Reference

Lanter BB, Sauer K, Davies DG. Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture. MBio. 2014;5(3). pii: e01206-14

Design

Samples of diseased carotid arteries were examined to determine the number of patients with bacterial DNA sequences associated with bacterial plaque. Five random samples from the whole group of 15 were further analyzed to determine the number of unique bacterial 16S gene sequences present in each sample.

Participants

The samples were obtained from 15 people with advanced atherosclerosis.

Outcome Measures

The 16S rRNA gene sequences were analyzed via polymerase chain reaction amplification of the visual area–2 and 3 region followed by gel electrophoresis. This technique is the current standard method used to “study bacterial phylogeny and taxonomy,”1 and 16S rRna is the most common genetic marker used for these studies.1

Key Findings

All carotid artery samples from all 15 patients were found to contain eubacterial rRNA sequences. Each of the 5 carotid arteries tested in greater depth were found to have 10 to 18 unique 16S rRNA gene signatures, indicating colonization by multiple types of bacteria. Comparison of the samples revealed 8 bacterial signatures that were common to all 5 patients and multiple additional sequences present in some but not all of the 5 patients.

Practice Implications

As esoteric as this study might appear at first glance, it has so many implications for our practices that we have decided to discuss it in detail even though this research is still at an early stage.

The data presented in this study strongly suggest that atherosclerotic plaques contain a variety of bacteria. This idea contrasts with our generally held belief that the interior of the body, in particular the bloodstream, is a fairly sterile place, at least when there is no infection involved. This study challenges that notion. The authors believe that the bacteria found in the arteries likely trigger plaque formation. Specifically, they speculate that the bacteria exist as biofilms and that the sudden dispersal of these biofilms into planktonic forms of bacteria is a major contributor to acute cardiovascular disease (CVD) events.

Before discussing clinical implications of this evidence further, let us first take a moment to discuss biofilms and then the general theory these researchers are hoping to validate with their study.

Biofilms

To begin, we need to briefly review several ideas in modern microbiology that are new enough to be unfamiliar to some readers. Certain microorganisms, in particular certain species of bacteria and yeast, will convert or coalesce from individual free-living single individuals into an aggregate of cells. These aggregates are bound together by a film of secreted material—“a hydrated polymeric matrix of their own synthesis to form biofilms”2—best described as slime.

Thus, it seems that bacteria (and some yeast) are capable of existing in 2 different life forms. In 1 form, the individual bacteria exist as single, independent, or planktonic cells and in the second form, the bacteria organize into sessile aggregates. The term planktonic usually refers to plankton, those organisms that live in water and cannot swim against the current. Since it comes from the Greek noun πλαγκτός or planktos, which is translated as “wanderer” or “drifter,”3 the term plankton also works well describing this bacterial phenotype. Further, sessile means “fixed in one place” in particular, as to a base. If plankton are drifters, going where the current takes them, the biofilm phenotype is the sessile form: It has an address.

Acute infections are assumed to involve planktonic bacteria and are generally treatable with antibiotics. As sessile biofilms, the same bacterial infections often develop into a chronic state, untreatable with antibiotics and capable of evading the body’s defenses.4 Biofilms adhere to a substrate or matrix that is not necessarily alive or even organic; they are notorious for forming on implanted medical devices made from inert plastics, such as indwelling catheters. Biofilm infections like pneumonia in cystic fibrosis patients; chronic wounds; chronic otitis media, prostatitis, or sinusitis; and implant- and catheter-associated infections affect millions of people, primarily in the developed world. Since biolfilms are so resistant to treatment, understanding their biology is vital.

