Abstract

The coaggregation ability of bacteria isolated from a freshwater biofilm was compared to those derived from the coexisting planktonic population. Twenty-nine morphologically distinct bacterial strains were isolated from a 6-month-old biofilm, established in a glass tank under high-shear conditions, and 15 distinct strains were isolated from the associated re-circulating water. All 44 strains were identified to genus or species level by 16S rDNA sequencing. The 29 biofilm strains belonged to 14 genera and 23.4% of all the possible pair-wise combinations coaggregated. The 15 planktonic strains belonged to seven genera and only 5.8% of all the possible pair-wise combinations coaggregated. Therefore, compared to the planktonic population, a greater proportion of the biofilm strains coaggregated. It is proposed that coaggregation influences biofilm formation and species diversity in freshwater under high shear.

Introduction

The microbial colonisation of interfaces and subsequent development of biofilms in both natural and man-made environments is ubiquitous [1]. Many of the processes leading to the development of biofilm, such as primary colonisation, the expression of extracellular polymeric substances and gross phenotypic changes are well described (for review see [2]). However, the possible role of coaggregation in the primary development of biofilm communities remains unclear. Whilst it is generally accepted that coaggregation may enhance biofilm development in the human oral cavity [3–5], there is no in situ evidence to suggest that coaggregation mediates biofilm development in environments such as freshwater ecosystems. Coaggregation interactions could enhance the development of biofilms in fast-flowing water systems [6] and mediate the integration of pathogens into biofilms [7]. Ultimately, coaggregation interactions could influence the bacterial diversity of freshwater biofilms.

Recent studies of coaggregating freshwater biofilm bacteria have demonstrated that coaggregation often occurs between bacteria that are taxonomically distant (intergeneric coaggregation) and occasionally between strains belonging to the same species (intraspecies coaggregation) [8,9]. In addition, coaggregation between freshwater bacteria has been shown to be mediated by growth-phase-dependent lectin–saccharide interactions, which are optimal in stationary phase cultures [10,11]. To date, however, studies have not compared the proportion of coaggregating strains in a biofilm to that in the surrounding bulk liquid. If coaggregation enhances biofilm development under high shear force then the proportion of coaggregating strains would be expected to be greater in the biofilm than in the surrounding bulk liquid. Therefore, the aim of this study was to compare the numbers and species of coaggregating bacteria isolated from an established freshwater biofilm on glass substrata, under high shear forces in a glass tank, to the numbers and species of coaggregating bacteria from the surrounding bulk liquid. In addition, the presence and numbers of intergeneric, intrageneric and intraspecies coaggregations between the strains derived from the biofilm and those derived from the bulk liquid were assessed.

Materials and methods

Strains and growth conditions

Biofilm and planktonic strains were isolated from a glass tank which contained 18 l of water at 28°C (±2°C) and pH 6.9. The tank contained a filter unit which re-circulated water at a rate of 2 l min−1 from the support base under 3 cm of gravel and impinged onto the glass wall. An established 6-month-old biofilm on the glass tank which was adjacent to the water filter-unit outlet, and subjected to a constant shear force from the filter, was sampled. Biofilm strains were isolated by lowering the level of the tank water and applying a cotton swab to the biofilm and then re-suspending the sample in 5 ml of autoclaved glass-tank water. Planktonic strains were isolated by taking 20 ml of the tank water and filtering through a Swinnex™ filter, with a membrane pore size of 0.2 µm, and re-suspending the membrane in 1 ml of autoclaved glass-tank water. Biofilm and planktonic strains were cultured on R2A agar [12] at 30°C. All strains that could be readily distinguished from each other by colony morphology on R2A agar were subcultured and stored at −70°C in 50% v/v glycerol for subsequent analysis. Prior to the visual coaggregation assay (below), strains were grown for 48 h in R2A broth at 30°C in a rotary shaker set at 200 rpm.

Characterisation of bacterial strains

All strains were characterised on the basis of colony and cell morphology after growth in R2A agar or liquid R2A at 30°C. In addition, strains were subjected to oxidase tests and Gram reactions.

