations influence Bacteroides fragilis with in the intestine, it may be probable to infer that such would also influence B. fragilis at sites removed such as within the atheroma. Additionally, given the ability of B. fragilis to utilize cholesterol, such inherent propensity would still be present wherever the organism is found – whether located within or outside of the intestine as the result of colonization, dissemination or infection.
Such variable and influence may provide insight into mechanisms relative the fate of cholesterol, buildup of cholesterol esters and the interplay of lipoproteins relative plaque foci and atherosclerosis in general. It should be noted however, that in reference to the observations made by Pence (1985) - the effect of cholesterol upon Bacteroides fragilis was suggested as attributed to an increase in bile acids within the test subject (rat) used – and not via a direct impact of the cholesterol molecule upon the organism, nor was such explored:
“…the data in the present study definitely indicate a role for increased bile acids in the selection growth of Bacteroides fragilis.”
Misinterpreting Cause & effect
- Empirical study performed without a ‘Control’
Perhaps a more compelling example of the effect of dietary cholesterol upon Bacteroides fragilis can be observed in a study conducted by Klurfeld et al (1979) -
Entitled:
“Alterations of host defenses paralleling cholesterol-induced atherogenesis. I. Interactions of prolonged experimental hypercholesterolemia and infections: published - Journal of Medicine, 01 Jan 1979, 10(1-2):35-48 PMID: 290725
In 1979…
an experiment was conducted to observe the alterations and interactions in host defenses specifically “normal repair processes in the arterial wall” resulting from prolonged experimental hypocholesterolemia. In the study, normal rabbits were administered cholesterol and also injected with human clinical isolates of Bacteroides fragilis, “a bacterium that is innocuous, even when multiple large doses are administered”.
After a period of cholesterol feeding, rabbits in the “cholesterol-fed, infected” group developed significant infections and complications ranging from interstitial pneumonitis, vasculitis to pulmonary hemorrhage and thrombosis - as well as a significantly higher rate of mortality.
Though, more than half of the rabbits were culturally tested as positive for Bacteroides fragilis at the time of autopsy, the results were interpreted and stated “suggesting that cholesterol may interfere with the functions of host cells involved in host defense”. However, such observations can be viewed in a different light:
(1) Though the authors stated exactly that observed, in what can only be considered a landmark study, the results, strangely, if not ironically, were neither interpreted or alluded to the possibility that the administration of cholesterol may increase the growth (as later observed by BC Pence, 1985) and/or subsequent virulence of the bacterial inoculum of B. fragilis administered.
Rather, it was simply stated - if not assumed or conjectured - that the observed increased morbidity and mortality in the experimental lab rabbits fed a high cholesterol diet plus B. fragilis (human clinical isolates) was most likely attributable to the effect of cholesterol upon decreased/altered - host defense.
(2) Curiously, none of the effects observed (such as those previously stated) in the “cholesterol-fed, infected” group were observed in the “cholesterol fed (non-infected)” group as it was stated “none of these entities were found in the normal or cholesterol animals that did not receive bacterial injections.”
Such findings tend to question the statement and premise “that cholesterol (administration) may interfere with the functions of those cells involved in host defense.” That is, as stated within the study, no group injected with Bacteroides fragilis was observed to have growth in obtained blood cultures indicating adequate initial clearance of bacteria from circulation.
Further, rabbits which comprised the test subjects are innocuous to the organism, and do not normally harbor Bacteroides fragilis species within the intestine.
An oddity presents within this study then, as it is assumed that the impact of cholesterol would not influence - in any manner - those commensal organisms normally comprising part of the regular gastrointestinal flora. Such is furthered by the fact that cholesterol fed non-infected rabbits did not produce identical findings.
It becomes questionable then if the influence of cholesterol truly represents an interference with “normal repair processes in the arterial wall”. It would present as more likely, that - the observed increased and advanced atherosclerosis was the product of a dissemination and/or increased load of bacteroides infection and/or toxin release concurrent to administration of a high cholesterol substrate feeding/diet – which would simply not be observed if all rabbits were otherwise healthy.
(3) As well, no differentiation was made regarding the specific strains of Bacteroides fragilis used and considered “innocuous”, especially compared to that regularly associated with “normal rabbits”. For example, a profile analysis of Bacteroides members regularly present and maintained within the intestine of rabbits conducted by De Rozas et al (2008) using REF-PCR, illustrates that the rabbit intestinal Bacteroides profile varies from members commonly found within the human intestine. Of specific note, within the studies “Materials and methods”section is the statement that
“…the bacterium chosen with Bacteroides fragilis subspecies fragilis, the anaerobe most commonly isolated from human infections; this organism is the predominant species among the normal flora of the gastrointestinal tract. Fresh clinical isolates were subcultured…”
It is noteworthy, however, based on the observations of the Rosa et al(2008), though Bacteroides fragilis subspecies comprise a component of the intestinal rabbit profile, such members include Bacteroides fragilis G2 and G5 - but not, Bacteroides fragilis NCTC 9343 and YCH46. So the statement years earlier that B. fragilis is innocuous to rabbits is (only partially) correct.
B. fragilis G2 and G5 without the production of the Bft virulence factor would likely be quite innocuous, however, given that most likely “Bft producing” B. fragilis clinical isolates were used in the study - the test animals would therefore succumb to the effect of Bft with the outcome of atheroma formations dispersed throughout the animals and most tissues.
In other words, though the observations and results obtained described the exact experimental events, it is altogether probable that the stated results - as interpreted - only partially explain the findings.
For example, it is probable that the results observed were not due to an interference in normal host repair processes but rather, more specifically - due to the direct and/or indirect effects of cholesterol upon Bacteroides fragilis NCTC 9343. Again, such mechanisms do not comprise regular members of the rabbit intestinal flora, and cannot be considered innocuous to the test subject.
It is logical and perceivable then, that the absence of disease development in rabbit subjects administered cholesterol but not infected with the Bacteroides fragilis subspecies fragilis isolates obtained from human clinical infections - is simply representative of their absence in relation to cholesterol. Alternatively, normal (non-cholesterol fed), infected rabbits injected in the absence of concurrent cholesterol administration would also not produce significant atherosclerosis and associated pathological results.
It may be more probable to state then, that the probability of atherosclerosis development and progression in normal rabbits without the administration of dietary cholesterol and injection of B. fragilis NCTC 9343 and/or YCH46 (i.e commonly obtained form found within clinical isolates) would be remote.
The probability of atherosclerosis in rabbits administered dietary cholesterol alone/only, would - in the absence of B. fragilis NCTC 9343 - also be remote.
However, the probability of atherosclerosis in rabbits colonized or infected with foreign members such as B. fragilis NCTC 9343 - would increase by virtue of their presence – but not develop or progress due to an otherwise lack of dietary cholesterol implicit to the herbivore diet.
Lastly, however, what the study observations may actually present is the observation of the concomitant administration of two elements which simultaneously are seldom – if ever - observed naturally or for any extended period within this ecological niche.
Simply, the presence of both B. fragilis NCTC 9343 and its virulent stimulation by cholesterol – result in the initiation and development of atherosclerosis and associated disease states – as opposed to a ‘weakening or interference of host repair processes’ - simply caused by cholesterol administration.
Again, a more important consideration, may in fact be the consequence, impact and significance of cholesterol upon the Bacteroides fragilis subspecies fragilis members isolated and obtained from human clinical infections - an otherwise uncommon member of the rabbit flora (De Rozas et al, 2008). In the absence of such consideration, it would likely be assumed as that the disease process was mostly attributable to the impact of cholesterol upon host mechanisms.
Unfortunately, an analysis to discern any potential interplay between cholesterol and B. fragilis virulence causing the results obtained was not conducted as may have been obviated by the co-administration cholesterol and killed bacteria.
Per Klurfeld et al, the interplay between live B. fragilis (nctc 9343/atcc 25285) cells and cholesterol resulting in atherosclerosis in a tested rabbit model - was observed.
NO CONTROL USED IN AN EMPIRICAL PEER REVIEWED EXPERIMENT.
IF "KILLED" etbf USED - THE SAME RESULTS WOULD BE OBSERVED
OBVIATING THAT THE CAUSE OF ATHEROSCLEROSIS WAS BFT (FRAGILYSIN TOXIN)
NOT A DECREASE IN IMMUNITY DUE TO CHOLESTEROL
If a comparative study (or arm of study) was used to compare the impact of cholesterol and nonviable B. fragilis cells in the rabbit model - otherwise referred to as a ‘control’ - could help define/deduce another mechanism (apart from the vague influence of viable cells to correlate with atheroma formation and cholesterol to inhibit host immunity. If conducted, some 45 years ago, a comparison between nonviable cells and cellular components (such as exotoxin - currently termed ‘fragilysin’) - could have further helped differentiate mechanism of atherosclerosis formation. Use of a control by Klurfeld et al - in an empirical, peer -reviewed, funded and published study of such significant outcome - could have both elucidated an etiological factor in atherosclerosis and illustrated the detrimental impact in the sacrificed rabbit test model.
It is the personal belief of the author that the failure to utilize a comparative control group to compare cholesterol administration with viable and nonviable B. fragilis - would have identified the cellular exotoxin - referred to as ‘fragilysin’. If the author is incorrect, observation of atherosclerosis in rabbit models fed cholesterol and non-viable B. fragilis - would have also allowed determination (i.e. via use of high protein denaturing temperatures) - to differentiate the possibility of influence of a lipophilic endotoxin or zwitterion (PSA/polysaccharide antigen).
