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The Ever Changing Oral Microbiome

Think about your last trip to the dentist. The vinyl chair, the bright lights, the tray of metal tools… And most likely the shame you feel when your dentist asks if you’ve been flossing. Now although this bi-annual trip to the dentist seems normal to most of us (I hope), dentist trips haven’t always been a part of the “routine” as far as human health goes. Why not? The answer lies in the composition of our ancestors’ oral microbiomes, which has been revealed through recent genome sequencing advancements.

The human oral microbiome can be persevered for an extensive period of time after death, unlike other microbial communities (such as those of the skin or mouth). This is a result of the formation of dental calculus, which thankfully has nothing to do with derivatives. Dental calculus forms in a sequential process, beginning with dense multi-species biofilms, known as dental plaque. This plaque can then become calcified by calcium phosphates precipitated from the saliva or tooth surface, causing the plaque to harden into dental calculus (tarter). Bacteria become “locked” in into the calculus, whose hard surface resembles bone. This is the perfect site for the formation of more biofilms, more plaque, and the accumulation of more dental calculus (Figure 1). Disgusting, right? Although we do out best to prevent this process from happening through regular oral hygiene practices, once tarter forms, it’s too hard to be removed using a toothbrush or floss. That’s where your dentist comes in with the scary tray of metal tools. If not removed it can lead to gingivitis (inflammation of the gums) or periodontitis (inflammation of the tissues and bone that supports the teeth).

Figure 1: The formation of dental calculus from dense oral biofilm (plaque) interacting with calcium phosphates from saliva and/ or the tooth surface facilitates the formation of more oral biofilm.

As you can probably imagine, our hunter/gatherer ancestors weren’t rinsing with Listerine or making regular dentist trips, so naturally they would have had a build up of dental calculus. Despite this, the structure of the bone supporting their teeth suggests that they had less periodontal disease than current human populations! By sequencing DNA persevered in ancient dental plaque, researchers have established that the oral microbiome of hunters/ gatherers was significantly more diverse than that of current human populations. Who knew plaque could actually be useful for something?! Major shifts in diet, such as those associated with the beginning of farming (10 000 years ago) and the industrial revolution (200 years ago), are thought to be the main contributors to this loss of diversity over the past 10 000 years.

By analyzing ancient human skeletons recovered from the last hunter/gatherers in Eastern Europe, as well as those of the first farmers of the agricultural era, a clear shift could be seen between the oral microbiomes the two populations. The beginning of farming was marked by increased consumption of fermentable carbohydrates, which changed the oral environment and lead to increased colonization by carbohydrate fermenting species, such as Porphyromonas gingivalis and Tannerella forsythia. These species are specifically adapted to a carbohydrate rich environment, which allowed them to outcompete other species and decrease the overall oral microbiome diversity.

Another significant decrease in diversity followed the industrial revolution about 200 years ago, which saw the large-scale production of foods containing both mono- and disaccharides. DNA sequencing of samples from before and after the revolution showed that the abundance of gram-positive anaerobic bacteria, such as Streptococcus mutans, increased greatly post revolution. The shift also correlated directly with an increased prevalence of dental calculus and periodontal disease, as well as a decrease in the overall diversity of the oral microbiome. Coincidence? I think not.

S. mutans is a gram positive anaerobic bacterium, that acts as the poster child for modern oral disease. It is known for its enhanced ability to alter its metabolic processes based on what nutrient sources are available in its environment. This makes it extremely competitive under conditions with varying nutrient sources, such as the oral biofilm. Using a process known as carbohydrate catabolite repression (CCR), S. mutans favourably metabolizes a preferred carbohydrate source in the presence of non-preferred sources. They are also able to use this process to repress of genes involved in the metabolism of non-preferred carbohydrates in order to optimize growth using the preferred carbon source until it runs out.

