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Cited by 350Bacterial biofilms and quorum sensingEscherichia coli research studiesBacterial Genetics and Biotechnology

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Role of Multicellular Aggregates in Biofilm Formation

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Kragh, Kasper N., Hutchison, Jaime B., Melaugh, Gavin, Rodesney, Chris, Roberts, Aled E. L., Irie, Yasuhiko, Jensen, Peter Ø., Diggle, Stephen P., Allen, Rosalind J., Gordon, Vernita, Bjarnsholt, Thomas·2016·mBio
DOI 10.1128/mbio.00237-16PMC PMC4807362
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Abstract

In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.

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Figures

FIG 1 Biofilm morphology shapes the oxygen concentration profile, with the fittest cells initially located at the top. (A and B) The oxygen concentration (in grams liter−1) (right-hand y axes) in a sample simulation after 30 h of growth of cells at low density (0.01 cell µm−1) (A) and high density (0.5 cell µm−1) (B). x and y (both in micrometers) are the spatial dimensions of the simulation domain. (C and D) Growth rate (μ) (right-hand y axes) for the resulting populations after 30 h of growth at low density (0.01 cell µm−1) (C) and high density (0.5 cell µm−1) (D). (E and F) 2D histograms representing the number of progeny, N (right-hand y axes), produced after 30 h of growth by individual bacteria as a function of their initial location in the aggregate: low density (0.01 cell µm−1) (E) and high density (0.5 cell µm−1) (F). These distributions were averaged over 40 simulations for each aggregate. Note that the gradient in the number of progeny is so large that a log scale is used for visualization purposes.
FIG 1 Biofilm morphology shapes the oxygen concentration profile, with the fittest cells initially located at the top. (A and B) The oxygen concentration (in grams liter−1) (right-hand y axes) in a sample simulation after 30 h of growth of cells at low density (0.01 cell µm−1) (A) and high density (0.5 cell µm−1) (B). x and y (both in micrometers) are the spatial dimensions of the simulation domain. (C and D) Growth rate (μ) (right-hand y axes) for the resulting populations after 30 h of growth at low density (0.01 cell µm−1) (C) and high density (0.5 cell µm−1) (D). (E and F) 2D histograms representing the number of progeny, N (right-hand y axes), produced after 30 h of growth by individual bacteria as a function of their initial location in the aggregate: low density (0.01 cell µm−1) (E) and high density (0.5 cell µm−1) (F). These distributions were averaged over 40 simulations for each aggregate. Note that the gradient in the number of progeny is so large that a log scale is used for visualization purposes.CC BY · PubMed Central
FIG 2 Simulations reveal that aggregates are relatively fitter than single cells at high density of competing cells on the surface and over long times. (A and B) Accumulated biomass normalized to initial biomass (N/N0) after 10, 30, and 120 h for single cells and for aggregates initiated at a low starting density (0.01 cell µm−1) (A) and a high starting density (0.5 cell µm−1) (B). For biofilms that were initiated at a low density, single cells produce more progeny than do cells in aggregates at all measured times. For biofilms that were initiated at a high density, single cells are more fit for early growth but aggregated cells produce more progeny than single cells do after 120 h.
FIG 2 Simulations reveal that aggregates are relatively fitter than single cells at high density of competing cells on the surface and over long times. (A and B) Accumulated biomass normalized to initial biomass (N/N0) after 10, 30, and 120 h for single cells and for aggregates initiated at a low starting density (0.01 cell µm−1) (A) and a high starting density (0.5 cell µm−1) (B). For biofilms that were initiated at a low density, single cells produce more progeny than do cells in aggregates at all measured times. For biofilms that were initiated at a high density, single cells are more fit for early growth but aggregated cells produce more progeny than single cells do after 120 h.CC BY · PubMed Central
FIG 3 In in vitro growth in flow cells, at low inoculum density, aggregates are less fit than single cells; at high inoculum density, aggregates are more fit than single cells. (A to C) Fitted exponential growth rates during the first 9 h of growth for initially aggregated cell and initially single-cell populations, starting with different cell densities in the inocula. Inoculum optical densities (OD) of 0.1 (A), 0.01 (B), and 0.001 (C) were used. (D to F) Measured biomass ratio N/N0 after 9 h of growth. Inoculum ODs of 0.1 (D), 0.01 (E), and 0.001 (F) were used. Values are means ± standard errors of the means (SEM) (error bars).
FIG 3 In in vitro growth in flow cells, at low inoculum density, aggregates are less fit than single cells; at high inoculum density, aggregates are more fit than single cells. (A to C) Fitted exponential growth rates during the first 9 h of growth for initially aggregated cell and initially single-cell populations, starting with different cell densities in the inocula. Inoculum optical densities (OD) of 0.1 (A), 0.01 (B), and 0.001 (C) were used. (D to F) Measured biomass ratio N/N0 after 9 h of growth. Inoculum ODs of 0.1 (D), 0.01 (E), and 0.001 (F) were used. Values are means ± standard errors of the means (SEM) (error bars).CC BY · PubMed Central
