biofilm studies

This week, the World Health Organization (WHO) is recognizing antimicrobial resistance (AMR) awareness week. AMR is a significant and growing problem and poses a grave public health risk. However, much of the dialogue surrounding AMR elides the importance of bacteria to human thriving. This week, I thought it would be interesting to talk about the fascinating way in which bacteria engender AMR by working together. Understanding how bacteria function can help us manage pathogens and achieve equilibrium with our resident biomes. After all, eradication of all microorganisms is not the goal. I think that sometimes in our pursuit of cleanliness, we forget that humans need bacteria. Ours is a symbiotic relationship, but one we tend to ignore most of the time unless it has gone terribly and tragically awry.

In the last few decades a growing body of research has emerged to show that bacteria actively cooperate in consortia known as biofilms. Many of the classical social behaviors observed in macroorganisms have correlates in bacteria, including domicile construction, communication and collective decision-making, division of labor, resource sharing, and perhaps even altruism.

The vast majority of bacteria live in sessile communities called “biofilms.” In fact, biofilms seem to be the preferred bacterial state. Most, if not all, wild-type (WT) bacteria can form biofilms—complex structures formed by layers of bacteria embedded in a self-produced matrix made of predominantly water, mixed with extracellular polymeric substances (EPS) that consist of carbohydrates (polysaccharides), proteins, lipids, and extracellular DNA (eDNA). The exact molecular composition of the matrix differs among strains and species, and depends upon environmental factors like temperature, pH, nutrient status, and shear forces. The EPS constitute a hydrated, secure microniche conducive to long-term bacterial interaction.

Within the larger matrix are smaller subdivisions or “microcolonies” separated by gaps or channels. These channels form a network that allows a biofilm-wide circulation of nutrients, metabolites, signaling molecules, waste products and other solutes. The results of formation of structures with one or more layers of varying morphologies; biofilms can be described, rather charmingly, as smooth, rough, fluffy or filamentous (Figure 1).

Figure 1. Say hello to my little friends! Various biofilm morphologies. From Fleming & Wingender (2010).

Five stages in biofilm formation have been proposed: 1) Reversible attachment, 2) irreversible attachment, 3) maturation I, 4) maturation II, and 5) dispersion. Biofilms begin with microbial adhesion facilitated by flagella or pili to practically any surface, organic or inorganic, with the corollary that they are found almost everywhere. This is not to imply that bacteria are lack discernment when it comes to selecting colonization sites; they are exquisitely sensitive to environmental conditions, particularly nutrient availability, although abundant nutrition does not guarantee the development of biofilms. Rather, bacteria seem more likely to congregate under adverse conditions.

Bacteria in biofilm communities display an interesting repertoire of social behaviors. For example, when resources in the present environment become depleted, Myxococcus xanthus cells within the biofilm leave vertical cell stacks to aggregate into spheres and form protective “fruiting bodies” containing spores. During this process, some of the rod-shaped M. xanthus biofilm residents will morph into rounded myxospores, but the majority of the cells in the aggregation will lyse, sacrificing themselves so that the spores might survive to spawn another swarm.

Communication among bacteria occurs at all stages of biofilm formation. Pseudomonas aeruginosa secrete a signal molecule when they visit a site that can bind to pili on its fellows, attracting more bacteria to facilitate biofilm creation. Similarly, when searching for new colonization sites, Escherichia coli and Proteus mirabilis use chemical signals to coordinate movement that resembles the swarming activity seen in insects and fish.

Once established, bacteria communicate within the biofilm through signaling systems known as quorum sensing (QS). By sending and receiving signals, bacteria can coordinate large-scale group activities such as bioluminescence, antibiotic production, or host cell invasion through simultaneous expression of appropriate genes. Indeed, the success of many behaviors is dependent upon majority participation. There are several systems of QS employed by different types of bacteria. The ability to communicate allows biofilm members to engage in cooperative behaviors.  Myxobacteria collectively attack colonies of prey bacteria through the coordinated release of lytic enzymes. Additionally, there is evidence of specialization and labor division. In fact much of biofilm resistance to antibiotics may be through the altruistic action of “persister” cells, which remain dormant while their more metabolically active kin flourish. By giving up the chance to reproduce for the ability to weather stressors such as antibiotics and host immune response, persistent bacteria can resuscitate colonies that would otherwise be destroyed. Perhaps unsurprisingly, bacteria which are regularly exposed to antibiotics produce populations containing a higher percentage of persisters. 

As with all things in life, there is a catch. Biofilms are not unequivocally advantageous arrangements. Perks are attenuated by accompanying disadvantages, including energetically costly biofilm entrance/exit, growth constraints, and within-biofilm competition. While EPS protect all biofilm dwellers, they can also be used to suffocate laterally adjacent neighbors and propel descendants to more nutrient-rich areas. Additionally, some species such as M. xanthus, Pseudoalteromonas tunicata, and Roseobacter gallaeciensis show territoriality, and produce microbicides that specifically inhibit competitors. There is also evidence of “cheating” behavior. For example, P. aeruginosa secrete iron-scavenging molecules called sidephores. Because siderophore production is energy-intensive, individuals have an incentive to take unfair advantage of shared resources by using the siderophores of others, while not producing their own. Given these and similar tendencies, one might expect that mono-species colonies would be favored to reduce the chances of conflict, assuming that bacteria are less likely to harm others with whom they share genetic material. In fact, it is more common for biofilms to have heterogeneous populations. More than 500 taxa have been found coexisting in oral biofilms. Some data indicate that multispecies biofilms are more robust and use energy more efficiently than do mono-species counterparts.

The widespread, categorical success of social bacteria implies that the benefits of the biofilm outweigh disadvantages. Migratory bacteria travel together and/or actively seek each other. They interdependently coordinate their gene expression/protein production to construct a shared habitat. Cellular organization and specialization occur in order to maximize resource utilization. Sacrifices are made, as in the case of persisters, where individual cells forego their chance at reproduction to ensure the survival of their fellows. There is still much to discover about biofilms. As our understanding grows, so too, I hope, will our ability to tame infectious disease, and harmonize with our prokaryote companions.