biofilm RNA seed by fanzhongqing

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									                 BIOFILM – OUR SUSTENANCE, OUR SIGNIFICANT OTHER, OUR SOURCE

Bacteria account for much of Earth's biological diversity. These small life forms play essential roles in
quite varied environments and we are experiencing an awakening in understanding about the
underlying importance of bacterial biodiversity, and how intertwined they (and we) are.

 Advances in molecular biology have provided astonishing insights into microbiological pleomorphic
variety, considerably improving our knowledge of the ever-changing morphological, physiological and
ecological features of our life partners, who mostly exist as colonial bacterial species in interactive
community enveloped by a mist of our ever-present shared viral as well as naked viroid DNA and RNA.

 Bacteria had been mostly examined and thought of by us as unchanging monomorphic individual free-
swimming species, captured and studied in their planktonic form. When these bacteria are grown in
culture, growth medium must be freshened daily in order for the organisms to maintain their
characteristics. When cultured in unchanged medium, bacteria begin to pleomorphically alter their
form and physiology depending on their new environment due to gradient changes in their surrounding
waste products and remaining nutrients.

 More importantly, 99% of all microbial species from most environments are ordinarily unculturable.
Microbiology as a discipline has traditionally focused by necessity on the 1% of cultivable species.
Attempts to culture more species in the lab by manipulating growth media have historically failed.

Studying a bacterium in isolation and understanding the biofilm is like trying to study a caged animal
             in a zoo or defining the human experience by studying a single sperm cell.

 In nature, 99% of bacteria live in structured biofilms, a kind of cooperative multi-cellular life form,
exhibiting many sophisticated survival strategies. As an organism composed of many integrated
microorganisms, it creates a small percentage of low-metabolism ‘persisters’ and exhibits hormetic
response to stressors, eliminating the weak cells within via apoptotic signaling, becoming globally more
resistant to death. Whatever does not completely eliminate biofilm makes it better able to survive.

 Pseudomonas aeruginosa, a biofilm-forming microorganism, is probably the most common life form on
the planet, as we animals and plants thrive, blanketed with our characteristic bacterial colonies.

 Cultured bacterial biofilm phenotype characteristics cannot be predictably correlated with the clinical
virulence of similar phenotypes (for example, E. coli isolates) living in the host within their biofilm. The
overriding influence of local environmental conditions provided by the host underscores the challenge in
developing better biofilm model systems to copy clinical situations (from cloudy contact lenses to tooth
decay, periodontal diseases, bacterial endocarditis, cystic fibrosis lung infections, avascular necrosis and
other non-healing wounds or implants as well as infections of joint synovia, nasopharynx, digestive
system or urogenital tracts).

 The goal of current microbiology research is to understand the molecular nature of uncultivability as
well as find new microorganisms. Roughly a third of bacterial domains do not have a single cultivable
representative. We know of their existence only from DNA recovered from the environment. Today the

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latest PCR (polymerase chain reaction) DNA tests imply that most chronic illness is really due to biofilm
infection, no matter what the cultures said.

 For example, the reverse transcriptase-PCR–based assay system can detect the presence of bacterial
mRNA in many culturally ‘sterile’ middle ear effusions. Bacterial mRNAs have a half-life measured in
seconds to minutes; therefore, detection of bacteria-specific mRNAs is evidence that metabolically
active organisms are present.

Careful adjustment of growth medium composition is an important first step. Incorporation of more
adequate attachment surfaces in the experimental design is an added measure; perhaps studying
biofilm formation directly on eukaryotic cells will help.

However, since multiple species are present in most natural environments, we also need models that
allow monitoring of possible antagonistic or synergistic interactions between community members (and
even these copies cannot take the state of an individual host’s immune system into account).

 A diffusion chamber has been designed for growing unculturable organisms. A simulated natural
environment is provided by growing microorganisms in a chamber that allows exchange of chemicals
with the environment, but restricts movement of cells. Many unculturable organisms will grow on
synthetic media in the presence of other, neighboring species that release substances that act as growth
promoters. Lack of bacterial growth on foreign media seems to be due to ‘choice’, rather than a missing
metabolic ability.

                                            Biofilm Formation

Biofilm becomes a collaborative community of sessile microorganisms now expressing different genes,
becoming metabolically distinct from their free-living, planktonic kindred. First bacteria become
attached, and then they accumulate, mature and then eventually detach to reproduce elsewhere. At
either extreme, nutrient-rich or very nutrient-poor conditions, greater numbers of cells exist in the
planktonic phase where they have greater access to local nutrients or can be distributed to a new
environment.

 The cryptic bacterial infection hypothesis holds that, many, if not all chronic diseases are initiated by
inflammatory events that release bacteria into the blood stream that become carried in phagocytic cells.
The cells migrate and take up residence at a region of inflammation. The bacteria produce molecules
that produce tissue hibernation and quell local inflammation in response to the bacteria. The bacteria
are, however, a source of ongoing irritation to the tissue and a chronic inflammatory disease results.

 The compromise of tissue inflammation in response to cryptic bacteria is similar to the physiology of
rodent hibernation. In both cases, systemic inflammation is suppressed. At the cellular level, this means
that other signaling pathways silence the inflammatory NFkB expression pattern. One of the major
nuclear receptors activated in hibernation is PPAR. PPAR is activated by opiods and sulfides (H2S),
which also induce hibernation in rodents.



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 An initial priming step in the biofilm forming process before attachment occurs. This describes an initial
interaction of the surface with its environment and is called conditioning. Conditioning happens, for
example, when a foreign body is placed in the bloodstream and the natural endothelial surface of the
vessel is altered by the adsorption of water, albumin, lipids, extracellular matrix molecules, complement,
fibronectin, inorganic salts or toxins. Once a surface has been conditioned, its properties are changed,
so that the attraction of a circulating organism for a normal or a conditioned surface can be very
different.

 Free floating or planktonic bacteria encounter a surface and form a reversible, sometimes transient
attachment, often within minutes. This attachment called adsorption is influenced by electrical charges
carried on the bacteria, by Van der Waals forces and by electrostatic magnetism (although the precise
nature of the pull is still debated). Van der Waals forces include attractions between atoms, molecules
and surfaces, differing from stronger covalent and ionic bonding since they are caused by harmonic
correlations in the frequencies of fluctuating polarizations of nearby particles (a vibrational effect of
quantum dynamics).

In some cases, as in the connection between a pathogen and the receptor sites on cells of its host there
may be a stereo specificity which though still reversible, is stronger than that achieved strictly by ionic or
electrostatic forces.

 A net repulsion between two surfaces can be overcome by specific molecular interactions orchestrated
by adhesins located on structures extending from the bacterial cell surface, such as pili (sticky hair-like
appendages bacteria use to adhere to surfaces). The longevity of primary adhesion depends on the sum
total of all variables, but surface chemistry pushes the equilibrium in favor of binding by predicting that
organic substances in solution will concentrate near a surface and that microorganisms tend to
congregate in nutrient-rich environs.

                                         Viruses change bacteria.

 Virus can enter bacteria via their pili. Pili are likely culprits since bacteria that lack them are rarely
pathogenic. Viruses then take over the cell and can cause normally benign bacteria to manufacture
toxin. Perhaps even pili themselves are expression of transmissible DNA, a viral infection turning a
friendly commensal without pili into a potential pathogen with pili, increasing stickiness and opening the
door for still more viruses.

 If the association between the bacterium and its substrate persists long enough, other types of
chemical and physical structures form which transform the reversible adsorption to a permanent and
essentially irreversible attachment. The final stage in the irreversible adhesion of a cell to an
environmental surface is associated with the production of extracellular polymer substances (EPS).

A glycocalyx is the glue that holds the biofilm fast to the colonized surface and is a complex of
exopolysaccharides of bacterial origin and trapped exogenous substances obtained from the local
environment, including nucleic acids, proteins, minerals, nutrients and cell wall material. Most of the


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EPS of biofilms are polymers containing sugars such as glucose, galactose, mannose, fructose, rhamnose,
N-acetylglucosamine and others.

 Bacteria mostly exist in their attached interconnected form with an altered genetic profile and
metabolism as part of the fabric of a highly-structured cooperative microscopic city, organized as a
sessile sentient organism with sophisticated forms of communication and a group strategy for survival.

 Virtually any surface, animal, mineral or vegetable becomes colonized with biofilm formation, including
contact lenses, ship hulls, dairy and petroleum pipelines, rocks in streams, as well as all varieties of
biomedical implants and transcutaneous devices. Free-swimming bacteria (which are what we capture
and culture) are mostly reproductive cells that are released from an established biofilm community that
has become mature, stressed or crowded.

 A dispersion-inducing molecule provokes genetic and physiological changes in the biofilm bacteria,
causing them to disperse and return to a planktonic state. The dispersion autoinducer now being
researched by David Davies (a halogenated furanone) has been effective in dispersing biofilms
containing Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans and Staphylococcus aureus,
whether those bacteria exist in a pure or mixed-culture biofilm. A surface protein-releasing enzyme (in
Streptococcus mutans) becomes actively involved in the degradation of attachment polymers on tooth
surfaces, releasing planktonic bacteria into the surrounding liquid.

 This is one of the few known examples anywhere in nature of a communication signal that remains
effective across species, family and phyla. Davies predicts the compound may also prove to have
communicative effect even across bacterial kingdoms. Since bacteria become easier to kill in their
planktonic state, this molecule shows promise in treatment of non-healing wounds.

Biofilm formation relies heavily on virus genes present within the bacteria, some injected by viral
bacteriophages. Viruses are made mostly of protein and RNA or DNA and ‘live’ primarily to inject their
message into a functioning chromosome, and then take over the cell’s metabolism and make the cell’s
physiology start working for the invading virus.

 A protein within bacterium called Hha has the ability to control whether virus genes are kept within the
organism or unloaded. When Hha is basically “turned on,” bacteria expel virus genes, opting for motility
over the ability to form biofilms. Likewise, when Hha is not expressed, bacteria move slower, but grow
biofilms at a much faster rate. The viral prophage (CP4-57) helps its host to attach to the surface to
form a biofilm, and also helps to generate and promote diversity inside the biofilm community.

                                     Viruses participate in biofilm too.

 The genes of E. coli have been sequenced and they contain nine or ten viral remnants that no longer
function as virus. Usually a bacterium will discard genes that it no longer needs, but in this case this life
form did not dump these leftover viral genetic fossils. E. coli has kept these tools that encourage
survival of its colony. These tools allow the cell to dismantle itself, preserving useful parts via apoptosis.



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This allows some planktonic cells to escape threatened biofilm or it can possibly protect itself by partly
dismantling, shrinking and entering a spore-like hypometabolic hibernation state for long-term survival.

 Bacteria in biofilm are connected to each other by a polymer, DNA, sucrose or protein. In order for
some reproductive cells to be released when conditions change, others near the surface must die by
apoptotic programmed cell death, where useful cellular parts are reclaimed.

 Another reason for the bacterium to keep and utilize viral DNA in order to commit apoptosis (kill itself)
is that when another virus attacks that particular cell it can eliminate itself ‘altruistically’ via cell suicide
before the infecting virus can take over its cellular machinery, replicate, multiply and spread its viral
progeny to the entire biofilm colony.

 Perhaps antibiotics do not primarily kill cells; more often metabolic derangements produced by
antibiotics actually trigger apoptotic suicide. The strongest argument for programmed death of
defective cells comes from the finding of genes (such as hip, vncS and sulA) that dramatically affect
survival to antibiotics without changing growth susceptibility.

 Antibiotics are low molecular weight products of bacteria and fungi that have long been used as
treatments for infectious and other diseases. It seems that these compounds have quite different roles
in the environment compared to therapeutic applications. The difference lies in the available
concentrations that determine inhibitory activity compared to transcription modulation. Low
concentrations may be responsible for cell-cell signaling activity in microbial communities.

 In flowing cold mountain streams, there might be only nine bacteria floating in a milliliter of water, but
a hundred million live in structured harmony in one square centimeter of biofilm slime covering the
rocks over which the water rushes. Attachment is possible because of catch bonds, which are
noncovalent biological adhesive bonds with the counterintuitive property that they are longer-lived
when mechanical force is applied to pull them apart.

A bacterial adhesive protein called FimH forms catch bonds. Bacteria binding through FimH will only
bind firmly at high levels of fluid flow, and will roll across the surface or detach if the flow is stopped.
FimH is expressed on most of our intestinal bacteria and is involved in urinary tract infections.

 Each person, each animal and every plant or rock provides ecological niches for its own characteristic
symbiotic, interactive and mostly beneficial microflora communities. A tree does not stand alone. It is
part of a whole ecosystem of virally, bacterially and insect laden soil containing yeasts decaying organic
matter as well as supporting the growth of weeds. Oxygen and carbon dioxide is exchanged. The
insects, birds and animals that seek shelter in the tree fertilize it and the soil as well.

                      We are not individuals, but cooperative symbiotic ecosystems.

 A typical mobile human host (supporting its ecosystems) consists of roughly a trillion cooperative
communicating eukaryotic ceils developed and organized by less than 30,000 genes. Each human also
hosts a biofilm which is an assemblage of surface-associated microbial cells enclosed in various
organized extracellular polymeric polysaccharide matrices consisting of about 10 trillion communicating

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cooperative prokaryotic cells, with their roughly 300,000 genes. If we add DNA and RNA viruses as well
as yeasts to the number of bacteria, our biofilms may total 100 trillion life forms.

Humans may be seen as super organisms in which many bacterial genomes (forming a metagenome)
work in tandem with our own. The NIH has estimated that 90% of the cells in Homo sapiens are
microbial and not human in origin.

 Perhaps the fundamental problem in our thinking is the concept that human tissue is sterile, free from
microorganisms, such as viruses, yeast or bacteria, unless there is overt infection. Part of the sterile
assumption derives from the intense inflammatory response to some microorganisms. In order for
bacteria to survive in tissue, they escape detection or suppress inflammation and the tissue must
tolerate the slow leaching of their inflammatory antigenic molecular metabolic markers.

 We can no longer assume that antibodies generated in autoimmune disease are created solely as
autoantibodies to human DNA. New evidence shows that the human microbiota accumulates during a
lifetime, and a variety of biofilm persistence mechanisms are now understood.

 In one model, obstruction of VDR nuclear–receptor-transcription prevents the innate immune system
from making key antimicrobials, allowing the microbes to persist. Genes from these microbial biofilms
must necessarily impact disease progression. Newly directed efforts to decrease this VDR-perverting
microbiota in patients with autoimmune disease have resulted in reversal of autoimmune processes.

Commensal and probiotic biofilms in the colonic mucosa act as a prominent stimulus for epithelial cell
development and differentiation, and that cross-talk among bacteria, and between bacteria and
epithelium provide fundamental signaling in gut physiology. All mammals are adapted to life in a
microbial world and are colonized by bacteria on all their body surfaces at birth.

 The stimulation by commensal bacterial antigens is critical for normal development of the mucosal
immunity and the maintenance of tolerance. In fact, animals that are kept germ-free from birth have
dysfunctional immune systems; development is somehow stunted and their energy requirements are
abnormally elevated.

 The intercommunication between the gut flora biofilms, the cells of the immune system juxtaposed
with the intestinal endothelium and cryptic bacteria/tissue biofilms produces stable chronic
inflammatory disease. Disrupting gut biofilms may permit a resumption of effective immunity and
remission.

 The human vermiform ("worm-like") appendix is a 5-10cm long and 0.5-1cm wide pouch that extends
from the cecum of the large bowel. The design of the human appendix is unique among mammals, and
few mammals other than humans have an appendix at all.

The function of the human appendix has been a matter of much debate (including its function as an
escape-valve for flatus). It is often considered a vestige of evolutionary development despite contrary
evidence based on comparative primate anatomy. The appendix seems to have some immune function
based on its association with substantial lymphatic tissue, and it can help make, direct and train white

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blood cells. Its removal increases risk to Chron’s disease, an autoimmune disease, in which the body's
deregulated immune system attacks its own gastrointestinal tract, causing inflammation; it is classified
as a type of inflammatory bowel disease.

 Based (a) on a new understanding of immune-mediated biofilm formation by commensal bacteria in the
mammalian gut, (b) on biofilm distribution in the large bowel, (c) the association of lymphoid tissue with
the appendix, (d) the ability of biofilms to protect and support colonization by commensal bacteria, and
(e) on the architecture of the human bowel, it seems that the human appendix functions as a "safe
house" for commensal bacteria. It provides support for commensal bacterial growth and for potential
re-inoculation of the colon in the event of purging of its contents following exposure to a pathogen.

                                 Immune recognition of friend and foe

Innate cellular immunity has generally been considered a non-specific immune response characterized
mainly by phagocytosis. However, we now know that innate immunity has substantial specificity, and is
capable of discriminating between individual species of microbes. Pathogens “seen” as dangerous to
the host elicit an inflammatory response capable of destroying the microbes, while commensals do not
bring forth such a response and their survival is tolerated or even encouraged by the host.