Identification of bacterial strains by partial 16S rRNA gene sequencing

Using the method of Rickard et al. [10], strains were identified by polymerase chain reaction (PCR) amplification and partial sequencing of the 16S rRNA gene fragment. Amplification of 16S rRNA was performed by taking one bacterial colony of each organism grown on R2A, boiling it in 100 µl of sterile nanopure water for 10 min and using 10 µl of the suspension as template DNA for PCR. Degenerate primers 806R [13] and 8FPL [14] were used to amplify a fragment of 16S rDNA which corresponds to nucleotides 8–806 in the Escherichia coli 16S rRNA gene sequence. PCR reactions were carried out in PCR reaction buffer (Boehringer Mannheim, Indianapolis, IN, USA) containing 3.2 µM of each primer, 0.5 mM dNTPs and 2 units of Taq DNA polymerase (Boehringer Mannheim) per 100 µl. The PCR cycles consisted of 35 cycles at 94°C (1 min), 53°C (1 min) and 72°C (1 min), plus a final cycle with a 15 min chain elongation step at 72°C. Amplified products were purified using the QIA-quick™ PCR purification kits (Qiagen, Warrington, UK) according to the manufacturer's instructions. PCR products were subsequently sequenced using the primers 806R and 8FPL. Sequencing reactions consisted of 30–100 ng of PCR product, 10 ng of primer, 4 µl of Big Dye™ (PE Applied Biosystems, Foster City, CA, USA) in a total volume of 20 µl. The samples were incubated at 94°C (4 min) followed by 25 cycles of 96°C (30 s), 50°C (15 s) and 60°C (4 min). Sequencing was performed in a Perkin-Elmer ABI 377 sequencer, and the sequences for each isolate were then compiled using Inherit™ (Perkin-Elmer). Compiled sequences of approximately 650 bases in length were obtained from each strain and compared to known sequences in the EMBL database using FASTA3 (http://www.ebi.ac.uk/FAST3/). Based on the criteria described by Stackebrandt and Goebel [15], partial 16S rRNA gene sequences that were 97–100% identical to speciated strains in the EMBL database were assigned the genus and species name. Sequences that possessed a sequence identity of less than 97% to speciated strains in the EMBL database were only assigned the genus name.

Construction of neighbour-joining tree

The closest relative species or genera were assigned based upon compiled partial 16S rRNA gene sequence comparisons with FASTA3 against sequences in the EMBL database. Unambiguous positions of representative sequences of closely related strains were then aligned by using CLUSTALX version 1.64b [16]. Neighbour-joining analysis was conducted with the correction of Jukes and Cantor [17] using TREECON version 1.3b [18] with Thermus thermophilus (X07998) as the outgroup and showing bootstrap values as percentages of 100 replications.

Visual coaggregation assay

A visual coaggregation assay, modified from Cisar et al. [19], was used to assess the ability of strains to coaggregate. Cells were harvested from batch culture and concentrated by centrifugation for 12.5 min at 3000×g and washed three times in sterile de-ionised water. Cells were re-suspended in de-ionised water to an O.D. at 650 nm of 1.0 and concentrated to give a calculated O.D. at 650 nm of 1.5. For coaggregation, pairs of strains were mixed at an O.D.650 of 1.5, in equal volumes (2×200 µl) at room temperature in 6×50 mm silica Durham tubes (Scientific Lab Supplies, Nottingham, UK). Mixtures were vortexed for 10 s and rolled gently for 30 s. The degree of coaggregation between each pair was scored using a semi-quantitative assay originally described by Cisar et al. [19]. The scoring criteria were as follows: ‘0’, no coaggregates in suspension; ‘1’, small uniform aggregates in a turbid suspension; ‘2’, easily visible coaggregates in a turbid suspension; ‘3’, clearly visible coaggregates which settle leaving a clear supernatant; ‘4’, large flocs of coaggregates that settle almost instantaneously leaving a clear supernatant. Control tubes containing separate isolates were included to assess autoaggregation (self-aggregation). Where present, autoaggregation was scored using the same criteria and the score was deducted from the coaggregation score.