FIG 4 The presence of multicellular aggregates at the start of biofilm growth is reflected in the structure of the biofilm a day later. Shown are perspective projections created from confocal microscope z-stacks of Pseudomonas aeruginosa biofilms. (A) Single cells attached to the surface at 0 h. (B) A preformed aggregate surrounded by single cells on the surface at 0 h. (C) Biofilm descending from single cells from panel A. (D) After 24 h of growth, a large biofilm structure descending from the preformed aggregate shown in panel B surrounded by biofilm descending from single cells.
FIG 4 The presence of multicellular aggregates at the start of biofilm growth is reflected in the structure of the biofilm a day later. Shown are perspective projections created from confocal microscope z-stacks of Pseudomonas aeruginosa biofilms. (A) Single cells attached to the surface at 0 h. (B) A preformed aggregate surrounded by single cells on the surface at 0 h. (C) Biofilm descending from single cells from panel A. (D) After 24 h of growth, a large biofilm structure descending from the preformed aggregate shown in panel B surrounded by biofilm descending from single cells.CC BY · PubMed Central
FIG 5 Fitted exponential growth rates for the first 9 h of growth of single cells of P. aeruginosa PAO1 either on the surface or elevated 100 µm above on a glass platform. The fractional relative fitness (w) is about 0.5 for all densities evaluated, indicating that cells on the step consistently have a growth advantage over cells on the surface. Low density was an initial inoculum OD of 0.001. Medium density was an initial inoculum OD of 0.01. High density was an initial inoculum OD of 0.1. Values are means ± SEM (error bars).
FIG 5 Fitted exponential growth rates for the first 9 h of growth of single cells of P. aeruginosa PAO1 either on the surface or elevated 100 µm above on a glass platform. The fractional relative fitness (w) is about 0.5 for all densities evaluated, indicating that cells on the step consistently have a growth advantage over cells on the surface. Low density was an initial inoculum OD of 0.001. Medium density was an initial inoculum OD of 0.01. High density was an initial inoculum OD of 0.1. Values are means ± SEM (error bars).CC BY · PubMed Central
FIG 6 Change in aspect ratio for aggregates after 6 h of growth at high competition (OD of 0.1). Mean change in aspect ratio (ΔH/ΔW where H represents height and W represents width) for motile (WT PAO1) and nonmotile (PAO1 ΔpilA) aggregates. Values are means ± SEM (error bars).
FIG 6 Change in aspect ratio for aggregates after 6 h of growth at high competition (OD of 0.1). Mean change in aspect ratio (ΔH/ΔW where H represents height and W represents width) for motile (WT PAO1) and nonmotile (PAO1 ΔpilA) aggregates. Values are means ± SEM (error bars).CC BY · PubMed Central
FIG 7 Oxygen content in inflow and outflow of media to the flow cells inoculated with bacterial cells at an OD of 0.001, 0.01, or 0.1. After inoculation, the cells were left without flow for 1 h before starting the flow at t0. From t0 to t9, the AUC values for outflow medium were 546.4, 854.7, and 874.3 for 0.1, 0.01, and 0.001 (% O2 saturation * h), respectively.
FIG 7 Oxygen content in inflow and outflow of media to the flow cells inoculated with bacterial cells at an OD of 0.001, 0.01, or 0.1. After inoculation, the cells were left without flow for 1 h before starting the flow at t0. From t0 to t9, the AUC values for outflow medium were 546.4, 854.7, and 874.3 for 0.1, 0.01, and 0.001 (% O2 saturation * h), respectively.CC BY · PubMed Central
FIG 8 Proposed revision of biofilm development. The five classical stages of development in the presence of a preformed, multicellular aggregate are shown. In stage 1, the surface can be seeded either by single cells in a planktonic phenotype or by a preformed aggregate. In stage 2, the single cells attach irreversibly, and the aggregated population grows. In stage 3, the biofilm matures with complete matrix. Descendants from the aggregate population reach out in an elevated structure. Stage 4 is the mature structured biofilm. The descendants of the aggregate tower several times higher than any surrounding structures descended from single cells. In stage 5, dispersal of single cells and sloughing off of biofilm aggregates occur.
FIG 8 Proposed revision of biofilm development. The five classical stages of development in the presence of a preformed, multicellular aggregate are shown. In stage 1, the surface can be seeded either by single cells in a planktonic phenotype or by a preformed aggregate. In stage 2, the single cells attach irreversibly, and the aggregated population grows. In stage 3, the biofilm matures with complete matrix. Descendants from the aggregate population reach out in an elevated structure. Stage 4 is the mature structured biofilm. The descendants of the aggregate tower several times higher than any surrounding structures descended from single cells. In stage 5, dispersal of single cells and sloughing off of biofilm aggregates occur.CC BY · PubMed Central