 This immune discrimination is attained through the recognition of multiple microbe-specific surface
molecules by pattern-recognition receptors (PRRs) present on mucosal cells. There are several types of
PRRs including the nucleotide binding oligomerization domain family of proteins (Nod1, Nod2), Toll-like
receptors as well as the receptors for complement, glucans and mannose.

 Innate immunity specifically recognizes molecular patterns of microorganisms through Toll-like
receptors (TLRs) and other pattern-recognition receptors, and use of pattern-recognition receptors
helps regulate the development of antigen-specific adaptive immune responses. TLR-dependent
recognition of commensal bacteria is required for reducing inflammation and maintenance of immune
intestinal homeostasis.

 The PRR-recognized molecules on the microbes include surface proteins, nucleic acids and
carbohydrates (e.g., lipopolysaccharide, peptidoglycan, lipoteichoic acids) and host factors that bind to
the microbial surfaces include complement fragments and salivary components. Such “patterns” are
called microbe-associated molecular patterns (MAMPs) and the innate immune system is thought to
recognize at least 1000 MAMPs. MAMPs include molecular patterns of commensals and pathogens
(PAMPs).

 Pattern-recognition receptors interactions with pathogen microbe-associated molecular patterns
trigger a complex set of intracellular signaling cascades that ultimately result in expression of
antimicrobial factors as well as pro-inflammatory molecules. These responses include activation of
complement, coagulation, phagocytosis, inflammation and apoptosis.

In addition, the innate immune system assists the adaptive immune system in recognition of microbial
antigens and production of a robust antibody response. Dysfunction in the discrimination between oral
or digestive commensal and pathogenic microbes could lead to either an ineffective immune response

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and infection by relatively harmless microbes, or a hyperactive immune response characterized by
inflammation and host tissue destruction.

 Among the adaptive immune cell populations in the intestinal mucosa, the best-characterized cells are
the secretory IgA-producing plasma cells. Normal intestinal mucosa contains abundant SIgA-secreting
plasma cells, and the secreted IgA plays a critical role in the host defense against pathogenic bacteria.
More importantly, SIgA regulates the ecological balance of commensal bacteria. SIgA and the mucus of
the large bowel may actually be involved in pro-microbial activity.

 EpiCor (dried yeast fermentate) can boost levels of secretory IgA (the crucial immunoglobulin that coats
mucosal pathways like mouth, nose and eyes) thus reinforcing your body’s defense against invaders
before they even enter your body. At the same time, EpiCor also enhances circulating natural killer (NK)
cells’ ability to destroy harmful pathogens with both speed and efficiency. It also provides significant
antioxidant protection and safe, natural inflammation control.

                                 Colonizing communication chemistry

 Quorum sensing genes respond to signals from numbers of bacteria present and environmental
changes as well as respond according to influences from ever-present local RNA’s interpretation of the
environment, and cause the bacteria to settle down and organize. Human and microbial cells both
genetically respond in order to optimize metabolism to meet current environmental needs based on
RNA messaging. Structured biofilm forms our outer protective barrier layer, teaches our immune
system tolerance and creates the digestive ‘soil’ that nurtures us.

 Furanones were first isolated from marine algae and are thought to be part of the plant’s natural
defense against microbial attack. Chemotaxis, motility and flagella gene expression in bacteria is post
transcriptionally repressed by furanones.

 Brominated furanones block quorum sensing by acyl homoserine lactones, signal molecules used by
Gram-negative bacteria (often pathogenic in humans since lipopolysaccharides in their cell walls mimic
endotoxin). Furanone-based quorum sensing inhibitors increase sensitivity of Pseudomonas aeruginosa
biofilms to antibiotics and improve clearance of these same bacteria from a mouse model of lung
infection.

 The indigenous, 'normal' microflora can in response to changed surroundings, become pathogenic and
then cause localized infectious diseases of the oral cavity (e.g., dental caries, alveolar abscesses,
periodontal diseases and candidiasis). This same microflora, when in balance, also protects the host
from exogenous pathogens by stimulating a vigorous immune response and providing colonization
resistance.

Symbiosis means 'life together.' Symbiosis is capable of continuous change as determined by selective
pressures of the environmental milieu. Mutuality symbiosis, where both the host and the indigenous
microflora benefit from the association may shift to a parasitic symbiosis, where the host is damaged
and the evolved indigenous microflora flourishes.


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 Complex microbial consortiums, existing as a biofilm, often provide the interfaces that initiate and
perpetuate infectious assault on host tissue. The ecological balance of the various microhabitats is
critical for the development of the appropriate selecting milieu for pathogens.

 The microbiota associated with dental caries progression is largely influenced by immune imbalance
and loss of pH buffering capacity, whereas periodontal diseases and pulpal infection appear to be due to
reduced energy production or redox potential, thus diminishing cellular immunity and heightening
destructive humoral immunity. Candidiasis results from immune-suppression host factors that favor
yeast overgrowth (like low vitamin C) or lack of bacterial competition caused by antibiotics.

 Oral health or disease as well as adequate digestion depends on microbial adaptation to prevailing
conditions; prevention of endogenous oral or digestive disease can occur only when we realize that
ecology is the heart of these host-symbiont relationships.

Enterohaemorrhagic Escherichia coli (EHEC) colonization will occur if it encounters a favorable signal
(e.g., the stress hormone norepinephrine). However, because the gastrointestinal tract nonpathogenic
flora is not uniform, it is also likely that the pathogen encounters a signal that inhibits colonization (e.g.,
indole). If the pathogen encounters both signals simultaneously, the extent of colonization will depend
on the dominant signal.

Autoinducer-2 (AI-2) is the signal utilized by E. coli for colonization inside a hot-blooded body. AI-2
helps warm E. coli produce more biofilm by making colonic acid, which is a sugar that forms the ‘mortar’
of the bacterial bedroom.

 Indole is used as a signal for biofilm growth outside of the body (for instance on cooler surgical
replacement parts yet to be implanted). E. coli can convert tryptophan into indole. A slightly modified
indole, 7-hydroxyindole, turns off the bacterium's ability to communicate and organize.

 Dehydration initiates exaggerated histamine production as a water-regulating control. Asthmatics have
excessive levels of histamines in their lung tissue, causing constriction of the bronchial passages and
increased mucus build-up, encouraging biofilm build up.

 Inflammation easily clears planktonic bacteria (who try to avoid triggering inflammatory response). In
contrast, sessile bacteria in biofilm engender inflammatory exudates for nutrition. Steroids rob biofilms
of food by reducing inflammatory exudates, allowing some chronic wounds to heal.

Planktonic bacteria are controlled by the inflammatory response, allowing a clot to seal and heal an
open wound. But an established biofilm in a wound creates a proteolytic environment that prevents
post-debridement blood clotting, creating a non-healing wound.

Fibrinogen and fibronectin both enhance Staphylococcus aureus binding and inhibit Staphylococcus
epidermidis or Gram-negative bacteria adherence, while whole blood promotes Pseudomonas
aeruginosa biofilm formation.




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                                  Body and biofilm are interconnected.

 Acidic polysaccharides are produced by bacteria and divalent cations cross-link the polysaccharides into
a matrix. The bacteria have agglutinins to attach to the matrix. Gut pathogens produce agglutinins that
they use to attach to the heparan sulfate (HS), the predominant acid polysaccharide of the intestinal
epithelium.

 Mast cells of the intestines normally release heparin, which is a mixture of HS fragments, to stick to the
agglutinins and block attachment to the HS of the epithelium. Numerous bacterial species form complex
communities on the polysaccharide matrix and prevent access by antibiotics.

 Several pathogenic intracellular micro-organisms that attack oral endothelium are now being
investigated for triggering the development of cardiovascular disease in the vascular endothelium.
Many mechanisms involved in the chronic intimal inflammatory process that is atherosclerosis are
mediated by the same cellular mechanisms that are "primed" to protect the body from chronic microbial
invasion.

 The herpes variant cytomegalovirus (CMV), carried in the body of 60-99 % of people, raises levels of
inflammation markers, increases blood vessel inflammation and causes high blood pressure in mice. An
enzyme that causes high blood pressure, rennin is elevated in the kidneys of mice and the blood vessels
of humans infected with CMV.

 Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immune
inflammatory mechanisms are involved. Inflammation is central at all stages of atherosclerosis. When
endothelium is activated, expressing chemokines and adhesion molecules, monocyte/lymphocyte
recruitment occurs with foam cell infiltration into the subendothelium, creating formation of early fatty
streaks.

 It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque
secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading
to rupture and thrombus formation. Cells involved in the atherosclerotic process secrete and are
activated by cytokines.

 New understanding of the mechanisms of atherosclerosis provided evidence that the immune
inflammatory response in atherosclerosis is modulated by regulatory pathways, in which the two anti-
inflammatory cytokines interleukin-10 and transforming growth factor- play a critical role.

 Regulatory (suppressor) T cells play a critical role in the control of the immune-inflammatory response
in atherosclerosis and substantially limit lesion development. Measles virus infection or vaccination is
associated with immune depression, in part through the induction of an anti-inflammatory response by
measles virus nucleoprotein.

 Repetitive administration of measles virus nucleoprotein to apolipoprotein E–deficient mice promotes
an anti-inflammatory T-regulatory-cell type 1–like response and inhibits macrophage and T-cell
accumulation within atherosclerotic lesions. Treatment with measles virus nucleoprotein significantly

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reduces the development of new atherosclerotic plaques and markedly inhibits the progression of
established lesions. The protective effects on lesion size are lost in mice with lymphocyte deficiency.

 Gut biofilms support system-wide chronic inflammation that leads to allergies, autoimmune diseases,
degenerative diseases and probably cancers. This attachment on the intestinal wall also produces a
leaky gut that supplies the bacteria that are moved by GI macrophages to all parts of the body. This may
be how Chlamydia pneumoniae colonizes sites of inflammation throughout the body.

 A strong association exists between coronary heart disease and Chlamydia pneumoniae, a Gram-
negative respiratory pathogen. Viable C. pneumoniae has been isolated from atherosclerotic plaques.
Whereas C. pneumoniae can be transported from the lung to the arteries through macrophages, oral
organisms are introduced into the bloodstream multiple times daily in individuals with periodontitis
through chewing or tooth brushing.

 The mouth holds a potentially large reservoir of Gram-negative pathogenic organisms that could readily
interact with cardiovascular tissues. P. gingivalis is the predominant species among anaerobic bacteria
in bacteremia after dental procedures. P. gingivalis heat shock protein-specific T-cell lines have also
been isolated from atheroma lesions.

 Invasive periodontal pathogens are present at sites of atherosclerotic disease. Their presence has been
demonstrated with DNA levels. Living P. gingivalis and A. actinomycetemcomitans can invade host cells.
Either exogenous microbial pathogen-associated molecular patterns and/or endogenous molecules
carrying antigenic messaging (heat shock proteins, ß2-glycoprotein-I, oxidized low-density lipoprotein
(LDL) and related phospholipids via molecular mimicry) may activate innate immune responses that
create the atherosclerotic process.

Accelerated atherosclerosis is associated with herpes viral infection both in transplant patients and after
balloon angioplasty. Marek's disease virus (MDV) is a herpes virus that induces accelerated
atherosclerosis associated with the development of invasive lymphoma in hyperlipemic roosters.
Striking, grossly visible atherosclerotic lesions were seen in large coronary arteries, aortas and major
aortic branches of infected normocholesterolemic and hypercholesterolemic chickens, but not in
matched uninfected chickens.

 Vascular endothelium may be a site of latent herpetic viral infection, and activation of such infection
might cause or aggravate atherosclerosis (think of herpetic mouth ulcers or of bubbling, crusting lesions
on the lips). There is widespread and persistent infection of human populations with up to five different
herpes viruses. Infected endothelium may be damaged by marginated inflammatory cells, and be
transformed from an anticoagulant to a pro-coagulant tissue.

 The benefit of vitamin D supplementation on reducing atherosclerotic plaque (a biofilm) is substantial.
Vitamin D was replaced using gel cap forms (for assured absorption, not obtainable with powder-based
tablet forms) as a nutritional supplement at a dose sufficient to maintain a serum level of 25-hydroxy
vitamin D of 50-60 ng/ml, requiring a mean dose of 3590 units per day. Participants were instructed to
take their dose in the morning with meals. Mean serum D3 levels increased 83%.

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 Vitamin D has known anti-viral properties. The mechanisms of vitamin D’s action with regards to
coronary atherosclerotic plaque are many. Vitamin D exerts suppressive effects at several points in
inflammatory pathways (e.g., suppression of matrix metalloproteinase-9, reduced C-reactive protein),
reduction in blood sugar and enhanced insulin responsiveness, reduced blood pressure via an
angiotensin-inhibiting mechanism. Vitamin D deficiency has been related to increased risk of
cardiovascular events and cardiovascular death repeatedly.

 Vitamin A helps regulate the immune system, which helps prevent or fight off infections by making
white blood cells that destroy harmful bacteria and viruses. Vitamin A also helps lymphocytes fight
infections more effectively. Vitamin D also helps maintain a healthy immune system and helps regulate
cell growth and differentiation, the process that determines what a cell is to become. Vitamin Es and Ks
are both also beneficial to intimal health.

                        Biofilm also houses yeast, a primitive eukaryocyte like us.

 Central to yeast infections is the biofilm, a population of microbial eukaryocytes, C. albicans cells, joined
together to form structured sheets of cells. Candida albicans is an opportunistic pathogen that infects
primarily immune-compromised hosts.

 The zinc-responsive regulatory protein Zap1 prevents the production of soluble b-1,3 glucan, a sugar
that is a major component of matrix. Other genes have been identified whose expression is controlled
by Zap1, called Zap1 target genes. These genes encode two types of enzymes, glucoamylases and
alcohol dehydrogenases, which both control the production and maturation of matrix components.

 Biofilms are very varied, with some areas being both highly hydrated and hydrophilic and others being
hydrophobic. They contain micro colonies of bacterial cells encased in their own extracellular polymeric
extracellular polysaccharide matrix and separated from other micro colonies by interstitial voids (water
channels). Some internal parts have low oxygen and are anaerobic. Surface areas may be aerobic.
Biofilm architecture is diverse both in space and time, constantly changing because of external and
internal communicated processes.



                              Biofilm is sentient and our ‘significant other’.

 Biofilm in its niche becomes a purposeful ecological community. As cells become sessile, diversification
is encouraged, seemingly an expression of altruism for the community. Each cell contributes to a larger
cooperative structure forming an organism with the will to survive, exhibiting consciousness and
seemingly even freedom of choice.

 Both we (and this group of ‘significant others’, our biofilm in its various recesses) meet the requirement
of sentience, the ability to feel pleasure and pain as well as the ability to communicate that perception.
Our biofilm seems to have its own mind, displaying surprising levels of creativity, intelligence, even
wisdom, self-awareness and intentionality.


                                                     12
 Biofilm has the ability to communicate its desires to us by making us crave and seek certain foods, thus
take action and alter our behavior. In Eastern philosophy, sentience is a metaphysical quality of
consciousness in all things (perhaps all surfaces covered by biofilm) that requires our respect and care.

 ‘Classification tree analysis’ through DNA samples of saliva microbiological composition showed that
98.4% of overweight women could be identified by the presence of a single bacterial species
(Selenomonas noxia) at levels greater than 1.05% of the total salivary bacteria.

 Likely the composition of salivary bacteria changes in overweight women, as the median percentage
difference of 7 of the 40 bacterial species measured was greater than 2% in the saliva of overweight
women. These bacterial species might serve as biological indicators of a developing overweight
condition. More intriguing, and the subject of future research, is the possibility that oral bacteria may
participate in the pathology that leads to obesity.

 Included among the many jobs performed by our intestinal bacteria is that of extracting calories and
nutrients from the foods we eat. They also store them for later use, as well as making sure there is
sufficient nourishment to produce new bacteria to perform the same job. Differences in our gut
microbial ecology may determine how many calories we are able to extract and absorb from our diet
and deposit in our fat cells.

 Individuals who are prone to weight gain tend to have greater numbers of gut bacteria called firmicutes
and fewer numbers of another type of bacteria called bacteroidetes. These two major groups account
for the majority of the microbes in the human digestive tract, more than 90%. As folks lose weight, the
ratio of bacteria shifts; bacteroidetes becomes more abundant, while the number of firmicutes lessens.

 A protozoan, toxoplasma gondii alters brain chemistry of rats so they are more likely to seek out cats.
This is a fatal mistake since the parasite (toxoplasma) reproduces in the cat. Roughly 60,000,000
‘symptom-free’ Americans carry toxoplasmas which are immune-suppressive, and alter
neurotransmitter function with effects from subtle shifts in behavior to schizophrenia (especially if there
are a lot of cats around). Behavioral changes in humans include slower reaction times and a 6-fold
increased risk of traffic accidents among those infected. Ummm, perhaps this explains some of the
habits of the ‘cat lady’ we all know.