An opportunity (unwillfully or otherwise) - with a tremendous burden of fallout relative morbidity and mortality - for mankind - was missed. This anomalous acceptance and publication of a peer reviewed study without implementation of a control precluded the opportunity to study the degree of magnitude of B. fragilis cellular components which have a tremendous impact upon the human host which otherwise harbors B. fragilis, it’s exotoxin (and endotoxin/LPS, PSA) during the life course from - approximately day 5 postpartum to demise/departure.
The findings published by Klurfeld - via the co-administration of both cholesterol and B. fragilis - were interpreted and communicated as the closest known model of human atherosclerosis - mimicking that found in man. The findings, as such would appear, was thereon used only to identify another factor contributing to atherosclerosis and further support the validity of an established cholesterol-rabbit model of atherosclerosis (by Nikolaj Nikolajewitsch Anitschkow) and to fund decades of further studies on atherosclerosis, utilized to develop drugs to treat atherosclerosis.
If the findings were correct that co-administration of cholesterol and B. fragilis resulted in the closest comparative model to that observed in atheroma specimens obtained from man - if not ‘identical’ - why was this avenue not further explored? The human consumes and generates the sterol ‘chole’-sterol and carries Bft/fragilysin producing B. fragilis NCTC 9343. The B. fragilis used in the experimental rabbit models, referred - were simply obtained and cultured from human clinical isolates.
5.3 – Cholesterol and B. fragilis virulence
If cholesterol does increase both the growth and virulence of Bacteroides fragilis then the resultant infections and complications may be as much attributable to mechanisms stemming from and associated with Bacteroides fragilis, as well as the impact of such consequently - resulting in a weakening of host defenses.
Perhaps, the addition of cholesterol by itself does not simply result in a weakening of host defenses, but in this particular case, two agents co-administered – one the causative agent the other a highly associated risk factor and substrate promoting the former - simultaneously resulted in the observed results and did not so much parallel cholesterol induced atherogenesis – but actually was an exact replication and verification of the exactmechanisms that occurs within the human host causing atherosclerosis. Such may be further verified by eliminating Bacteroides fragilis from the rabbit test subject through vaccination and re-conducting the exact experiment as well as alternatively using cholesterol plus another unrelated bacteria to which the host (normal rabbit) is innocuous – as a further verification.
Such experiment would likely produce different results, especially in light of the study conducted by Rozas et al (2008) using REP – PCR analysis of rabbit microbiota, observed that though various species of Bacteroides are common commensals within the rabbit microflora, however, particular strains such as Bacteroides fragilis NCTC 9343 and YCH46 - were not observed as a regular member of the anaerobic ecology.
This is significant, as Bacteroides fragilis NCTC 9343 and YCH 46 different from other Bacteroides strains in their ability to produce - aminopeptidase N (CD13)-like enzyme. Aminopeptidase N, within voluminous literature, has been linked to endothelial ingression and uptake of cholesterol containing vesicles in atherosclerosis, and may, perhaps, provide further insight into the observations made by Klurfeld et al (1979).
That is, it is probable that aminopeptidase N, though generally viewed primarily as a protease, may have other primary functions such as endocytosis and signal transduction (Mina-Osorio, 2008). Further, it is also possible that aminopeptidase N may play a role in protein binding - specifically, at glycyl-cysteinyl (gc) linkages - the opposite of which results in the hydrolysis of cysteinyl glycine (cg) linkages, serving an adaptive and efficacious role through one single receptor.
Perhaps an example can be taken from the formation of bile cholesterol stones. It is been observed that aminopeptidase N is associated, in addition to other factors such as cholesterol saturation, with the formation of what, to the host, presents as maladaptive obstructions without evolutionary value that may acutely and/or chronically impact the host's systems.
If, however, such scenario is viewed from the vantage point of Bacteroides fragilis containing cells which possess aminopeptidase N, such as Bacteroides fragilis NCTC 9343 and YCH46 - the formation of cholesterol deposits represents an adaptive and responsible organization, storage and allocation of an entity, which facilitates the growth and long-term survival of the organism, especially in the absence of fermentable carbohydrates as a means of energy production.
It may be no surprise then, that Ballal et al (2001) observed B. fragilis as the predominant anaerobic species present (58.8%) of patients studied in relation to chronic cholecystitis with cholelithiasis, being frequently associated with common duct stones or strictures (Nielson & Justesen, 1976). This would support the observation made by Méndez-Sánchez N et al (2008) that gallstones are and associated risk for carotid atherosclerosis in particular, stroke and myocardial infarction.
If such is the case, that CD13 is correlated with gallstone and biliary cholesterol as well as atheroma development, then it may be probable that cholesterol not only increases the growth of B. fragilis NCTC 9343, but increases also the upregulation of virulence mechanisms such as aminopeptidase N, 7-alpha-hydroxysteroid dehydrogenase and perhaps putative export protein.
Further, such mechanisms would lend support to The Lipid Research Clinics Coronary Primary Prevention Trial results. JAMA 1984; 252: 351-374 – wherein, it stated a 2% decrease in the rate of coronary artery disease for each 1% reduction in circulating blood cholesterol level.
If such bacterial mechanisms occur concurrent to that of the host it is possible that various host mechanisms may inadvertently be observed or mistaken as related to the occurrence of such mechanisms. Further, it is possible that exposed to such bacterial mechanisms, the host compensates by down-regulating particular associated and/or homologous mechanisms. Further, it is possible that without factoring in such bacterial mechanisms and adjusting and standardizing for interplay of Bacteroides fragilis variables, such may be attributable to host related processes and perhaps polymorphisms and less observed host produced proteinaceous aberrations and alterations, seldom seen but again originating from an orchestrated by the host.
As an example, b. fragilis seven alpha Dehydroxysteroid hydrogenase may also be upregulated and active in the presence of high cholesterol substrates but go relatively unnoticed regarding studies on atherosclerosis. Such perhaps representing or further contributing to background noise or artifact.
5.4 - Cholesterol: Bile acid production
Connecting dietary chole-sterol, B. fragilis and atherosclerosis
Factoring in Bacteroides fragilis as a variable in atherosclerosis may be important bridge connecting sterol structures in disease progression as well as associated conditions and diseases such as, non-insulin-dependent diabetes mellitus, obesity, Alzheimer's disease and the positive correlation with disorders of the central nervous system.
The metabolic activities of the intestinal bacteria are extremely diverse in that microbes, through a multitude of pathways and enzymes, can use as substrate virtually any compound in the intestinal lumen that consumes orally or entering into the host intestine by secretion through the biliary tract or directly across the mucosa.
As a case in point, the ability of the intestinal commensal flora to conjugate compounds that were formerly conjugated in the liver with agents such as glucuronate, glycine, taurine, glutathione and sulfate (as example) – help maintain the enterohepatic circulation, and reabsorption of the original parent compounds back across the intestine. Further examples of the importance of the host microflora can be seen relative endogenous compounds such as: bilirubin, bile acids, cholesterol, estrogens, and metabolites of vitamin D as well as administered pharmaceuticals , including morphine diethylstilbestrol, digitalis, colchicine, and antibiotics such as rifampin and chloramphenicol.
It is of note that particular antibiotics which influence the intestinal Flora may alter or block the enterohepatic circulation. Ironically, many such compounds also have a direct or indirect effect upon of atherosclerosis. And using such model in reference to the protective effect of estrogens against atherosclerotic progression as but one an example, it becomes obvious that agents such as antibiotics, which may be expected to decrease atherosclerosis if truly of a bacterial origin, may in fact increase its progression by increasing the rate of fecal excretion and thereby increasing the half-life of estrogen in the bloodstream.
It may be important then to consider atherosclerosis not only as a disease process hypothetically caused by the organism Bacteroides fragilis, but one that is influenced as well by the organisms proximity within and dissemination from the upper intestine. This may be especially probable given that higher numbers of the resident Bacteroides fragilis group are observed within portions of the upper G.I. tract (Brook, Pediatric anaerobic infections) and specifically relative its proximity to food substrates within the duodenum and the release of hepatic and gallbladder components along the common bile duct into the intestine and until the distal regions of the terminal ileum which composes the enterohepatic pathway, and substrate recycling through the portal circulation.
Equipped with the enzyme 7-alpha hydroxysteroid dehydrogenase, the Bacteroides fragilis organism is well-equipped and suited to deconjugate the two main bile acids synthesized by the human liver - cholic acid and chenodeoxycholic acid which becomes conjugated with taurine or glycine, prior to secretion in the bile. It is then conceivable, if not forcastable, that alterations such as a decrease or increase (overgrowth) of Bacteroides fragilis in the proximal colon may be associated respectively with decreased and increased levels of the conjugation. It is perceivable, for example that high levels of the conjugation within the proximal colon caused by Bacteroides fragilis overgrowth may result in the reuptake of higher concentrations of deconjugated bile acids, which may influence, if not, promote higher serum cholesterol levels.
Though such microbially aided reabsorption of deconjugated, sterols would initially appear to benefit the host, the value of such evolutionary and preservational mechanism may pale in comparison to the detrimental effect such overgrowth may have upon the host. That is, the same deconjugation mechanism was initially evolved to provide an energy source to Bacteroides fragilis in the absence of fermentable carbohydrates, and likely functioned as a symbiotic mechanism relative B fragilis and members of fungi - prior to it’s adapted role within the niche of the human host. Such may also reflect the effectiveness and propensity of compounds such as statin groups - originally derived from fungi - and contemporarily utilized to control serum cholesterol levels and atherosclerotic progression. It must be questioned by contemporary society whether such commensalism is truly a benefit or of detriment to the human host.
