15 - What are Biofilms (w/ Gregory Broussard!)
15. What are Biofilms (w/ Gregory Broussard!)
Last week we spoke of bacteria, a major contributor to biofilms. What are biofilms and why do microbes make them? How is human health impacted by this mechanism of microbial life? Why do my teeth feel so fuzzy? Let’s learn to be scientifically conversational.
General Learning Concepts
1) What are microbes? Why do they make biofilms?
a. What is a bacterium? Microscopic, single-celled organisms. They do not have different internal compartments like Eukaryotic cells (human cells, plant cells, yeast cells, for example). They have a cell wall of some thickness, unlike human cells. They can be found in all manners of locations (mountains, oceans, inside of animals, frozen in ice, just about everywhere).
b. What is a microbe? A microbe, or “microscopic organism,” is a living thing that is too small to be seen with the naked eye. We need to use a microscope to see them. This includes bacteria, archaea (a different domain of life, look similar to bacteria but are incredibly different; thermoproteus), fungi (yeast), protists (paramecia, amoebas), viruses (polio, phage), and microscopic animals (mites)
c. What is a biofilm? A paper from 2002 by Rodney Donlan (CDC head of the biofil laboratory at the Centers for Disease Control and Prevention) was entitled “Biofilms: Microbial Life on Surfaces”. To you and I, they may be more easily represented by the slimy-mats that cover rocks in a stream or a river. They’re communities full of microbes and typically are complex mixtures of fungi, algae, yeasts, protozoa, or other microorganisms (though single biofilm mats can form from simply bacteria). The structural component to all of this madness is given by things called “extracellular polymeric substances” (which are chemically dissimilar and have resulted in various publications defining their makeup; think of it as a house for the microbes that live in the biofilm) and can result in biofilms microns in thickness to inches thick. Biofilms can impact fundamental environmental conditions like nutrient availability, spacial availability, and oxygen availability.
d. Do all bacteria make biofilms? Unclear; there is a wealth of different types of bacteria in our world (likely 1 trillion microbes, 99.999 percent undiscovered or unculturable). It seems as if most bacterial species are willing to settle down and form biofilms if offered the chance or a long enough period of time. Still, there are bacteria that this is unknown for; planktonic types that live in the free, open ocean.
e. What is the process of formation of a biofilm? There are characteristics that are important to start a biofilm; the surface might need to be rougher (more surface area) or of a particular type (hydrophobicity). The cells themselves dictate how well they stick to each other. This defines the process of attachment where bacteria adsorp to both the surface and each other. Growth of this biofilm continues by replication, which could be faster or slower depending on the availability of nutrients. These cells can or will begin using entirely different sets of genes because of the signaling from cell to cell and the nutrients available. As this process continues, further colonizing cells from the liquid surrounds can bind and attach. This can be dependent on what kind of molecules are being expressed in the first place. Individual members can become detached from a multitude of factors including erosion, human intervention, predatory grazing, abrasion, or starvation. Those detached members may start another biofilm elsewhere.
2) Biological relevance of biofilms
a. In the environment: Communication; just as humans need to communicate to accomplish tasks (making a sandwich, running a government) microbial species need to be able to speak with one another. These microbes do not communicate by talking but use chemical signals (similar in theory to using hormones in our bodies). There are inherent fitness disadvantages in the environment (because rarely are things as nutrient rich as we make them in the lab) and forming biofilms imparts reproductive benefits to the community as a whole, including defense, favorable habitats, community, and a default lifestyle. 
i. Defense: Biofilms are resistant to physical forces (shear forces of liquids in environments like lakes or blood). Harder for bacteria to be “eaten” by the body or environmental predators.
ii. Colonization: Once again, favorable environments are required for the best fitness of an organism.
iii. Community: Some researchers go as far to imply that we should consider biofilms multicellular organisms and that biofilms exhibit cooperative, unselfish behavior. Much of this is purely philosophical but research is being done to model cooperativity between bacterial and microbial species. Gene expression can become heterogenous (division of labor) for metabolism and beyond, for example. Genes can be transferred in these populations, leading to antibiotic resistance and greater fitness of the population as a whole.
iv. Default mode of growth: The lab does not mimic how bacteria live in the greater world around us; proper substrate and nutrient conditions may more accurately show how these organisms live.
b. Teeth: Biofilms can form on teeth in the mouth (and are more commonly known as plaque); the intercellular matrix is often derived from in/organic materials from the organism’s saliva and other bacteria products. Over time, these bacteria can cause periodontal disease (inflammation affecting the bone and tissues of the teeth), gingivitis (inflammation of the gum at the necks of the teeth), cavities (softening of tooth enamel to cause holes in teeth; teeth decay). Often it is because of their natural metabolic pathways (releasing acids, causing inflammation due to immune responses, etc).   
c. Medical equipment: Problematically, biofilms can form on medical equipment like catheters (central venous and urinary), orthopedic devices like hip replacements, and other devices like artificial heart valves and pacemaker leads (which we will discuss further in the episode about the artificial heart). This leads to infection from medical devices. Furthermore, a Journal of Indian Society of Periodontology publication from 2011 claimed that “organisms in a biofilm are 1000-1500 times more resistant to antibiotics than in their planktonic state” though other sources have said this is too high an estimation. It becomes incredibly difficult to eliminate biofilms by antibiotics than when a bacterium (bacteria) is free floating.    
i. Mechanisms to prevent biofilms in medical equipment: Materials that prevent initial attachment (material sciences); preventing bacteria from being able to communicate to know they could form a biofilm; pursuing greater asepsis (Lawson Tait, episode 11, totally sterile environments); designing molecules or proteins to degrade the biofilm matrix.
3) Fun Tidbits
a. Quorum sensing: Originally a term to use for a committee when enough members are present to legally take action; quorum sensing is often used to describe Vibrio fischeri which produce light at sufficient populations. There are quorum sensing opportunities for other bacteria, like to produce toxins when at a certain population, to protect themselves. These chemical molecules that are used to do this communication are kept at high concentrations because of the sticky matrix that the biofilm provides.
b. When were biofilms discovered? Biofilms were first described by Antonie van Leeuwenhoek (Dutch, 1632-1723, “Father of Microbiology”, early microscopist). While he observed bacteria, RBCs, and other single cellular organisms he also observed biofilms:
i. "The number of these animalcules in the scurf of a man's teeth are so many that I believe they exceed the number of men in a kingdom."
Still, much of biofilms or the process of formation was not traditionally understood until 1978, when Bill Costerton (Director of the Center for Biofilm Engineering in Montana State University) showed that biofilms acted differently than their free-floating relatives.  
4) Solicited Naïve Questions
a. What is Pseudomonas aeruginosa? A bacterial species that acts as an opportunistic pathogen that can exploit numerous environmental niches; commonly found in cystic fibrosis patients (like me if I wasn’t only a carrier) but can also be seen in those with burn wounds or implanted biomaterials. P. aeruginosa is a major cause of nosocomial infections (infections derived from the hospital) which affect more than 2 million patients every year and are accounted for around 90,000 deaths annually, due to the things we’ve already discussed in this episode.