As with many other bacterial species, the preferred carbon source of S. mutans is glucose, whose uptake is facilitated by a phosphotransferase system (PTS). What’s special about S. mutans is that it has at least 15 identified PTSs, specific to different mono- di- and oligosaccharides, that can be expressed based on the carbohydrates available in the environment. This allows S. mutans cells to shift almost seamlessly between different carbon sources, and outcompete other species who are not able to do so. PTSs generally work as a phosphorelay system, consisting of non-specific EI (enzyme 1), a heat resistant kinase (HPr) and specific EII (enzyme II). EII is a transmembrane protease that recognizes specific carbohydrates, transports them into the cell and phosphorylates them to keep them from being transported back out. If glucose is present, its phosphorylated form is able to bind and active transcription factors that repress genes for other PTSs (Figure 2). This mechanism demonstrates one of the ways S. mutans is able to outcompete other species in the oral biofilm, which contributes to its pathogenicity.

Figure 2: The phosphotransferase system (PTS) of S. mutans used in carbohydrate catabolite repression (CCR), where the EII enzyme can be any of 15 mono-, di- or oligosaccharide specific transmembrane proteases. A phosphate group is transferred via a phosophorelay to an incoming carbohydrate (glucose), which can then repress genes involved in alternative metabolism.

The ability of S. mutans to adjust its metabolic processes so rapidly is only partially responsible for drop in diversity that was seen as a result of the adoption of a carbohydrate rich diet. The lactic acid producing ability of this species through anaerobic fermentation lead to an increasingly acidic environment in the oral biofilm, which allowed it to further outcompete other members of the biofilm. This acidity also causes damage to the tooth surface, one of the direct factors implicated in how this species causes oral disease.

Although maintaining good oral health today is somewhat demanding, this was not always the case. Analysis of dental plaque from ancient human remains has allowed researchers to identify that major shifts in human diet changed the oral environment enough to alter the composition of the oral microbiome. This was primarily driven by the introduction of large amounts of fermentable carbohydrates, which lead the less diverse and disease associated oral microbiome of today’s human population. So even though our ancestors didn’t have to floss, you still do!

Thanks for reading!

Jac

Here are some additional readings for you to check out:

Alder CJ et al. (2013) Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions. Nature Genetics, 45: 450-455.

Moye ZD, Zeng L, Burne RA (2014) Fueling the caries process: carbohydrate metabolism and gene regulation by Streptococcus mutans. Journal of Oral Microbiology, 6.

Pihlstrom BL, Michalowicz BS, Johnson NW. (2005) Periodontal diseases. Lancet, 366(9499): 1809-1820.

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Treating bacterial infections with oral bacteria

The paper I decided to share this week is all about the interaction between commensal oral streptococci species and the pathogen Pseudomonas aeruginosa. P. aeruginosa is the most common cause of chronic lung infections in cystic fibrosis (CF) patients, where it releases toxins that damage lung tissue. These infections are often very difficult to treat, as P. aeruginosa forms biofilms that protect it from traditional antibiotic therapies.

Interestingly, recent evidence has shown that when P. aeruginosa is cocolonized with oral streptococci species in the lungs of CF patients, lung function is improved. The streptococci is part of the biofilm, where it has an inhibitory effect on the P. aeruginosa.

In order to understand this interaction, this study looked at how hydrogen peroxide (H2O2) producing oral streptococci are able to inhibit the growth of P. aeruginosa, in the presence of nitrite, a compound whose levels are elevated in CF patients. Hydrogen peroxide in the presence of nitrite forms reactive nitrogenous intermediates (RNI), such a peroxynitrite, which act to inhibit the pathogenic P. aeruginosa. If streptococci species were not able to produce H2O2 (due to mutation), P. aeruginosa was no longer inhibited. This same effect was observed if nitrite was not present. This lead researchers to conclude that the modulation of H2O2 and nitrite levels in CF patients, with the help of oral bacteria, could potentially serve as a defense mechanism against chronic P. aeruginosa infections.

You can check out the full paper, Oral Streptococci and Nitrite-Mediated Interference of Pseudomonas aeruginosa, here. 

Jac 

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The World of Oral Biofilms!