On the whole, the portion of the intestine between the duodenum and terminal ileum, comprising the enterohepatic circulation may, in the presence of Bacteroides fragilis, be viewed collectively and analogously - as a result of the diffusion and active transport that occurs within the region – as a second stomach within the host (so to speak). For example, in the absence of bacterial deconjugation, a higher percentage of bile acids/bile ‘salts’ produced by the metabolism of liver hepatocytes, such as cholic acid and chenodeoxycholic acid utilized by the host and - bound with glycine or taurine molecules to form water-soluble primary conjugated bile acids - would otherwise be carried along the intestine and excreted of no in higher concentrations in fecal matter. However, given that intestinal anaerobes can deconjugate such compounds, much of what is excreted from the liver into the intestine becomes reabsorbed and therefore recycled.
Though such cyclic pathway is viewed as an adaptive mechanism which supports the human host, such needs to be viewed relative to its primary ecological value of supporting the host during times of food and substrate scarcity, as opposed to the human’s contemporary existence wherein food and substrate abundance is commonplace and substrate scarcity represents an anomaly.
That is, the adaptive utility of such “enterohepatic pathway” may have served, if not outlived its evolutionary place and purpose, and actually present as maladaptive, and incongruent when overlaid upon current societal behaviors and needs – especially since the mass production and availability of food sources subsequent to the industrial revolution.
Unfortunately, the involvement and ability of the B fragilis group of species to deconjugate bile acids which has historically provided an adaptive survival advantage to the human, may now serve only to further its accelerated demise on an individual level. That is, it was noted by Duerden et al (1976) that the growth of Bacteroides fragilis was stimulated by sodium taurocholate and additionally by Holdeman et al (1977) , who observed an increase in growth of the B. fragilis group of species - by human bile.
As such, the inherent ability of Bacteroides fragilis to recycle deconjugated sterols presents an energy saving mechanism for the human host, especially when considering the additional provision of short chain fatty acids utilized by the host epithelial layer within the intestine and the diffusion of additional byproducts which may be converted or directly utilized by the hosts tricarboxylic acid (krebs) cycle.
Given, however, that B. fragilis can garner energy through deconjugation reactions, and become stimulated by particular bile acids as well as effectively eliminate byproducts into its environment that are eventually removed and utilized by host cells - growth of Bacteroides fragilis increases concurrent to that of the host.
Within the context of societies contemporary abundant and available food supply, such effectively represents a maladaptive ‘loop’ or the perpetuation of an energy-saving/calorie saving system within the host that is utilized and perhaps beyond the hosts basic and basal needs, can essentially equate to a system that is then being ‘used’ or ‘exploited’ by B. fragilis in that above the minimum threshold of daily survival and baring periods of starvation – realistically only serves of purpose and utility to the perpetuation and growth of bacteroides fragilis.
Further, if virulent mechanisms are increased as a result of such growth by B. fragilis, beyond that otherwise matched by the human host’s innate, cell mediated and humoral immunological components – then such enterohepatic pathway may essentially represent the ‘breeding grounds’ for the perpetuation of acute and chronic infection – resulting from increased total bacterial load, increased virulence, dissemination and decreased host capacity to respond immunologically, especially if such bacterial load additionally results in the continued depletion of various host substrates essential for response such as cysteine, H2S or reduces dissolved oxygen – to name but few.
Such may be an important consideration regarding atherosclerosis as Sakai et al (1980) observed an increase in Bacteroides population levels in rats fed cholic acid or deoxycholic acid which illustrated a relationship not only between bile acid in the intestinal microflora but with the specific bile acid composition itself.
Further, Hylemon & Sherron (1975) observing that “oxidation of the seven alpha hydroxy group occurred only after glucose was nearly depleted from the growth medium” suggested that physiological significance of the multiple forms of seven alpha hydroxysteroid dehydrogenase possessed by particular strains of Bacteroides fragilis may afford the organism to utilize bile acids as a potential energy source in the absence of fermentable carbohydrates.
It is likely then, that the possession of 7 alpha hydroxysteroid dehydrogenase which increases growth resulting in findings such as that observed by Sakai et al – represents a primary, albeit microbial risk factor which fundamentally connects cholesterol and a multitude of cholesterol derivatives (ranging from bile acids to various steroidal hormones) to Bacteroides fragilis NCTC 9343 and its impact upon atherosclerosis on a larger level.
In particular, with specific reference to the NAD and NADP dependent activity and rate of reaction (relative Vmax ) ,of seven alpha hydroxysteroid dehydrogenase possessed by Bacteroides fragilis, a particular pattern emerges regarding bile acids which reportedly are positively and negatively associated with the progression of atherosclerosis.
Bile acid sterol groups predominantly utilized by Bacteroides fragilis and having substrate specificity for seven alpha hydroxysteroid dehydrogenase are coincidentally those also associated with increased progression of atherosclerosis. The general order of bile acid substrate specificity associated with atherosclerosis and stimulatory for the growth of Bacteroides fragilis, in particular Bacteroides fragilis NCTC 9343, is based upon the presence of hydroxylation of groups on the sterol structure at sterol carbon positions 3, carbon 7 and carbon 12 – with atherosclerotic association and use of such sterol by B. fragilis decreasing, respectively.
Such can be overgeneralized using the following three examples:
(1) the bile salt cholate, contains 3 hydroxyl groups located at positions: carbon 3, carbon 7 and carbon 12. It possesses a strong substrate specificity for 7 alpha hydroxysteroid dehydrogenase – undergoing a higher rate of 7 alpha dehydroxylation and is stimulatory relative the growth of Bacteroides fragilis. As well, cholate has been observed as positively associated with atherosclerosis progression given that cholate fed rats resulted in higher liver and serum cholesterol concentrations as compared to controls and those fed deoxycholate (Yuangkiang et al, 2005).
a. (Similar results would likely be observed regarding the association with atherosclerosis under higher NAD concentrations. Further, higher to no difference in association may be observed for taurocholate and glycocholate relative higher NADP concentrations, but a relatively lower association of atherosclerosis progression associated with taurocholate as compared to glycocholate under increased NAD concentrations).
b. Similar results may be expected with chenodeoxycholate under high NAD conditions and taurochenodeoxycholate under higher NADP – possibly attributable to the presence of hydroxyl in the carbon 13 position in both compounds – though both contain no hydroxyl group at the carbon 12 atom.
c. Lastly, given that glycochenodeoxycholate also has high substrate specificity for 7-alpha hydroxysteroid dehydrogenase under high NAD concentrations, such may further provide that the fit within the enzymatic pocket is the ultimate determinant (as opposed to more generalized patterns of carbon hydroxylation – which nonetheless serves as a good representation amidst the plethora of steroidal and sterols to which B. fragilis may be exposed) and therefore the greatest determinant of relevance relative NAD/NADP concentrations regarding association with atherosclerosis.
Nonetheless, such illustration relative the interplay of substrate specificity and avidity for the enzymatic ligand pocket and presence of carbon hydroxylation can be provided when citing that both glycochenodeoxycholate and deoxycholate contain only 2 hydroxyl groups. The presence of the hydroxyl group at the carbon 7 atom of glycochenodeoxycholate and its conformity to 7 alpha hydroxysteroid dehydrogenase results in a high ‘relative Vmax’ and stimulation of B. fragilis (and thus association with atherosclerosis). Deoxycholate, however, with no hydroxyl group at the carbon seven atom , confers low substrate affinity to seven alpha hydroxysteroid dehydrogenase and significantly reduces it's relative Vmax.
(2) The bile salt deoxycholate, contains only 2 hydroxyl groups located at positions: carbon 3 and carbon 12. It possesses a moderately low substrate specificity for seven alpha hydroxysteroid dehydrogenase – undergoes a lower rate of 7 alpha dehydroxylation and is only minimally stimulatory relative the growth of Bacteroides fragilis. As well - deoxycholate is observed as significantly associated with decreased atherosclerosis progression.
Consumption of deoxycholate is negatively correlated with serum cholesterol concentrations in the rat (Yuangidang et al, 2005) as well as was observed to increase the intestinal flora while also decreasing synthesis of cholesterol during its administration in human subjects, was observed with significant decreases in serum cholesterol in human subjects – measurable in days - and was therefore “concluded to have an important role in the regulation of cholesterol metabolism in humans” (Galio et al, 1979). Further, Shimada et al (1970) observed that deoxycholate which can minimally support the growth of Bacteroides fragilis completely inhibited its growth in the presence of bile - a result which may prove to have significant relevance regarding the absence of such organism and decreased progression of atherosclerosis in humans.
Deoxycholate as a bacteriostatic - may also serve as an effective therapy in atherosclerosis used to lower Bacteroides fragilis cell load and accompanying virulence factors such as Bacteroides fragilis toxin (Bft, fragilysin) preventing circulation through the upper enterohepatic circulation, and perhaps, lower intestine. Such compound may serve of value versus the lytic release of increased concentrations of Bacteroides fragilis toxin, etc that may be associated with the administration of particular antibiotics, especially considering the property “of sodium deoxycholate to separate” antigenic components from other membrane components of thickly encapsulated B. fragilis cells (Hofstad, 1979).
