Microbial biofilms: life in a community

Part 1 - Know thy enemy

  🕑 10 min read.

This is the first in the series of 3 blog posts exploring biofilms, their role in periodontitis and our ways to fight them.

“No man is an island,
Entire of itself,
Every man is a piece of the continent,
A part of the main.”
John Dunne

First thing we learn in dental school is that both caries and periodontal disease, our greatest woes, are biofilm-associated diseases. And even though the curriculum requires from us to learn and shoot biofilm-related facts (What are the extracellular polymeric substances that constitute its matrix? / Explain the biofilm formation and maturation process. / What are microbial complexes and what bacteria constitute them?), how come then that “calculus removal appointments” and prescribing antibiotics for treatment of gingivitis without picking up the ultrasonic are still a thing in the daily practice of many clinicians? And so, the aim of these posts on biofilm is to gradually, through a learning discovery process, build understanding of it and put it to clinical practice.

Brief lesson in history

We have not known much about bacteria until 350 years ago. One ingenious mind of the 17th century, Antonie van Leeuwenhoek, in 1676 observed a sample of pond water under a self-made microscope, seeing what we today know were protozoa and which he had named “animalcules” (derived from Latin, meaning “little animals”). A few years later, in 1683, he gave an indisputable description of bacteria, observed from his own dental plaque, which he also went on later to define as “white matter, batter-like consistency”, which, even after “cleaning the tooth, can be seen in some places with a magnifying glass, where it stays or grows between molars”.

The miasma theory, holding that diseases are caused by miasma, i.e. bad air/night air, was the prevalent theory of disease in Europe up until, I would dare to say, recent history of humankind.  It wasn’t until the middle and the end of the 19th century, the time of Louis Pasteur and Robert Koch, that doctors and scientists associated germs (pathogens) with the occurrence of certain diseases. Louis Pasteur was the one that proposed the germ theory of disease, while Robert Koch validated it with his experiments. His 4 basic criteria identifying a certain pathogen as the disease’s cause became known as Koch’s postulates. These proved to be inapplicable to certain diseases, such as periodontitis, it being an opportunistic rather than a classic infection, as I will further discuss in the part 2 of this series of posts on biofilm. Thus, Sigmund Socransky and Anne Haffajee proposed modified postulates for periodontal pathogens in year 1992, hundred years after Koch. With many infectious agents identified and newly acquired knowledge to halt the spread of microorganisms and epidemics, the late 19th and early 20th century became the Golden Age of Microbiology. Following Alexander Flemming’s accidental discovery of penicillin in 1928, the end of World War II brought a profound advancement in medicine with the widespread use of antibiotics. Modern society is still obsessed with fighting germs. Ironically, the practice of mindless prescription of antibiotics in healthcare, as a result of sometimes irrational fear (and ignorance?) has directly contributed to the emergence of pathogens resistant to them. And, while we are still living in the modern era of microbiology, the post-antibiotic one seems to be dawning upon us.

microbiology eras periocampus
The 4 eras of microbiology - are we entering a 5th, post-antiobiotic one?

What are we?

Even with the revised estimated numbers, we are more bacteria than we are human. There are 30% more bacterial cells inside and on our bodies than there are our own. In 2008, the United States National Institutes of Health launched a project called the Human Microbiome Project, which aimed to sequence the genome of the human microbial community and identify changes in its composition in health and disease. Published results show that our body is home to over 10,000 different types of microorganisms.

Even though these figures may seem daunting, the fact is that the connection between the host, the human, and its microbial flora is very often mutually beneficial. Microbial flora plays an important role in certain body functions. One of the best-known examples is the production of vitamin K2 (menaquinone), which is synthesized by the bacteria in the gut, particularly in the ileum. Vitamin K is necessary for the synthesis of clotting factors, prothrombin (factor II), factors IV, IX and X. Although vitamin K can partially, in the form of K1 (phylloquinone), be ingested through food, studies suggest that the bacterial production contributes the most to the body’s need for this vitamin, preventing clinically significant coagulopathy, especially during periods of reduced dietary intake.

Furthermore, the microbial flora influences the development of the body’s immune system. Newborns at birth, after growth in the sterile environmental conditions of the uterus, are exposed to invasion by various microorganisms, particularly bacteria. This process is estimated to be happening in the first four years of life, after which the child’s microbiome stabilizes. This microbial invasion is by far the strongest stimulus for the immune system and has an important impact on its maturation. Many developed countries have seen an increase in the prevalence of allergic diseases such as atopic dermatitis, asthma and atopic rhinitis in recent decades. Due to improvements in hospital conditions, more frequent cesarean sections, better hygiene standards in general, widespread and irrational use of antibiotics, fewer family members and other factors, there’s been a change in exposure to bacterial species of newborns and children. Th-2 response (humoral immunity) typical of the newborn, of which Th-2 lymphocyte-producing cytokines are associated with enhanced IgE and eosinophil response typical in atopic diseases, decreases after exposure to bacteria. The intestinal flora is thought to serve as a counter-regulator and promotes Th-1 (cellular immunity) differentiation.