 Geography of T. gondii infection (preference for raw meat in France) shows links with ‘neuroticism.’ In
the human host, the parasites form tissue cysts within cells (escaping immune detection), most
commonly in skeletal muscle, myocardium, brain and eyes. These cysts may remain throughout the life
of the host.

 A high titer of T. gondii antibodies in mothers skews newborn’s sex ratio more strongly to the male
(260/100) with normal being (104/100). Congenital infection occurs if mom is infected during
pregnancy. Toxoxplasma gondii tachyzoites can cross the placenta to the fetus which may lead to
spontaneous abortion, stillbirths or severe birth defects, especially of the female child. Early diagnosis
and treatment of the mother may reduce the probability of congenital infection.



                                                    13
 Fibrin limits hemorrhagic blood loss during infection by the protozoan parasite Toxoplasma gondii,
thereby performing a host-protective function essential for survival. Surprisingly, fibrin does not simply
protect against vascular damage caused directly by the parasite, but rather, protects against
hemorrhagic damage triggered by IFN-gamma, a messenger molecule of our immune systems.

Coagulation has a beneficial role during immunity. Fibrin protects our tissue from collateral damage
caused by our immune response to infection, by limiting hemorrhagic pathology as well as smothering
growth and dissemination of bacteria. Pathological coagulation may result from dysregulation of
cytokine pathways that are meant to function protectively.

 Fibrin's dense mesh-like structure likely physically traps bacteria, thereby curbing dissemination.
Interestingly, this fibrin-mediated restraint of bacterial growth and dissemination applies both to
infection by bacteria that predominantly replicate extracellularly (ex. Staphylococcus aureus) and
intracellularly (ex. Listeria monocytogenes).

 Host resistance to this protozoan parasite depends on a Th1 immune response with potent production
of the cytokines interleukin-12 and interferon . Although Toll-like receptor 11 (TLR11) plays a major
role in controlling Th1 immunity to this pathogen in mice, this innate immune receptor is nonfunctional
in humans, and the mechanisms of TLR11-independent sensing of T. gondii remain elusive.

 Oral infection by T. gondii triggers a TLR11-independent but MyD88-dependent Th1 response that is
impaired in TLR2xTLR4 double knockout and TLR9 single knockout mice. These mucosal innate and
adaptive immune responses to T. gondii rely on the indirect stimulation of dendritic cells by normal gut
microflora.

 Thus, in humans, gut commensal bacteria serve as molecular adjuvants during parasitic infection,
providing indirect immune stimulation that protects against T. gondii in the absence of TLR11.
Encouraging the production of our natural gut flora through probiotic use tends to help stimulate our
immune systems to protect us against the ubiquitous protozoan parasite Toxoplasma gondii.

 When microbes communicate with any of the thousands of sensory nerve cells that lie within the gut
walls, either directly or through nearby mobile macrophages, it would influence immune response and
the emotions. Supporting evidence has been found in rodent experiments. Beneficial biofilm boosts
our general sense of well being due to friendly bacterial communication via ‘toll receptors’ on immune
cells and the vagus nerve system. Kefir, the ancient Turkish word for goat milk fermented with
beneficial flora, literally means ‘feel good.’

                                            Pathogenic biofilm

 Virulence characteristics of biofilms that influence infectious disease processes include detachment of
cells or biofilm aggregates that may result in bloodstream or urinary tract infections or in the production
of emboli; cells may exchange virulence and resistance plasmids, a form of horizontal gene transfer
within biofilms; cells in biofilms have dramatically reduced susceptibility to antimicrobial agents; biofilm-



                                                     14
associated gram-negative bacteria may produce endotoxins and biofilms are resistant to host immune
system clearance.

 Variants of the species E.coli are adapted to various host organisms, such as humans, monkeys, horses
and birds, in which they belong to the normal intestinal flora. In addition, pathogenic strains (perhaps
virally infected) have the capacity to cause sepsis or local infections of the intestines, as well as of the
kidney, bladder and brain, in different hosts.

 On the other hand, high doses of non-pathogenic E. coli bacteria can function as a strong, direct
inhibitor of mast cell degranulation. This suggests a basis for antiallergic treatment (modulating
exaggerated humoral immunity) or prevention, with commensal bacteria.

 German professor Alfred Nissle, in 1917, isolated a strain of E. coli from the feces of a World War I
soldier who did not develop enterocolitis during a severe outbreak of shigellosis. In those days,
antibiotics were not yet discovered, and Nissle used the strain with considerable success in acute cases
of infectious intestinal diseases (salmonellosis and shigellosis). Escherichia coli Nissle 1917 is still in use
(as Mutaflor) and is one of the few examples of a non-lactobacillus probiotic.

 Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA
elements contributing to specific virulence traits and also in the presence and absence of additional
genetic information. Horizontal gene transfer and gene reduction represent two mechanisms
contributing to the evolution of prokaryotic genomes “in quantum leaps”. Thus, the acquisition of
plasmids and phages, as well as large DNA regions called “genomic islands,” plays an important role in
the development of new species, subspecies as well as pathogenic characteristics of known species.

Many microbes can accelerate the rate at which their genes mutate. This allows them to obtain new
abilities that may be helpful when conditions get tough. Escherichia coli mutates more rapidly when
under stress, and yeast can perform the same trick.

The localization of many virulence-associated genes on mobile genetic elements, such as
bacteriophages, plasmids and pathogenicity islands (PAIs), reveals that horizontal gene transfer plays a
major role in the evolution of different bacterial pathogenic types. Genetic diversity among pathogenic
and commensal E. coli isolates is very high with respect to genomic alterations.

 Plasminogen (Plg) is a proenzyme of the serine protease plasmin, which is involved in several important
physiological and pathological processes such as fibrinolysis, degradation of the extracellular matrix
(ECM), eukaryotic cell migration, tissue remodeling, embryonic development and inflammation as well
as tumor metastasis.

 Plasmin is central for cell migration as it directly degrades laminin, the major glycoprotein in basement
membranes, and indirectly enhances tissue damage by activating latent matrix metalloproteases
(MMPs) which are capable of degrading collagens and other constituents of ECM.




                                                       15
Basement membranes form important tissue barriers and also offer a setting where components of the
plasminogen system are present and can be activated, thus the proteolytic activity of plasmin is
efficiently targeted to these physiological barrier structures.

 Invasive bacterial, yeast and parasitic pathogens can intervene with and usurp our plasminogen system
by expressing receptor molecules which enable the microorganisms to gain surface-bound invasive
proteolytic activity. On the other hand, plasminogen is modified in the presence of beneficial L.
crispatus and Lactobacillus johnsonii cells into internal plasminogen fragments which included
angiostatin, known to suppress endothelial cell proliferation and tumor metastasis.

                                Really small life forms exist in the biofilm.

 Mycoplasmas (L-form or cell-wall deficient bacteria) sometimes originate from bacteria which have
shed all or part of their cell wall or had it destroyed by antibiotics. Many chronic diseases may be due to
smaller infectious bacteria which transform into the mycoplasma state (kind of small bacteria acting like
a virus) and live intracellularly to better survive inside the body (often even infecting our immune cells).

 Infected phagocytes circulate in blood and tissues. In the extreme case of sarcoidosis they clump
together and form granulomas, clusters of phagocytes without normal supporting structure. They are
also capable of accumulating in regions of inflammation such as joints. When a phagocyte becomes
infected, intra-cellular bacteria can manufacture a lot more proteins, cytokines and toxins than they
could if they had infected a red cell, as the cell nucleus and mitochondria allow them access to Homo
sapiens mRNA transcription, and Homo sapiens nutrients.

 Regular bacteria stressed with starvation pack their DNA into tight bundles, shut down virtually all their
activities & shrink down to about 1/3 normal size. These ultra micro bacteria can remain in this dormant
state for decades, or possibly even centuries, floating around in the deep oceans or buried far
underground (and they have surprisingly survived years of outer space travel).

 These self-mummifying bacteria outmaneuver death by transforming themselves into a metabolically
suspended quasi-crystal: the endospore. The endospore is a downward spiral that can go upward, a
tautological emergency exit into blissful core energy, a survival strategy that only prolonged boiling can
tear apart. Once environmental conditions again favor normal metabolism, the endospores return to
life as if the last 60 hours or 6000 years never happened.

 The beta-lactam antibiotics (cephalosporins and penicillins) that attack cell walls are deadly to free-
swimming blood-borne bacteria but ineffective against Cell Wall Deficient (CWD) bacteria. In fact, they
actually promote formation (by destroying bacterial cell walls) of the tiny L-form CWD bacteria which
have been seen living inside phagocytes. Because the beta-lactams actually protect the CWD bacteria,
they are even used in test tube when culturing and growing the tiny L-forms.

  CWD mycoplasmas are associated with many different conditions as chronic fatigue syndrome,
fibromyalgia, TB, Gulf War Illness, AIDS, rheumatoid arthritis and even certain forms of cancer. The tests
used to identify mycoplasmal infections, Forensic Polymerase Chain Reaction and Nucleoprotein Gene


                                                     16
Tracking, are very sensitive and highly specific. Mycoplasmal biofilm infections can explain much if not
most of the chronic signs and symptoms found in these challenging, chronically ill, difficult to diagnose
patients.

 The pleomorphic cancer causative agent has been described as having virus-like and fungus-like, as well
as bacterial mycoplasma-like phases. Such a “life cycle” that would allow survival of microorganisms
under attack is considered nonsense and microbiologic heresy by conventional approach, but reflects
real observations of the biofilm. Could a simple deficiency of friendly commensal microflora caused by
taking antibiotics or anti-infective botanicals/minerals, or experiencing overwhelming stress at any point
in one's life, promote disease-causing chronic inflammation, biofilm micro bacterial infections and ignite
the process of carcinogenesis?

                                Beneficial viruses and childhood diseases

 The 3-D structure of Seneca Valley Virus-001, shows that it is different from any other known member
of the Picornaviridae viral family. It is dissimilar from other known picornaviruses such as poliovirus and
rhinoviruses (which cause the common cold). The ‘new’ Senecavirus has an outer protein shell that
looks like a craggy golf ball (one with uneven divets and raised spikes) and the RNA strand beneath it is
arranged in a round mesh shaped like a whiffle ball.

 The Seneca virus does not affect normal human cells, but can infect certain solid tumors, such as small
cell lung cancer, the most common form of lung cancer. Several areas on the viral protein coat likely
hook onto receptors on cancer cells in the process of infecting them. This virus has a cancer-killing
specificity that is 10,000 times higher than that seen in traditional chemotherapeutics, with no overt
toxicity.

 Mumps is a common childhood disease which is benign in the in the vast majority of cases. It is
preferable that mumps be contracted in early childhood because, when experienced in adulthood, the
disease may cause meningitis and/or damage to the testes, ovaries, auditory nerves or pancreas. Also,
notably, women are less likely to experience ovarian cancer if they were infected with mumps during
childhood.

 Adults who had had natural measles with a rash have a decreased incidence of various cancers,
including cervical. After contracting measles and other childhood illnesses (e.g.. chickenpox, scarlet
fever, whooping cough, rubella, mumps and perhaps others), it has been widely accepted by many
health practitioners, including experienced orthodox pediatricians that this is most often beneficial for
the general health of our children. Children contracting measles naturally become less likely to suffer
later from allergic conditions such as asthma, eczema and hay fever.

 An acute inflammatory childhood illness (measles, mumps, rubella, chicken pox, scarlatina or whooping
cough) develops the cell-mediated immune system, while a vaccine activates the humoral immune
system. The difference is crucial because it is the cell-mediated response that is first line of response to
protect from future illness and that provides, in effect, the deeper immunity. Humoral immunity fosters
antibodies, allergy as well as auto-immune disease like diabetes, migraine and asthma.

                                                    17
 Physicians who practice Anthroposophical medicine foundationally believe that having acute but limited
inflammatory diseases as a child helps protect one as an adult against more serious, long-term, chronic
illnesses. Not having these childhood illnesses (because of multiple vaccinations) can lead to diminished
cellular immunity and a greater incidence of adult health problems. The same result occurs when
childhood illnesses are routinely suppressed with antibiotics rather than helping the cell-mediated
immune system to eliminate the illness with fever, sweats, rash or mucous discharge.

                         Periodontitis, nanobacteria and immune dysregulation

 Periodontitis is thought to arise from complex microflora consisting of putative disease causing bacteria,
such as P. gingivalis, T. denticola, Tannerella forsythia (formerly Bacteroides forsythus or Tannerella
forsythensis) and Fusobacterium spp. These bacteria have been co-isolated from diseased periodontal
sites, and periodontitis is maintained by cooperation between the different species.

 Perhaps these oral bacteria are simply attracted to low-oxygen inflammatory sites of auto-immune
destruction. These tooth-bound avascular pockets of periodontal devastation are created by
uncontrolled upregulated humoral response to antigenic cellular immune messaging left behind on
forming root surfaces (neuroectodermal cells called rests of Malasez that remain due to disabled
developmentally-planned apoptotic elimination of these enamel-like root guiding cells).

 In addition to P. gingivalis, oral treponemes have been implicated in periodontitis and oral malodor.
These helically shaped spirochetes (which can also exist in very small cyst or cell wall deficient forms) are
present in significantly elevated numbers in plaque samples from deep-pocket sites of patients with
severe periodontitis. Most oral spirochetes either cannot yet be grown or are extremely difficult to grow
in culture because they have lost the ability to synthesize many essential molecules that they normally
obtain from their host.

 Even smaller are the mycoplasmas which may have the smallest of the prokaryotic genomes and seem
to lack complex gene-regulatory systems. Biofilm formation by Mycoplasma pulmonis is dependent on
the length of the tandem repeat region of its variable surface antigen (Vsa) protein. Mycoplasmas that
produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and
glass (with an extracellular matrix containing Vsa protein, lipid, DNA and saccharide). Mycoplasmas that
produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the
medium.

 Even smaller are the nanobacteria, perhaps the most primitive organisms on Earth. Nanobacteria are
structurally and physiologically unique in many ways: they are 20-200 nanometers in size; they have a
unique “cellular” and membrane structure. They replicate very slowly (every 3-5 days) and by different
methods. In contrast, typical bacteria mitotically double about every 20 minutes.

Nanobacteria are pleomorphic, assuming different life forms and sizes for different phases and activities
of their lives; they can go dormant in a self-made calcium shell. They are saprophytic in humans, feeding
on dead or decaying organic matter. They are “the toughest of bugs” resistant to being killed both In



                                                     18
vitro and In vivo and may be at the root cause of many human diseases. They are structurally and
functionally simplistic, unbelievably small and genetically unique.

 Nanobacteria excrete a slick calcium biofilm around themselves that subsequently hardens, creating a
calcium shell “igloo” around them. They can build upon themselves in this calcified form much like coral
formation.

 Pathological calcification in humans, like coronary artery heart plaque, vascular plaque, soft tissue
calcifications, dental tartar and pulp stones, kidney stones, polycystic kidney disease, arthritis as well as
chronic prostatitis and even dermatology disorders (eczema, psoriasis, lichen planus and scleroderma),
may be biofilm-generated calcification, plausibly created by nanobacteria or mycoplasma in their
microscopically mixed pleomorphic concreted colonies. Nanobacteria have even been found in the
calcified debris found in tissue biopsies from ovarian cancer patients.

 Biologic nanoparticles (NPs) isolated from human arterial calcifications and kidney stones form an
adherent film on the bottom of flasks when placed in standard cell culture conditions. Since NPs have
certain features consistent with living microorganisms, perhaps antibiotics and/or RNAse might inhibit
this biofilm formation.

 NPs isolated from calcified human arterial tissue were cultured onto glass cover slips, plus either no
treatment (control), RNase, tetracycline or gentamicin added. At 1, 7 and 14 days after inoculation,
cover slips were fixed, mounted and analyzed. In controls, the percent cover slip covered by biofilm
increased by 120% from day 1 to day 14, compared to 47% and 35% in wells treated with RNase and
gentamicin, respectively.

 Wells treated with tetracycline showed a 3% decrease in biofilm by day 14. Since RNase should not
enter intact cells, these results indicate that extracellular RNA plays a role in biofilm formation by NPs.
Although tetracycline can chelate calcium, it also inhibits ribosomal function, as does gentamicin,
indicating that intact ribosomal function is required for NP-associated biofilm growth. Biofilm formation
by NPs then consists of biologically driven physicochemical processes.

 Nanobacteria can easily cross the blood-brain barrier to cause brain calcification disorders and “brain
sand”. Small self-replicating structures containing nucleic acids were cultured from filtered
homogenates of two calcified aneurysms. Almost 100% of atherosclerotic patients have anti-
nanobacteria antibodies in their serum, while in healthy blood donors anti-nanobacteria antibodies are
seen in about 15%.

 Nanobacteria cause “apoptosis” or cell-death in tissues they contact. The antigenic properties of the
structures identified as Nanobacterium sanguineum might be explained as humoral antibody response
to a number of biomolecules simply adsorbed onto the surface of the biofilm’s hydroxyapatite
microcrystals. However, arteries without atherosclerotic plaque have 20-50 fold increase in Vitamin K2
concentration over arteries with plaque in the same human body.