(3) As a final case in point - the bile salt lithocholate, contains only 1 hydroxyl group located at position: carbon 3. It possesses a significantly weak substrate specificity for 7 alpha hydroxysteroid dehydrogenase – undergoes a dramatically lower rate of 7 alpha dehydroxylation and is inhibitory relative the growth of Bacteroides fragilis subspecies fragilis (Stellwag & Hylemon, 1976). As well, lithocholate administration is observed as positively correlated with an increase in high density lipoprotein (HDL) a significant marker associated with decreased atherosclerotic progression (Loo et al, 1992).
Not only does such obviate the importance of a carbon 7 hydroxyl group in order to undergo 7 alpha dehydroxylation, but the additional presence of a hydroxyl group as carbon 12 additionally increases the substrate specificity as well as stimulates the growth of Bacteroides fragilis, and is positively associated with atherosclerosis. The lack of a hydroxyl group at carbon 12 confers low substrate specificity as well as inhibits the growth of Bacteroides fragilis, and is negatively associated with atherosclerosis.
It is possible that such insight may also provide potentially valuable insight regarding the relevance of cholesterol 7 alpha-hydroxylase, which catalyzes the rate determining step converting cholesterol to bile within the liver of the human host. The first and fundamental step in the conversion of cholesterol to bile is the addition of a hydroxyl group at the pinnacle carbon 7 position - capable of undergoing 7 alpha dehydroxylation, by seven alpha hydroxysteroid dehydrogenase as possessed by Bacteroides fragilis NCTC 9343 and YCH46.
Therefore, any probable influence by Bacteroides fragilis upon the product formed (7 alpha hydroxycholesterol ) at this step of bile acid production - could have a pivotal if not, far-reaching impact upon the growth, long term survival and influence of Bacteroides fragilis in atherosclerosis. Further, and curiously, Bjorkhem et al (1994) observed that 7-oxocholesterol and 27-hydroxycholesterol - known to be a potent inhibitor of cholesterol synthesis and precursor to chenodeoxycholic acid (Martin et al, 1997 - J Lipid Res. 1997 May;38(5):1053-8.) as well as 7-alpha-hydroxycholesterol and 7 beta-hydroxycholesterol (B. fragilis utilized?) constituted a major percentage of oxysterol distributed within human atherosclerotic femoral arteries – perhaps representative of products formed by B. fragilis and reminiscent of mechanisms presented by host cells toinhibit such organism and reactions with the atheroma.
Regarding the relevance and further support of such potential association, it is noted that low levels of 7-dehydrocholesterol, or provitamin D3, have been associated with atherosclerosis, and specifically peripheral vascular disease. The compound naturally found in human breast milk, and capable of influencing the early development of the intestinal microflora - may also be linked to Bacteroides fragilis inhibition as 7 alpha hydroxysteroid dehydrogenase antagonist by virtue of the low substrate specificity and low Vmax inherent to such compound.
This, resulting from the lack of a hydroxyl group at the sterol positions: carbon seven and carbon 12. Such may provide insight into the negative association of vitamin D3 with colorectal cancer in african americans relative the observed reduction in Bacteroides flora (Mai et al, 2009) as well as atherosclerosis upon increased dietary consumption of vitamin D.
Another example, regarding the potential association of seven alpha hydroxysteroid dehydrogenase antagonist may be observed with the estrogen: estrone. Increased estrone levels have been associated with decreased incidence of breast cancer and atherosclerosis - and contains no hydroxyl group at steroid carbon atoms: carbon seven and carbon 12.
As such, estrone would serve as an antagonist, and inhibitory to the growth of Bacteroides fragilis NCTC 9343 and YCH46. Further examples, include betulinic acid and glycyrrhetinic acid (Shibata, 1962) both of which contain no hydroxyl group at carbons 7 and 12 and have been associated with decreased risk of atherosclerosis as well as colon cancers in which Bacteroides fragilis has also been predominantly implicated.
5.6 - Cholesterol:
Conclusions & inferences regarding B. fragilis and atherosclerosis
Lastly, antibiotic studies conducted to date, relative atherosclerosis, have observed only weak associations and limited reduction in disease progress. However, it may be important to state that antibiotics utilized such as ciprofloxacin (as a particular example) which limit inhibitory bile acids such as lithocholic acid by the destruction of competing Bacteroides fragilis species members (such as B. fragilis ss. thetaiotaomicron) as well as various other members of the intestinal flora such as Citrobacter, Peptostreptococcus (Kelsey & Thompson, 1976), (Toda et al, 2009) – may account, among other variables, for such findings.
Additionally, the positive value of various compounds utilized and observed to decrease intestinal and or serum cholesterol (relative other described mechanisms) – such as, but not limited to: niacin, flavonoids, sterols (Kudchodkar et al, 1972), inulin (), EDTA, fibrates (Powanda & Canonico, 1976) and statins - may also reflect antagonism of particular enzymes such as 7 alpha hydroxysteroid dehydrogenase as well as bacterial metabolism and load in general. That is, successes in decreasing coronary artery disease, blood pressure and cholesterol in some ways may also be reflective of an otherwise overlooked impact upon Bacteroides fragilis metabolism and its contribution to atherosclerosis.
For example decreases in intestinal bile concentrations are also directly related to decreased intestinal NAD-dependent enzyme activity within Bacteroides fragilis organisms resulting in a bacteriostatic effect during the period of use of such therapeutic agents. Such therapies, in addition to interventions aimed at other variables such as reduced high glycemic intake - decreasing intestinal and circulating glucose (compounding reductions in cholesterol) - may, in effect, limited availability of additional substrates or alternative carbon energy sources otherwise derived for use by Bacteroides fragilis. The combined effect may result in the cessation or decreased progression of atherosclerosis.
Such may represent an important aspect for research as compounds and therapeutic agents which specifically impact upon the metabolic pathways of Bacteroides fragilis may prove as efficacious an approach to that directed at the host to reduce cholesterol levels. For example, it may be probable to consider if increases in serum cholesterol values are simply the result of increased host production. Using the isoprenoid pathway as an example, and barring feedback mechanisms inherent to such, it is theoretically probable that increases in squalene intake would promote cholesterol production. However it is also probable that an increase in dietary squalene may also influence the isoprenoid pathway of particular members of Bacteroides fragilis resulting in increased growth and virulence.
If dysregulation in host cholesterol is subsequent to an interaction between Bacteroides fragilis NCTC 9343 and YCH46, as proposed, then it may be possible that particular dietary fibers, statins, and fibrates compensate (within the host) for the impact of B. Fragilis, as well as possibly and directly influence mechanisms within B fragilis. That is, current therapeutic approaches may affect the interaction between B fragilis and host as orchestrated from the micro-organism’s strategic foci/positioning along the proximal intestine and enterohepatic pathway, and probable sites of dissemination - including but not limited to: the biliary system, liver, distal colon and perhaps dissemination into the circulatory system, vascular bed and central nervous system.
Additionally, repleting specific nutrients that may otherwise be scavenged and depleted within the host by Bacteroides fragilis may be an important approach. Host squalene for example, may be utilized by Bacteroides fragilis and unabated over time may invariably have an impact on vitamin D2 and vitamin D3 production and levels within the host.
5.7 – Bile Acids:
Bacteroides fragilis and bile acid stimulated growth…
Cholesterol reduction is a primary intervention therapy directed at preventing, eliminating, controlling and decreasing the progression of atherosclerosis in millions of patients. However, in the also be of significance that decreases in dietary cholesterol, serum cholesterol and increases in the concentrations of specific bile acids also dramatically affects (stimulates or inhibits) the growth of Bacteroides fragilis subspecies fragilis.
Is it not possible then, that one of the underlying influences of cholesterol restriction via dietary modification and therapeutic interventions upon atherosclerotic progression - is the ultimate impact upon the growth of pathogenic dynamics of B fragilis subspecies fragilis? That is, with reference to B. fragilis - decreasing cholesterol may result in decreasing bile acid release into the intestine resulting in decreased concentrations of Bacteroides fragilis subspecies fragilis and 7 alpha hydroxysteroid dehydrogenase among other virulent factors - ultimately resulting in decreased atherosclerotic progression.
Exploring such possibility may present some degree of insight into the atherosclerotic process. Pumbwe et al (2008), for example when studying the effect of bile salt treatment upon Bacteroides fragilis NCTC 9343 observed that bile salt treated bacteria conferred increased resistance to structurally unrelated antimicrobial agents, a significant increase in bacterial co-aggregation, a significant increase in the adhesion of Bacteroides fragilis to intestinal epithelial cells, a significant increase in biofilm formation and increase in outer membrane vesicles - which, taken in totality was suggested to enhance Bacteroides fragilis intestinal colonization.
5.8 - Bacteroides fragilis survive/susceptibility - Decreases in Cholesterol…
If bile predominantly stimulates Bacteroides fragilis growth, it may be that decreases in cholesterol also decrease bile, concentration or specific bile acids that simply the growth of Bacteroides fragilis subspecies fragilis. i.e. Cholesterol depletion is associated with cellular membrane lipid rafts. Using plating patterns of susceptibility to bile antibiotic disks: lowering cholesterol and bile content makes Bacteroides fragilis more susceptible to particular antibiotics.
