Dawei Ren:
Synergistic Interactions in Multispecies Biofilms

Date: 07-02-2014    Supervisor: Søren Johannes Sørensen & Mette Burmølle

The coexistence of hugely diverse microbes in most environments highlights the intricate interactions in microbial communities, which are central to their properties, such as productivity, stability and the resilience to disturbance. Biofilm, in environmental habitats, is such a spatially structured aggregation consisting of multiple species of bacteria whose function relies on a complex web of cooperative and/or competitive interactions between community members, indicating that research in “whole-entity” should not be based on the assembled results from “mono pieces”. As one of the best multispecies biofilm models, oral microbial community, also known as “dental plaque” is thoroughly investigated as a focal point to describe the interspecies interactions [1]. However, owing to the lack of a reliable high throughput and quantitative approach for exploring the interplay between multiple bacterial species, the study to elucidate the impact of interaction networks on the multispecies biofilms in natural ecosystems, especially in soil, is still at an early stage. The diverse patterns of interactions within the mixed communities as well as the predatorprey relationship between protozoa and biofilm are summarized in Sections 1, 2 and 3 of this thesis, where the state-of-the-art techniques developed to exploit such interactions, including precisely quantifying the numbers of individual species by quantitative PCR (qPCR) and monitoring gene expression changes during interactions by transcriptomic analysis are also presented.

Due to the poor reproducibility of most biofilm quantification assays, the first part of my work is to develop a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, followed by species specific qPCR based on SYBR Green I fluorescence to measure the relative proportion of individual species in mixed-species biofilms. The reported approach was described in Manuscript 1 which can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. By use of this valuable tool, a more than 3-fold increase in biofilm formation and dominance of Xanthomonas retroflexus and Paenibacillus amylolyticus over the other two species Stenotrophomonas rhizophila and Microbacterium oxydans were demonstrated, indicating the strong synergistic interactions in this four-species biofilm model community.

Manuscript 2 presents the further application of this developed approach on evaluating the synergistic/antagonistic interactions in multispecies biofilms composed of seven soil isolates. 63% of the four-species biofilms were found to interact synergistically, indicating a prevalence of synergistic interaction in biofilm formation among these strains. Hereafter, the population dynamics in a multispecies biofilm composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was assessed using qPCRs with species specific primers. Despite of the high prevalence of X. retroflexus (> 97% of total biofilm cell number), the presence of the three other strains was indispensable for the strong synergism that occurs in this mixed-species biofilm. The dramatically increased cell numbers of each strain at 24 h proved all the individual strains gained benefits in the multispecies biofilms compared with in monospecies biofilms, that is, they would rather cooperate than compete with each other.

The significant synergistic interaction observed in the biofilm consisting of four soil bacteria make this consortium a powerful model to study development and interactions in multispecies biofilms. In Manuscript 3, the gene expression profile of Xanthomonas retroflexus in a single-species biofilm was compared to its expression profiles in dual-species biofilms with Stenotrophomonas rhizophila, Microbacterium oxydans or Paenibacillus amylolyticus as well as in a four-species biofilm. The strongest change in expression profile was observed in the dual-species biofilms of X. retroflexus and P. amylolyticus, while a distinct expression pattern (non-linear response) was detected in the four-species biofilm, indicating the significant effect of interspecies interactions on gene expression. This is consistent with the results presented in manuscript 2 where each species was demonstrated to be indispensable for the synergistic interactions in the biofilm formation. 70 genes were found differentially expressed when co-culturing X. retroflexus with other species, which include genes involved in membrane bound efflux system and MazE/MazF toxin-antitoxin system, suggesting the enhanced resistance of multispecies biofilms.

Despite of the widespread existence of biofilms and protozoa in nature, the predator-prey relation between biofilms and protozoa is still poorly studied. Moreover, this relationship could be affected by interspecies interactions within multispecies biofilms. The study presented by Manuscript 4 was to test whether these interactions in the developed multispecies biofilm model are involved in the defense mechanism of bacterial biofilms against protozoan grazing. The presence of the flagellate Neocercomonas jutlandica was shown to increase or reduce the bacterial abundance in biofilms, depending on the co-cultured bacterial prey, which suggests the grazing ability is closely related with the predator-prey interactions, whereas, the synergistic interactions in the multispecies biofilm model did not confer more protection against predation compared with single-species X. retroflexus biofilm. The same ratio of cell numbers between three species regardless of protozoan grazing suggests they were spatially arranged in integrated communities in multispecies biofilm. However, these conclusions are based on the assumption that this flagellate predator prefers surface attached cells which needs to be confirmed by further studies.

Horizontal gene transfer by conjugation occurs more efficiently in biofilms. The connection between plasmid host range and composition of the recipient community was investigated in Manuscript 5 by comparing plasmid permissiveness in single populations and in a microbial community composed of 15 soil strains. By use of flow cytometry (FCM) and 16S rRNA gene sequencing, the IncP1 plasmid, pKJK10, was found only to transfer from Pseudomonas putida to Stenotrophomonas rhizophila in a diparental mating. However, when hosted by Escherichia coli, transfer of this plasmid occurred only in the mixed community, with Ochrobactrum rhizosphaerae as the dominating plasmid recipient. This study demonstrates that the plasmid host range can be greatly affected by the surrounding bacterial community. This needs to be taken into account as many antibiotic resistance and virulence determinants are plasmid-encoded, which can spread further and raise antibiotic-resistant bacteria in soil.