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High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions, Useful for Exploring Interspecific Bacterial Interactions

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Abstract

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.

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Acknowledgments

This research was supported by funding to Mette Burmølle by The Danish Council for Independent Research, Technology and Production, ref no: 09-090701, to Søren Sørensen by The Danish Council for Independent Research, Natural Sciences and the Danish Innovation Consortium, SiB, ref no: 11804520 to Jonas Stenløkke Madsen.

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Authors and Affiliations

  1. Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Dawei Ren, Jonas Stenløkke Madsen, Claudia I de la Cruz-Perera, Lasse Bergmark, Søren J. Sørensen & Mette Burmølle

  2. Section of Microbiology, University of Copenhagen, Universitetsparken 15, bygn 1, 2100, Copenhagen Ø, Denmark

    Mette Burmølle

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  1. Dawei Ren
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Correspondence to Mette Burmølle.

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Supplementary Fig. 1

Standard curves used to measure the copy numbers of 16S rRNA genes of strains 2: Stenotrophomonas rhizophila, 3: Xanthomonas retroflexus, 5: Microbacterium oxydans, and 7: Paenibacillus amylolyticus in multispecies biofilm. See text for further details and discussion of RSq and efficiencies (Eff). (DOCX 122 kb)

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Ren, D., Madsen, J.S., de la Cruz-Perera, C.I. et al. High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions, Useful for Exploring Interspecific Bacterial Interactions. Microb Ecol 68, 146–154 (2014). https://doi.org/10.1007/s00248-013-0315-z

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