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. 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.