                                                     19
Calcium deposits are not random. They aggregate around the remnants of dead (”apoptotic”) smooth
muscle cells. Vitamin K2 supports at least two proteins that are likely to protect against atherosclerosis:
matrix Gla proteins and growth arrest specific gene product 6 (Gas6).

Gas6 increases cell survival and helps clear away any fragments left behind by cells that do happen to
die. When vitamin K2 levels are adequate, two of the Gla-proteins that are activated are: (1)
osteocalcin, the protein responsible for anchoring calcium within bone, and (2) matrix Gla-protein,
which prevents calcium from depositing in the heart, arteries, breast and kidneys.

 Nanobacteria do not grow in standard bacteriologic culture conditions. They alter RNA and DNA and
transcription expression of the cells they grow on or in. Their small size, slow grow rate, and unusual
culture requirements explain why Nanobacterium sanguineum has until recently eluded detection, as
they can only be visualized frozen under an electron microscope. They are calcifying self-propagating
nanoparticles that may be part of a larger bacterial life cycle.

 Known ultra micro-bacteria are sized at about 200-300nm. The theoretical minimum size for a free-
living organism (capable of holding the minimal molecular group of about 250-450 proteins, genes and
ribosomes) would be 250-300nm in diameter, about the same size of the ultra micro-bacteria. Indeed,
even a single ribosome, if surrounded by membrane and wall, would occupy a viral-sized sphere of
57nm in diameter

 Nanobacteria may represent a primitive Archaea symbiont that requires cell contact or lipids from other
cells for growth, and are only about 1/100th the size of conventional bacteria, at 20-150nm.
Coincidentally, this is also the standard size of commercially produced hydroxyapatite nanocrystals.
Perhaps the particles identified as the living organism Nanobacterium sanguineum might actually be
non-living, but self-generating inorganic particles of calcium hydroxyapatite (once they have been
complexed with nucleic acids, proteins and other ionic molecules of the biofilm).

 Organic materials play key roles as nucleating surfaces, triggering crystal growth in the bio
mineralization of apatite, in addition to modulating and finally inhibiting the process. Such crystal
growth is enhanced markedly in low gravitational environments (and astronauts returning to earth are
very prone to calcific atherosclerosis).

 Nanobacteria cause production of human antibodies in response to their biofilm, but when our cellular
defense systems arrive, they cannot “see” anything because these bacteria are too small; it’s like
fighting a war against invisible men. Nanobacterial biofilm triggers an immune system hyperalert
process, which causes chronic inflammation to spread systemically from tissues locally affected by the
bacterial antigens.

 These parasitic bacteria are also called pleomorphic (many shapes) or L-form (named for the Lister
Institute where they were discovered) or Cell Wall Deficient (CWD) or cell wall divergent or cell-wall
defective or large bodies or cryptic or nanobacteria, micro bacteria or spores.




                                                    20
 We are exposed to CWD pathogens in our food/milk (they are not killed by pasteurization), water (they
are not killed by chlorination), intimate contact (spouses are at higher risk), before birth (via sperm), at
birth (mother to child transmission) and biologic (injectable) medicines (they are too small to be filtered
during the 'purification' processes used in pharmaceutical manufacturing procedures). They have even
been cultured from dry soil. L-forms of Bacillus anthracis (Anthrax) are known to survive in dry soil
indefinitely.

 The Th1 (T-helper) inflammatory response occurs in reaction to the invasion of cells by extremely tiny
variant bacteria. The body’s defenses trigger several medical markers of inflammation, including C-
reactive protein (CRP). Elevated CRP levels are a major risk factor and harbinger of coronary artery
disease and stroke. Many human degenerative disease processes including cancer, have been
associated with chronic inflammatory pathological calcification, likely due to biofilm containing
nanobacteria.

 Dr. Gary Mezo has proposed that anti-nanobacterial therapy might help treat coronary artery disease,
as well as other disease states associated with abnormal, extra-skeletal calcification. His study protocol
involves the nightly administration of 1500mg of a rectal EDTA suppository (a calcium and heavy metal
chelating agent), coupled with an oral prescription of 500mg tetracycline (an antibiotic with metal
chelating and anti-inflammatory properties) taken orally for three to twelve months. (This therapy
would be effective at debulking and dismantling most biofilms.)



                                            Immune tolerance

The difference between friendly commensal biofilm and harmful pathologic biofilm is its local
environment, partly controlled by the host’s individual DNA and RNA heredity with resultant physiology,
hygiene habits, stress levels, diet, sleep, daily rhythm, heavy metals and other toxicities as well as
onboard viruses.

 A newly found gene, pims has been found to be expressed during bacterial infection in the gut. It
disrupts the pro-inflammatory Imd pathway, signifying a role in modulation of this pathway. Pims is a
negative immune-regulator triggered when specific Imd activation thresholds are reached, after which
the immune response is suppressed. It is the existence of this immune-reactivity threshold that allows
coincident existence of tolerance to commensal gut microorganisms while maintaining immune-
reactivity against infective pathogens.

The mechanism of oral tolerance partly exists because mucosal tissues are replete with a unique subset
of antigen-presenting dendritic cells (macrophages) that secrete factors such as, TGF-beta1 and retinoic
acid, that induce immunosuppressive regulatory T cells or Tregs (foxp3+ regulatory T cells).

 T cell regulation is partly mediated by secretion of soluble IL-10 (Tr1 cells) and/or TGF-b (from Th3
suppressor or regulatory cells) after antigen-specific triggering. Tr1 cells, which suppress colitis and are
induced by IL-10, mediate regulation, in part, by secretion of TGF-b. Both Th3 and Tr1 cells are induced


                                                     21
via the mucosal route, thru different compartments. IL-10 and TGF-b are important regulatory cytokines
(TGF-b-deficient animals have widespread inflammation and IL-10-deficient animals develop colitis).

 The primary putative pathogens causing periodontitis are Porphyromonas gingivalis and Bacteroides
forsythus. Half of young adults with severe inflammatory periodontitis are seronegative for antigens of
their infecting bacteria. In treating inflamed gingiva, removal of bacterial deposits from roots of teeth
via scaling and root planning results in marked clinical improvement.

 Prophylaxis induces gingival bleeding resulting in bacteremia. This causes seronegative patients to
seroconvert and produce higher titers of biologically more effective serum antibody (mostly IgG2
subclass), thus reducing biofilm accumulation and gingival inflammation.

  Dietary proteins are taken up preferentially by gatekeeper dendritic cells in the lamina propria of the
small intestine. These then migrate to interact with antigen-specific CD4+ T cells in the mesenteric
lymph nodes, inducing tolerance to the mucosal immune system under physiological conditions, but
being sufficiently responsive to inflammatory stimuli to allow T cell priming and protective immunity
when necessary.

 Expression profiles of human mucosal gene expression patterns display striking differences in
modulation of pro-inflammatory NF-kB-dependent pathways, notably after consumption of living L.
plantarum bacteria in different growth phases. These cellular pathways correlate with immune
tolerance in healthy adults.

 Classically, commensal microbiota establishes tolerance at mucosal surfaces, and invasive pathogens
cause stereotypic inflammation. The reality is more complex, marked by three emerging concepts: (1)
pathogens take advantage of inflammation to cross the epithelial barrier, (2) pathogens reduce the
commensal flora to invade their niche, and (3) pathogens express dedicated effectors that modulate
inflammation.

 Gut pathogens in combination with stimulation by cytokines such as TNF-alpha (tumor necrosis factor)
can cause cells of the intestinal epithelium to respond by releasing more proinflammatory messenger
molecules like interleukin-8 (IL-8). On the other hand, probiotic strains, Bifidobacterium longum and
Lactobacillus bulgaricus, can suppress IL-8 secretion in intestinal epithelia when stimulated by
proinflammatory cytokines, down-regulating inflammation in the gut.

 In mice, pre-existing persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13
prevents the induction of tolerance, mixed chimerism and donor-reactive T cell deletion. Mice continue
to be refractory to tolerance induction even after viremia has been resolved and virus is present only at
very low levels in peripheral tissues.

 Conversely, the full tolerance regimen, or co stimulation blockade alone (CD28 and/or CD40 blockade-
based strategies to induce tolerance), specifically inhibits already ongoing antiviral immune responses,
leading to an inability to control viremia. Ongoing T cell responses continue to depend on co stimulatory



                                                    22
interactions in the setting of a chronic infection and provide insight into potential risks following co
stimulation blockade posed by chronic or latent viral infections such as hepatitis C, EBV and CMV.

 Innate immune responses and inflammation are regulated in part by neural mechanisms. Innate
immunity and inflammation are controlled by the vagus nerve, classically known as a regulator of other
vital physiological functions. When vagus nerve function is diminished, inflammation soars, inviting
biofilms to grow and become pathogenic. (The relaxation effects of neurally derived NO appear to
inhibit ongoing vagal cholinergic activity. The primary site of action of nitrergic mechanisms on gastric
fundic tone is at a presynaptic site on vagal cholinergic efferent nerves.)

 Activation of vagus nerve cholinergic signaling inhibits TNF (tumor necrosis factor) and other pro-
inflammatory cytokine overproduction through ‘immune’ α7 nicotinic receptor-mediated mechanisms.
This efferent vagus nerve-based ‘cholinergic anti-inflammatory pathway’ is a critical regulator of
inflammation in several experimental models of diseases.

 Acupuncture, meditation, hypnosis and relaxation therapies can stimulate vagus nerve activity.
Exercise raises vagus nerve firing and decreases inflammatory cytokine levels. Fish oil, soy oil and olive
oil increase vagus nerve activity mediated by cholecystokinin. Choline helps to form acetylcholine, the
neurotransmitter of the efferent vagus.

 Nicotine is anti-inflammatory by acting on the acetylcholine receptors normally responsive to
acetylcholine released by the vagus nerve. Macrophages also have receptors for the neurotransmitter
acetylcholine that is released by branches of the vagus nerve in the intestines. Nicotine blocks
inflammation triggered by LPS. (LPS typically signals a macrophage, NF-kB becomes activated,
inflammatory genes are expressed, mediators are secreted and tissue inflammation ensues.)

                                      Restore an imbalanced biofilm.

 Chrysanthemum tea contains choline, vitamin A, B1, glycosides, adenine, amino acids, flavonoids,
pigments and volatile oils. It has an inhibiting effect on bacteria, including Staphylococcus aureus,
Streptococcus hemolyticus B, Pseudomonas aeruginosa, Shigella dysenteriae, tubercle bacillus and
dermatomycosis. The tea also has antivirus and antispirochete qualities.

  Honeysuckle flower is one of the most potent anti-bacterials of nature’s “antibiotics”. Honeysuckle
flowers (from what some consider an invasive weed) are effective against bacteria, viruses, fungi and
parasites. Honeysuckle is also be used to treat infections caused by staphylococcal or streptococcal
bacteria.

 Chlorogenic acid, an ester of caffeic acid and quinic acid, found in honeysuckle, is also a major phenolic
compound in coffee, and can be isolated from the leaves and fruit of many plants. This well known
antioxidant also slows the release of glucose into the bloodstream after a meal.

Chlorogenic acid can be used as anti-infectious active ingredient, it has wide anti-virus, anti-bacteria
effects and has relatively low toxicity or side-effects. Some Japanese health food products include


                                                     23
honeysuckle for treating bloating, nausea, and vomiting caused by hepatitis C. It has obvious anti-
infectious effects, and unlikely to lead to anti-microbial resistance.

  Honeysuckle is best used for acute conditions, and is not generally used in the treatment of chronic
illnesses. This herb can be used for all infections and inflammations, including urinary tract infections,
respiratory tract infections and some gastrointestinal tract inflammations. It is also used for fevers, the
common cold, sore throat and influenza. Honeysuckle flowers act as natural antihistamine, although they
are more useful for treating rashes and inflammation than for treating coughing and sneezing.

 Honeysuckle is a very useful remedy for persistent acne, and all sorts of skin eruptions. Simply add to a
cup of hot/boiling water. Add a little honey if you wish for taste. To eliminate a skin boil, mash
honeysuckle flowers into a paste, place on top of the boil with a gauze covering the area.

 In comparison between semi (black tea) and non-fermented tea shoots, biofilm growth inhibitory
concentration of oral streptococci was lower for semi fermented Camellia sinensis extract and its
antimicrobial activity was better as compared to green tea. Bactericidal effect of both tea extracts was
clear. Drinking green tea while taking antibiotics appears to increase the action of antibiotics and
reduce the drug resistance of bacteria, even in superbug strains.

 Community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) , necrotizing (“flesh
eating") pneumonia, can be fatal in many cases. Besides causing a high fever, CA-MRSA pneumonia can
sometimes cause blood pressure to drop and progress to septic shock, requiring patients to be placed
on mechanical respirators in order to breathe. Potentially deadly CA-MRSA pneumonia seems to occur
most commonly following a flu-type illness, like the H1Ni virus.

 When bacteria are exposed to antibiotics, some cells that are resistant to these drugs live on to
reproduce. Thus, while antibiotics are important for disease treatment, their use creates stronger,
more-resistant strains of bacteria over time. The amount of antibiotics used non-therapeutically in
animal agriculture is eight times greater than the amount used in all of human medicine.

 Many of the antibiotics used in animal agriculture are also used in human medicine. While overuse in
human medicine is a significant part of the problem, 70% of all U.S. antibiotics and related drugs are fed
non- therapeutically to livestock, poultry and fish on industrial farms (as a routine feed additive to
promote slightly faster growth and to compensate for unsanitary and crowded conditions). This is an
idiotic way to encourage bacteria to develop antibiotic resistance that can easily be spread about.

 In comparison between non-fermented and semi fermented Camellia sinensis extracts, the black tea
(with more volatile components) has faster effect on growth inhibition of oral streptococci. In case of 3
mg mL-1 of black tea extract, after 30 minutes the count of viable cells was below 1 log of cfu mL-1 and
this amount achieved by same amount of green tea extract took 40 minutes.

 Epigallocatechin gallate (EGCg), the primary polyphenol of green tea, has several antibacterial qualities.
Tea catechins can fight or even destroy the bacteria that cause cholera, pneumonia, abscesses,
botulism, dysentery and food poisoning, as well as those that cause cavities and bad breath. Catechins
can inhibit the action of the flu virus, herpes simplex, polio, HIV and others.

                                                     24
 Catechins inhibit the growth of the disease-causing bacteria, especially Clostridium perfringens (a
common cause of food poisoning), Clostridium difficile (which is linked to killer diarrhea and colitis), and
Bacteroides (which can cause abscesses if bacteria escape from the intestines). But the gut’s “friendly”
bacteria, including Bifidobacterium and Lactobacillus, are relatively unaffected by the tea catechins.

 EGCg interferes with the polysaccharides that form the glycocalyx, disrupting their interactions either
reciprocally or with the cell wall, thus reducing the amount of biofilm slime that accumulates. However,
EGCg also binds to peptidoglycan, breaking the integrity of the bacterial cell wall, and could therefore
interfere with the initial docking phase of biofilm formation, which requires hydrophobic interactions
between the bacterial cell wall and the surface to be colonized.

                                Nature’s acids versus pathogenic bacteria

 Lactic acid fermentation is used by some fungi and bacteria. The most important lactic acid producing
bacteria is Lactobacillus. The presence of lactic acid, produced during lactic acid fermentation is
responsible for the sour taste and for improved microbiological stability and safety of food.

 Re-establishing predominance of these bacteria in the biofilm helps return a pathogenic microbial
community to its symbiotic commensal role. Breast-feeding can be a significant source of lactic acid
bacteria to the infant gut. Lactic acid bacteria present in breast milk has an internal origin (not
contamination from the surrounding breast skin).

 Lactic acid fermentation is responsible for the sour taste of dairy products such as cheese, yogurt and
kefir. Lactic acid fermentation also gives the sour taste to fermented vegetables such as traditionally
cultured sauerkraut and pickles. The sugars in the cabbage are converted into lactic acid and serve as a
preservative.

 One common lichen (which is a symbiosis of algae and fungus) metabolite, usnic acid inhibits carotenoid
synthesis. Usnic acid has antihistaminic, antiviral and antibacterial activities.

 Usnea (usnea longissima, hair                       lichen) combines the properties of both lichen and algae.
It is anti-viral, antiseptic, antibiotic, antibacterial and anti-tumor. It is more powerful than penicillin
against both staphylococcus and streptococcus bacteria and works by changing cellular metabolism. As
usnea has no known contraindications or side effects it is a popular herbal antibiotic remedy (often
combined with golden seal, wild indigo, myrrh or witch hazel depending on the nature of the problem
treated).

 Lechenya Meera (a proprietary blend of lichens, mosses, fungi and algae) which is raw harvested and
cold-pressed with other natural substances, is a natural anti-septic and disinfectant. Lichen is a moss-
like plant that grows primarily in cold and moist environments. The lichen and other ingredients are
blended when the fungus and alga in their dual relationship produce active acids, which create anti-
microbial byproducts, effective against various bacteria (staph and strep). It is also effective as a surface
treatment for mildew and molds in the more humid areas of the world.