Reversal of coaggregation with simple sugars

The ability of sugars to reverse coaggregation was determined by the addition of lactose, galactose, N-acetyl-d-galactosamine, methyl-a-d-galactopyranoside, and galactosamine (Sigma, Dorset, UK) to coaggregating pairs. Filter-sterilised solutions of each of these sugars were added to coaggregating pairs to give final concentrations of 50 mM. Mixtures were then vortexed and tested for coaggregation using the visual assay.

Results

Comparison of biofilm and planktonic populations

Preliminary characterisation of the strains isolated from the biofilm and planktonic samples resulted in the differentiation of 29 heterotrophic biofilm strains and 15 heterotrophic planktonic strains (Table 1a,b). All the strains were unique with respect to their colony morphology. All were oxidase-positive and the vast majority were rod-shaped cells in R2A broth at 30°C. The relative numbers of Gram-positive and Gram-negative strains from the biofilm and from planktonic samples were significantly different. The majority of the 29 biofilm strains were Gram-positive (20 of 29 strains) whilst only six of the 15 planktonic strains were Gram-positive.

1a

Identification of freshwater biofilm strains by alignment with 16S rRNA gene sequences of organisms in the EMBL database

Strain Highest percentage identity to sequence in database Proposed identity EMBL accession code 
B1 100 Shewanella sp. AJ491823 
B2 98.61 Flavobacterium columnare AJ491824 
B3 100 Shewanella sp. AJ491825 
B4 100 Bacillus sororensis AJ518822 
B5 98.95 Leifsonia aquatica AJ518811 
B6 98.64 Brevibacterium sp. AJ491826 
B7 99.32 Leifsonia aquatica AJ518812 
B9 99.66 Stenotrophomonas maltophilia AJ518813 
B10 99.63 Bacillus sp. AJ491827 
B11 99.46 Flavobacterium sp. AJ518814 
B13 99.84 Leifsonia aquatica AJ518823 
B14 99.46 Leifsonia aquatica AJ518824 
B15 99.30 Leifsonia aquatica AJ518815 
B16 99.64 Corynebacterium ulcerans AJ491828 
B17 99.50 Brevibacterium sp. AJ491829 
B18 99.58 Brevibacterium sp. AJ491830 
B19 99.45 Brevibacterium sp. AJ491831 
B21 99.71 Leifsonia aquatica AJ518816 
B24 99.33 Vibrio sp. AJ491832 
B25 99.66 Leifsonia aquatica AJ491833 
B26 99.43 Kocuria rhizophila AJ518817 
B27 99.31 Acidovorax delafieldii AJ518818 
B28 99.67 Brevibacterium sp. AJ491834 
B30 99.15 Pseudomonas sp. AJ491835 
B31 99.30 Bacillus licheniformis AJ518819 
B32 99.58 Bacillus sp. AJ518820 
B33 99.39 Streptococcus intermedius AJ491836 
B34 99.69 Micrococcus luteus AJ491837 
B35 98.02 Methylobacterium sp. AJ491838 
Strain Highest percentage identity to sequence in database Proposed identity EMBL accession code 
B1 100 Shewanella sp. AJ491823 
B2 98.61 Flavobacterium columnare AJ491824 
B3 100 Shewanella sp. AJ491825 
B4 100 Bacillus sororensis AJ518822 
B5 98.95 Leifsonia aquatica AJ518811 
B6 98.64 Brevibacterium sp. AJ491826 
B7 99.32 Leifsonia aquatica AJ518812 
B9 99.66 Stenotrophomonas maltophilia AJ518813 
B10 99.63 Bacillus sp. AJ491827 
B11 99.46 Flavobacterium sp. AJ518814 
B13 99.84 Leifsonia aquatica AJ518823 
B14 99.46 Leifsonia aquatica AJ518824 
B15 99.30 Leifsonia aquatica AJ518815 
B16 99.64 Corynebacterium ulcerans AJ491828 
B17 99.50 Brevibacterium sp. AJ491829 
B18 99.58 Brevibacterium sp. AJ491830 
B19 99.45 Brevibacterium sp. AJ491831 
B21 99.71 Leifsonia aquatica AJ518816 
B24 99.33 Vibrio sp. AJ491832 
B25 99.66 Leifsonia aquatica AJ491833 
B26 99.43 Kocuria rhizophila AJ518817 
B27 99.31 Acidovorax delafieldii AJ518818 
B28 99.67 Brevibacterium sp. AJ491834 
B30 99.15 Pseudomonas sp. AJ491835 
B31 99.30 Bacillus licheniformis AJ518819 
B32 99.58 Bacillus sp. AJ518820 
B33 99.39 Streptococcus intermedius AJ491836 
B34 99.69 Micrococcus luteus AJ491837 
B35 98.02 Methylobacterium sp. AJ491838 

Highest percentage identity to speciated or non-speciated strains in the EMBL database.

