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Hello. My name is Mette Burmølle and I am an associate Professor in Microbiology at
University of Copenhagen. In this lecture I will introduce you to this last theme of
the course, which focuses on evolution in biofilms and chronic infections.
The general principles of evolution are independent of the specific environment, but some very
specific conditions relating to time and space are faced by bacteria in chronic infections
- and this affects evolution and thereby shapes the bacterial community.
As you know, bacteria in chronic infections are found as biofilms, so we can learn a lot
from biofilm models when identifying the evolutionary drivers shaping chronic infections. In several
cases, biofilms have shown to give rise to a higher frequency of cells with varying colony
morphology as opposed to planktonic cultures. These can be smooth, wrinkly and small, and
are caused by either transient changes in gene expression or mutations in the genetic
material. The main difference between these two modes of responding to a specific environment
– also called adaptation - is that where gene regulation is a transient change, mutations
in the genetic material are irreversible and will be passed on to the later generations
– and this latter mechanism is the foundation of evolution.
In order to describe the principles of evolution it is necessary to introduce a few terms.
I will briefly explain these here:
The first term is fitness: Fitness describes the ability of an organism to survive and
reproduce in a specific environment or under specific conditions. The fitness will depend
on the phenotype and the underlying genotype of an organism. The genotype is the term describing
the genetic background, giving rise to certain, specific traits expressed by this specific
genotype, thereby determining the phenotype. So those individuals with the geno- and phenotype
giving rise to the best ability of reproducing and surviving will have the highest fitness.
Another important term is adaptation: Adaptation is an evolutionary change in the genotype
– and thereby phenotype – of an organism that leads to a higher fitness in a particular
environment. The underlying genetic changes are most often caused by mutation in single
base pairs of DNA or acquisition and stabilization of longer DNA fragments. Such changes are
often unfavourable or even lethal to the organism, but sometimes they lead to higher fitness
and they will then be selected for by natural selection – which is the last term:
Natural selection describes the process in nature leading to the dominance of the more
fit phenotypes over the less fit ones, due to their abilities of surviving better or
reproducing more or at a faster rate. So by natural selection, the organisms with the
highest fitness will be selected for and will - over time - dominate the population.
Evolution is the process of adaptation leading phenotypes with higher fitness that will be
favoured by natural selection, leading to elevated frequency of this or even complete
dominance over time.
So back to the biofilm and the phenotypic variants I briefly introduced earlier in this
session. We often observe the occurrence of such variants in high frequencies in biofilms
as opposed to planktonic cultures, where they don’t arise. By further investigation, it
can be showed that the variants are phenotypically irreversible, which indicates that they represent
adaptations to the specific biofilm environment that have been selected for by natural selection.
The specific conditions in the biofilm may select for variants better at adhering to
surfaces, or for those able to get access to oxygen or nutrients, which may be limiting
factors. In chronic infections, very specific conditions are faced by the bacteria present,
so here also the ability of tolerating the host immune defence system or antimicrobials
may be strongly selected for.
As you already know, chronic infections are very often caused by more than one bacterial
species and this affects evolution. When evolution is triggered by the presence of another organism,
this is described as co-evolution. The drivers of co-evolution are highly diverse as the
intension can be either to inhibit or escape the other species – such as a predator-prey
interaction – or to get advantage of it – such as symbiosis. Examples of co-evolution
in the bacterial world are production of toxins that inhibit other species or optimized utilization
of metabolites and waste products – where one species are living on the by-products
of another – also know as syntrophy. Because of the high cell density and structural organisation
in biofilms, the conditions for co-evolution are present and has been documented in this
environment.
Very recent results from my own research group at University of Copenhagen, indicate that
bacteria in natural environments co-evolve through long-term co-existence. In order to
investigate this, we have isolated bacterial strain collections from soil, freshwater and
marine environments, and evaluated their ability of biofilm formation when grown alone and
as co-cultures containing combinations of 2 to 8 species isolated from the same environment.
We found that – on average – more than half of these co-cultures produced more biofilm
than that expected from the biofilm formation of each individual strain when grown alone.
This means that there seems to be a tendency of bacterial strains to stimulate biofilm
formation of each other, when mixing them in co-cultures.
We then decided to test strain collections from environments where co-existence is less
pronounced due to regular cleaning, and we therefore isolated bacterial strains from,
a day-care and a food processing facility. When testing these as mono- and co-cultures
we found that the frequency of enhanced biofilm formation by co-cultures was much lower – only
in approximately 10% of the combinations tested co- cultures formed more biofilm than expected
based on the mono-culture biofilms. So there seems to be a tendency of co-existence in
natural environments to lead to enhanced biofilm formation by co-cultures.
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We have not identified whether this enhanced
biofilm formation among co-cultures is caused by bacterial cooperation or competition – or
a combination of both. But we do have data from another study, where we have investigated
the mechanism of biofilm synergy in more detail. In this study we can see that when we mix
four species in a co-culture, they all achieve higher cell counts in the mixed biofilm community,
compared to their biomass as monospecies biofilms. This strongly indicates that, in this case,
the biofilm synergy is caused by cooperative forces.
Of course the time frames of co-existence in natural environments and chronic infections
are very different, but nevertheless some of the evolutionary forces driving co-evolution
in natural systems may also apply in chronic infections.
To summarize, evolution in chronic infections are driven by adaptation to the physical environment,
resulting in specialized variants that are selected for. The presence of multiple species
may result in co-evolution, so that the infecting species are shaping each other, and long-term
co-existence may facilitate this process.
Thomas BjarnsholtProfessor
