So, what initiates biofilm formation in the first place? Quorum sensing, bacterial density, autoinducers?

How do 10, 20, or 1000+ individual bacteria all minding their own business at a specific threshold, 1001, for example, initiate quorum sensing?

Has anyone studied the mechanism behind population density? Why 100 bacteria can peaceful bacteria cohabitate, but at 101, it becomes too much to handle, and the whole environment changes, and not for the better.?

This was posed to Dr. Gregory Anderson, a biofilm researcher, in an email exchange. The more data I have, the better I’m able to come up with an effective and comprehensive, natural biofilm-busting protocol. I truly appreciate the time and effort Dr. Anderson put into the information he shared with me.


The numbers really don’t matter. What matters is the population density. You could have ten bacteria in a really small space, and they would be experiencing all sorts of quorum-sensing behavior. On the other hand, you could have 10^9 bacterial cells in a huge volume and no quorum sense at all. This is because, at high cell density, you also have a high density of the autoinducer molecule (the molecule produced by the bacteria that they then sense to initiate quorum sensing).

Def. Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria.
Bassler BL, et al., Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012 Nov 1;2(11). pii: a012427.

In fact, in the early days of quorum sensing research, they took cell-free supernatants from dense cultures and added them to low-density cultures, and they saw quorum sensing regulated gene expression. This was because the culture supernatant contained high concentrations of the autoinducer molecules.

Def. Autoinduction (AI), the response to self-produced chemical signals, is widespread in the bacterial world. This process controls vastly different target functions, such as luminescence, nutrient acquisition, and biofilm formation, in different ways and integrates additional environmental and physiological cues.
Burkhard A. Hense, et al., Core Principles of Bacterial Autoinducer Systems. Microbiology and molecular biology reviews

In a growing culture, though, you are right; at low numbers (or, more accurately, low cell density), there is no quorum sensing. But once that threshold density is achieved, then that pathway is initiated. What’s happening is that the autoinducers, which are steadily pumped out of each cell, finally accumulate to a sufficient level to be simultaneously recognized by each cell.

Imagine two people standing on opposite sides of a tennis court, tossing out tennis balls at random. The probability is low that any tennis balls will hit either person. If there are 300 people on the tennis court throwing balls, the probability is high that each person will be hit constantly. So as you increase from low density (2 people) to high density (300), the probability steadily increases that each person will be hit (4 people, probably not; 8 people, probably not; 16 people, maybe; 32 people, likely). Once they reach that threshold density, then each person will be hit constantly.

Similarly, in quorum sensing, as bacteria increase in number in a confined space, they will eventually reach a threshold density at which they each constantly receive the quorum sensing signal (autoinducers). Is it possible that they get hit before then? Yes. But since they are free-floating, as they diffuse away, it is less likely that they will get continual autoinducer signals until the threshold density is achieved. Thus, they will only experience transient quorum sensing activated gene transcription.

In a biofilm, however, bacteria are stuck to each other. Thus, in a growing biofilm microcolony, there is a local high density of bacteria, which is ideal for constant quorum sensing activation. So once more than one bacterium starts to bind to a surface (or one binds and then divides), quorum sensing will play a large role in that biofilm development. It’s all based on probability and statistics.

I’m not sure I have the best article to describe quorum sensing, but the attached review is from one of the greats in the field.

Bacterial Quorum Sensing: Its Role in Virulence and Possibilities for Its Control



Dr. Gregory Anderson, PhD
Assistant Professor Biology Department at Indiana University–Purdue University Indianapolis

My laboratory studies the interactions between bacterial pathogens and the host epithelium.  Specifically, we are interested in understanding how Pseudomonas aeruginosa exploits underlying lung dysfunction in individuals with cystic fibrosis (CF) to establish and maintain chronic lung infection.  After CF lung colonization, P. aeruginosa undergoes genetic regulatory changes leading to the formation of antibiotic-resistant biofilms, which persist in the lung for the life of the patient despite aggressive antimicrobial therapy.  We have developed a novel system for the development of P. aeruginosa biofilms on human CF-derived airway epithelial cells in vitro.  Using this model, we are identifying factors that impact biofilm antibiotic resistance as well as bacterial virulence in the context of CF lung infection.

