Intra-Species Interactions in Streptococcus pneumoniae Biofilms

Abstract

Streptococcus pneumoniae is a human pathogen responsible for high morbidity and mortality worldwide. Disease is incidental and is preceded by asymptomatic nasopharyngeal colonization in the form of biofilms. Simultaneous colonization by multiple pneumococcal strains is frequent but remains poorly characterized. Previous studies, using mostly laboratory strains, showed that pneumococcal strains can reciprocally affect each other's colonization ability. Here, we aimed at developing a strategy to investigate pneumococcal intra-species interactions occurring in biofilms. A 72h abiotic biofilm model mimicking long-term colonization was applied to study eight pneumococcal strains encompassing 6 capsular types and 7 multilocus sequence types. Strains were labeled with GFP or RFP, generating two fluorescent variants for each. Intra-species interactions were evaluated in dual-strain biofilms (1:1 ratio) using flow cytometry. Confocal microscopy was used to image representative biofilms. Twenty-eight dual-strain combinations were tested. Interactions of commensalism, competition, amensalism and neutralism were identified. The outcome of an interaction was independent of the capsular and sequence type of the strains involved. Confocal imaging of biofilms confirmed the positive, negative and neutral effects that pneumococci can exert on each other. In conclusion, we developed an experimental approach that successfully discriminates pneumococcal strains growing in mixed biofilms, which enables the identification of intra-species interactions. Several types of interactions occur among pneumococci. These observations are a starting point to study the mechanisms underlying those interactions.

Keywords: Streptococcus pneumoniae; biofilms; co-colonization; colonization; competition; intraspecies interactions; multiple carriage.

Figure 1

Representative fluorescently labelled strains used in the study (remaining strains are shown in Figure S1 ). Strains’ reference and serotype are indicated on top of panel (A). (A) Imaging of GFP and RFP-labelled strains by fluorescence microscopy. GFP- and RFP-labelled strains were grown until exponential phase and imaged by fluorescence microscopy with filters for contrast phase optics, FITC and TX2. (B) Viability of wild-type and fluorescently labelled variants in the biofilm model. Each WT strain and its correspondent GFP- and RFP-labelled variants were grown under biofilm-promoting conditions (see the Materials and Methods section), after which viability (CFU/mL) was determined. