Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

TLDR

Biofilms in natural settings are typically multispecies consortia whose members interact synergistically or antagonistically, yet few studies have examined these interactions. The authors screened 17 epiphytic bacterial strains from the marine alga *Ulva australis* to identify synergistic biofilm interactions among different strain combinations. Synergy was shown to depend on both species‑specific physical contacts and extracellular secreted factors, as demonstrated by replacing individual strains with their cell‑free culture supernatants. Four strains (Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii) formed a biofilm with >167 % greater biomass and exhibited markedly higher resistance to hydrogen peroxide, tetracycline, and invasion by *Pseudoalteromonas tunicata* than single‑species biofilms.

Abstract

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.