1b

Identification of freshwater planktonic strains by alignment with 16S rRNA gene sequences of organisms in the EMBL database

Strain Highest percentage identity to sequence in database Proposed identity EMBL accession code 
P1 99.85 Variovorax paradoxus AJ491705 
P2 100 Bacillus thuringiensi AJ491706 
P3 99.83 Bacillus sp. AJ491707 
P4 99.46 Bacillus macroides AJ491708 
P5 99.86 Staphylococcus epidermidis AJ491709 
P6 98.24 Exiguobacterium acetylicum AJ491710 
P7 100 Bacillus thuringiensi AJ491711 
P8 100 Aeromonas sp. AJ491712 
P9 98.79 Aeromonas sp. AJ491713 
P10 96.76 Exiguobacterium sp. AJ518821 
P11 100 Aeromonas sp. AJ491714 
P12 99.72 Shewanella sp. AJ491715 
P13 100 Aeromonas sp. AJ491716 
P14 98.50 Aeromonas hydrophila AJ518825 
P15 99.81 Methylobacterium sp. AJ491717 
Strain Highest percentage identity to sequence in database Proposed identity EMBL accession code 
P1 99.85 Variovorax paradoxus AJ491705 
P2 100 Bacillus thuringiensi AJ491706 
P3 99.83 Bacillus sp. AJ491707 
P4 99.46 Bacillus macroides AJ491708 
P5 99.86 Staphylococcus epidermidis AJ491709 
P6 98.24 Exiguobacterium acetylicum AJ491710 
P7 100 Bacillus thuringiensi AJ491711 
P8 100 Aeromonas sp. AJ491712 
P9 98.79 Aeromonas sp. AJ491713 
P10 96.76 Exiguobacterium sp. AJ518821 
P11 100 Aeromonas sp. AJ491714 
P12 99.72 Shewanella sp. AJ491715 
P13 100 Aeromonas sp. AJ491716 
P14 98.50 Aeromonas hydrophila AJ518825 
P15 99.81 Methylobacterium sp. AJ491717 

Highest percentage identity to speciated or non-speciated strains in the EMBL database.

All the planktonic and biofilms strains were identified by partial 16S rRNA gene sequencing, to at least the genus level (Table 1a,b). The 29 biofilm strains belonged to 14 genera and the planktonic strains belonged to seven genera. The numerically dominant culturable genera from the biofilm were Leifsonia sp. (seven strains) and Brevibacterium sp. (five strains). The numerically dominant culturable genera from the bulk liquid were Aeromonas spp. (five strains) and Bacillus spp. (four strains). Shewenella spp., Bacillus spp. and Methylobacterium spp. were isolated from both the biofilm and bulk liquid. The other strains were unique either to the biofilm or to the bulk liquid.

Coaggregation ability of freshwater biofilm and planktonic strains

The coaggregation assay demonstrated that 28 of the 29 biofilm strains (all but Leifsonia aquatica B21) coaggregated with one or more partner strains and visual coaggregation scores ranged from 1+ to 4+ (Table 2). A total of 95 of a possible 406 coaggregation partnerships (23.4%) occurred between bacteria isolated from the biofilm. The coaggregating biofilm strains with the most partners were L. aquatica B25 (15 coaggregation partnerships), Shewanella sp. B3 (13 coaggregation partnerships), L. aquatica B15 (12 coaggregation partnerships) and Methylobacterium sp. B35 (12 coaggregation partnerships). Conversely, only eight of the 15 planktonic strains coaggregated with one another with a maximum visual coaggregation score of 1+. A total of seven, of a possible 105, coaggregations (5.8%) occurred between the planktonic strains. The coaggregating planktonic strain with the most partners was Shewanella sp. P12 (five coaggregation partnerships).

