Henriette Lyng Røder:
Co-existence in multispecies biofilm communities

Date: 20-04-2017    Supervisor: Mette Burmølle & Søren J. Sørensen

Bacteria have traditionally been viewed as individuals and studied as such. However, there is currently consensus that bacteria predominantly exist in biofilm. Bacteria residing in biofilm are organized in a matrix enabling them to adhere to each other and to surfaces. They are often provided with an increased tolerance against e.g. antibiotics, which could otherwise have killed the individual bacteria. In natural environments biofilm consists of several distinct species increasing the complexity of interactions between the species. When several bacterial species co-exist, they influence each other leading to emergent properties that cannot be predicted from studies of mono species biofilm. These emergent functions can lead to e.g. enhanced tolerance or increased biomass production compared to mono species equivalents. Understanding multispecies interactions can expand our knowledge of these emergent properties which are relevant to as diverse areas as clinical settings and natural systems.

In this thesis, I have attempted to contribute to our knowledge on the multispecies interactions with a special focus on biofilm communities. I was especially interested in how co-existing species affect each other and in understanding the key mechanisms and interactions involved. In the introduction of this thesis the most important concepts of multi-species interactions and biofilm development are explained. After this the topic changes to the various ways of examining community interactions in biofilm and their potential. Towards the end of this introduction, I present my conclusions and thoughts on the future perspectives of this research area.

This PhD thesis has resulted in 3 published manuscripts in peer-reviewed journals and the production of 3 draft manuscripts. The manuscripts follow the order of my work on describing the effects of multispecies interaction with a focus on biofilm formation.

Manuscript 1 is a review that emphasizes the importance of considering which bacterial strains to combine when studying multispecies biofilm and the complexity level that follows as the number of member species increases. After this, various approaches taken by different studies when investigating multispecies communities are discussed, and different techniques for studying multispecies biofilm are described.

In manuscript 2, a diverse group of bacteria was co-isolated from a meat processing environment to evaluate their biofilm formation capability. It was found that multispecies consortia could lead to increased biofilm formation compared to mono species growth. This shows how co-localized isolates are able to influence biofilm production in a community with high relevance for food safety and production.

The analysis was further extended in manuscript 3, in which the effect of social interac-tions on biofilm formation in multispecies co-cultures isolated from a diverse range of environments was examined. The question raised was whether the interspecific interactions of co-existing bacteria generally lead to enhanced biofilm formation in complex communities. We showed that bacteria, expected to have co-existed for a long time, generated more biofilm biomass in co-culture. This was further tested against random co-cultures of bacteria, which supported the conclusion.

Discovering that some bacterial species responded to the presence of other species led to the study presented in manuscript 4. Here, the influence of co-existence on two species was further evaluated by examining how bacterial co-evolution affected diversification and how this in turn influenced their relationship. It was found that two facultative mutualistic bacteria, Paenibacillus amylolyticus and Xanthomonas retroflexus promoted a distinct phe-notypic variant of X. retroflexus. Co-cultures containing this wrinkled colony were more productive than those containing the wild type; assessed as enhanced cell numbers of both strains. This illustrates the importance of considering interspecific interaction when evaluating factors that shape intraspecific diversification in a bacterial community.

The discovery of the wrinkled variant of X. retroflexus prompted manuscript 5, where we examined the ability of this variant to produce biofilm. The main aim of the study was to test if changes in the matrix produced by the wrinkled X. retroflexus could explain the in-creased productivity of the two strains. It was discovered that the changes in the biofilm matrix, induced by the wrinkled variant of X. retroflexus, stabilized the community. The increased biofilm biomass produced by the new wrinkled variant of X. retroflexus lead to a more positive or neutral co-existence with P. amylolyticus compared to the wild type X. retroflexus.

Manuscript 6 investigated how a multispecies biofilm was affected from grazing by the heterotrophic protist, Tetrahymena pyriformis. The biofilm community consisted of Pseudomonas aeruginosa PAO1, Pseudomonas protegens and Klebsiella pneumoniae. P. aeruginosa PAO1 was able to protect the other, sensitive strains by producing rhamnolipids. Interestingly, a rhamnolipid defective P. aeruginosa PAO1 was still able to maintain grazing tolerance for the whole community. Furthermore, P. aeruginosa PAO1 without neither rhamnolipids nor the type III secretion system was also able to resist grazing, but the overall grazing resistance was reduced in this case. This demonstrates that residing in a multi-species biofilm can be an advantageous survival strategy for species susceptible to grazing.