6.0 Steroids
B. fragilis & Steroid Groups
6.0.1 - Steroidal FRP substrate substitutions and Interplay
Research into Bacteroides fragilis involvement in atherosclerosis may be particularly relevant given that it has been observed by Kornman & Loesche (1981) that bacteroides members have been observed to increase uptake of estradiol and progesterone during the second trimester of pregnancy and that radiolabeled estradiol and progesterone substituted for menadione along the fumarate reduction pathway.
What makes this significant regarding atherosclerosis was the observation that steroid uptake was further increased by the addition of fumarate which is further reduced to succinate - providing cellular energy. It was suggested by the authors that estradiol and progesterone - both of which have been implicated in atherosclerosis and obesity - are actively involved in the fumarate reductase system of Bacteroides and that such may represent (within particular limits) stimulatory growth factors for Bacteroides due to the interchangeability of menadione and gonadal steroids.
Such could explains estrogen inhibitors in cancers, reason why human males collectively die earlier than females, the mechanism of bioflavonoid conferred health benefits. This may also provide an explanation of sex-related differences regarding protection or progression of atherosclerosis – relative various disease states and the administration of steroidal agents including, but not limited, to birth control.
Further, it has been observed that mixed cultures of human fecal bacteria can not only dehydroxylate or dehydrogenate bile salts, steroids and sterols - but as observed by Aries and Hill (1970) B fragilis which possesses the enzyme 7- alpha-hydroxysteroid dehydrogenase, in pure cultures is capable of reducing estrone to estradiol. As such, particular forms of vitamin K (mk-4 etc.) or an analog such as menadione which is essential for the growth of particular strains of Bacteroides, may be substituted by various steroid hormones (Kornman & Loesche, 1981).
Regarding Bacteroides fragilis, this may have significance, as such similar mechanisms could increase growth rate and yield of this pathogen by increasing electron transfer along the fumarate reduction pathway - contributing to the organism’s growth, virulence and hypothesized progression of atherosclerosis. Reduction of menadione via substitution and/or inhibition by molecules such as: phylloquinone (plant based diet), menaquinones of various length and competing compounds such as quinines, quinones, sesquiterpenes such as berberine may confer health benefit (attenuates, abrogates, ameliorates, inhibits - atherosclerosis, cancer and otherwise).
Additionally, if the specific affinity, optimal concentrations and utilization of various estrogens by particular species - can be further discerned - such may not only explain the protective effect of particular steroids compared to others, but could possibly be further exploited to decrease the impact of Bacteroides fragilis upon the host relative atherosclerotic progression (i.e. tamoxifen in cancer and atherosclerosis). Further, if “the uptake of steroids appears to reflect an active metabolic process” as suggested by Kornman & Loesche (1971), such may also reflect the protective effect of particular sterols (stigmasterol, trodusquemine, etc) upon atherosclerosis as consumed within the diet.
4.4 - B. fragilis …FRP Steroidal energetics
Given that the endocrine system has an enormous impact on atherosclerosis and it’s subsequent progression or regression, it may then be relevant to factor in the hypothetical interplay of hormonal influences on Bacteroides fragilis as well as the reciprotive influence Bacteroides fragilis may have upon endogenous hormones relative their influence in atherosclerosis. As mentioned, steroidal compounds may serve a substitutional utilization along the fumarate reductase pathway within Bacteroides fragilis - essentially equating to an overlooked form of ‘steroidal energetics’ relative the organism.
Again, steroidal compounds may directly influence Bacteroides fragilis substitution, synthesis and utilization of vitamin K2. Further, such may give merit to, while providing a possible explanation regarding the decreased prevalence of atherosclerosis within subjects whose diets are supplemented with particular forms of vitamin K. Perhaps such represents the antagonistic effect upon energy production by Bacteroides fragilis along the FPR.
If such hypothesis regarding the interplay of B. fragilis upon atherosclerosis and related states such as NIDDM is correct, this may give merit to the relevance of potential inhibitory, and yet, stimulatory effects of various forms of vitamin E upon the host - which is similar in structure to the oestrogen, oestradiol (Behl et al, 1994). Further, such substitution in the FRP and influence upon B. fragilis may also insightfully reflect the relevance of the observed negative impact of beta-carotene upon atherosclerosis and its contribution to increased progression.
Further as observed by Rothery et al (1998), hydroxylated naphthoquinones can serve as substrates for E. coli anaerobic reductases. It may not then, be improbable that such mechanisms may also occur within Bacteroides fragilis subspecies contributing to an increased risk in atherosclerosis and cancer given similar host exposures.
Consideration of influences upon B. fragilis fumarate reductase pathway and its relevance to growth and influence upon atherosclerosis, may also give credence regarding the beneficial effect of vitamin D3 upon atherosclerosis as a competitive antagonist for various sterol groups otherwise substituting along the FPR as well as competitively antagonizing sterol structures which have an avidity for seven alpha dehydrogenase steroid? possessed by Bacteroides fragilis NCTC 9343 and YCH46.
6.1 - Steroidal energetics:
Estrogenic and androgenic steroidal moieties
It may be of value to assess the impact of estrogenic and androgenic steroidal moieties not only upon atherosclerosis and diabetes but also their direct impact upon Bacteroides fragilis subspecies -in this regard. For example it has been observed that various estrogens have a particular impact upon the progression of atherosclerosis.
As a case in point, Mikamo et al (1998), when studying the pathogenicity of anaerobes in a rat pyometra model observed that Bacteroides fragilis was isolated and a high-frequency in the luteal phase demonstrating that growth is promoted by estrogen but not progesterone. It was also noted, and reiterated, that the isolation rate of anaerobes to aerobes is high in premenopausal women and in postmenopausal women who take estrogen (Osborne et al). Further it was suggested that the growth of bacteria might be partially regulated by sex steroids.
Such may reflect the protective effect conferred by chronic administration of particular progesterone based birth control and hormone replacement therapies relative atherosclerosis and non-insulin-dependent diabetes mellitus. Supporting such, Dwyer and Nordstrom (2002) observed that intima media thickness (IMT) was reduced in women who had undergone hysterectomy versus those who had a hysterectomy and bilateral oophorectomy despite the high prevalence of hormone replacement therapy (HRT) within the latter.
It was suggested “that endogenous hormones may offer a protection that currently prescribed replacement hormones do not”. It was also suggested that the loss ovarian hormones may impact atherosclerosis by pathways relative HDL. However it is also possible, that particular estrogens have an inhibitory effect upon Bacteroides fragilis which was not factored into the scope of this particular study.
Further, such may allude as well to a possible influence inherent the route of administration of HRT relative it's impact upon disseminated Bacteroides fragilis subspecies at the site of atheroma - therein influencing IMT. That is, it may be possible that the administration of particular estrogens stimulates growth of Bacteroides fragilis subspecies fragilis in the upper intestine thereby negating its potential protective benefits in atherosclerosis.
As example, Park et al (2006) observed that particular strains of Bacteroides fragilis were able to hydrolyze daidzin to daidzein and calycosin, and suggested that such inherent ability may potentially activate potent estrogenic activities which have an influence upon estrogen related c-fos mRNA and PR protein expressions within the host. Again, such may have an intrinsic association relative B fragilis influence upon atherosclerosis development and progression.
As well, beyond its immediate impact upon the fumarate reductase pathway it should be noted that Seli et al (2001) observed, while studying the potential regulatory effect of aminopeptidase N upon this cycle – dependent bioavailability of interleukin 8 (IL-8) in the endometrium, that estradiol inhibited aminopeptidase N activity in a concentration dependent manner and that progesterone did not have a significant effect.
This may be of relevance considering the high degree of homology between aminopeptidase N (CD13) within the human host and the aminopeptidase N possessed and expressed by Bacteroides fragilis NCTC 9343 and YCH 46. Given the proatherogenic effect of interleukin 8 in atherosclerosis - Apostolakis (2009), as well as the compounded ability of Bacteroides fragilis endotoxin (Bft) to initiate an inflammatory response via secreted interleukin 8 upon human colonic epithelial cells (Sanfilippo at al 2000 - IL-8 slide Pro inflammation and HSP 90) - it may be of relevance and worthwhile to explore such relations between estrogens and Bacteroides fragilis - beyond surface knowledge.
As well, there is a close relation between corticosteroids, stress and atherosclerosis.If one considers the observation of Plager et al (1964), it was observed that the concentration of bound and unbound plasma cortisol levels were “appreciably elevated” in estrogen treated subjects and that such was reflective of adrenal cortisol production. Further, Robertson et al (1959), who earlier studied the influence of estrogen on the secretion, disposition and biological activity of cortisol - suggested that patients receiving estrogen had a diminished transformation of particular steroidal metabolites and greater retention of estrogen within the intravascular and extracellular fluid compartments.
Overlaid upon the potential availability, exposure and influence of estrogens and glucocorticoids on Bacteroides fragilis growth, it can be hypothesized that such hormones may be related to the organism’s impact on atherosclerosis development.
6.2 - Steroidal energetics: Mineralocorticoids…
As an extension of the potential implications of estrogen upon Bacteroides, it has been observed that there is a close relation between corticosteroids, stress and atherosclerosis. If one considers the observation of Plager et al (1964), it was observed that the concentration of bound and unbound plasma cortisol levels were “appreciably elevated” in estrogen treated subjects and that such was reflective of adrenal cortisol production.
Further, Robertson et al (1959), who earlier studied the influence of estrogen on the secretion, disposition and biological activity of cortisol - suggested that patients receiving estrogen had a diminished transformation of particular steroidal metabolites and greater retention of estrogen within the intravascular and extracellular fluid compartments. Overlaid upon the potential availability, exposure and influence of estrogens and glucocorticoids on Bacteroides fragilis growth, it can be hypothesized that such hormonal relations may be relevant to the organism’s impact on atherosclerosis development.