Scientific attention is now focused on what triggers conversion from biofilm to planktonic form. Bacteria sense each other’s presence through quorum sensing (QS) and will coalesce at points into a biofilm or disperse and go their individual ways again. According to Solano et al:

QS is a cell-cell communication mechanism that synchronizes gene expression in response to population cell density. Intuitively, it would appear that QS might coordinate the switch to a biofilm lifestyle when the population density reaches a threshold level. However, compelling evidence obtained in different bacterial species coincides in that activation of QS occurs in the formed biofilm and activates the maturation and disassembly of the biofilm in a coordinate manner.5

In some situations, triggering the biofilms to disperse back into planktonic forms makes the bacteria susceptible to antibiotic treatment, and current research focuses on drug and antibiotic combinations that do exactly that.6 One particular approach under investigation to treat infections that are resistant to multiple drugs is to use QS inhibitors that will break down the biofilms.7,8 A number of natural substances interfere with QS and break down biofilms, including horseradish,9 nitric oxide,10 L-arginine,11,12 garlic,13 grapeseed extract,14 berberine,15 curcumin,16 quercetin,17 tannins,17 and eugenol.18 A 2-step treatment using reserpine followed by linoleic acid was the most potent biofilm inhibitor noted in one report that compared many of these agents.19

Even so, the conversion from biofilm to plankton is not always a good thing for the host. For example, dispersion of Streptococcus pneumonia biofilms converts a benign flora to a disease-causing organism. There are a variety of natural processes that signal this conversion. Viral infections can trigger biofilm into this dispersion. Infection with influenza A is an interesting example. It is unclear whether this flu-triggered dispersion is initiated by chemical signals from the virus or from chemical signals generated by the host’s response to the infection (eg, fever, norepinephrine, increased nutrients).20 The bacteria that are dispersed from biofilms triggered by influenza infection are more virulent than those grown in laboratory settings. One must ponder patients with “double-pneumonia” and whether some of the botanical medicines that are in common usage to inhibit QS might impact the progression from viral bronchitis to bacterial pneumonia.

The Theory: Norepinephrine Releases Iron, Inducing Sudden Biofilm Dispersion

This particular, largely unnoticed study reporting on genetic traces of various bacteria in arterial plaques fits into a larger theory on the etiology of heart disease that the study authors hope to illuminate.

The authors believe that the bacteria found in arterial plaques are aggregated in the body as biofilms, which, as mentioned, are resistant to antimicrobial treatments. The authors hypothesize that if biofilms are present in atherosclerotic lesions, they may be susceptible to induction of a dispersion response, which may affect the stability of the plaque deposit. Biofilm dispersion is characterized by the coordinated release of enzymes liberating individual cells from the biofilm matrix and a sudden increase in growth rates. As the biofilm disperses, the plaque may break up and trigger a cardiovascular event.

If and when this theory becomes accepted, we will see a paradigm shift in our understanding of cardiovascular disease prevention and treatment. For those of us who like to stay ahead of the curve, this study is important.

One possible trigger to a biofilm dispersion event can be the sudden increase in a previously rate-limiting nutrient. The authors theorize that the sudden availability of free iron could be such a trigger, a response that in turn could be caused by the sudden acute elevation of norepinephrine in the blood.

It is well understood that emotional or physical stress can trigger a once stable plaque to rupture, precipitating a thromboembolytic event. In the Multicenter Investigation of Limitation of Infarct Size Study, triggers of acute CVD were identified in almost half of the events. The most common trigger was emotional upset (18.4%) followed by moderate physical activity (14.1%).21 Leor reports that sudden fear after earthquakes precipitates CVD events.22 Mittelman reported that relative risk of an acute MI more than doubles in the 2 hours after an episode of anger.23 Other studies report similar effects with work-related stress.24 These stressors cause sudden increases in the plasma concentration of norepinephrine.25 This in turn acts on serum transferrin, causing it to release iron.26 Transferrin normally restricts the amount of free iron in the body to a level low enough that it inhibits bacterial growth. A sudden increase in free iron can allow a surge in the growth rate of resident pathogens in an infected host.

This is a big hypothesis, and the present study tested only part of it, dealing specifically with whether atherosclerosis may be a biofilm-related chronic disease. The results so far support the bigger hypothesis.

Now let’s consider in detail how much of this theory is supported by the data in this study. Fifteen out of the 15 plaques studied contained signs that bacteria were present, and each of the 5 samples that underwent more detailed analysis were heavily populated by multiple different organisms. This should put to rest any idea that our blood vessels or plaques are sterile. In detailed micrographs of these plaque samples, about three-fourths of the microcolonies of bacteria were proximal to the internal elastic lamina or within the tunica interna associated with fibrous tissue. About 6% of the bacterial colonies were associated with the external elastic lamina.

