Tag Archives: dental plaque

New test maps acidity in the mouth to spot cavities before they form

The phrase ‘prevention is better than the cure’ is a fundamental principle of modern health, and your oral health should be no different. One of the best ways to prevent cavities is by brushing and flossing correctly. But by now, most people do this and they still end up with some caries eventually. Taking prevention to the next level, scientists at the University of Washington have now developed an optical-based method that can identify the most at-risk teeth by mapping high acidity in the dental plaque that covers the teeth.

Shining light on teeth covered with a florescent dye solution can reveal where the enamel is most at risk from acidity. Credit: University of Washington/IEE Xplore.

Dental plaque is produced by bacteria that live in our mouths as a byproduct as they consume sugars, starches, and other bits of foods that haven’t been properly cleaned from the teeth. If plaque stays on the teeth for more than a few days, it hardens and becomes a substance called tartar. In time, the microorganisms that form on the plaque release acids that wear down the tooth enamel, then the next layer called dentin, before reaching the pulp. When acid attacks the pulp, you’ve officially gotten a new cavity.

But what if we could monitor this acidic activity and stop it before it crosses a point of no return that triggers the cavity formation? That’s exactly what researchers at the University of Washington set out to do. They’ve devised a system, which they call O-pH, that measures the pH levels, or acidity, of the plaque covering each tooth under inspection.

In order to map the acidity of the plaque, a person’s teeth are first covered in a non-toxic, safe chemical dye that reacts with light to produce fluorescent reactions. An optical probe then detects these fluorescent reactions, whose signals can reveal the exact acidity of the underlying dental plaque.

The proof of concept was demonstrated on a small sample of 30 patients, aged 10 to 18. Children and teenagers were selected because their enamel is much thinner than that of adults, which makes detecting any sign of erosion — and consequently a potential cavity — early on very important. The tooth acidity was read before and after sugar rinses, as well as pre- and post-professional dental cleaning.

In the future, this acidity test could be standard practice in dental practices. Eric Seibel, senior author and research professor of mechanical engineering at the University of Washington, says that when a patient comes in for routine teeth cleaning, “a dentist would rinse them with the tasteless fluorescent dye solution and then get their teeth optically scanned to look for high acid production areas where the enamel is getting demineralized.” The dentist and patient can then form a treatment plan to reduce the acidity and avoid costly cavities.

“We do need more results to show how effective it is for diagnosis, but it can definitely help us understand some of your oral health quantitatively,” said Manuja Sharma, lead author and a doctoral student in the UW Department of Electrical and Computer Engineering.  “It can also help educate patients about the effects of sugar on the chemistry of plaque. We can show them, live, what happens, and that is an experience they’ll remember and say, OK, fine, I need to cut down on sugar!”

The O-pH system was described in the journal IEEE Xplore.

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

What are biofilms and how do they form?

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

Thousands of years ago, when no one was safe from famine, dangerous wild animals, or diseases, some of our early ancestors got a bright idea: let’s band together! By forming communities made of people with different talents and skills, the survival rate of individuals grew tremendously. There’s strength in numbers but let’s not pat ourselves on the back too hard — we’re not the only ones who got this idea. Ants and bees do it. Microbes too — they group together into communities called biofilms.

The microorganisms that form biofilms include bacteria, fungi, and protists. Perhaps the most common biofilm familiar to most is the dental plaque — that sticky, colorless film of bacteria and sugars that constantly forms on our teeth. That slime on the surface of water, particularly ponds, is also biofilm.

According to this paper, a bacterial biofilm is defined as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.” In plain English, this means that bacteria sometimes join together, cling to essentially any surface, and form a protective matrix around the group. Indeed, we’ve found biofilms almost anywhere; on mineral, metals, inside our gut etc. In fact, biofilms have been around for at least 3.3 billion years. However, it’s in wet and moist environments that you’ll the most biofilms. They love wetness.

A vast number of pathogens are grouped as biofilms. Like humans, they’ve learned this configuration enhances their survival rate as they are better able to combat the cells of our immune system bent upon destroying them.

How biofilms form

The life cycle of a biofilm. Credit: Bay Area Lyme Foundation.

The life cycle of a biofilm. Credit: Bay Area Lyme Foundation.