2

Visual coaggregation scores between freshwater biofilm strains after growth in liquid R2A for 48 h

Strain Source Autoaggregation Coaggregation Total coaggregations 
Shewanella sp. B1 B9(1), B14 (1), B17(1), B25(1), B32(1), B31(2), B33(3) 
Flavobacterium columnare B2 B3(2), B15(2), B19(1), B28(1), B31(1), B33(2) 
Shewanella sp. B3 B2(2), B4(1), B9(1), B13(1), B14(2), B15(1), B16(1), B24(1), B25(3), B26(1), B30(2), B35(3) 13 
Bacillus sorensis B4 B3(1), B25(2), B31(1), B16(1), B17(1), B18(1) 
Leifsonia aquatica B5 B14(1), B15(1), B24(1), B26(1), B35(2), B31(2) 
Brevibacterium sp. B6 B9(1), B10(1), B11(2), B14(2), B15(1), B16(1), B19(2), B25(1), B30(1), B31(1)B33(1) 11 
Corynebacterium aquaticum B7 B14(1), B15(1), B25(2), B27(2), B30(1) 
Stenotrophomonas maltophilia B9 B1(1), B3(1), B6(1), B18(1), B25(4), B28(4), B30(1), B31(1), B33(1) 
Bacillus sp. B10 B6(1), B15(1), B18(1), B25(2) 
Flavobacterium sp. B11 B6(2), B35(1), B31(2) 
Corynebacterium sp. B13 B3(1), B15(2), B26(1), B27(1), B35(3), B33(2) 
Leifsonia aquatica B14 B1(1), B3(2), B5(1), B6(2), B7(1), B35(1) 
Leifsonia aquatica B15 B2(2), B3(1), B5(1), B6(1), B7(1), B10(1), B13(2), B19(1), B25(3), B28(1), B35(1), B31(2) 12 
Corynebacterium ulcerans B16 B3(1), B4(1), B6(1), B25(2) 
Brevibacterium sp. B17 B1(1), B4(1), B28(1), B35(2) 
Brevibacterium sp. B18 B4(1), B9(1), B10(1), B25(1), B35(1) 
Brevibacterium sp. B19 B2(1), B6(2), B15(1), B24(1), B25(1), B35(1) 
Leifsonia aquatica B21 None 
Vibrio sp. B24 B3(1), B5(1), B19(1), B27(1), B28(1) 
Leifsonia aquatica B25 B1(1), B3(3), B4(2), B6(1), B7(2), B9(4), B10(2), B15(3), B16(2), B18(1), B19(1), B26(1), B30(2), B35(1), B34(1) 15 
Kocuria rhizophila B26 B3(1), B5(1), B13(1), B25(1), B28(1) 
Acidovorax delafieldii B27 B7(2), B13(1), B24(1), B31(2) 
Brevibacterium sp. B28 B2(1), B9(4), B15(1), B17(1), B24(1), B26(1), B34(3) 
Pseudomonas sp. B30 B3(2), B6(1), B7(1), B9(1), B25(2), B33(1) 
Bacillus licheniformis B31 B1(2), B2(1), B4(1), B5(2), B6(1), B9(1), B11(2), B15(2), B27(2) 
Bacillus sp. B32 B1(1), B33(1), B34(1), B35(1) 
Streptococcus intermedius B33 B1(3), B2(2), B6(1), B9(1), B13(2), B30(1), B32(1), B35(2) 
Micrococcus luteus B34 B25(1), B28(3), B32(1) 
Methylobacterium sp. B35 B3(3), B5(2), B11(1), B13(3), B14(1), B15(1), B17(2), B18(1), B19(1), B25(1), B32(1), B33(2) 12 
Variovorax paradoxus P1 P12(1), P13(1) 
Bacillus thuringiensis P2 None 
Bacillus sp. P3 None 
Bacillus macroides P4 P12(1) 
Staphylococcus epidermidis P5 None 
Exiguobacterium acetylicum P6 P12(1) 
Bacillus thuringiensis P7 None 
Aeromonas sp. P8 P14(1) 
Aeromonas sp. P9 None 
Bacillus sp. P10 None 
Aeromonas veronii P11 P12(1) 
Shewanella sp. P12 P1(1), P4(1), P6(1), P11(1), P13(1) 
Aeromonas veronii P13 P1(1), P12(1) 