While the authors readily admit that theirs is “the first direct observation of biofilm bacteria within a carotid arterial plaque deposit,” we think their theory and the implications of it are worth our consideration even at this early stage of proof.

Not only are biofilm infections different from planktonic bacterial infections and the routine treatments far less effective, there is an additional worry: Antibiotic treatment of Pseudomonas aeruginosa triggers a response that makes the bacteria even more resistant to eradication, leading to drug resistance, development of a thicker biofilm, and an increased rate of mutations.27 In the study under review, P aeruginosa was identified in 5 of the 15 plaque samples and was demonstrated to undergo biofilm dispersion when challenged with elevated levels of free iron. Biofilm dispersion by this microorganism was also shown here to be inducible by the addition of norepinephrine to transferrin-containing culture medium. Thus, under laboratory conditions, an in vitro spike in hormone concentration was shown to induce biofilm dispersion.

Serum iron levels are already associated with CVD risk, and in fact, high serum iron is associated with triple the MI risk.28 Elevated iron is also associated with quadruple all-cause mortality in coronary patients. As the authors of one study note:

Catalytic iron was associated with a stepwise increased risk of death, with the highest quartile at an almost 4-fold risk compared with baseline (hazard ratio: 3.94, P=0.035), which persisted after adjustment for age, diabetes, prior myocardial infarction (MI), prior congestive heart failure, ST-segment deviation, creatinine clearance, B-type natriuretic peptide, smoking, and Killip class (adjusted hazard ratio: 3.97, P=0.036).29

(Killip class, for those unfamiliar with the term, is a classification system long used to describe individuals with acute MI in order to stratify them according to risk. Individuals with a low Killip class are less likely to die within the first month after their MI than individuals with a high Killip class.30) Additionally, a 2012 article reported that “high ferritin was significantly associated with [acute] MI.”31

As a supportive corollary, consider the cardioprotective effect of the flu vaccine. Influenza-A infections can trigger biofilm dispersion; it turns out that avoiding the flu decreases risk of cardiovascular events. Getting vaccinated against the flu is associated with lower CVD mortality rates.32 Incidentally, vaccinated mice have more stable plaques.33

It is interesting that many of the natural agents known to have some impact on CVD (eg, garlic, arginine, nitric oxide, berberine) may be doing so by inhibiting biofilm formation or by encouraging gradual dispersal, rather than sudden dispersal, that may rupture plaques. Even some of the cardiovascular drugs may affect biofilms; for example, propranolol inhibits Candida biofilm formation.34

We have been using arginine for heart patients thinking that it acted by increasing nitric oxide and blood flow. It may also be interacting in a useful manner with plaque biofilms.10 So might nuts, which are high in arginine, so their protection against CVD may be thanks to their biofilm actions. Extracts of English walnuts are reported biofilm inhibitors.35

It is also interesting to note that some of the agents we have long selected for chronic conditions perpetuated by biofilms may be effective due to their ability to disperse biofilms through QS inhibition. Examples would include horseradish for sinusitis, quercetin for prostatitis, or berberine for cystitis, to name a few.

Conclusions

If and when this theory becomes accepted, we will see a paradigm shift in our understanding of CVD prevention and treatment. For those of us who like to stay ahead of the curve, this study is important.

So what are the key takeaways? One is that our blood is not as aseptic as we thought. Bacteria, under the cover of biofilms, apparently take up residence along our arterial walls with disconcerting ease. Yet as long as we don’t shake things up, this relationship can be a symbiotic one because biofilms exert plaque-stabilizing effects. However, events that trigger bacterial dispersion, namely viral infections and stress-induced acute elevations of iron, can destabilize plaques and lead to thrombosis and acute cardiac events. Such a sequence of events underscores the importance of stress management as a disease-prevention strategy. It also becomes critically important for us to both inhibit and to gently and gradually disperse biofilms with natural agents such as arginine, quercetin, berberine, nitric oxide, garlic, grapeseed extract, curcumin from turmeric, tannins, and eugenol. There is established benefit associated with these agents in the prevention and management of many chronic diseases, diseases that are now associated with biofilms. This pathophysiology ultimately lends further argument to the unique value of lifestyle strategies and natural agents in the prevention and management of chronic disease.