Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies 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.

FIG. 1.

Biofilm formation in microtiter wells by the 17 epiphytic isolates (including Pseudoalteromonas tunicata) isolated from the marine alga Ulva australis. After 24 h of incubation, the biofilm formation was quantified by staining with crystal violet followed by spectrophotometric absorbance measurements (OD₆₀₀). The ratio of biofilm absorbance/planktonic absorbance was calculated, and this value is presented as the “biofilm formation” on the y axis. Isolates with a biofilm formation of less than 2 (indicated by a line) were chosen for further studies on interactions in biofilms. Bars represent means ± standard errors for four replicates.

FIG. 2.

Biofilm formed by the four epiphytic isolates, 2.04 (Microbacterium phyllosphaerae), 2.12 (Shewanella japonica), 2.3 (Dokdonia donghaensis), and 2.34 (Acinetobacter lwoffii), when incubated in microtiter wells in various combinations of one to four isolates. Equal total cell densities were inoculated in each well. The plate was incubated for 24 h, followed by crystal violet staining and spectrophotometric absorbance measurements (OD₆₀₀). The ratio of biofilm absorbance/planktonic absorbance was calculated, and this value is presented as the “biofilm formation” on the y axis. Bars represent means ± standard errors for four replicates.

FIG. 3.

Activities of biofilms composed of one or four strains of the epiphytic isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, when exposed to hydrogen peroxide (1,700 μg/ml) or tetracycline (20 μg/ml). After 24 h of incubation in microtiter wells, the biofilms were exposed to the antimicrobial agent in VNSS medium or to plain VNSS. After 1 h of exposure, the respiratory indicator TTC was added and the plates were further incubated for 15 h. The metabolic activity was determined by the absorbance of reduced (red) TTC at 490 nm. The activities of the hydrogen peroxide- or tetracycline-exposed biofilms were related to the activities of the corresponding, unexposed biofilms and are presented as “percentage activity”. Bars represent means ± standard errors for four replicates.

FIG. 4.

Bacterial invasion of one- and four-species biofilms by Pseudoalteromonas tunicata. Biofilms composed of one or four strains of the epiphytic isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were established in glass flow cells inoculated with equal total cell densities. After 2 h of growth in the presence of medium flow, the antibacterial protein-producing P. tunicata was introduced to the biofilms. This strain constitutively expressed green fluorescent protein. At various time points, the fraction of the surface covered by P. tunicata biofilm was determined by staining of the biofilm cells followed by confocal laser scanning microscopy, image analysis, and comparisons to corresponding biofilms not subjected to P. tunicata invasion. This is presented as “percentage invasion by Pseudoalteromonas tunicata”. Bars represent means ± standard errors for eight replicates.