We are also interested in understanding the mechanisms of biofilm formation in chronic wound infections. While a number of different pathogens have been identified in chronic wounds, P. aeruginosa is found in the most severe. We are investigating how this microbe colonizes these sites and maintains long-term infection.

The overall goal of our research is to better understand the nature of chronic infections so that new and better therapies can be developed. Toward that end, we are testing novel compounds for antimicrobial and antibiofilm activity.

3-6-19 Update with a personal note:

Quorum sensing in bacteria.
Miller MB, Bassler BL.

Yes, I’m a science and truth geek. This research journal article (abstract below) was written with love and much research. Very few people will ever read it and even fewer will grasp the totality of what it says. I don’t expect anyone to go further than this paragraph. but if you can or choose to research this you will actually know more about the basics of life and how we operate from bacteria to groups of humans. It is one of the common denominators behind how you can take a harmless bacteria, a human being, or a civilization and go from passive to aggressive in the blink of an eye.

“Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum-sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of autoinducers leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum-sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that bacterial autoinducers elicit specific responses from host organisms. Although the nature of the chemical signals, the signal relay mechanisms, and the target genes controlled by bacterial quorum sensing systems differ, in every case the ability to communicate with one another allows bacteria to coordinate the gene expression, and therefore the behavior, of the entire community. Presumably, this process bestows upon bacteria some of the qualities of higher organisms. The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.”

03-15-2020 Update

3-13-2020 Katherine Unger Baillie, University of Pennsylvania

For their study, they used the microbe Streptococcus mutans, an oral pathogen responsible for causing cavities when it forms a biofilm known more commonly as dental plaque and releases acids that decay tooth enamel.

They distributed the bacteria on a tooth-enamel-like material and followed hundreds of individual microbes for several hours as they divided and grew.

Overall, the growth patterns were reminiscent of the formation of urban areas, the team found. Some individual “settlers” grew, expanding into small bacteria “villages.” Then, as the boundaries of the villages grew and, in some cases, met, they joined to form larger villages and eventually “cities.” Some of these cities then merged to form larger “megacities.”

Surprisingly, the researchers’ results showed that only a subset of the bacteria grew. “We thought that the majority of the individual bacteria would grow,” says Koo. “But the actual number was less than 40%, with the rest either dying off or being engulfed by the growth of other microcolonies.”

They also didn’t expect a lack of inhibition when this engulfment took place. They thought that, as different microcolonies met, they might compete with one another, causing the two edges to repel, perhaps.

“Instead, they merge and begin to grow as a single unit,” says Koo.

On both the individual bacteria and biofilm-wide scale, the researchers confirmed that the gluelike secretion known as extracellular polymeric substances (EPS) enabled bacteria to pack together closely and firmly in the biofilm. When they introduced an enzyme that digested EPS, the communities dissolved and returned to a collection of individual bacteria.

“Without EPS, they lose the ability to pack and form these ‘cities densely,'” says Koo.

Finally, the researchers experimented to see how adding a microbial “friend” or “foe” would influence the original bacteria’s growth. The “foe” was Streptococcus oralis, a bacteria that can inhibit the growth of S. mutans. This addition dramatically impaired the ability of S. mutans to form larger “cities,” like disruptive neighbors that can affect the collective growth of the community.

The “friend”—the fungus Candida albicans, which Koo and others have found to interact with S. mutans in biofilms and to contribute to tooth decay—did not affect the biofilm’s growth rate but did help bridge adjacent microcolonies, enabling the development of larger “cities.”

Koo cautions about taking the urbanization metaphor of biofilm growth too far but underscores the useful lessons that can result from studying the system holistically and by looking at the events under both “close-up” and “bird’s eye” views.

“It’s a useful analogy, but it should be taken with a grain of salt,” Koo says. “We’re not saying these bacteria are anthropomorphic. But taking this perspective of biofilm growth gives us a multiscale, multidimensional picture of how they grow that we’ve not seen before.”

So, now you know a little more about what initiates biofilm and its relationship to quorum sensing, bacterial density, and autoinducers.

Nitric Oxide Activation For Biofilm Dispersal – A Novel Approach