Further it was observed by Johnson & Engel (1986), “stress which is long been known to be associated with disease processes, appears to play a role through induction of increased cortisol and catecholamine levels…compromising host immune responses’. Within the review, published regarding “Acute necrotizing ulcerative gingivitis”, it was suggested that cortisol may serve as a nutrient source for Bacteroides bacteria - thus illuminating the balance between bacterial interplay and host response which may pivot upon nutrient/metabolic source.
Such may be of significance regarding the purported association between psychological stress and atherosclerosis, given that the cerebral cortex, as example, synthesizes and secretes glucocorticoids (i.e. cortisol), mineralocorticoids (i.e. aldosterone) and androgens (i.e. testosterone) which beyond their inherent signaling capacity may simultaneously influence Bacteroides fragilis growth and balance within the host. Bokkenheuser et al (1973or5?) Demonstrated that Bacteroides fragilis as part of the intestinal flora can convert steroids into a variety metabolites including the hydrolysis of conjugates and 21-hydroxylation of corticoids.
An interesting example of the influence of stress upon atherosclerosis is alluded to in the text “Coronary heart disease epidemiology; from etiology to public health” - where the effect of societal transition on coronary heart disease mortality was noted as dramatically increased after the breakup of the Soviet Union in 1991 and again during the ruble crisis in 1998 (J. Shapiro).
Though these mortality trends are not completely understood, such was attributed, at the very least, to a probable change in societal dietary habits. Perhaps, important also to factor in his the influence of stress and resulting influence of cortisol upon both host and Bacteroides bacteria.
As a compounding example, which may further illustrate the effect of stress upon both host and Bacteroides fragilis, Belay et al (2003) further “extended upon previous observations that showed a differential capability of catecholamines to enhance (in vitro) bacterial growth” and specifically utilizing norepinephrine and epinephrine to influence the growth of Bacteroides fragilis.
Such may be an important finding as factors affecting the host personally ranging from personality type, sleep cycle, shift work, occupational noise and physical workload (Virkkunen et al, 2007) as well as emotional responses and behaviors ranging from hostility and anger expression are also predictors of cardiovascular disease – as reflecting personality and psychosocial risk factors (Haukkala et al, 2010).
Such findings are not surprising, however, considering that Holdeman et al (1976) when studying human fecal flora in reference to the variation in bacterial composition relative emotional stress - observed that “an anger stress situation tends to produce a high count for the species Bacteroides fragilis”. Within this study, it was also noted that “anger or fright and depression reportedly produce distinct effects upon the colon and upper gastrointestinal tract and that emotional stress which affects epinephrine and gastrin secretion, which in turn affect intestinal motility, blood flow, mucin secretion as well as nutrient and bile derivative absorption and secretion”.
Also it has been noted that Bacteroides fragilis is capable of dynamically responding to various environmental stress conditions through the up regulation of various cell envelope proteins that afford adaptive ability and utility drawing adverse events and within adverse environments (Avelar et al, 1998). Likely such adaptation occurs by B. fragilis within the host. As a chronic member of the commensal flora and resultantly exposed to the direct and indirect effects of various stress hormones – it has perhaps evolved to utilize what is otherwise, an adaptive response by the human host for it’s own survival.
A further example, of the interplay of stress upon the ‘host commensal dynamic’ may be observed in Cushing's syndrome, which “is associated with an increased rate of cardiovascular morbidity and mortality”, where endothelial dysfunction associated with hypercortisolism – and, an early event incidence of atherosclerosis - can actually be reversed (Akaza et al).
It is probable to speculate that the chronic glucocorticoid excess associated with the condition may not only increase Bacteroides fragilis growth but do so while impacting and decreasing the host’s innate defense mechanisms such as the integrity of tight junctions, decreasing endothelial NO synthase activity while increasing oxidant stress - which, taken together, would promote the organism’s dissemination.
6.3 - Steroidal energetics:
Endocrines and B. fragilis = ‘opportunistic interplay’…
Further, if endocrine secretions can affect the growth and survival of commensals such as B. fragilis, an intriguing extension of thought is the possibility of outcome regarding an ‘opportunistic interplay’ regarding the alteration of various endocrinological feedback mechanisms as potentially influenced and affected in presence of B. fragilis as an opportunistic pathogen. That is, it may be probable and relevant to consider, for example, the possibility of host receptors which may regulate endocrine secretions and levels - but which may also be vulnerable to Bacteroides fragilis virulent mechanisms such as the proteolytic activity of aminopeptidase N and the protease activity of BFT (fragilysin) - which can invariably influence such.
A simple example may be observed when comparing the amino acid sequences of the estrogen and progesterone receptors, the latter of which contains 5 cysteinyl leucine linkages and 2 cysteinyl lysine linkages not observed in the former. Such may result not only in a disruption of hormone feedback mechanisms, but conceivably an advantageous endocrinological environment preferrably in favor of progesterone which has been observed to have a more profound effect upon bacteroides members (Kornman & Loesche, 1981) as opposed to lesser and more the inhibitory effect of particular estrogens upon both the FRP and aminopeptidase N activity - which may adaptively promote the survival and growth of Bacteroides fragilis.
If such occurs in nature, it may further compliment and reflect the observations of de Medina et al, 2004 regarding the estrogen receptor inhibitor tamoxifen as a potent inhibitor of cholesterol esterification and its ability to prevent the formation of foam cells beyond its capacity as an inhibitor of Acyl-CoA:cholesterol acyl transferase (ACAT) - by influencing, in addition to other factors, the growth of Bacteroides fragilis subspecies such as NCTC9343 and YCH46.
The administration of tamoxifen among a host of other mechanisms, may for example, increase circulating estrogens – which, besides possessing a less stimulatory effect by substituting for menadione along the B. fragilis fumarate reductase pathway, may dynamically affect the host’s coagulation and fibrinolysis. Such elevation in estrogen levels subsequent to therapeutic administration of tamoxifen may compensate for its lower basal concentration and effects due to unaltered and regulated levels relative that influenced by the effect of B. fragilis upon progesterone.
For example, elevated estrogen levels may be responsible for increased coagulation by increasing cell mediated coagulation mechanisms further compounding that resulting from the presence of disseminated viable and nonviable Bacteroides fragilis cells to decrease clotting times (increased clotting) within the host (Rosenthal et al, 1988). Additionally, elevated estrogen levels which induce coagulation resulting from activated Hageman factor (intrinsic coagulatory pathway) may as well serve to further increase thrombus formation subsequent to the dissemination of cell wall components of B. fragilis ( such as lipopolysaccharide and lipid A) observed to accelerate blood coagulation ( Bjornson & Hill ,1973), (Bjornson, 1984).
Further, given that estrogen can substitute for menadione only within a narrow concentration range (Kornman & loesche, 1982) it is probable that the administration of the estrogen receptor antagonist tamoxifen administered during breast cancer therapy and - and observed to decrease atherosclerosis progression via the inhibition of cholesterol esterification and foam cell formation (de Medina et al, 2004) – may also result from an elevation in levels of serum estrogens - estrone, E1 and estradiol, E2 - (as well as elevated DHEA) subsequent to such therapy (Lum et al, 1997).
Such dynamic may be significant, as Kornman & Loesche (1982) observed that levels of estradiol only three times higher than that observed to stimulate the growth of Bacteroides species such as B. intermedius, could conversely and “markedly inhibit (it’s) growth”. Similar observations were associated with B. melaninogenicus ssp. relative estradiol. As well, unlike estradiol, no growth inhibition was observed upon B. intermedius with progesterone at higher concentrations and elevated progesterone was actually required to stimulate B. melaninogenicus. This was in stark contrast to B. gingivalis which showed no growth regardless of whether estradiol or progesterone was substituted for menadione.
Though no such study has been performed to date to evaluate the effect of estrogen or progesterone upon Bacteroides fragilis NCTC 9343, it is probable that estrogenic steroids may have an influential affect upon this organism, and potentially explain the protective effect of tamoxifen, hormone replacement therapy and birth control therapy relative atherosclerosis. For example, it is possible that similar to the effect of estradiol upon Bacteroides intermedius, continuous and elevated circulating levels of estradiol resulting from therapeutic administration may decrease the acute and chronic effects of Bacteroides fragilis 9343 and YCH 46 upon the host.
6.4 - Steroidal energetics: Androgenic steroids…
Kornman and Loesche (1982) stated that previous to their study on the effect of estrogens upon Bacteroides, not only was there no previous report of an enhancement of bacterial growth by steroids, but that previous studies on gonadal steroids had been observed to inhibit growth of various gram-positive organisms and N. gonorrhoeae. Again, this may have relevance in atherosclerosis as DHEA has been observed to decrease the disease process.
As well, serum DHEA becomes significantly elevated during the period of tamoxifen administration, as previously discussed (Lum et al, 1997). It is probable then, in addition to the effects of DHEA upon the host, that such steroid may also have a protective impact by decreasing Bacteroides fragilis.
As mentioned, Bacteroides fragilis expresses the oxidoreductase enzyme 7 alpha hydroxy steroid dehydrogenase (McDonald et al, 1975), which catalyses the oxidation of the 7alpha-hydroxy group of bile acids and alcohols both in their free and conjugated forms and can adaptively catalyse the production of various energy sources in the absence of sugar (Hylemon and Sherrod, 1975).