The human microbiome consists of trillions of bacterial, fungal, viral and archeal cells that outnumber our own eukaryotic cells by more than 10 to 1. Although the human genome has been sequenced thousands of times, the genomes of many of these tiny organisms that call our bodies home are still unknown or poorly understood. Home to over 700 species of commensal microorganisms and opportunistic pathogens, the human oral cavity contains a very complex microbial ecosystem. Changes in the composition of the oral microbiome have recently been linked to a variety of health conditions, such as diabetes and cardiovascular disease. This illustrates the potential for fluctuations in microbiome composition to serve as an indicator of overall health, which has lead to the hypothesis that there is an ideal oral microbial composition associated with overall health in the human body. To investigate this theory, current research is focused on characterizing the complex interactions between microorganisms that call our mouths home. I’ll be focusing on bacteria for the sake of this post, simply because they are one of the better understood inhabitants of the mouth.

The establishment of an overall microbiome begins at birth, where microbes from the mother begin to colonize the newborn child almost instantaneously, and develop into a unique and complex community. The establishment of the oral microbiome specifically is not quite understood, but direct contact with family members and objects in the infant’s environment are suspected to be the major contributors.

In adulthood, the oral microbiome remains relatively consistent in healthy individuals, despite the chemical and physical stressors the microbes encounter on a daily basis. Eating and drinking alter the pH of the mouth, while brushing and flossing can physically disrupt the bacteria that live on our teeth. These organisms must also withstand temperature fluctuations, evade our immune defenses and survive antibiotic treatment (Figure 1). That’s a long list of challenges to overcome for bacteria that want to call our mouths home! So what’s the big secret to long-term residency in the oral cavity? Biofilms!

 Figure 1: Physical and chemical stressors encountered by microbial inhabitants of the human oral cavity.

Biofilms are essentially large numbers of cells that adhere together on a surface, inside a mucous-like matrix they produce. Any hard surface bathed in liquid is prime real estate for a biofilm. As you can probably imagine, that makes our mouths the Boardwalk on the Monopoly board of potential habitats that is our bodies. Our saliva acts as the perfect carrier of nutrients to the biofilm in the form of sugars, water and proteins that come from our food to create a perfect habitat for microbes.

Oral biofilms begin with only a few primary colonizing cells, which attach to oral surfaces, such as teeth, that are inherently covered by glycoproteins produced by our own cells. Some of the most common primary colonizers are members of the Streptococci and Actinomycetes genera, which use outer membrane recognition proteins, and/or pili to facilitate attachment to the glycoprotein-covered surface. Once initial attachment has occurred, the cells begin to produce extracellular polymeric substances (made up of mostly polysaccharides) that act as adhesins between themselves and other incoming cells. An essential contributor to this development process is Fusobacterium nucleatum, a gram-negative bacterium that mediates the coaggregation between the early colonizers and late colonizers of the biofilm. In the presence of this species, coaggregation occurs between species that are not able to coaggregate on their own. This leads to the maturation of the biofilm into a multi-species community of bacteria that interact with each other via signaling molecules to share information and control growth. The protective biofilm matrix consisting of polysaccharides, proteins, lipids and DNA is able to withstand chemical and physical stressors in order to foster a happy community of neighborly microbes (Figure 2).

Figure 2: The process of biofilm formation on the surface of teeth (adapted  from O’Toole et al. 2000).

The residents we really want to move into these biofilms are known as commensal bacteria, with whom we have a symbiotic relationship. To be symbiotic simply means that both parties in the relationship benefit, in a “you scratch my back, and I’ll scratch yours” way. We give commensal bacteria a home, and they protect us from their nasty pathogenic neighbors. This is based on the theory that under normal conditions, the environment inside the biofilm favors the commensal species, which outcompete the pathogenic species for resources. So although present, the pathogens simply do not have the numbers to cause any real disease.  

Biofilms are not completely stable however; and can be disrupted by the use of antibiotics, or long-term poor oral hygiene. These factors have the capacity to disrupt the normal biofilm environment, leaving room for pathogens to thrive and cause disease. An example of this can be seen with the relationship between Lactobacillus reuteri, a commensal gram-positive species, and Tannerella forsythia, an opportunistically pathogenic gram-negative species that is known to cause periodontitis (an inflammation of the tissue supporting the teeth). Both of these species are normally found in the biofilm between nestled between our gums and our teeth, where L. reuteri produces lactic acid that inhibits T. forsythia growth. Under poor oral hygiene conditions, changes in the oral environment stimulate virulence genes in T. forsythia to be turned on, which protect it from the lactic acid and open the door to unpleasant oral disease (so make sure to floss!).