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  1. Characklis WG, Marshall KC. 1990. Biofilms. Wiley Interscience, New York, NY.

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Metadata

Authors
Kragh, K. N., Hutchison, J. B., Melaugh, G., Rodesney, C., Roberts, A. E. L., Irie, Y., Jensen, P. Ø., Diggle, S. P., Allen, R. J., Gordon, V., & Bjarnsholt, T.
Affiliations
  • University of Copenhagen (DK)
  • The University of Texas at Austin (US)
  • University of Edinburgh (GB)
  • University of Nottingham (GB)
  • University of Bath (GB)
  • Rigshospitalet (DK)
Corresponding author
Bjarnsholt, Thomas
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2016-05-04
Volume
7
Issue
2
Pages
e00237-e00237
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.00237-16
PMC
PMC4807362
OpenAlex
W2307656886
Cited by
350
References
48
Citations by year
2026: 17 · 2025: 29 · 2024: 28 · 2023: 37 · 2022: 49 · 2021: 36 · 2020: 40 · 2019: 36 · 2018: 28 · 2017: 32 · 2016: 17
FWCI
19.58
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Repository@Nottingham (University of Nottingham) (publishedVersion, cc-by)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • Repository@Nottingham (University of Nottingham) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Nottingham ePrints (University of Nottingham) (submittedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
  • Texas ScholarWorks (Texas Digital Library) (submittedVersion, cc-by)
  • Texas Digital Library (University of Texas)
Funders
  • Human Frontier Science Program (RGY0081/2012)
  • Royal Society
  • Lundbeckfonden
  • University of Texas at Austin
  • Exxon Mobil Corporation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
biofilm formation, multicellular aggregates, Pseudomonas aeruginosa, biofilm initiation, cell competition, planktonic cells, biofilm dispersal, antibiotic resistance, immune evasion
MeSH terms
Cell Adhesion, Computer Simulation, Pseudomonas aeruginosa, Biofilms

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