The slimy films start forming when initially free-floating bacteria adhere to surfaces in aqueous environments and start ‘laying their roots’. To stay sticky, the bacteria excrete a glue-like substance that’s effective at anchoring them to all kinds of materials, from plastics to soil to medical implants such as pacemakers. This glue is known as an extracellular polymeric substance (EPS) and is comprised of sugars, proteins, and nuclei acids like DNA.

In time, layers upon layers of EPS are added. After a period of growth, a complex 3D structure emerges which is packed with water channels on the inside that facilitate the exchange of nutrients and waste products.

A fascinating thing about biofilm formation has to do with how the bacteria communicate. Pathogens can instruct each other where to position themselves through quorum sensing. Basically, this phenomenon allows a single-celled bacteria to sense how many other bacteria are there in its close proximity. If the bacteria senses there’s a dense population surrounding it, it will be inclined to join them. Remember, strength in numbers.

“Disease-causing bacteria talk to each other with a chemical vocabulary,” says Doug Hibbins of Princeton University.

“Forming a biofilm is one of the crucial steps in cholera’s progression,” said Dr. Bonnie Bassler, a microbiologist also at Princeton. “They [bacteria] cover themselves in a sort of goop that’s a shield against antibiotics, allowing them to grow rapidly. When they sense there are enough of them, they try to leave the body.”

Sometimes clumps of biofilm can break away from the main mass and establish themselves on a new surface. These new pioneers will continue to extend their slimy film until they form a new, bigger colony.

How big can a biofilm get

Most biofilms are very thin — just a few cell layers thick. That’s too thin to see with the naked eye. In fact, your kitchen counter almost certainly has a biofilm layer on it. You just can’t see it. Some biofilms, however, can grow many inches thick and are obviously noticeable. You’ll find these thick slime molds growing as algae on rocks in a streambed.

The thickness of biofilms depends on several environmental factors. Some organisms can produce large amounts of EPS and hence grow a thicker biofilm. Water flow is also an important factor or, to be more precise, shear stress is. If a biofilm forms in a creek where there’s a high flow of water, it ought to be fairly thin. Biofilms formed in slow flowing water, like a pond, can grow quite thick.

Why biofilms form

Biofilm on teeth, commonly known as dental plaque. Credit: Mead Family Dental.

Biofilm on teeth, commonly known as dental plaque. Credit: Mead Family Dental.

As mentioned, bacteria band together because as a community they enhance their chance of survival, but what threats do they face and how does living a slime mold protect them? Some of the stressors bacteria face are the lack of water, high or low pH, or the presence of ‘toxic’ substances, i.e. antibiotics or antimicrobials.

The EPS layers act as the first line of defense against these threats. It can prevent dehydration or shield the bacteria against UV light. When they come in contact with the EPS, antimicrobials, bleach or even metals become bounded and neutralized by the sticky EPS.

Antibiotics can certainly destroy biofilm but not always because biofilms employ another line of defense. For instance, despite antibiotic substances might penetrate the EPS layer, they can be met dormant bacteria. Because these bacteria lack cellular activity, the antibiotics don’t work their magic because there’s nothing to disrupt.

Another line of defense against antibiotics are the ‘persisters’ or special bacteria that do not divide. These bacteria produce substances that block the targets of many antibiotics, according to a 2010 paper. Compared to free-floating bacteria, those growing as a biofilm can be up to 1,500 times more resistant to antibiotics

Finally, living inside a community, often made of different bacterial species, means its members can reap the benefits that come with having a multi-skilled network. For instance, some biofilms are made of both autotrophic and heterotrophic microorganisms. The autotrophs produce their own food using photosynthesis and available organic material while heterotrophs don’t make their own food and require external sources of carbon. As such, in these biofilms, the microorganisms will often cross-feed. It’s a sort of division of labor.

Biofilms, humans, and disease

Scanning electron images of the surface of a mouse bladder infected with urinary tract infection show large intracellular communities of biofilm bacteria inside pods. Uninfected bladders appeared smooth, but infected bladders had bumps all over them. Credit: The Marshall Protocol Knowledge Base.

Scanning electron images of the surface of a mouse bladder infected with urinary tract infection show large intracellular communities of biofilm bacteria inside pods. Uninfected bladders appeared smooth, but infected bladders had bumps all over them. Credit: The Marshall Protocol Knowledge Base.