No bacterium is an island

I did not quote John Donne in the beginning by chance. Bacteria have actually long been thought to be unicellular, planktonic organisms. In 1943 though, Claude ZoBell, an American father of maritime microbiology, discovered that the number of microorganisms adhering to a solid surface was far greater than of those in the surrounding liquid medium. ZoBell’s contribution to our understanding of biofilm formation is remarkable; he discovered that biofilm formation is a rapid, active process influenced by the amount of nutrients available, as well as correctly recognizing that it is the bacteria themselves that produce the adhesive matrix of the biofilm. While ZoBell used the term “attached films”, the term “biofilm” we use today was only coined and described in 1978 (!!!).

Biofilm up-close-and-personal. Traditionally there’s been an over-emphasis on the removal of calculus (hard deposits), while it is the mechanical disruption of biofilm that is the cornerstone of periodontal treatment. More about periodontal biofilm and calculus in Part 2.

Why do bacteria prefer living in community rather than solo?

Bacterial biofilm can be defined as an aggregation of one or more distinct groups of microorganisms embedded in a matrix which is attached to a solid surface. Biofilms are ubiquitous; bacteria can form them on a wide variety of surfaces, natural or artificially produced, in naturally humid environments, on living tissues, medical instruments, water supply systems… The formula is very simple – as long as you have bacteria in a wet or humid environment and a solid surface, you will have biofilm. Recent studies have shown that biofilms can also be found on Antarctic glaciers. They are not least bothered by high temperature and/or high pressure because they inhabit thermal waters at depths of 60m and temperatures of 35° to 50°C. Even the most unwelcoming conditions, such as extreme acidity, high concentrations of metals and nutrient deficiencies cannot stop bacteria from forming and thriving inside the biofilm.

High or low temperatures, toxic metals, absence of nutrients and/or desiccation... bacterial communities inside of the biofilm could not be bothered in the slightest.

Keeping that in mind, the logical question is what makes the biofilm so resilient? The answer is – its structure. Bacterial microcolonies account for only 15 – 20% of the biofilm’s volume, while the rest, 75 – 80%, is the matrix in which they are embedded. The biofilm’s matrix consists of a mixture of different natural polymeric compounds, (exo)polysaccharides (EPS), mostly produced by the biofilm’s microorganisms themselves. EPS is called the “dark matter of biofilms” because of the large variety of matrix biopolymers and the difficulty to analyze them. EPS vary among different biofilms, depending on the present bacteria, temperature and available nutrients. Recently, the term exopolysaccharides was replaced with the term extracellular polymeric substances (also EPS), as it more accurately captures the complex structure constituting of proteins, nucleic acids, lipids and other biopolymers, not just polysaccharides. The structure of bacteria-produced exopolysaccharides may differ even within strains of the same bacterial species. Pseudomonas aeruginosa, one of the best studied models for biofilm formation, produces as many as three structurally distinct exopolysaccharides that contribute to biofilm development and architecture. Mutant strains that cannot synthesize exopolysaccharides are not able to form a mature biofilm. In biofilms composed of different bacterial species, bacteria that synthesize exopolysaccharides allow the integration of bacteria of other species that cannot do the same. In conclusion, the diversity of exopolysaccharides in mixed biofilms does not necessarily reflect the diversity of cells present, nor do different exopolysaccharides contribute equally to the structure and properties of the biofilm.

Favelas remind me of biofilm structure. Densely populated, with internal sets of rules, unwelcoming for the outsiders (external conditions!) and law enforcement (antibiotics!).

EPS surround and immobilize bacterial cells, holding them in close proximity, allowing them to interact and enabling their intercellular communication which is discussed in part 3 of the biofilm post series. The biofilm matrix is also a recycling ground. It retains the components of degraded bacterial cells, including their DNA which can serve as a potential gene reservoir for horizontal gene transfer (i.e. expression of certain virulence factor)! It also retains extracellular enzymes which, in a sort of a “digestive” system of the biofilm, break down individual nutrients and allow bacteria to use them as a source of energy. Further functions of the biofilm’s matrix substance are water retention (- prevents biofilm from drying), donor and electron acceptor functions (- enables redox activity in the matrix), biofilm cohesion, absorption of organic constituents and inorganic ions, and surface adhesion in the first stages of biofilm formation.

Know thy enemy

With all of this in mind, it is far easier to answer the question why bacteria prefer life in biofilms. From a perspective of a single, planktonic bacterium, the external environment and surrounding conditions are often and rapidly changing. A bacterium that passes through the human gastrointestinal tract will experience pH changes from highly acidic (pH 2-3) to normal (pH 8-9) within just a few hours. The daily life of bacteria in Iceland’s underwater hydrothermal springs presumes presence of metals and temperatures ranging from + 100 ° C to freezing temperatures within minutes. Finally, bacteria are exposed to constant changes in the amount of nutrients such as nitrate, phosphate, sulfate and the presence / absence of water and oxygen. They are exposed to many different external stresses that compromise their ability to survive and grow. Formation and life in biofilms are in fact an extraordinary, ingenious adaptation strategy that makes it easier to reduce the impact of environmental factors.

 

 

To be continued…

In part 2 in the series of posts on biofilms we will explore periodontal pathogens and how knowledge of biofilms directs our periodontal treatment.