It is through the formation of biofilms that oral bacterial species are able to withstand the chemical and physical stressors they encounter by calling our mouths home. These biofilms offer a protective matrix of polysaccharides, proteins, lipids and DNA, that protects the coaggregation of cells within them. By characterizing the interactions that occur between the microbial inhabitants of these biofilms, we can investigate how changes environmental conditions alter these interactions and induce bacteria that are normally not pathogenic to cause disease. Some researchers hypothesize that this understanding could lead to the potential for changes in the oral microbiome composition to serve as a representation of overall human health and indicator of disease. In other words, our microbes can detect disease before we can! I think it’s about time we start giving these guys some more credit!

Did this make anyone else want to brush their teeth?

Jac

References:

Avila, M., Ojcius, D. M., & Yilmaz, Ö. (2009). The Oral Microbiota: Living with a Permanent Guest. DNA and Cell Biology, 28(8), 405-411.

Baca-Castañón, M. L. et al. (2015). Antimicrobial Effect of Lactobacillus reuteri on Cariogenic Bacteria Streptococcus gordonii, Streptococcus mutans, and Periodontal Diseases Actinomyces naeslundii and Tannerella forsythia. Probiotics and Antimicrobial Proteins, 7 (1), 1-8.

O'Toole, G., Kaplan, H. B., & Kolter, R. (2000). Biofilm Formation as Microbial Development. Annual Review of Microbiology, 54, 49-79.

Sharma, A. (2010). Virulence mechanisms of Tannerella forsythia. Periodontology 2000, 54(1), 106–116.

Wade, W. J. Has the use of molecular methods for the characterization of the human oral microbiome changed our understanding of the role of bacteria in the pathogenesis of periodontal disease?. Journal of Clinical Periodontology, 38 (11), 7-8.

Zaura, E., Nicu, E. A., Krom, B. P., & Keijser, B. J. F. (2014). Acquiring and maintaining a normal oral microbiome: current perspective. Frontiers in Cellular and Infection Microbiology, 4 (85).

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What am I?

This week I got creative and decided to write a poem/ riddle. Can you figure out what I am given the clues below?

What am I?

To a surface I am stuck

Without me you'd be out of luck

Try to get rid of me and you will find

I am an essential part of mankind

On both living and non-living surfaces I form

I help those within me weather the storm

At first I attach and then I mature

I'll develop and disperse you can be sure

Bathed in liquid is my exterior

To some antibiotics I am superior

Made of members not all the same

Many infections have me to blame

With many microorganisms I am dense

They use quorum to help them sense

I contain molecules of different types

But they all aren't here to help, yikes!

I know this isn't an exam,

But can you guess what I am?

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Kissing: Sharing more than just feelings

When it comes to microbiomes, those of family members are often more similar than those of unrelated people, which has been attributed to both genetic and environmental factors . This same trend has also been observed the oral microbiomes of couples, who don't necessarily have genetic similarities, but rather similar diets and lifestyles. In light of Valentine's Day coming up this weekend, I wanted to share a study that investigated a behavioural influence on the similarity of couple's oral microbiomes, kissing! 

A 2014 study of 21 Dutch couples found that as many as 80 million bacterial cells are transferred from one partner to another during a 10 second intimate kiss! The samples taken post-kiss showed that the microbiomes of the partners were most similar right after the kiss had occurred, and became less similar over time. This is because the majority of bacterial transfer occurs via the exchange of saliva, meaning the incoming bacteria are often flushed out a few hours post make-out session. So essentially, kissing was not found to be the major factor in explaining why the partners had similar oral mircobiomes. It was also found however, that couples who reported higher kissing frequencies showed more similar oral microbiomes than those with lower frequencies, even a few hours after a kiss. Perhaps consistent exposure to a new environment (one partner's mouth) gives the bacteria the opportunity to colonize the mouth more permanently. So, if partners kiss enough, the kissing does have an effect on their microbiomes!

Although not the biggest factor in microbiome similarities, kissing is still a factor to be considered, especially while you're getting your romance on this weekend. Happy kissing/ bacteria exchanging!