Biofilms seem to be able to form and cling to just about any external surface as long as it’s wet. This may naturally beg the question — does that mean they can form inside the human body as well? It certainly is wet enough and, indeed, we find that the answer is ‘yes’. According to the National Institutes of Health, more than 65% of all microbial infections are caused by biofilms. That might strike you as a lot but you need to keep in mind that the vast majority of infections are common like urinary tract infections, catheter infections, common dental plaque formation and so on.

However, biofilms can be involved in a range of diseases and medical problems. One example is kidney stones which are caused by biofilms. Some 15 to 20 percent of kidney stones form as a result of urinary tract infections, produced by the interplay between infecting bacteria and mineral substances from the urine.

Then there’s endocarditis, a disease that involves inflammation of the inner layers of the heart. Endocarditis seems to be triggered by a complex biofilm made from bacterial and host component located on a cardiac valve. This type of biofilm is known as a vegetation. The vegetation can disrupt valve function, produce a near-continuous infection of the bloodstream, and can block blood circulation through a process known as embolization.

Pathogenic biofilms also plague prostheses and various medical implants like artificial joints and heart valves or pacemakers. This first came to the medical community’s attention in the 1980s when bacterial biofilms were found on intravenous catheters and pacemakers.

“When people think of infection, they may think of fever or pus coming out of a wound,” explains Dr. Patel from the Mayo Clinic. “However, this is not the case with prosthetic joint infection. Patients will often experience pain, but not other symptoms usually associated with infection. Often what happens is that the bacteria that cause infection on prosthetic joints are the same as bacteria that live harmlessly on our skin. However, on a prosthetic joint they can stick, grow and cause problems over the long term. Many of these bacteria would not infect the joint were it not for the prosthesis.”

Biofilms have been poorly understudied until recently but evidence suggests they’re involved in many human diseases, including debilitating chronic infections. According to Dr. Trevor Marshall, a biomedical researcher at Murdoch University, Australia, some large microbiota of chronic biofilm like L-shaped bacteria can evade the immune system because, long ago, they evolved the ability to reside inside macrophages. Ironically, these are the very white blood cells of the immune system which are supposed to kill the invading pathogens. Marshall also says that biofilm infections occur with great ease in immunocompromised hosts.

Targeting biofilm infections

Research carried out over the past three decades suggests that biofilms are either extremely difficult or impossible to eradicate from the human body. What’s certain is that administering antibiotics in a standard manner (high dose, constant) does not work.

After high doses of antibiotics are administered, it may seem the biofilm infection has disappeared. However, it will reappear because the biofilm was not destroyed, only weakened. It seems that while antibiotics can penetrate the biofilm matrix and kill bacteria, a number of cells called ‘persisters’ are left behind. These are able to survive the onslaught of antibiotics and gradually allow the biofilm to form again.

Dr. Kim Lewis of Tulane University, however, says that it is possible to destroy some biofilms. His treatment involves using pulsed, low dose antibiotics to break up the biofilm. For instance, research suggests this technique is effective at destroying P. aeruginosa biofilm bacteria in a manner that is indistinguishable when the same antibiotic concentrations are administered to single planktonic cells.

When the low, pulsed dosing of antibiotics is applied, the first application eradicates the bulk of the biofilm cells, leaving the persisters behind. Because the antibiotics are stopped, the survival of the persisters is not enhanced. Lewis believes this causes the cells to lose their shape and biochemical properties, making them unable to restart the biofilm formation process. A second application of the antibiotic after a certain time should then completely eliminate the persister cells.

The efficacy of this method depends on the ability to manipulate the antibiotic concentration. Furthermore, not all biofilms can be broken down this way.

Useful biofilms

Biofilms can cause serious medical conditions and, as we’ve seen, they can be very difficult to get rid of. But there are instances when biofilms can be useful, for bioremediation purposes. Biofilms are used, for instance, in treating waste water or contamination with heavy metals or radioactive substances. Another practical use for biofilms is in microbial fuel cells. In such fuel cells, microbes that live on the surface of an electrode break down nutrients and transfer electrons through a circuit, providing electricity. Microbial fuel cells can be very useful if you need to remotely generate power for sensors in wastewaters or landfills.