Aeromonas hydrophila P14 P8(1) 
Methylobacterium sp. P15 None 
Strain Source Autoaggregation Coaggregation Total coaggregations 
Shewanella sp. B1 B9(1), B14 (1), B17(1), B25(1), B32(1), B31(2), B33(3) 
Flavobacterium columnare B2 B3(2), B15(2), B19(1), B28(1), B31(1), B33(2) 
Shewanella sp. B3 B2(2), B4(1), B9(1), B13(1), B14(2), B15(1), B16(1), B24(1), B25(3), B26(1), B30(2), B35(3) 13 
Bacillus sorensis B4 B3(1), B25(2), B31(1), B16(1), B17(1), B18(1) 
Leifsonia aquatica B5 B14(1), B15(1), B24(1), B26(1), B35(2), B31(2) 
Brevibacterium sp. B6 B9(1), B10(1), B11(2), B14(2), B15(1), B16(1), B19(2), B25(1), B30(1), B31(1)B33(1) 11 
Corynebacterium aquaticum B7 B14(1), B15(1), B25(2), B27(2), B30(1) 
Stenotrophomonas maltophilia B9 B1(1), B3(1), B6(1), B18(1), B25(4), B28(4), B30(1), B31(1), B33(1) 
Bacillus sp. B10 B6(1), B15(1), B18(1), B25(2) 
Flavobacterium sp. B11 B6(2), B35(1), B31(2) 
Corynebacterium sp. B13 B3(1), B15(2), B26(1), B27(1), B35(3), B33(2) 
Leifsonia aquatica B14 B1(1), B3(2), B5(1), B6(2), B7(1), B35(1) 
Leifsonia aquatica B15 B2(2), B3(1), B5(1), B6(1), B7(1), B10(1), B13(2), B19(1), B25(3), B28(1), B35(1), B31(2) 12 
Corynebacterium ulcerans B16 B3(1), B4(1), B6(1), B25(2) 
Brevibacterium sp. B17 B1(1), B4(1), B28(1), B35(2) 
Brevibacterium sp. B18 B4(1), B9(1), B10(1), B25(1), B35(1) 
Brevibacterium sp. B19 B2(1), B6(2), B15(1), B24(1), B25(1), B35(1) 
Leifsonia aquatica B21 None 
Vibrio sp. B24 B3(1), B5(1), B19(1), B27(1), B28(1) 
Leifsonia aquatica B25 B1(1), B3(3), B4(2), B6(1), B7(2), B9(4), B10(2), B15(3), B16(2), B18(1), B19(1), B26(1), B30(2), B35(1), B34(1) 15 
Kocuria rhizophila B26 B3(1), B5(1), B13(1), B25(1), B28(1) 
Acidovorax delafieldii B27 B7(2), B13(1), B24(1), B31(2) 
Brevibacterium sp. B28 B2(1), B9(4), B15(1), B17(1), B24(1), B26(1), B34(3) 
Pseudomonas sp. B30 B3(2), B6(1), B7(1), B9(1), B25(2), B33(1) 
Bacillus licheniformis B31 B1(2), B2(1), B4(1), B5(2), B6(1), B9(1), B11(2), B15(2), B27(2) 
Bacillus sp. B32 B1(1), B33(1), B34(1), B35(1) 
Streptococcus intermedius B33 B1(3), B2(2), B6(1), B9(1), B13(2), B30(1), B32(1), B35(2) 
Micrococcus luteus B34 B25(1), B28(3), B32(1) 
Methylobacterium sp. B35 B3(3), B5(2), B11(1), B13(3), B14(1), B15(1), B17(2), B18(1), B19(1), B25(1), B32(1), B33(2) 12 
Variovorax paradoxus P1 P12(1), P13(1) 
Bacillus thuringiensis P2 None 
Bacillus sp. P3 None 
Bacillus macroides P4 P12(1) 
Staphylococcus epidermidis P5 None 
Exiguobacterium acetylicum P6 P12(1) 
Bacillus thuringiensis P7 None 
Aeromonas sp. P8 P14(1) 
Aeromonas sp. P9 None 
Bacillus sp. P10 None 
Aeromonas veronii P11 P12(1) 
Shewanella sp. P12 P1(1), P4(1), P6(1), P11(1), P13(1) 
Aeromonas veronii P13 P1(1), P12(1) 
Aeromonas hydrophila P14 P8(1) 
Methylobacterium sp. P15 None 