As suggested by Hylemon and Sherrod, (1975) during their analysis of seven alpha hydroxysteroid dehydrogenase within selected strains of Bacteroides fragilis: “The physiological significance of the 7 alpha hydroxysteroid dehydrogenase reaction(s) to B.fragilis is not yet clear. However, the fact that oxidation of the 7-a-hydroxy group occurred only after glucose was nearly depleted from the growth medium (Fig. 5) suggests that bile acids might serve as potential energy sources in the absence of fermentable carbohydrates.”
It is indeed probable, that such enzyme is directly influenced by estrogenic and androgenic steroids such as DHEA and accounts for the positive and negative associations of various sterols and steroids upon the progression of atherosclerosis -reflecting a binding affinity for sterol and steroidal substrates and their subsequent impact, both positive and negative, upon the metabolism, growth or survival of Bacteroides fragilis subspecies fragilis.
It should also be noted, that particular Bacteroides strains contain the steroid desulfating enzyme arylsulfatase. As observed by Eldere et al (1988), hydrogen sulfide required for the optimal growth of Bacteroides could be produced by the action of arylsulfatase upon the addition of particular, sulfites taurine and moderately by the female estrogen estrone sulfate. However, three alpha sulfates, and three beta sulfates of androgens and particular bile salts, administered to Bacteroides species produced no hydrogen sulfide - as there was (only trace to) no substrate specificity of these steroid desulfating strains for such compounds.
Further, with the exception of estrone-3-sulfate upon one particular Bacteroides strain, estrone three sulfate as well as beta-estradiol-3-sulfate was observed to have low substrate specificity for the Bacteroides strains tested. Additionally, only trace substrate specificity could be observed for estriol-3-sulfate relative the steroid desulfating enzyme arylsulfatase, and no substrate specificity could be observed for any Bacteroides strains tested regarding beta estradiol-17-sulfate.
This is potentially significant, especially when, overlaid upon the beneficial effects (negative correlation) of androgens (Manolakou et al, 2009) and particular estrogens (Kolovou et al, 2011) observed in atherosclerosis . Such may reveal additional processes that are ultimately affected and involved in the interplay of disease progression, such as the important influence upon the organism Bacteroides fragilis.
Such administration may also allude to the pivotal role of hydrogen sulfide (H2S) production upon the organism’s growth and virulence - especially in terms of it's total load and burden upon the host organism at any one time. This interplay may be of particular importance specifically within the hypoxic environment of arterial plaques - where carbohydrate substrates may be limited and reliance on otherwise secondary mechanisms (relative to the more diverse and abundant substrate availability within the intestine) such as seven alpha hydroxysteroid dehydrogenase and arylsulfatase may become an important determinant of the organism’s survival.
It is possible then that steroids and sterols observed as anti-atherosclerotic (anti-progressive or ‘protective’ in nature), in fact, represent substrates non-metabolizable or antagonistic to the growth and survival of Bacteroides fragilis. Again as overviewed by Manolakou et al, (2009) many androgenic steroids such as the DHEA, DHEA sulfate, androstenedione and indices such as the free androgen index, free testosterone and total testosterone - are generally, negatively correlated as well with decreased blood pressure, post load insulin and diabetes. Such observational associations regarding the importance of steroid sulfatase and estrogen sulfotransferase, albeit with reference only to that produced by the human host, supports what was observed as positively correlated with the progression of atherosclerosis within the human aorta by (Nakamure et al, 2003).
6.5 - Steroidal Energetics: Demographics, Culture, Race and B. fragilis…
Demographics, cultural practices, race and atherosclerosis is strongly associated. Newton et al in 2001, concluded that bacterial organisms and race have a consistent association with the presence or absence of cervical-vagina organisms. Such may have profound implications, regarding a predominance of Bacteroides fragilis subspecies fragilis among members of a particular race (shared genealogy) or birth origin and the association of such as a purported nonmodifiable risk factor for atherosclerosis - further relating disease mechanisms such as non-insulin-dependent diabetes mellitus, obesity, hypertension - referred: metabolic X.
6.6 - Steroidal and sterol entities:
A generalization of the hierarchy relative Bacteroides fragilis…
When viewing the positive and negative impact (correlations) of steroids upon atherosclerosis and comparatively overlaying such structures relative Bacteroides fragilis – a picture begins to emerge. In particular, if one considers for example, Bacteroides fragilis strains NCTC 9343 and the impact of such steroidal structures upon the organism’s: fumarate reductase pathway, 7- alpha-hydroxysteroid dehydrogenase and steroid desulfating arylsulfatase – as prototypical pathways of analysis - a general, but by no means definitively exclusive, hierarchy can be discerned, at present.
That is, it may be relevant to suggest that, particular: estrogens, androgens (such as testosterone and DHEA) as well as bile salts such as taurolithocholate, glycolithocholate, lithocholate, taurodeoxycholate, glycodeoxycholate and perhaps deoxycholate are more negatively correlated with atherosclerosis because they negatively impact (antagonize) the growth of exotoxigenic Bacteroides fragilis (or further elucidated or modified/manipulated strains).
Though, also an overgeneralization, it may be of utility regarding further insight into the atherosclerotic process, to suggest that steroidal agents which promote, increase or stimulate the growth and virulent capacity of Bacteroides fragilis such as: glycochenodeoxycholate, glycocholate,taurochenodeoxycholate, taurocholate, chenodeoxycholate, cholate, cholesterol, cortisol, and particular forms of progesterone as well as squalene and pregnenolone – are therefore positively correlated (strongly associated) with atherosclerosis, as observed within the voluminous literature regarding such steroidal compounds.
7.0 Sulphur:
Hydrogen Sulfide & Cysteine
7.1 – Atherosclerosis, B. fragilis media and Sulphur …
Bacteroides fragilis & Sulfur: Cysteine - A reducing agent
Generally, when referring to cysteine such is mentioned relative to the compounds classification as a sulphur containing amino acid. Unique from the host however, and as demonstrated by Tamimi et al (1960), B. fragilis utilizes cysteine not only as a sulphur containing structural amino acid but also as a preferential substitute for thioglycolate in culture (1960) – itself a reducing agent creating anaerobic conditions by reducing molecular oxygen to water. As such the addition of cysteine to culturable media resulted in significantly observable increases in the growth of strains of B. fragilis – within particular concentrational margins.
It may be, that the possession of Bft/fragilysin metalloprotease (and CD13-like protease), which can cleave, liberate and procure cysteine for redox potential as well as the metabolic and structural needs of B. fragilis - essentially provides for the establishment of anaerobic conditions locally (and disseminated/distal regions of infection: host occupation/establishment/colonization).
Proteolytic ‘titration’/opportunism - may help propagate the optimal conditions required for fullest growth rate - pending immediately sensed host circumstances by B. fragilis – and it’s consequent, reactionary response by the upregulation and activity of CD13 and the release of Bft - both of which cleave specific cysteine linkages.
Such may underlie studies highlighting the beneficial impact of dietary cysteine administration in atherosclerosis – such as those involving acetyl-cysteine and the purported health benefits and improvements regarding sources of cystiene such as garlic, fish etc. As well, given that cysteine and sulfide each are capable of serving individually as sole sources of sulfur for B. fragilis Subsp. fragilis (Varel & Bryant, 1974) it may not be surprising to note that disease states strongly associated with atherosclerosis and also likely caused by B. fragilis NCTC 9343 - such as type II diabetes (non-insulin dependent diabetes melitus) – correlate with low serum concentrations of both and that insulin signalling pathways, as well as glucose tolerance - is increased by administration of cysteine and sulfide (7 - Manna & Jain, 2011).
7.2 - Bacteroides fragilis & Sulfur: Exogenous vs. endogenous cysteine
In addition to the above, cysteine has not been observed as an essential component for B. fragilis, but rather “dispensable”(Tamimi et al, 1960). Such may have merit regarding atherosclerosis as the evolutionary and biologically adaptive metabolism of B. fragilis - may serve not only to determine reduction potential of the environment upon proteolytic liberation of cysteine from host proteins - but could conceivably provide a means to conserve ATP by consuming and utilizing host derived cysteine as required. This mechanism of survival would allow B. fragilis to reserve the innate capacity to synthesize cysteine during exogenously low environmental concentrations within the host.
7.3 - Bacteroides fragilis & Sulfur: Cysteine and homocysteine
If such hypothesis is correct, such could account for the serologically disproportionate balance observed relative high homocysteine and low cysteine concentrations. As cysteine is an ‘excellent sole source of sulphur’ for B. fragilis NCTC 9343 (6 - Varel & Bryant, 1974) it is probable that high serum homocysteine may also reflect production by B. fragilis as well.
It is interesting to note that in a 2005 study conducted by Davis et al relative folate levels and homocysteine in young women, homocysteine synthesis was elevated regardless of dietary folate restriction and an associated methylene-tetrafolate reductase polymorphism. Also, though radio-labelled [13C1] serine was utilized as a substrate within the study, the observers “were unable to detect cysteine”. Such is an example that perhaps draws into question the further possibility of an unknown variable such as B. fragilis and it’s involvement in intermediary cysteine consumption as well as relevance – as being a culprit responsible for a great proportion of homocysteine produced in atherosclerosis.