                                                      25
  Total Solution 2000 (containing Lechenya Meera) attacks the microbe, bacteria or virus, by suffocating
it and especially by depriving it of "biofilm," the primary protection and food source of bacteria. It
affects both bacteria and viruses by stopping their reproductive processes and sterilizes the bacterial
spore or the virus so that it cannot reproduce, mutate or cross-contaminate. With both feeding and
reproduction shut down, bacteria are neutralized so that they are no longer harmful and they soon die.

RESPIRATORY MIST is a special solution prepared for use on mucous membranes, ears and the eyes. It
relieves sinus congestion, bronchial disturbances and stuffed up nose. It is useful for treating filters for
furnaces and air conditioners creating a residual effect that acts like an air freshener. It contains Witch
Hazel, Lechenya Meera, Stabilized Oxygen, Citronella and Pennyroyal in an alkaline water base.

FIX (2004): is a special fermented Lechenya Meera to be used to relieve upset stomach, diarrhea, protect
against toxins in food, counteract indigestion, correct allergic reactions replace and restore electrolytes
and beneficial flora to the digestive system. Call Suzy Star, at (541) 736-5150 / suzystar@earthlink.net.

  The black seeds of papaya are edible and have a sharp, spicy taste. They are sometimes ground up and
used as a substitute for black pepper. Both fruit and seed extracts have pronounced bactericidal activity
against Staphylococcus aureus, Bacillus cereus, Escherischia coli, Pseudomonas aeruginosa and Shigella
flexneri. After a course of antibiotic therapy, papaya juice rapidly returns intestinal bacteria to normal.

                                          Life-threatening sepsis

  Lipopolysaccharides (which mimic endotoxin) from the bacterial cell wall are a potent stimulus for
Tumor Necrosis Factor-alpha (TNF-α) synthesis. TNF-α acts as a key mediator in the local inflammatory
immune response by initiating a cascade of cytokine release, recruiting macrophages to the site of
infection, and cause blood clotting to attempt to contain the infection. TNF-α amplifies and prolongs
the inflammatory response.

 Originally, sepsis was believed to result from invading bacteria itself, but it was later found that the
host’s system proteins, like TNF-α and HMGB1, induced sepsis as an exaggerated immune response.
Sepsis is caused either when cytokine production exponentially increases to an extent that it escapes the
local infection or when uncontrolled infection enters the circulation. Victims of septic shock experience
fever, plummeting blood pressure, myocardial suppression, headache, dehydration, kidney failure and
respiratory arrest. This exaggerated immune response causes body organs to fail and death may result
from lethal septic shock.

 Staph aureus is a normal commensal inhabitant of skin and mucus linings. When stressed by a low zinc
or magnesium environment, it changes its metabolism, making more lipopolysaccharides to create a
thicker cell wall. These cell wall lipopolysaccharides mimic one of our own cytokines, endotoxin, which
even at extremely low concentrations is a major immune-system messenger of alarm.

  Women whose diets are low in zinc or magnesium or may be a bit bulimic or have diarrhea or be
taking mineral-wasting diuretics will have lower tissue levels of these important minerals. If a super-
absorbent tampon is used, that further lowers level of zinc and magnesium locally causing the normal


                                                     26
commensal staph aureus to change its metabolism in response to this stress and thicken its
lipopolysaccharide cell wall, making many more mimics of endotoxin. Such lipopolysaccharide signaling
can create an overwhelming crescendo of immune response, sometimes leading to toxic shock
syndrome and death.

   Exhaustion of reduced glutathione is the septic switch that shuts down cellular immunity, while
   encouraging uncontrolled inflammation, which signals biofilm to become pathogenic triggering
            responsive unbridled allergic response as well as auto-immune destruction.

 Zinc and magnesium are also critical for recreating reduced glutathione (GSH). Lack of GSH, this
primary hydrogen ion supplier, reduces energy production dimming the electromagnetic barrier
produced by our aerobic cells while at the same time slowing activity of immune white blood cells.

 Lack of reduced glutathione shuts down phagocytic cellular immunity since the white blood cell
becomes sluggish and can neither turn on its ‘killer’ metabolism nor protect itself from its own purifying
oxidative burst. Blocks in the ability to recycle glutathione become the ‘septic switch’ that encourages
pathogenicity of one’s resident biofilm. Then, to compound the damage, commonly prescribed
antibiotics often further wipe out the friendly flora designed to compete against yeast overgrowth or
viral attacks like influenza.

 Activation of central (brain) cholinergic transmission by selective muscarinic receptor ligands results in
lower systemic TNF levels in rodents and indicates that the efferent vagus nerve provides a functional
brain-to-immune system connection. Central cholinergic signaling significantly activates the cholinergic
anti-inflammatory pathway.

When cellular immunity becomes depressed, humoral immunity is then responsively turned on, (and
untethered due to lack of reduced glutathione) with its hypersensitivities and allergies caused by
increased antibody production, along with heightened expression of normally suppressed onboard
bacteria, yeasts and viruses as well as unbridled auto-immune destruction.

 Reduced glutathione is so critically foundational for cellular function that multiple genes and three
major chemical pathways create, regenerate and support its production. Multiple toxicities (mercury
poisons a key enzyme) or deficiencies must be present before GSH (reduced glutathione) stores are
compromised. Vitamins C and E recycle oxidized glutathione. Hardly anybody eats enough fresh fruit or
whole grains with vitamin E. ‘Which straw broke the camel’s back?’

 Recycling oxidized glutathione to reduced glutathione is heavily dependent on all methylating B
vitamins as well as foundationally on methionine, niacin, riboflavin and pyridoxine. Zinc, selenium or
magnesium are also critical cofactors. NAC (n-acetyl cysteine), glutamine, glycine, taurine, selenium,
milk thistle, regular and fermented garlic, ginger, turmeric and its curcurmin, balloon flower root, guggal
and Hawthorne all help recycle glutathione.

 Lipoic acid at (100-200mg/day) independently helps recycle glutathione and increases levels 30%. Take
lipoic acid along with biotin for best effect, at 50mcg biotin for every 100mg lipoic acid.


                                                    27
 Larger doses of lipoic acid (like NAC) in ranges of 600-3,000mg/day may also mobilize heavy metals and
toxins creating irritability, low blood sugars, rashes and cognitive difficulty. Reduce dose to lessen
negative symptoms of redistribution; drink lots of water; stoke multiminerals; eat seaweeds, greens,
avocados and raw egg yolks; use clays and activated charcoal to sop up poisons. Friendly bacteria and
extra fiber detoxify and open up excretion channels.

                            Biofilm life-forms want glutathione and SOD too.

 Immunity often weakens due to opportunistic onboard RNA viruses chronically replicating and
encoding to produce their own protective glutathione peroxidase, reducing our supplies. Many viruses
and bacteria either encode for and make glutathione or acquire (steal) it or its ingredients directly from
their host.

 Most life forms use glutathione as a universal mechanism for protection from free radicals, toxicity and
radiation. Included are HIV 1 & 2, Coxsackie B, Hepatitis B & C, some herpes and other viruses along
with many forms of bacteria.

 Next among the most important regulators of reactive oxygen species levels are the superoxide
dismutase (SOD) enzymes: copper/zinc SOD functioning in the cytoplasm and outer mitochondrial space,
and manganese SOD existing exclusively in the inner mitochondrial space. Superoxide is converted to
hydrogen peroxide (H2O2) and O2 by SOD. Peroxiredoxins and abundant catalase enzyme then scavenge
the hydrogen peroxide, converting it to molecular oxygen and water.

 Microorganisms can also hijack SOD for their own use, then making our mitochondria reject the
normally sustaining, but now damaging oxygen (due to SOD lack), disabling energy production, creating
chronic fatigue, fibromyalgia with compromised detoxification and altered immunity along with multiple
environmental sensitivities.

 When a phagocytic cell attacks defective cell, virus, bacteria or antigenic food information, its
metabolism revs up a thousand fold, using peroxisome produced peroxides to dismantle its foe(s). If
short of pivotal glutathione reserves, the white blood cell cannot generate an explosion of energy or
protect itself from its own peroxidative burst and cellular immunity shuts down.

 The cellularly-undefended allergic or hypersensitive state called humoral immunity then
compensatorily switches on. Plaque begins accumulating in the mouth or the yeast itch and/or burning
begins; or prodromal awareness starts with a brewing expression of herpes outbreak that may create
ulcers in mucus membranes, fissures in the tongue or clusters of painfully bubbling hives on the lips or
skin. Next, that injured joint or tooth that was just a minor annoyance begins to add to the
inflammatory cacophony.

 Virus replication competes for and removes onboard glutathione precursors: selenium, glutamine,
cysteine and tryptophan or glycine from the host. This creates deficiencies that disable immune and
neuroendocrine function at multiple levels. Virally infected ‘putative’ bacterial periodontal pathogens



                                                    28
(also found in arterial plaque) are five times (if Epstein-Barr co-infected) to thirty times (if Herpes-6 co-
infected) more pathogenic.

 The herpes virus (human cytomegalovirus, HCMV and Epstein-Barr virus, EBV) are often involved in
periodontal diseases. Genomes of HCMV and EBV occur at high frequency in aggressive, HIV-associated,
ANUG, and advanced type periodontitis associated with medical disorders. HCMV infects periodontal
monocytes/macrophages and lymphocytes, and EBV infects periodontal B-lymphocytes. Herpes virus-
infected inflammatory cells may elicit tissue-destroying cytokines and thus create diminished ability to
defend against bacterial challenge.

 Viruses and viroids are the RNA ribosomal world responding to the environment we are both in,
choosing various expressions of our species’ DNA and our individual biofilm’s DNA. Virally-induced
nutritional deficiency is expressed as spiraling-downward worsening disease. Aggressive periodontal
disease is a significant ‘septic’ break down in immunity, not that different from other AIDS-related
infections, requiring significant changes in diet and lifestyle to restore rhythm and repair cellular
immunity for real reversal.

                                      More biofilm control strategies

 The therapeutic effect of hyperbaric oxygen (HBO) is related to elevated partial oxygen pressure in the
tissues. HBO both alone and in combination with scaling and root planning of the teeth (SRP) reduced
the Gingival Index value to zero and gingival health persisted for 3 months at least. Thus, in parallel with
the loss of periodontal pathogenic bacteria, a substantial improvement in oral health was observed.

 The combination of HBO and SRP substantially reduced (by up to 99.9%) gram-negative anaerobe loads
of subgingival microflora. The low values of pathogens persisted for at least two months after the
therapy. HBO or SRP alone produced a temporarily more limited effect on periodontal anaerobes. HBO
both alone and in combination with SRP reduced the Gingival Index value to zero and gingival health
persisted for 3 months at least.



                             Phosphate sequestration theory of dental caries

 Both bacteria and we use large amounts of phosphorous to make ATP (adenosine triphosphate), the
biochemical currency of energy. We make roughly half our body weight in ATP each day. When we
consume large amounts of sugar, lots of ATP is generated, reducing the amount of available needed
phosphorous for the benign biofilm plaque living on teeth. Taking large doses of calcium can also
deplete available phosphorous.

 By changing its metabolism to generate more acid, dental plaque, the mouth’s cooperative biofilm can
conveniently release and obtain newly needed phosphorus by dissolving the calcium phosphate
hydroxyapatite crystal of adjacent teeth.




                                                      29
 Besides lowering available phosphorus, excessive sugar (or just too many calories) fuels the germs while
it creates a spiked release of cortisol and other stress hormones. This causes patrolling mobile white
blood cells to sequester themselves in the liver and spleen, thus removing them from actively policing
the barrier tissues and tubes (arteries, veins, digestive system, ducts, bladders as well as oral and
nasopharyngeal membranes). Stress in all of its guises shuts down cellular immunity, responsively
encouraging hypersensitivities, auto-immunity and development of pathogenic biofilm.

                                      Other anti-plaque strategies

 Polyphenols occurring in cocoa, coffee and tea have a role in the prevention of cariogenic processes,
due to their antibacterial action. Cocoa polyphenol pentamers significantly reduce biofilm formation
and acid production by Streptococcus mutans and S. sanguinis. In the same way, trigonelline, caffeine
and chlorogenic acid occurring in green and roasted coffee interfere with S. mutans adsorption to saliva-
coated hydroxyapatite beads.

Studies on green, oolong and black teas show that tea polyphenols exert an anti-caries effect via an
anti-microbial mode-of-action, and galloyl esters of (-)-epicatechin, (-)-epigallocatechin and (-)-
gallocatechin show increasing antibacterial activities. The anti-cariogenic effects against alpha-
hemolytic streptococci showed by polyphenols from cocoa, coffee and tea suggest use of these
beverages in the prevention of caries.

 Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are three major etiological
agents of chronic periodontitis. The strong proteolytic activities of these bacteria are critical to their
survival since their energy source is obtained from peptides and amino acids derived from proteins. In
addition, proteases are important factors potentially contributing to periodontal tissue destruction by a
variety of mechanisms, including direct tissue degradation and modulation of host inflammatory
responses.

Non-dialyzable material prepared from cranberry juice concentrate reduces either the proliferation of P.
gingivalis, T. forsythia and T. denticola in periodontal pockets or their proteinase-mediated destructive
process occurring in periodontitis.

 Ethanol extracts from the peels of mangosteen (Garcinia mangostana L.) showed strong inhibition of
acid production by S. mutans. Garcinia mangostana L extract in ethanol strongly inhibited the biofilm
formation of S. mutans. Its deep purple rind contains fulxanthones and polysaccharides, which have
powerful antibacterial effects, with in vitro activity against a range of antibiotic-resistant bacteria.
Mangosteen compounds also help inhibit IgE-mediated histamine release and inhibit development of
pre-neoplastic lesions.

                   Biofilm survival strategy creates non-healing wounds and cancer.

 Once established to critical mass, biofilm infections circumvent the host immune system to promote
chronicity in wounds. The biofilm in wounds can actually hijack host immunity to help its pathogens
survive and remain in their niche within the wound. In contrast to the most commonly accepted


                                                    30
hypothesis of host-centered pathology, it is quite possible that bacteria in an unclean wound, not host
dysfunction, cause the chronicity and perpetual inflammation associated with chronic non-healing
wounds.

 Biofilm formation in wounds is the best unifying explanation for the failure of a variety of chronic
wounds to heal. Clinical evidence has shown improved healing when chronic wounds are treated with
the assumption that biofilm is the cause of the failure to heal.

 Cancer is a non-healing wound, and shares many characteristics with pathogenic biofilm. It turns out
that the promising anti-cancer angiogenesis (VEGF) inhibitors do reduce blood supply to tumors and
thus shrink them, extending life a few months. But then the strategies of the tumor changes (just like a
biofilm), and it seeds and metastasizes.

 Dr. T. Simoncini, an oncologist in Rome, Italy has pioneered sodium bicarbonate (NaHCO3) therapy as a
means to treat cancer. The fundamental theory behind this treatment is that, despite a number of
variable factors, the formation and spreading of tumors seems simply the result of the presence of an
invasive fungal biofilm. Dandruff, yeasty dental plaque, athlete’s foot and nail fungus are common
‘contained’ fungal biofilm infections.

For about 100 years, the fundamental theory behind cancer has been based on the theory that it is a
malfunctioning of the genes. This point of view implies that cancer is primarily genetic and intracellular.
Dr. Simoncini thinks that cancer is often a chronic fungal biofilm infection, and therefore primarily an
extra cellular phenomenon, then triggering genetic changes in responsive stem cells.

 Sodium bicarbonate, unlike other anti-fungal remedies to which fungus can become tolerant or
resistant, is extremely diffusible and retains its ability to penetrate the tumor, due to the speed at which
the sodium bicarbonate disintegrates the tumor. This speed makes fungi’s adaptability impossible,
rendering it defenseless.

 The sodium bicarbonate solution is administered directly on the tumor, if possible. Otherwise, it can
be administered by selective arteriography, which basically means selecting specific arteries through
which the solution is administered, which subsequently dissolves the tumor.

 Molecular analyses of chronic wound specimens divulge diverse polymicrobial communities and the
presence of bacteria, including strictly anaerobic bacteria, rarely revealed by culture. Bacterial biofilm
prevalence in specimens from chronic wounds relative to acute wounds provides evidence that
established bacterial biofilms occur commonly in chronic wounds and are unusual in acute wounds.

                                         Natural biofilm therapy

Honey may be the best antibacterial dressing for a surface wound, infected or not. Manuka is the best
healer of the honeys. Garlic kills antibiotic-resistant bacteria too in its fermented or cooked forms (raw
garlic or undiluted garlic oil can burn).




                                                     31
 Aloe is another famous skin dressing for soothing burns or controlling infections. Aloe mucilaginous
polysaccharide (AMP) molecules from the gel promote healing and balance immune response of barrier
tissues for internal use. The gel and leaves of the plants contain natural irritants and anthraquinones
which speed healing topically.