For strains that autoaggregated, the autoaggregation score was deducted from the coaggregation score.

Coaggregation partnerships: Strain number plus the visual coaggregation score in brackets.

In order to show the phylogenetic affiliations of the coaggregating planktonic and biofilm strains a neighbour-joining tree was constructed (Fig. 1). The tree demonstrates that coaggregation occurred between both distantly and closely related strains from either the biofilm or the bulk liquid. For example, the biofilm strain Shewanella sp. B3 coaggregates with the distantly related biofilm strain L. aquatica B25 as well as the relatively closely related biofilm strain Pseudomonas sp. B30 (Fig. 1). Intergeneric, intrageneric and intraspecies coaggregations occurred between the freshwater strains. Intergeneric coaggregations were the most common between bacteria isolated from the biofilm or the bulk liquid. Between strains isolated from the biofilm, 85 of the 97 coaggregation partnerships were at an intergeneric level. Between strains isolated from the bulk liquid, six of the seven coaggregation partnerships were at an intergeneric level. Intrageneric coaggregations were less common and, between the biofilm strains, eight of the 95 coaggregation partnerships were at an intrageneric level. In the bulk liquid, there was no intrageneric coaggregation partnerships. Intraspecies coaggregation was least common and only four interspecies coaggregation partnerships were detected. Similar to intrageneric coaggregation, intraspecies coaggregation only occurred between strains isolated from the biofilm.

1

Phylogenetic tree showing bacteria isolated from the freshwater biofilm and bulk liquid together with speciated reference strains based on 540-bp-long sequences of 16S rRNA genes. The tree was constructed using the neighbour-joining method of Jukes and Cantor [17]. The scale bar represents one estimated substitution for every 10 nucleotides. The bootstrap values indicate confidence limits of the phylogenies, based on percentages of 100 replications. T. thermophilus (X07998) was used to root the tree.

1

Phylogenetic tree showing bacteria isolated from the freshwater biofilm and bulk liquid together with speciated reference strains based on 540-bp-long sequences of 16S rRNA genes. The tree was constructed using the neighbour-joining method of Jukes and Cantor [17]. The scale bar represents one estimated substitution for every 10 nucleotides. The bootstrap values indicate confidence limits of the phylogenies, based on percentages of 100 replications. T. thermophilus (X07998) was used to root the tree.

Inhibition of coaggregation with simple sugars

The ability of five simple sugars to inhibit coaggregation was tested by adding them to five coaggregation partnerships between biofilm strains and three coaggregation partnerships between planktonic strains. In all eight cases, coaggregation was partially or completely inhibited following the addition of at least one of the sugars (data not shown). Galactosamine and N-acetyl-d-galactosamine were the most effective inhibitors and inhibited coaggregation between four of the coaggregation partnerships (three biofilm coaggregation partnerships and one planktonic coaggregation partnership). There was no significant inhibition of coaggregation when lactose or methyl-α-d-galactopyranoside was added to any of the coaggregating strains.

Discussion

This is the first study to compare the numbers of coaggregating freshwater bacteria present in a biofilm to the numbers of coaggregating bacteria in the surrounding bulk liquid. In this report we have shown that the numbers of coaggregation partnerships between biofilm strains from the biofilm were far greater than the numbers of coaggregation partnerships between planktonic strains from the bulk liquid (23.4% compared to 5.8%). In addition, this study supports previous observations by Rickard et al. [8,10] that intergeneric, intrageneric and intraspecies coaggregation occurs between freshwater bacteria and these interactions are likely to be mediated by lectin–saccharide interactions.