Biofilms are right now the subject of intense research. Biofilms cause billions in damage every year due to disease, equipment damage, energy loss or contaminations, and as such finding ways to get rid of them is a priority. The resilience of biofilms is a big challenge and requires contributions from different sciences such as biochemistry, engineering, mathematics, and microbiology.

 

Fossilised dental plaque (calculus) on the teeth of a middle-aged man from the Medieval site of Dalheim, Germany, ca. AD 1100. Photo credit: Christina Warinner.

Microbial tomb discovered in 1,000 year-old human teeth

An international team of scientists believe they have across the  “microbial Pompeii” after they found preserved bacteria and microscopic particles of food on the surface of teeth more than 1,000 years old. The findings were made after the dental calculus or plaque that covered the ancient teeth was analyzed. Some very important discoveries were made in the process: ancient oral bacteria had already by then basic genetic machinery for antibiotic resistance even though the first antibiotics would only arrive eight century later; humans then as now were affected by the same oral bacteria, causing numerous potentially deadly diseases, despite diet has significantly shifted since ancient times.

Researchers from  University of Zürich, the University of Copenhagen, and the University of York were involved in the study which  applied shotgun DNA sequencing to dental calculus for the first time. Unlike bone which when buried, per ritualistic customs,  rapidly loses much of its molecular information, dental plaque grows slowly in the mouth and enters the soil in a much more stable state helping it to preserve biomolecules.

Fossilised dental plaque (calculus) on the teeth of a middle-aged man from the Medieval site of Dalheim, Germany, ca. AD 1100. Photo credit: Christina Warinner.

Fossilised dental plaque (calculus) on the teeth of a middle-aged man from the Medieval site of Dalheim, Germany, ca. AD 1100. Photo credit: Christina Warinner.

Estimates of the number of bacterial species in the oral cavity vary between 500 to 650 different species, but truth is they may be a lot more. In all, some 20 billion oral microbes live in the average human mouth. Some are helpful, some are nasty. For instance, the oral microbiome is responsible for the development of periodontal disease that causes distinctive   proteomic changes in your tooth. The decay is characterized by inflammation and tooth loss.

Both in ancient and present time, humans had lousy teeth

One in ten people in the world suffer from periodontal disease, which at its own has linked with  to diverse systemic diseases, including cardiovascular disease, stroke, pulmonary disease, and type II diabetes.  It’s really common in humans, zoo animals, pets, domesticated farm animals and such, but never in wild animals. Clearly, there’s something in the human lifestyle, which fringes upon the rest of the animals that depend on humans, that causes this changes.

“We knew that calculus preserved microscopic particles of food and other debris but the level of preservation of biomolecules is remarkable.  A microbiome entombed and preserved in a mineral matrix, a microbial Pompeii,” said Professor Matthew Collins, of the BioArCh research centre in the Department of Archaeology at York.

Many believe that these changes occurred once with the introduction of our now modern lifestyle and diet. The analysis of the ancient teeth, and subsequent lurking bacteria, shows however that  ancient human oral cavity carries numerous opportunistic pathogens and that periodontal disease is caused by the same bacteria today as in the past, despite major changes in human diet and hygiene.

Ancient dental calculus magnified 1,000 times reveals the presence of millions of Gram positive (blue) and Gram negative (red) oral bacteria fossilised in situ. Photo credit: Natallia Shved.

Ancient dental calculus magnified 1,000 times reveals the presence of millions of Gram positive (blue) and Gram negative (red) oral bacteria fossilised in situ. Photo credit: Natallia Shved.

“As we learn more about the evolution of this microbiome in response to migration and changes in diet, health and medicine, I can imagine a future in which most archaeologists regard calculus as more interesting than the teeth themselves,” says Professor Collins.

Analyzing the ancient micriobiome was no easy task, mind you. The researchers had to shift through  millions of genetic sequences like puzzle pieces in order to reconstruct the complex biology. Besides the novel use of shotgun DNA sequencing, the study also performed for the first time Raman spectroscopy  on ancient dental calculus.

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“Raman spectroscopy which involves the interaction of light and matter does not destroy archaeological samples. By comparing modern and ancient dental calculus, Raman spectroscopy confirmed its very high preservation helping to verify it as a key archaeological material,” said Dr Yvette Hancock, of the Department of Physics at York.

The findings were presented in a paper published in the journal Nature Genetics.