High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation

Abstract

Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.

Figure 1

Four-species biofilm formation of seven soil isolates. The observed data points (dark-gray dots) were collected by quantifying multispecies biofilm formation of all possible four-species cocultures using the CV assay. Error bars represent standard deviations of four replicates. Gray bars indicate the amount of monospecies biofilm produced by the best biofilm former present in the specific combination. Data points (including standard deviations) above gray areas indicate synergistic effects in four-species biofilms (see text for further details). The data obtained from combinations containing strain 3 are shown in a, whereas other combinations (without strain 3) are shown in b in order to visualize the data points at the lower end of the range. Strains: 1: P. lutea, 2: S. rhizophila, 3: X. retroflexus, 4: O. rhizosphaerae, 5: M. oxydans, 6: A. nitroguajacolicus, 7: P. amylolyticus.

Figure 2

Biofilm formation of four selected strains in monocultures and cocultures. Biofilms formed by four isolates 2: S. rhizophila, 3: X. retroflexus, 5: M. oxydans and 7: P. amylolyticus when equal aliquots of the diluted cultures were incubated in cocultures of two, three and four isolates. Assays for the detection of synergistic effects were performed three times (experiments 1, 2 and 3) with four replicates each time. Error bars represent±s.e.m. of four replicates.

Figure 3

Cell numbers of biofilm (light gray lines) and planktonic (dark gray lines) fraction of each strain in four-species cocultures. The estimated cell numbers per peg or well, respectively, of each strain in four-species biofilms and associated planktonic fractions of S. rhizophila, X. retroflexus, M. oxydans and P. amylolyticus at six time points (4 h, 8 h, 12 h, 16 h, 20 h and 24 h after coincubation) based on qPCR. MP, multispecies planktonic cells; MB, multispecies biofilms. Each point represents the mean of three replicates, with vertical lines representing±s.d.

Figure 4

Cell numbers of each strain in single and multi species biofilms. The estimated cell numbers of each strain (S. rhizophila, X. retroflexus, M. oxydans and P. amylolyticus) in multispecies (gray bars) and single-species (striped bars) biofilms at 24 h. Bars represent means±s.d. for three replicates.