Alternatively, it is important to consider whether imbalances regarding homocysteine may also culminate subsequent to the virulent impact of B. fragilis cysteine proteases upon host mechanisms. For example, the hypothetical cleavage of cysteine linkages upon influential and concentration determining host enzymes such as the cysteinyl-glycine bonds located at positions 324-325 and 1153-1154 or the effect of fragilysin up cysteinyl-lysyl bond at 1081-1082 (N2 Q99707.2; GI:2842762) within methionine synthase, could hypothetically denature such – altering host cysteine/homocysteine balance.
Induced high serum homocysteine may be ‘maladaptively advantageous’ if indeed caused by B. fragilis given the fact that homocysteine can be utilized in transsulfuration reactions by the organism to produce H2S (Yoshida et al, 2009). Further, homocysteine conversion to cystathionine would contribute to the formation of cysteine by the host – wherein cysteine could be utilized by B. fragilis as a preferential source of sulfur. Proteolytic (or LPS) involvement by B. fragilis that can influentially shunt the flow of reaction in the direction of homocysteine and limit conversion to methionine – would invariably increase the flow of cysteine available to opportunistic commensals within the host.
Another interesting perspective relative the host regarding the observation of high levels of homocysteine as observed in atherosclerosis is that “cysteine and its disulfide form cystine are relatively insoluble and toxic in excess”. As such, one of the pathways of catabolism involves the formation of cysteine sulfinate via the oxidation of molecular O2 to cysteine sulfonate - which itself is converted to decarboxylated to taurine a component of bile salts (Masella and Maza, Book).
What is interesting here, especially in light of the relevance of atherosclerosis as being hypothetically proposed as caused and progressed by an anaerobes such as B. fragilis NCTC 9343 is that:
(1) the oxidation of cysteine would reduce dissolved O2 within the host, therein producing increased probability of serous survival of B. fragilis cells. Also, (2) increased cysteine within the host can inevitably become converted to taurine - a component of bile (taurocholate, taurchenodeoxycholate). This is significant as taurocholate and taurochenodeoxycholate are preferable forms of bile sterol utilized by B. fragilis via 7-alpha-hydroxysteroid dehydrogenase promoting it’s growth within the intestine - serving as a potential energy source in the absence of fermentable carbohydrates and glucose (10 - Sakai et al, 1980), (11 - Pumbwe et al, 2007), (12 - Hylemon & Sherrod, 1975).
Supporting such statement, it has been observed that taurine increases the fecal 7a-dehydroxylase activity in Western-style diets (14 - Garbutt et al, 1971), (15 – Jacobsen & Smith, 1968). Further, Van Eldere et al, (1998) observed and suggested that taurine may be “an important mediator in the effect of a Western diet on the bile acid metabolism of the intestinal flora” as it was observed that the Bacteroides species strain studied, “did not grow unless taurine or another appropriate reducible sulfur source was present” and stated that “increased 7 alpha-dehydroxylation of tauroconjugated cholic acid depends on the reduction of taurine to H2S; H2S of which - is a necessary growth factor for 7-alpha-dehydroxylating bacteria”.
Following such path, H2S deficiency has in recent decades been cited as potentially associated with hypertension and atherosclerosis – which may also be indicative of chronic utilization of such (cardiovascularly associated endogenous gas) within the host - by B. fragilis (Perna et al, 2010).
Also, increased cysteine production by the host from homocysteine could be utilized by B. fragilis via a heretofore under-recognized putative membrane exported protein (i.e. B. fragilis: YP_212126, YP_099703 – analogous to the Radical SAM Superfamily proteins and, specifically, the host’s functional cysteine dioxygenase; GenBank: BAA12873.1) utilized to bind molecular oxygen with cysteine to produce hypoxic conditions at specific loci – such as at the site of an atheroma.
It is interesting then, that the balance of homocysteine may (among other mechanisms) represent a commonly competitive sulfur pathway by the host organism and B. fragilis. It may be probable that an adaptive preservatory ability of the host to limit cysteine availability to B. fragilis by maintaining high homocysteine levels (or alternatively the suspended conversion of host homocysteine to cysteine) – is ultimately a compensatory response – which decreases the creation of anaerobic conditions as well as further growth of B. fragilis within and beyond the intestine.
Compounding such, cysteine dioxygenase (cysteine oxidase) which catalyzes in the conversion of cysteine plus molecular oxygen to cysteine sulfinic acid is feedback inhibited by homocysteine (Yamaguchi et al, 1978) – which, though disbalanced, may reflect a counter mechanism by which high concentrations of homocysteine are maintained within the host, also limits the loss of molecular oxygen during subclinical colonization and dissemination of anaerobes such as B. fragilis.
Compounding the above regarding cysteine oxidase were the unique observations by Misra (1983) who studied the effects of cysteine analogs and other compounds on in vitro cysteine oxidase in rat brain microsomes. It was noted that maximum inhibition of cysteine oxidase was by obtained by alpha,alpha'-dipyridyl and the least inhibition by dithiothreitol. This is peculiar given that both inhibit and promote B. fragilis growth, respectfully. Also, L-homocysteine was found to be a competitive and reversible inhibitor of cysteine oxidase – which likely supports the aforementioned. Further, pyridoxal HCl activated cysteine oxidase at the same concentration and recently has been questioned regarding its association with diabetes – which is strongly associated with atherosclerosis.
Lastly, it was observed that particular divalent ions such as Zn2+ and Cu2+ - which circumstantially also decrease atherosclerosis risk also inhibited cysteine oxidase activity and that reversal of the ranked order of metal ions inhibiting cysteine oxidase parallels that required for optimal B. fragilis growth.
Perhaps such alludes in a small way to the possibility that various means utilized to tangibly assess risk factors and various correlates relative atherosclerosis such as host derived agents, biomarkers, biochemical reactions - more aptly represent the interplay of at least four competing domains. This would include: disease stage of atherosclerosis, the host milieu against the backdrop of nonmodifiable factors, modifiable host variables and B. fragilis – relative the micro-organisms plethora of variables. On a finite level, B. fragilis survival, virulence, responsive biochemical reactions are host manipulated/modifiable and nonmodifiable (possesses a set genome) – relative, if not pursuant environment circumstances of immediate, acute &/or chronic selective pressure.
Compounding that mentioned, homocysteine is also a zwitterion molecule at a serum pH of 7.4 and may effectively be utilized to help protect against the B. fragilis zwitterion capsular polysaccharide A (PSA-1,2) by molecular polarizing orientations to CP as well as LPS - and effectively screening and/or minimizing its effects – especially upon the arterial vasculature, to name but one. This is a variable to consider given that zwitterionic polysaccharide A can stimulate cellular immunity and such motifs activate TLR2 on APCs – perhaps contributing to the ongoing pro-inflammatory state of the host in atherosclerosis. (Kalka-Moll et al, 2000),(Gallorini et al, 2007).
If homocysteine can also be utilized by the host to neutralize LPS, this may prove of significant relevance regarding the hypothetical proposal of B. fragilis as the causal agent in atherosclerosis. Given that B. fragilis LPS has been observed early in life in amniotic fluid (Beckmann et al, 1994) and coursing throughout the host for the duration of the host’s life (Delahooke et al, 1995), it is probable then that high homocysteine levels may not only correlate with such continued occurrence but also help protect the host against what perhaps represents - a significant production of background zwitterionic PSA and general LPS originating from the organism within the intestine and chronically observed within the bloodstream (and chronic stimulating T)…
(to copy/paste 2nd half of text at later date, as time permits)
~ End - Paper condensed ~
(Reference section omitted)
SUPPLEMENTAL - BACKGROUND…
Ennis, O. (2011–2012). Atherosclerosis & Bacteroides fragilis (nctc 9343 / atcc25285) fragilysin/Bft - A union of theories [Zenodo]. https://doi.org/10.5281/zenodo.15356078
Interesting: Virulence: ETBF, Vaccines, pbk-cmV, hiV-1 https://zenodo.org/records/15331014
all same promoter - but was that the only reason for use?
2025... STILL WAITING... ETBF: BFT/FRAGILYSIN (EC3.4.24.74) & ATHEROSCLEROSIS PROBABLY THIS WILL BE PUBLISHED AFTER COVID VACCINE DISCONTINUATION - IN THE EVENT THAT COVID VACCINES CAN CAUSE RELEASE OF BFT/FRAGILYSIN (EC3.4.24.74)
ATHEROSCLEROSIS & ETBF…
HUMANS CAPABLE OF TELLING THE TRUTH...
2024…
2022…
2012 - After receiving little to no interest by researchers in north america - decided to forward to researchers in China who were working on similar ideas - proof of sending should still reside on Telus Servers… or some government server/repository : ) I doubt this ever reached them or that they didn’t already know the difference.
But some examples of work developed:
Forwarded to innumerous research institutions (2011-2012) - for consideration
2012…
After forwarding thesis to major Canadian Research University in Western Canada:
“THANK YOU FOR SENDING THIS MATERIAL - IF WE EVER FIND ANY EVIDENCE IN THE LITERATURE CONNECTING B. FRAGILIS AND ATHEROSCLEROSIS WE’LL BE SURE TO LET YOU KNOW…
Forwarded copy of research personally conducted since 1996 and commenced writing on in Saskatchewan 2006-07, Calgary 2008 - 2012…
2006…
CHOLESTEROL & HEART DISEASE CONNECTION: HIGH CHOLESTEROL = HIGH BFT BINDING
Our data suggest that the BFT receptor is a membrane protein sensitive to depletion of membrane cholesterol