Relatively few planktonic strains from the bulk phase coaggregated whilst a greater proportion of the strains from the biofilm coaggregated. Furthermore, the identities of the majority of the numerically dominant strains from the bulk phase were markedly different from those found in the biofilm (Fig. 1). Taken together, the data suggest that two distinct populations have developed. A predominantly non-coaggregating planktonic population composed mostly of Gram-negative strains accumulated in the bulk phase whilst a coaggregating population of predominantly Gram-positive strains, composed predominantly of bacteria from the genera Leifsonia, Brevibacterium and Bacillus (Fig. 1) formed in the biofilm. Whether or not the two populations exchange strains has not been determined, although it is likely that this occurs as the biofilm must have developed from cells originally in the bulk phase. In addition, it is well known that bacteria migrate from biofilms as single cells and as sloughed aggregates (reviewed by [20]). The biofilm developed in a high-shear flowing environment and coaggregating bacteria may well have been selectively incorporated because coadhered bacteria could more easily resist sheer. Similar differences in the composition of dominant species in planktonic and coexisting biofilm populations in freshwater ecosystems have been reported previously by Jones et al. [21] in bottled water. A biofilm will act as a microniche for many organisms including bacteria [1] and the majority of the micro-organisms will have physiological and biochemical characteristics that support the integration and survival within the biofilms. The ability to coaggregate may be a significant physiological characteristic of bacteria in biofilms under high shear, as non-coaggregating bacteria are less likely to successfully integrate into developing biofilms [22]. Therefore, coaggregation may influence biofilm development and the diversity of bacterial species present in a freshwater biofilm.

The inferred identities of the strains by 16S rRNA gene sequencing showed that intergeneric and intrageneric coaggregation occurred between bacteria from the biofilm and also between bacteria from the bulk liquid. Intraspecies coaggregation was only observed between strains within the biofilm. Whilst intergeneric and, to a lesser extent, intrageneric coaggregation occurs between oral dental plaque bacteria [23,24], intraspecies coaggregation has previously only been observed between bacteria from a potable water biofilm [8]. It is not clear why intraspecies coaggregation has only been detected in freshwater biofilms, although it may relate to the constantly changing environmental conditions and the possibility of bacteria from a wide variety of other niches coming into contact with the biofilm. In addition, the bacteria in this study were isolated from a biofilm that was subjected to high shear forces from a pump-filter unit within the glass tank and this may well modulate the numbers and types of coaggregation interactions between the bacterial strains.

Previous studies of coaggregation between freshwater bacteria have shown that coaggregation is growth-phase-dependent, mediated by lectin–saccharide interactions, and maximal in early stationary phase [10,11]. In this study cells were harvested after 48 h growth in batch culture when all the cells were in early stationary phase. Initial studies of four coaggregating pairs have shown that coaggregation is growth-phase-dependent (data not shown) with little or no coaggregation occurring between strains harvested from exponential-phase and higher visual coaggregation scores being expressed by the same pairs harvested from stationary phase. In addition, coaggregation between maximally coaggregating pairs was inhibited by the addition of simple sugars. In the oral cavity and in other freshwater ecosystems, coaggregation often occurs as a result of lectin–saccharide interactions [4,10,19]. Coaggregation between the freshwater strains was similarly inhibited by the addition of simple sugars.

Coaggregation was not the only type of cell–cell interaction common to the strains derived from the biofilm population. Autoaggregating strains were also numerically dominant in the biofilm population (14 of 29 strains), whereas only one planktonic strain (of a total of 15 strains) autoaggregated. Similar to coaggregation, autoaggregation may also enhance the development of freshwater biofilms. However, as opposed to coaggregation, autoaggregation may be a ‘selfish’ mechanism whereby a strain within the biofilm will express polymers to enhance the integration of genetically identical strains.

In summary, this work has shown that coaggregation is a phenomenon that occurs between a greater proportion of bacterial strains in a natural multi-species biofilm than bacteria from the coexisting planktonic population. This is the first evidence to be presented to show that coaggregation is likely to enhance the development of freshwater multi-species biofilms and may influence biofilm species diversity in the natural environment.

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