Jac

You can check out the full study by Kort et al. here:

http://www.microbiomejournal.com/content/2/1/41

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Curing halitosis requires the right balance of oral microbes.

Halitosis, the scientific term used to describe chronic bad breath. Although not exactly life-threatening, having bad breath can definitely be social life-threatening. Oral hygiene products, such as Excel gum, use marketing techniques that link having fresh breath with success at work or with happy interpersonal relationships. Their well-known tv ads portray bad breath as the result of cute cartooned coffee and onions, and boast the slogan "Don't let bad breath get in the way". Check out this 30 second Excel commercial to see for yourself: https://www.youtube.com/watch?v=VPCoXWYctQM 

Although eating stinky foods can often be a cause of bad breath, there are people who suffer chronically from halitosis despite brushing, flossing and using mouthwash in a way that would make any dentist proud. This article provides a brief overview of how an imbalance in a person's oral microbial community can be the cause of halitosis, rather than his/her diet. Foul smelling sulfur compounds (think rotten eggs...ew) produced by an overabundance of mouth dwelling gram negative bacteria have been linked with chronic bad breath. Fortunately, the treatment of patients with bacteria that don't produce nasty smells can serve as a possible solution to bad breath woes. 

By coupling good oral hygiene with a probiotic supplement of Streptococcus salivarius K12, a malignant strain of gram positive bacteria often found in the respiratory tract, bad breath was significantly reduced in halitosis patients. Why? The gram positive species outcompetes the gram negative species, which reduces the amount of foul smelling compounds being produced. Instead of covering up bad breath with nice smelling compounds, it can be treated by creating the right balance oral bacteria! Keeping the microbes in your mouth happy, and I'm sure your colleagues and friends will be happy too. 

I found this article interesting because it describes a problem many people can relate to, and offers a somewhat atypical solution. The idea of treating bad breath with bacteria seems strange at first, but by simply touching the surface of the microbiology that's going on, it becomes clear that it's not strange at all! I'll be anxiously awaiting the Excel ad that markets probiobiotic gum... Cute cartoon bacteria perhaps? 

Enjoy!

Jac

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Pleased to meet you!

Hey everyone,

Welcome to my blog, so nice of you to join me!

My name is Jacqueline, and I am a 4th year Biology and Chemistry student at the University of New Brunswick. After completing two years of my science degree as a Biology-Psychology major at the UNB Saint John campus, I came to realize that the degree was not the right fit for me. I found myself excited about an organic chemistry class I took as an elective, and bored by my mandatory psychology courses. A few short months later I was packing up to move away from my hometown, to attend the UNB Fredericton campus as a Bio-Chem major. I couldn't be happier with the decision I made to switch, school is so much more fun when you love what you study!

Aside from my love of science, my biggest love is traveling. I spend a lot (maybe too much) of my time watching travel videos, reading travel blogs and dreaming of where my next trip will take me. Nothing quite compares to the excitement of seeing a city for the first time. Here's a photo of me taken minutes after arriving in Amsterdam, one of my favourite cities. I think the smile says it all! 

As far as everyday interests go, running, tea, good food, and fine wine top my list. My guilty pleasures include sleeping in and bad reality TV, but in moderation. I also work in IT during my summers off of school, which is hilarious considering it took me twenty minutes to figure out how to follow my classmates on Tumblr.

Now that you've heard a bit about me, let's dive into what this blog is really all about: The Oral Microbiome. As many of you know, our bodies are home to trillions of bacterial, fungal, viral and archeal cells that outnumber our human cells by 10 to 1! But don't let that gross you out! This community of microbes, known as the microbiome, is essential in human health. The oral microbiome in particular, is compromised of microorganisms that call your teeth, tongue, cheeks and tonsils home. To put it simply, they are the microbes in your mouth! This blog will explore current knowledge on how humans establish an oral microbiome, how it benefits us, as well as how changes in its composition have been linked to disease. I stumbled upon this topic somewhat accidentally, but the more research I do the more excited I get about what's living in my mouth. Is that a weird thing to say?

Stay tuned!

Jac

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My blog will focus on the human oral microbiome. 

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Day 1

Hi everyone, welcome to my blog for Biology 4272!