 Small chains of aloe mucilaginous polysaccharides provide anti-Inflammatory messaging, helping heal
colitis and arthritis. Medium chains of AMPs act as beneficial antioxidants for the heart and arteries.
Large chains are anti-bacterial and anti-viral. Very large chains improve and support immune system:
healing properties by attracting white cells, releasing chemo tactic factor, tumor necrosis factor as well
as increasing natural killer cells.

 Aloe speeds digestive tract healing. Aloe mucilaginous polysaccharides are not broken down in the
digestive tract; rather they are completely engulfed in the cell wall and transported across the intestinal
cell wall, which protects the length of the chain by providing the beneficial properties a way to move
directly into the bloodstream.

 Staphylococcus epidermidis is one of the main causes of medical device-related infections owing to its
adhesion and biofilm-forming abilities on biomaterial surfaces. Berberine is an isoquinoline-type
alkaloid isolated from Coptidis rhizoma (huang lian in Chinese) and other herbs (Barberry and Oregon
grape root) with many activities against various disorders. Modest concentrations of berberine (30–45
μg/mL) seem sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly.

                                        We are all in this together.

Most prokaryocytes (viruses, bacteria and life forms in between) live in their tissue-specific ecological
niche as integral parts of structured symbiotic biofilms along with their characteristic viruses, both
beneficial and parasitic interlaced with some beneficial (yet potentially pathogenic) yeast, the most
primitive eukaryocyte along with some protists.

All of these life forms have their characteristic parasites as well, waiting to take advantage of the weak.
Bacteria within biofilms may be subject to predation by free-living protozoa, bacteriophages and
phagocytic polymorphonuclear leukocytes. Amoebae act similarly in the sea as immune cells in the
human body; they look for and feed on bacteria.

 The protists include a variety of unicellular, coenocytic, colonial and multicellular eukaryocytic
organisms, such as the protozoans, slime molds, brown algae and red algae. Protistan grazing enhances
yeast biofilm metabolism and increases biofilm biomass and viability. The extracellular polymeric matrix
of biofilms may act as an interface regulating interaction between predator and prey, while serving as
source of nutrients and energy for protists.

                                Control is a fantasy necessary for health.

 Since large doses of antibiotics cannot eradicate biofilm infections (and cultures were mostly clinically
useless) low-dose pulsed or periodic dosing of antibiotics is now currently being medically
recommended to diminish the bulk of biofilms and lower their pathogenic expression.

                                                    32
 In the mouth, the topical antibiotic chlorhexidine gluconate (CHX) reduces plaque formation. Exposure
to 0.2% CHX first causes the biofilm to defensively contract (perhaps creating dormant persister cells
that are “essentially invulnerable to killing”) and also reduces viability profiles through the biofilms after
a delay of 3 to 5 minutes.

 Anti-candidal activity of carvacrol and eugenol, the major phenolic components of oregano and clove
essential oils, respectively, were tested in an animal model, and effectively eliminated vaginal
candidiasis. Oregano essential oil showed a good in vitro activity in inhibiting and eradicating biofilm
produced by staphylococci strains commonly involved in medical-devices related infection.

The effect of eugenol on adherent cells and subsequent biofilm formation is dependent on the initial
adherence time and the concentration of this compound. Eugenol can also inhibit filamentous growth
of C. albicans cells. Eugenol shows low hemolytic activity, signifying low cytotoxicity (using human
erythrocytes) and is beneficial in treating biofilm-associated candida infections.

                                            Helicobacter pylorii

In the mucus-rich stomach, urease-negative H. pylori planktonic growth is favored over pathogenic
biofilm formation. The herb marshmallow or the powdered inner bark of slippery elm has mucilaginous
qualities, as do soaked flax or chia seeds. Folks carrying H. pylori strains with CagA-negative genes have
double the risk to esophageal adenocarcinoma as those with commensal CagA-positive H. pylori.

Flagella (perhaps also virally encoded into the formerly friendly bacterial genome) have a significant role
during initial step of pathogenic H. pylori biofilm formation. Dense, mature biofilms become attached to
the cell surface by urease-positive ammonia-producing H. pylori-positive specimens (that buffer stomach
acid).

 The production of a water-insoluble biofilm by H. pylori is important in enhancing resistance to host
defense factors and antibiotics, as well as providing micro environmental buffering of pH, thus
facilitating the growth and survival of H.pylori in vivo. Curcurmin has a significant effect against H. pylori
biofilm and shown a significant inhibitory action on adherence of H. pylori to human cancer lines of HEp-
2 cells.

 While 25-30% of people in the U.S. carry H. pylori in their stomachs, 80% show no obvious signs of
distress. ‘Infection’ rates are much higher in Japan, at nearly 90%, due in part to crowding. Fifty people
in Japan ate either 2.5 ounces of broccoli sprouts or 2.5 ounces of alfalfa sprouts each day for two
months. Broccoli, a cruciferous vegetable, contains high levels of the protective phytochemical
sulforaphane. Alfalfa is not a cruciferous vegetable and contains no sulforaphane.

 Among those who ate broccoli sprouts, H. pylori levels decreased 40%. They were then told to stop
eating broccoli sprouts. After another two months, H. pylori levels had returned to pre-study levels.
Consumption of alfalfa sprouts had no effect on H. pylori levels.




                                                      33
 Helicobacter pylori infection along with consumption of red meat and dairy products increases the risk
to gastric cancer. Epidemiologically, high intake of Allium vegetables and fruit, especially citrus fruit and
consumption of fresh fish seems significantly protective.

In some countries, as many as 90% of the population are ‘infected’ with H. pylori, yet the frequency of
gastritis and peptic ulcer disease in these countries is rather limited. The presence of H. pylori in the
majority of peptic ulcer patients does not necessarily mean that these bacteria cause ulcers.

 These bacteria are invited by a changed local ecology, really caused by zinc, selenium or magnesium
deficiency or other factors uncoupling recycling of reduced glutathione (GSH). Lack of GSH encourages
viral expression, replication and mutation, thus creating altered immune messaging, ulcers or fissures in
the barrier tissues, as well as changed gene expression in commensal microorganisms, converting
friendly tolerance-inducing symbiotic biofilm to pathogenic inflammation-inciting parasitic biofilm.

 Zinc is necessary for the functioning of more than 300 different enzymes and plays a vital role in an
enormous number of biological processes. Zinc is a cofactor for the antioxidant enzyme superoxide
dismutase (SOD) and is in a number of enzymatic reactions involved in carbohydrate and protein
metabolism. Its immune-enhancing activities include regulation of T lymphocytes, CD4, natural killer
cells, and interleukin II. In addition, zinc has been claimed to possess antiviral activity.

 In herpetic geometric glossitis, linear fissures on the dorsum of the tongue are characteristic of herpes
simplex virus (HSV) type 1 infection in immune compromised patients. The fissures can present in
longitudinal, crossed, or branched geometric patterns. HSV infection of the cornea involves a
characteristic branching pattern of the corneal epithelium called a dendrite, similar to tongue fissures,
the corneal dendrites represent areas of lost epithelium with concomitant superficial ulceration of the
underlying corneal tissue.

 Zinc is likely a safe and effective alternative treatment for herpes type I and II. It also plays a role in
preventing ulcers and in promoting wound healing, especially following burns or surgical incisions.
Studies have reported significant reduction in dental plaque (biofilm) accumulation following treatment
with zinc rinses and dentifrices. Zinc citrate dentifrice tends to reduce the severity and occurrence of
supragingival calculus formation (which may result from calcifying nanobacteria in oral biofilm).

 Reduced glutathione (GSH) plays a major role in tissue protection against ulceration. Glutathione likely
plays a role in the anti-ulcer effect of black tea. Many studies have shown the benefit of free radical
scavengers (antioxidants) on promoting the healing of gastric and duodenal ulcers resistant to therapy.

 Helicobacter pylori bacteria overgrowth simply contributes to the general free radical overload
(significant when unavailable reduced glutathione turns the ‘septic switch’)by producing their acid-
buffering enzyme urease, which also eventually leads to the release of more free radicals as well as acid,
causing further damage to the epithelium.

 As a result, the gentle, fragile mucosal lining of the stomach and duodenum becomes one of the first
tissues to suffer from the damaging chain reactions induced by oxidative free radicals. A dietary healing


                                                     34
antioxidant complex, abundant in very potent and effective free radical scavengers, is present in pine
nut oil and many other natural substances.

In February 2005, Dr. Martin Blaser published a counter intuitive article about H. pylori, entitled An
Endangered Species in the Stomach, in Scientific American. He convincingly demonstrated that the
decline of H. pylori in developed countries over the past 100 years has paralleled an upsurge in
potentially fatal diseases of the esophagus.

 "The possibility that this bacterium may actually protect people against diseases of the esophagus has
significant implications. Current antibiotic treatments that eradicate H. pylori from the stomach need be
reconsidered to ensure that the benefits are not outweighed by potential harm.

To fully understand H. pylori's effects on health, we must investigate the complex web of interactions
between this remarkable microbe and its hosts. Ultimately, the study of H. pylori may help us
understand other bacteria that colonize the human body, as well as the evolutionary processes that
allow humans and bacteria to develop such intimate relations with one another."

 Mastica, the resinous gum of a species of Greek pistachio tree, Pistacia lentiscus, has been used in
Greece for hundreds of years as a remedy for a broad range of gastro-intestinal disorders. It has been
found to be an effective alternative to pharmaceuticals in the treatment of gastritis, gastro-esophageal
reflux disease (GERD) and many types of intestinal inflammation.

Mastic gunm is a safe and effective alternative to antibiotics in the treatment of stomach and duodenal
ulcers and the clearance of the bacteria so frequently the cause of these conditions, H. pylori. Unlike
antibiotics, mastica does not eradicate the populations of friendly bacteria in the intestines, so crucial to
health and well-being.

Consume seven to eight bananas on a daily basis (banana powder is available) for a fast recovery from
peptic ulcer. A glassful of germinated almond milk will help cure peptic ulcer, or it can be relieved by
drinking chamomile tea at least three times in a day.

 Gastritis results from inflammation in the linings of the stomach, causing indigestion, nausea, vomiting
sensation and vertigo. Coconut water is an effective home remedy for gastritis. Consume a glassful of
coconut water at least four times in a day to achieve relief from gastritis.

Zinc is anti-viral, anti-inflammatory and immune modulatory. Zinc-carnosine provides a synergistic
benefit and is an anti-ulcer drug of membrane protective type.

                                            Bacteroides fragilis

 Bacteroides fragilis, which is part of the normal intestinal flora as well as an important opportunistic
pathogen, can set the stage for malignancy in the mouse colon. Although unencapsulated B. fragilis
seems benign, lipopolysaccharide encapsulated varieties of these bacterium trick immune cells (T cell-
dependent) into allowing colon tissue to be continuously inflamed, setting the stage for cancer.



                                                     35
 Enterotoxigenic Bacteroides fragilis (ETBF) causes diarrhea and is implicated in inflammatory bowel
diseases and colorectal cancer. The only known ETBF virulence factor is a 20 kDa protein, the
Bacteroides fragilis toxin (BFT), which induces E-cadherin cleavage, IL-8 secretion and epithelial
proliferation.

 ETBF induces robust, selective colonic signal transducer and activator of transcription-3 (Stat3)
activation with colitis characterized by a selective T helper type 17 (TH17) response distributed between
CD4+ T cell receptor- (TCR)+ and CD4–8–TCR+ T cells. Antibody-mediated blockade of IL-17 as well as
the receptor for IL-23, a key cytokine amplifying TH17 responses, inhibits ETBF-induced colitis, colonic
hyperplasia and tumor formation. A Stat3- and TH17-dependent pathway for inflammation-induced
cancer by a common human commensal bacterium provides new insight into human colonic
carcinogenesis.

 Bacteroides fragilis is able to modulate its surface antigenicity by producing at least eight distinct
capsular lipopolysaccharides (a greater number of LPSs than previously reported for a bacterium) and is
able to regulate LPS expression in an on–off manner by the reversible inversion of DNA segments
containing the promoters for their expression.

This means of generating surface diversity allows the organism to exhibit a wide array of distinct surface
polysaccharide combinations, and likely has broad implications for how the predominant human colonic
microorganisms, the Bacteroides species, maintain their ecological niche(s) in the intestinal tract.

 In a rat model of intra-abdominal sepsis, the capsular polysaccharide complex (CPC) from B. fragilis
promotes abscess formation. The binding of CPC to murine peritoneal macrophages stimulates TNF-
production, which when transferred to monolayers of murine mesothelial cells (MMCs) elicited
significant ICAM-1 expression by these cells. This response results in enhanced polymorphonuclear
leukocyte attachment to MMCs, leading to the development of abscesses associated with experimental
intra-abdominal sepsis.

(When administered sub-cutaneously with a part of CPC, polysaccharide A, before infection with typical
multiple live ‘sepsis species’, a T cell-dependent immune mechanism protected mice against abscess.)

 Bacteroides fragilis is considered by some the ‘H. pylori’ of colon cancer. H. pylori are bacteria ‘proven’
to cause stomach ulcers. B. fragilis infections are often polymicrobial (with notably E. coli), and abscess
formation is common. These bacteria (widely known to cause diarrhea) have been linked to 40% of
colon cancers in a Turkish study. Some people experience no symptoms and others develop diarrhea
and colon inflammation.

 Altered intestinal permeability is a key pathogenic factor of idiopathic bowel inflammation. In the rat
colon, colonization with Escherichia coli, Klebsiella pneumoniae and Streptococcus viridans significantly
increased lumen to blood clearance of mannitol. Colonization with Lactobacillus brevis had the opposite
effect and reduced permeability to mannitol. This Bacteroides fragilis did not induce significant
changes, although strains of Bacteroides fragilis producing B. fragilis toxin (fragilysin) are associated with
diarrhea in animals and humans.

                                                     36
 When growing in the wrong place, bacteroides is the most frequent anaerobic pathogen in man (80% of
anaerobic infections). However, bacteroides species are common in the terminal ileum and prolific in
the colon (10 11 organisms per gram). Fecal matter is 30-50% B. fragilis!

Bacteroides species produce anti-carcinogenic short chain fat6ty acids (butyrate, acetate and
propionate) anaerobically, providing about 70% of the energy supply of colonic enterocytes.
Bacteroides plays a key role in enterohepatic bile acid recirculation and bile acid biotransformation.

 Bacteroides competes with pathogenic micro-organisms for colonic resources - diminishing the food
supply and receptor sites for agents such as Salmonellae and Shigellae, and making the environment
unfavorable by deconjugating bile salts and changing colonic pH. Some vitamin K2 may also be
produced by these commensals.

                                        Multidrug resistance pumps

 Antibiotic resistance to drug therapy is a worldwide problem. Antibiotics, chemotherapy, pesticides,
disinfectants and household cleaners as well as other toxins turn on bacterial multidrug resistance
pumps (MDRs). This genetic survival pumping ability can be transferred to progeny or other species by
accessory genes with ease.

 MDRs protect microbial cells from both synthetic and natural antimicrobials. Spraying a houseplant
with insecticide may upregulate gene transfer and paradoxically promote colonies of antibiotic-resistant
living bacteria in one’s home. Multidrug (MDR) efflux is increasingly reported and has been described
for many organisms, including bacteria, fungi and protozoa, even as a mechanism of resistance in
mammalian tumor cells.

 Mercury leached from amalgam fillings increases antibiotic resistance of oral and intestinal bacteria.
Triclosan, which is in soaps, toothpastes, mouthwashes, skin disinfectants and household cleaners as
well as cutting boards and children’s’ toys turns on the same resistance genes. Triclosan can select for
bacterial mutants that overproduce multidrug resistance pumps.

 Prescription drugs clofibrate (for hyperlipidemias) and ethacrynic acid (a diuretic) chemically resemble
the herbicide 2-4-D. Excreted through the urine, these pharmaceuticals can incite antibiotic-resistant
pathogenic activity of resident E. coli in the urinary tract, bladder or kidneys.

  Corn silk is used to treat urinary tract infections and kidney stones in adults. Corn silk tea is regarded
as a soothing diuretic and useful for irritation in the urinary system, since today, physicians are more
concerned about the increased use of antibiotics to treat infections, especially in children. However,
overuse can lead to drug-resistant bacteria. Adding ginger not only flavors corn silk tea, it compounds
its antibacterial activity, cools inflammation and boosts one’s energy levels naturally.

Amphipathic cations are preferred substrates of MDR pumps. Berberine alkaloids, which are cationic
antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal
plants (like barberry or Oregon grape root) that produce berberine also synthesize an inhibitor of the
NorA MDR pump of a human pathogen, Staphylococcus aureus. The inhibitor was identified as 5′-

                                                      37
methoxyhydnocarpin (5′-MHC), also a minor component of chaulmoogra oil, a traditional therapy for
leprosy.

Chaulmoogra contains strongly antibacterial chemicals, two of which, hydnocarpic and chaulmoogric
acids, are responsible for destroying the bacterium, Mycobacterium leepra, that causes leprosy. In
Ayurvedic medicine, the oil has also been used to treat intestinal worms and skin diseases.

 A new method of bioactivity-directed fractionation, based on multidrug resistant pump (MDR)
inhibition in Staphylococcus aureus resulted in the isolation, from berberine-containing Berberis species,
of two compounds that are themselves devoid of S. aureus antibacterial activity, but form potent
synergistic couples with a subinhibitory concentration of berberine. The bacterial MDR pump inhibitors
were identified as the flavonolignan 2 and the porphyrin 3. Another natural flavonolignan, silybin from
milk thistle (Silybum marianum), was also shown to be a bacterial MDR pump inhibitor.

 5′-MHC (a flavonolignan) is an amphipathic weak acid and is distinctly different from the cationic
substrates of NorA MDR pumps. 5′-MHC has no antimicrobial activity alone, but strongly potentiates the
action of berberine and other NorA substrates against S. aureus.

 MDRs of Gram-positive species are strongly biased toward hydrophobic cations and do not present a
serious problem for the penetration of most clinically relevant antibiotics. The NorA (norfloxacin A)
major facilitator MDR of S. aureus pumps out norfloxacin (Noroxin) and ciprofloxacin (Cipro), which have
cationic forms, increasing the minimum inhibitory concentration (MIC) 2-4 times.

 However, the latest fluoroquinolones bypass the MDRs of Gram positive bacteria. The cytoplasmic
membrane of Gram-positive species, like a simple lipid bilayer, is not a barrier for most amphipathic
compounds and can be readily traversed by antibacterials.

The last class of broad-spectrum compounds effective against that were discovered by the
pharmaceutical industry, the fluoroquinolones, were developed 40 years ago. Interestingly, this class
was derived from nalidixic acid (NegGram), a synthetic inhibitor of DNA gyrase that was synthesized as a
precursor of quinine.

 In Brazilian herbal medicine quinine bark is considered tonic, stomachic and febrifuge. It is used for
anemia, indigestion, gastrointestinal disorders, general fatigue, fevers and malaria as well as an appetite
stimulant. Other folk remedies in South America cite quinine bark as a natural remedy for cancer
(breast, glands, liver, mesentery and spleen), amoebiasis, cardidtis, colds, diarrhea, dysentery,
dyspepsia, fevers, flu, hangover, lumbago, malaria, neuralgia, pneumonia, sciatica, typhoid and varicose
veins.

 In contrast, Gram-negative bacteria have evolved a sophisticated permeability barrier. The surface of
the additional, outer membrane of Gram-negative species comprises lipopolysaccharide, and this highly
hydrophilic layer restricts penetration of hydrophobic and amphipathic compounds. The only known
plant antibacterial with high potency against Gram-negative bacteria is the fairly toxic pyrithione.

 The Gram-negative cell envelope is designed to restrict penetration of all molecules; nutrients enter
through porins and specialized transporters. Evolution produced antibiotics (e.g., aminoglycosides,


                                                    38
tetracycline and chloramphenicol (Chloromycetin)) that can largely bypass the dual barrier-extrusion
mechanism of Gram-negative bacteria, but single synthetic compounds almost always fail.

 MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely
inhibited by 5′-MHC. Berberine levels within bacterial cells strongly increase in the presence of 5′-MHC.
This plant compound effectively disables the bacterial resistance mechanism against the berberine
antimicrobial (or the antibiotic Cipro). Goldenseal contains a mix of antimicrobial alkaloids (including
berberine) which are active in concert against oral and digestive pathogens.

 Plants have faced the problem of microbial multidrug resistance for far longer than we have, and their
solution evolved to produce and use a combination of chemicals, often including an antibiotic along with
an MDR inhibitor. Copying Nature's strategy and potentiating antibiotics with MDR inhibitors can be a
more effective tactic in treating drug-resistant microorganisms.

                                  Dormancy, persisters and spore forms

 Biofilm tolerance to eradication is partly due to the presence of a small fraction of persister cell
essentially invulnerable to antibiotics. Persisters are not mutants, but phenotypic variants of the wild
type. The gene profile of persisters points to a dormancy program that is turned on when these
bacterial cells are stressed.

 Proteins known as “toxins” that form toxin/antitoxin modules participate in persister formation.
“Toxins” appear to be the exact opposite of what their name suggests. They reversibly block important
processes such as translation, protecting the now dormant cell from bactericidal antibiotics that are
effective against active metabolic processes in their targets.

 This dormancy program is likely responsible for the tolerance of M. tuberculosis to antibiotics, leading
to a latent form of the disease which can then later relapse into a life-threatening infection.

 Resistance mechanisms can prevent an antibiotic from binding to the target, which leads to an increase
in minimum inhibitory concentration. Bactericidal antibiotics act by corrupting the target, producing a
toxic product that kills the cell or causes it to commit apoptosis. Tolerance occurs when a persister
protein blocks the targeted molecule, preventing formation of a toxic product.

 Viruses quickly develop drug resistance and have become a serious health issue as well. Practically all
plants and plant extracts help change the pathogenic dynamic of the biofilm and have safely exhibited
profound virucidal activity against viruses as well as strong effects against bacteria and yeast. They
include glycyrrhizin, monolaurin and several Phyllanthus species.

                                                Useful herbs

 The Achilles’ heal of biofilms is the ionic interaction between the acidic polysaccharide and divalent
cations. This interaction can be attacked by both small fragments of similar acid oligosaccharides, by
organic acids that can solubilize the cations, e.g. acidic acid in vinegar, or by chelators, such as EDTA.




                                                     39
All of these treatments can remove the calcium, magnesium and iron that is essential to the matrix.
Small molecules, such as glucosamine, chondroitin sulfate fragments, heparin, and pectin can disrupt
biofilms. Molecules that bind to heparin or nucleic acids, e.g. berberine, quinine (tonic), methylene
blue, should also be effective in disrupting biofilms. Lactoferrin is effective, since it both binds iron and
binds to acidic polysaccharides via its heparin-binding domains.

 Human studies showing efficacy of glycyrrhizin include: hepatitis B, hepatitis C, HIV, upper respiratory
tract infections, cytomegalovirus and SARS. In vivo animal studies of glycyrrhizin effectiveness include:
influenza, herpes and encephalitis as well as versus Candida albicans. In vitro studies of glycyrrhizin
show value against hepatitis A, herpes I, II, EBV, zoster, Vaccina virus, Newcastle disease, Vesicular
stomatitis, Flaviviruses, respiratory syncytial virus, Kaposi sarcoma-associated herpes virus and H. pylori.

Phyllanthus urinaria has been used traditionally in Indonesia to cure kidney and gallbladder stones, and
at the same time help those with liver disease. Phyllanthus blocks DNA polymerase, the RNA enzyme
needed for the hepatitis B virus to reproduce. It provides clear-cut relief and even a cure in viral
diseases like HIV, various hepatitis viruses, the simple flu, bird flu as well as herpes.

 Phyllanthus has been used in Ayurvedic medicine for over 2,000 years. It is taken internally for pain,
jaundice, gonorrhea, frequent menstruation and diabetes as well as applied topically as a poultice for
skin ulcers, sores, swelling or itchiness. Concoctions of the young shoots of the plant are infused to treat
chronic dysentery, another biofilm problem, as are common other bacterial and viral expressions
responsive to phyllanthus.

 Lauric acid was discovered as the most active antiviral and antibacterial substance in human breast milk
and is also richly present in coconut oil. Monolaurin is the glycerol ester of lauric acid and is more
biologically active than lauric acid. Fresh coconut juice has been used to relieve bladder and kidney
infections since recorded time in SE Asia. Coconut water has been a traditional remedy for
strengthening kidneys and cooling hot urine.

 Monolaurin has been shown to be active against (in vitro): Influenza virus, Pneumovirus, Paramyxovirus
(Newcastle), Morbillivirus (Rubella), Coronavirus (Avian Infectious, Bronchitis virus), herpes simplex I &
II, CMV, EBV, HIV, measles, leukemia virus, Simliki forest virus, HPV, Visna virus, Vesicular stomatitis
virus, respiratory syncytial virus, Dengue virus (type 1-4) and lymphocytic choriomeningitis.

 Monolaurin is effective against Gram Positive Bacteria including: Anthrax, Listeria monocytogenes,
Staphylococcus aureus, Groups A, B, F, and G streptococci, Streptococcus agalactiae, Mycobacteria
Clostridium perfringens as well as Gram Negative Bacteria including: Chlamydia pneumonia, Neisseria
gonorrhoeae, H. pylori, Mycoplasma pneumonia and Vibrio parahaemolyticus.

 Monolaurin is also successful against yeast, fungi and molds including Aspergillus niger, Saccharomyces
cerevisiae, Ringworm/Tinea, Malassezia species, Penicillium citrinum and Candida utilis. A number of
protozoa like Giardia lamblia are also inactivated or killed by Monolaurin.




                                                      40
 Monolaurin acts by disrupting the lipid bilayer of virus and prevents the attachment to susceptible host
cells. It prevents replication and removes all measurable infectivity by directly disintegrating the viral
envelope making the virus more susceptible to host defense.

  Undecylenic acid is an organic unsaturated fatty acid derivative of castor oil that is anti-bacterial, anti-
fungal and anti-viral. Castor oil is effective against candida fungus, dandruff, keratoses, diaper rash, jock
itch, ringworm, nail fungus, denture stomatitis, prickly heat, psoriasis and herpes simplex. Undecylenic
acid is fungicidal against Candida albicans, thus promoting a healthy balance of normal vaginal and
intestinal flora.

 Richly present in breast milk, egg white, saliva, nasal secretions, mucus and tears, lysozyme protects us
from bacterial infection. As an enzyme in major salivary secretions, it helps regulate the oral flora.
Enzymes possessing lysozyme activity have been found in bacteria, bacteriophages and plants as well as
in human leukocytes.

 Lysozyme is a small enzyme that attacks the protective cell walls of bacteria. Bacteria build a tough skin
of carbohydrate chains, interlocked by short peptide strands, that braces their delicate membrane
against the cell's high osmotic pressure. Lysozyme breaks these carbohydrate chains, destroying the
structural integrity of the cell wall. The bacteria then burst from their own internal osmotic pressure.

 Lysozyme is a large unpatentable natural molecule that is not particularly useful as a drug. It can be
applied topically, but cannot rid the entire body of disease, because it is too large to travel between
cells.

Ocean Aid Spray’s (http://naturalwoundcare.com) patented formula is synergistically comprised of
Reverse Osmosis Filtered Water, Coral Reef Sea Salt, Lysozyme and Sodium Benzoate. Sodium Benzoate
has been used by food manufactures for over 80 years as a source of benzoic acid to inhibit microbial
growth and can prevent the growth of almost all microorganisms (yeast, bacteria and fungi).

 Antibody-rich colostrum in ImmuneCE works in concert with EpiCor to ignite an even more active and
more intelligently balanced immune response. In-vitro testing indicates that this blend trains immune
cells to identify, target, and destroy specific pathogens with greater accuracy, acting as a homing device
for NK cells, cytokines and phagocytes. In this way, ImmuneCE boosts EpiCor’s powerful virus-fighting
abilities, making harmful invaders even more vulnerable to immune system strategic attack.

There are no known side effects of Phyllanthus or monolaurin and resistance has not been seen with
any of these natural compounds including glycyrrhizin. There is likely synergy in combining these three
natural compounds enhancing their virucidal, bactericidal and fungicidal effects, resulting in lower
effective doses by combination of therapeutic agents.

 Local ribozymal signals that amplify and encourage bacterial quorum sensing also enhance virulence.
These RNA viruses or enzymes can be blocked by an extract of witch hazel bark, called hamamelitannin.
In veins, arteries, ducts, bladders and other tubes and linings of the body, hamamelitannin also inhibits



                                                     41
monocyte TNF-mediated endothelial cell death (without altering the tumor necrosis factor-induced
upregulation of endothelial adhesiveness).

The observed anti-TNF activity of hamamelitannin may explain part of the anti-hemorrhagic use of
Hamamelis virginiana in traditional medicine and its use as a protective agent against UV radiation.
Witch hazel has been used historically to relieve hemorrhoids, injuries, tumors and ulcers.

 In Germany, extracts are approved to treat mild diarrhea, inflammation of the gingiva and mucous
membranes of the mouth, as well as mild irritation or local inflammation of the skin, hemorrhoids and
varicose veins (all of which can be considered biofilm problems). Witch hazel also helps speed healing of
viral cold sores with several daily topical applications.

                                       Attack the protective matrix

The largest part of biofilms' antimicrobial resistance is due to the properties of its extracellular
polymeric substances (EPSs), creating a protective structured slime with diffusion, sorption, water
binding, mass transport and mechanical stability.

 EPSs contain lots of polysaccharides, proteins, nucleic acids and lipids, which maintain the structural
integrity of the biofilm as well as provide an ideal matrix for bacterial cell growth and communication.
Intermolecular interactions between the various functional groups within these macromolecules serve
to strengthen the overall mechanical stability of the EPSs as well as the survivability of the enveloped
microorganisms.

Fluoride kills planktonic bacterial cells at one part per million in the water and weakens the associations
between neighboring cells in a biofilm with the effect of opening up the film rendering it more
permeable (while it competes with essential iodine, nauseates and poisons us just a bit less).

 The amine fluoride (AmF) N'-octadecyltrimethylendiamine-N,N,N'-tris(2-ethanol)-dihydrofluoride is a
cationic antimicrobial which can reduce plaque formation. Biofilm viability decreases significantly after
AmF treatment, but fluoride hurts the host too.

The electrostatic interaction between cationic AmF and negatively charged bacterial cell surfaces is
pivotal in establishing reduced biofilm formation by AmF through a combination of effects on initial
adhesion and killing. The major effect of AmF treatment was a reduction in biofilm viability. There are
much safer poisons for the host.

When silver ions bind to biological molecules containing thio, amino, carboxylate, imidazole or
phosphate groups, they inhibit activities that are vital to bacteria's regulatory processes and cause
bacterial inactivation. Silver ions also act by displacing other essential metal ions, such as Ca2+ and Zn2+.

 Silver cations exhibit broad antimicrobial action at low concentrations, and they are used for treatment
of burn wounds and traumatic injuries. While silver ions at ppb concentrations are effective
antimicrobial agents against most planktonic cells, they have been shown to be ineffective against
sessile cells residing within the biofilm.

                                                     42
 A small dosage of silver ions is insufficient to release excess unbound silver ions for antimicrobial
action, suggesting that low concentrations of silver ions are unsuitable for the treatment of biofilm
infections. Although higher silver concentrations have increased effectiveness against sessile cells, they
face the challenge of maintaining their ionic form in applications containing large amount of halides and
other ions, like Cl−, HCO3− and CO3− as well as protein anions due to the production of the insoluble silver
salt, which causes inactivation of the silver ion.

 Silver ions have a positive charge that kills microorganisms, with low levels of toxicity to humans, and
are safe agents for enhancing removal of biofilms. Due to the reactivity of silver ions with electron
donor groups, silver ions have a considerable effect on reducing the overall stability of EPSs. Several
clinical variables influence the incidence and microbiology of catheter-associated urinary tract infections
(UTI s) and a silver coated catheter (more effectively than silver-oxide coated) significantly reduces risk
to UTIs among women not receiving antimicrobials.

 The silver iontophoretic catheter has a broad spectrum inhibitory activity against Gram-positive
bacteria, Gram-negative bacteria and Candida albicans. The silver iontophoretic catheter provides a
long-term electrochemical barrier against the migration of organisms from the external contaminated
environment into the sterile intravascular compartment.



                             Attack the matrix with enzymes and chelators

 One attractive, and still underdeveloped, strategy for biofilm control is to target the gelatinous matrix
of the biofilm rather than the cells themselves. If the extracellular polymers that hold the biofilm
together could be disrupted or degraded, the biofilm would disperse and its innate defenses would be
subverted.

 Protein and starch digesting enzymes (proteases and amylases) can help break down the proteoglycan
structure of biofilm, which is often strengthened with purloined fibrin. Enzymes might come from
mucostop, lumbrokinase, nattokinase, serratiopeptidase, SPS30, papaya or pineapple.

  Digestive enzymes have excellent history in the treatment of viral diseases. Viruses may enter the body
by a variety of paths. An invading virus should be subdued and immobilized by the immune system,
lying dormant and harmless in the body.

 In the gut, certain agents of the immune system in the mucosa lining usually conquer any viruses.
However, if the intestinal mucosa is damaged or is deficient this can leave an opening for a virus to be
reactivated, get out of control and become industrious in the gut, even spreading to other parts of the
body.

Viruses live and hide intracellularly, thus may directly and indirectly cause symptoms and complications.
The virus may lead to gastrointestinal and/or neurological problems, forcing the immune system to work
at a higher level constantly. It becomes overburdened on a daily basis, yet cannot completely destroy or


                                                    43
subdue its foe. Viruses may include the stealth virus, herpes virus, measles, chicken pox, viral
encephalitis as well as others.

Viruses can cause central nervous system dysfunction and damage the myelin, sheathing the nerves.
This leaves the nerves exposed and susceptible to damage. Viruses are suspect as agents in many
destructive autoimmune diseases.

 A hexosaminidase that specifically cleaves the primary extracellular polymer of many staphylococcal
biofilms has been recently described. This enzyme, called Dispersin B, has demonstrated remarkable
efficacy in disrupting and removing Staphylococcus epidermidis biofilms. An enzyme cocktail could be
considered for topical wound treatment as a way of loosening and removing biofilm.

 When structurally incorporated, xylitol and other alcohol sugars (which are made from a five-sided ring
molecule), create a weaker proteoglycan biofilm matrix than the usual six-sided rings of most sugars
(which form a strong tenacious proteoglycan structure for the biofilm community).

 Persistent pathogenic biofilm may lead to chronic symptoms as the body’s immune responses are
repelled and resultant inflammation chronically damages tissue. In the gut, biofilm formed by
Helicobacter pylori, Clostridium difficile and Candida albicans may be pathogenic. Biofilm can be
disrupted by enzymes such as cellulase and Serratia peptidase combined with metal chelating agents
such as disodium EDTA and lactoferrin. The beneficial yeasts saccharomyces boulardii or cerevisiae
effectively compete with and control Clostridium difficile and Candida albicans.

                               Sequester iron desired for bacterial growth

 Iron is emerging as an important signal in normal biofilm development. Iron concentrations are
generally very low in vivo. The availability of iron appears to govern the rate of staphylococcal biofilm
development on venous catheters. An early stage of Pseudomonas aeruginosa biofilm development
involves the association of bacteria with the surface in an active state of twitching motility. When
sufficient iron is available, the bacteria become sessile and begin building stationary cell clusters.

 When there is not sufficient iron available, for example in the company of added lactoferrin or some
other iron chelator, the bacteria continue their twitching motility and never settle down to construct a
three-dimensional biofilm. The use of natural or synthetic iron scavenging compounds could be a way of
limiting biofilm formation in wound in patients.

 Lectins contribute to the virulence of the opportunistic human pathogen P. aeruginosa by their
involvement in the production, adhesion and pathogenic effects of its biofilm on host cells. Human milk
glycoprotein saccharides (lectins) show high efficiency in blocking P. aeruginosa lectins and likely protect
the infant against infections.

  Lactoferrin is a protein belonging to the iron transporter family that occurs naturally in several bodily
fluids such as tears, saliva, mucus and milk. Richly found in mother's milk (up to 15%) produced 24-48
hours after birthing, lactoferrin provides the body's first immune defense.

                                                     44
Lactoferrin is a powerful anti-microbial that inhibits a wide range of pathogenic bacteria and other
microbes, because of lactoferrin’s extremely high tendency to bind iron, which most pathogenic bacteria
need to multiply. In the presence of lactoferrin, these bad bugs are strongly inhibited or killed.

 Exaggerated immune response to an endotoxin (lipopolysaccharide) is known to cause severe septic
shock and death. Feeding lactoferrin to mice or pigs dramatically reduces the lethality of endotoxin
while improving immune response parameters.

 Lactoferrin strongly inhibits the toxic bacteria heliobacter pylori, as well as inhibiting a wide range of
gram positive and gram negative bacteria, yeasts and even certain intestinal parasites. Cholera,
escherichia coli, shigella flexneri, staphylococcus epidermidis, pseudomonas aeruginosa, candida
albicans and others have all been found to be strongly or partially inhibited in the presence of
lactoferrin. Lactoferrin also improves the efficiency of antibiotic treatments in the fight against
pathogenic microbes.

 Lactoferrin is manufactured by mucosa lining the mouth and intestinal tract as well as by white blood
cells. Lactoferrin benefits intestinal health by promoting the growth of commensal lactobacilli and
bifidobacteria. Lactoferrin discourages bacteria from clumping into biofilms. Lactoferrin stimulates
'twitching,' which causes bacteria to wander around rather than form harmful clusters.

 Whey protein, containing glutathione and lactoferrin as well as multifermented whey (in yogurt and
kefir) are good candidates as dietary inhibitors of oxidative stress and should be considered as potential
medicinal foods in various pathologies such as HIV infection and cancer. Whey is a complex protein
made up of many smaller protein subfractions (peptides), including beta-lactoglobulin, alpha-
lactalbumin, immunoglobulins (IgGs), glycomacropeptides, bovine serum albumin (BSA), as well as minor
peptides such as lactoperoxidases, lysozyme and lactoferrin.

 There is little doubt that lactoferrin is a powerful natural nontoxic treatment in an array of human
ailments. Several companies concentrate lactoferrin. The harder to find apolactoferrin (iron depleted)
form may be the superior supplement.

                                        Mechanically cleanse biofilm

S. epidermidis obtained from infected implants forms thicker biofilms than that of healthy volunteers.
Hydrogen peroxide, at a concentration of 3% and 5%, and alcohols rapidly eradicate S. epidermidis
biofilms, whereas povidone-iodine is less effective.

 Chitosan (derived from shellfish chitin), a polymer of n-acetyl glucosamine (NAG) thins biofilm, and is
especially effective versus strep. It is effective against viruses, yeasts and moulds as well as Gram-
positive bacteria and finally Gram-negative bacteria. An important property of chitosan is its positive
charge in acidic solution.

 Food grade diatomaceous earth detoxifies and absorbs methyl mercury, e-coli, endotoxins, viruses
(including poliovirus), organophosphate pesticide residues, drug resides and protein (perhaps even the


                                                      45
proteinaceous toxins produced by some intestinal infections). Diatomaceous earth has a negative
charge and heavy metals and bacteria have a positive charge, helping sweep them out of the body.

It is both digestive aid and colon cleanser. The microscopic honeycomb skeletal form of diatomaceous
earth is found to become filled and clogged with hard debris such as intestinal scale. Food grade
diatomaceous earth has not been found to cause any insult to the mucosa or barrier wall.

 By combining two or more disinfection agents, it may be possible to lower concentrations of each
component, reduce exposures, minimize the formation of toxic and undesirable disinfection by-products
(DBPs) as well as minimize the health risks associated with disinfection. Bacteria that survived after 48
hours disinfection with hydrogen peroxide and the combined disinfectant showed high catalase activity,
hinting that hydrogen peroxide and the combined disinfectant may have a rather limited effectiveness in
continuous operation.

 Biofilm can be physically removed and destroyed by physical treatments such as hot water (greater
than 80 degrees Celsius) and mechanical scrubbing. Biofilm can also be compromised by chemical
biocides, most of which are either oxidizing or non-oxidizing. Chlorine is an oxidizing biocide and is likely
the most effective and inexpensive. Chlorine works against planktonic and biofilm bacteria, and also
destroys the polysaccharide web and its attachments to the surface. By destroying the extra-cellular
polymers, chlorine breaks up the physical integrity of the biofilm.



                                        Ozone and chlorine dioxide

Non-oxidizing biocides are chlorine dioxide and ozone, both of which fight biofilm in similar fashions.
Both are unstable though, and must therefore be prepared on site. Systems that use ozone must be
made with ozone-resistant materials.

 The addition of the secondary disinfectant following the use of chlorine as a primary disinfectant
produces very dramatic reductions in undesirable by-product formation, like trihalomethanes [THMs]
and haloacetic acids [HAAs]. This effect is due to the reduction of chlorine to chloride by H2O2, which
halts further reaction of chlorine with dissolved organic matter and other DBP precursors. When used
with ozone, H2O2 also quenches formation of THMs and reduces, though not as strongly, formation of
inorganic byproducts, like bromate.

 Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were
grown in planktonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were
exposed to ozone, sodium hypochlorite (NaOCl: 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%),
hydrogen peroxide (H2O2 - 3%) and phosphate buffered saline (control) for 1 min and the remaining
colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings,
resembling root canal areas.

 Concentrations of gaseous ozone down to 1 g m(-3) almost and aqueous ozone down to 5 microg mL(-1)
completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and

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lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and
strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by
high-concentrated ozone gas and by NaOCl after 1 min or a lower gas concentration (4 g m(-3)) after at
least 2.5 min. High-concentrated aqueous ozone (20 microg mL(-1)) and CHX almost completely
eliminated the biofilm cells, while H2O2 was less effective.

 Plasma, the fourth state of matter, consists of electrons, ions, and neutral species and is the most
common form found in space, stars and lightning is usually hot. The cooler nature of an experimental
plasma generator from USC comes from its pulsed power supply. Instead of employing a steady stream
of energy to the plasma probe, the pulsed power supply sends 100-nanosecond pulses of several
kilovolts to the probe once every millisecond, with an average power less than 2 Watts.

 According to plasma emission spectroscopy, atomic oxygen (a single atom of oxygen, instead of the
more common O2 molecule) becomes the antibacterial agent in plasma. Ozone (O3) also provides
singlet oxygen. Oxygen free radicals disrupt the cellular membranes of the biofilms in order to cause
their demise and the plasma plume's adjustable, fluid reach allowed the disinfection to occur even in the
hardest-to-reach areas of the root canal.

Viruses are small, independent particles, built of crystals and macromolecules. They can multiply only
within the host cell. Ozone destroys viruses by diffusing through their protein coat into their nucleic
acid core, resulting in damage of the viral RNA.

 Viruses have little protections against oxidative stress. Normal mammalian cells, however, possess
complex systems of enzymes (i.e., glutathiones, superoxide dismutases, catalases, peroxidases), which
tend to defuse the damaging effects of free radical species and oxidative challenge. One can effectively
treat infected tissues with ozone, respecting the homeostasis derived from their natural defenses, while
neutralizing offending and attacking pathogen devoid of similar defenses.

At higher concentrations, ozone destroys the virus’s capsid or exterior protein shell by oxidation. Ozone
also tends to break apart lipid molecules at sites of multiple bond configuration. So, once the lipid
envelope of the virus is fragmented, its DNA or RNA core cannot survive.

 Like ozone and chlorine, chlorine dioxide is an oxidizing biocide and not a metabolic toxin. Thus,
chlorine dioxide kills microorganisms by disruption of the transport of nutrients across the cell wall, not
by disruption of a metabolic process. Stabilized chlorine dioxide is ClO2 buffered in an aqueous solution.
Adding an acid to the required concentration activates the disinfectant.

The Mineral Miracle Supplement (MMS) solution is 28% sodium chlorite in distilled water. When it is
activated as instructed (1:5 drop solution using a 10% solution of citric acid), chlorine dioxide is
produced.

Of the oxidizing biocides, chlorine dioxide is the most selective oxidant. Both ozone and chlorine are
much more reactive than chlorine dioxide, and they will be consumed by most organic compounds.



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Chlorine dioxide however, reacts only with reduced sulfur compounds, secondary and tertiary amines,
and some other highly reduced and reactive organics.

 This allows much lower dosages of chlorine dioxide to achieve a more stable residual than either
chlorine or ozone. Chlorine dioxide, generated properly, can be effectively used in much higher organic
loading than either ozone or chlorine because of its selectivity.

                                            Survival strategies

 Besides the pleasurable endorphin reward, the advantage of sexual reproduction is that it jumbles the
genes, creating a new, unique life-form with each succeeding generation, a survival strategy and
advantage that helps this newly reproduced unique organism stay one step ahead of its constantly
evolving familial parasites.

 Although they reproduce mitotically, even bacterial and earlier Archaeal populations in biofilm are not
truly clonal (identical copies of one another). These ‘simple’ microorganisms generationally change RNA
interference survival strategy to subvert evolving pleomorphic viral attack.

 RNA silencing or RNA interference (RNAi) is a highly conserved process in which the introduction or the
production of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs containing
homologous sequences by sequence-specific cleavage of the mRNAs

 Bacteria typically snip up invading viruses and stick these bits in the CRISPR area of their genome, using
so-called "spacers" to produce RNA to silence similar viruses. Viruses shuffle their genome frequently,
leading to rapid rearrangement of their DNA, evidently gambling that one of the mutated genomes will
contain a mismatch with bacteria’s interfering RNA. Because RNA interference works only if the spacer
RNA exactly matches a virus's messenger RNA, a single base pair difference can neutralize RNA silencing.

                              ‘RNA world’ coordinates us and our biofilms.

 Taking ecology theories further might take us back in time before our DNA world existed to the ‘RNA
world’ (which is still present). We carry about our familial and personal sentient biofilms’ cooperative
and varied mix of pleomorphic microorganisms (viruses, bacteria and yeasts) with their ability of
communication and cooperation, even to the level of virtually exchanging DNA and RNA. We cannot
exist very long without our biofilm. The primordial slime can go on without us however, but perhaps not
thrive in the same way.

 Our biofilm (composed primarily of more-primitive prokaryotic viruses and cells) symbiotically forms
the outer layer of our larger cooperative eukaryotic cells (with their exponentially more sophisticated
intelligent inner and outer convoluted membranes forming nuclei). Bacteria form the inner and outer
boundaries of our immune system, crowding out unfavorable life forms.

When you add the oral and intestinal bacteria, one’s biofilms become an organ system that comprises
about 3% of our body weight. In addition, eukaryotic cells themselves contain so many mitochondrial



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organelles (still carrying parts of their early prokaryotic genome) that those symbiotic primitive bodies
alone make up about 10% of our mass.

 Friendly commensal life forms communicate with and teach tolerance to our immune barrier cells. Gut
biofilm promotes digestion of our food and helps make vitamins for us. Bacteria manufacture vitamin
B12. Yeast thrives when we are low in vitamin C (likely to beneficently make it for us). Biofilm
communities also help us by detoxifying poisons and sequestering heavy metals.

 Ever-present in and floating around all these life forms are the free RNA ribozymes, molecules which
have both memory and enzyme activity. These overlooked and mostly ignored ribozymes exist
everywhere in intracellular and extracellular spaces and within our structured biofilm communities,
barely visible at highest magnifications of visible light microscopy, viroids seen, but not seen, usually
ignored, like the specks of dust suspended by Brownian motion in sunlit air.

 Messenger RNA levels for individual genes are not uniformly distributed throughout biofilms but may
vary by orders of magnitude over small distances (usually being higher near the aerobic surface). The
metabolic activities of bacteria growing in biofilms result in spatial gradients of oxygen, nutrients, and
waste products. Because bacteria respond to local environmental conditions through changes in gene
expression, mRNA levels of individual genes vary spatially among bacteria within biofilm.

 RNA III inhibiting peptide (RIP) is known to inhibit S. aureus pathogenesis by disrupting quorum-sensing
mechanisms in Gram-positive bacteria. RIP was tested for its ability to inhibit S. aureus biofilm
formation in a rat Dacron graft model. The activity of RIP was synergistic with those of antibiotics for
the complete prevention of drug-resistant S. aureus infections.

 Viruses are relatively basic, consisting of naked DNA or RNA and a protein coat, the bare essentials for
invading a cell. Bacteria and Archaea are larger and more complex than viruses and like us, provide the
machinery for viral reproduction. Viruses have probably been invading larger cells and using their
enzymes to reproduce for 3-4 billion years.

 Viruses have developed very powerful, and very disruptive, strategies for circumventing our simplistic
central DNA dogma. Every known virus is primarily a device that delivers a genetic message to the cell
that it infects. Virus "particles" do not possess cellular machinery and for that reason are not "living"
particles. At the same time, the genetic information carried by the virus contains the instructions
necessary to convert, or subvert normal cell processes into a different focused set of activities that may
lead to the production of many more viruses.

 A sophisticated microbial "immune system" spits out bits of RNA to silence viral genes. Viruses have a
counterstrategy. Viruses shuffle their DNA (or RNA) until their genome sequences becomes scrambled
enough to evade bacterial RNA silencers.

                           Are we androids manipulated by the ‘RNA world’?

 DNA is a scaffold of possibilities that creates species. However, DNA has no inherent enzyme activity.
DNA is a book containing many potential responses to different environmental conditions. DNA is read

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and activated by versions of RNA. RNA determines individuality by selecting certain DNA segments to
decipher depending on RNA’s interpretation of degrees of environmental scarcity or abundance.

 The seemingly savage simplicity of bacteria is not a sign of stupidity but proof of their long term
commitment to survival. When looking at the tree of life in terms of flexibility or creative ability it is
clear that it is not the bacteria that are primitive; it are the branches 'above' them that are caged in an
ancient, conservative, over-elaborate and therefore more rigidly fragile textual DNA heritage.

 Free ribozymes are the ever-present unseen hand of the enveloping sentience of the ‘RNA world’,
creating enzymes and ‘viral’ messaging, thus activating by messenger RNA’s choice, specific parts of our
personal DNA repertoire. Perhaps we DNA-based bacteria, insects, plants and people are simply
biological androids, created and directed by the awareness and intelligence of the all-encompassing RNA
world to fulfill its aims and aspirations on a larger scale.

Steven N. Green, DDS, 10261 SW 72 St., #106,

Miami, FL 33173, 305-273-7779, August 29, 2009




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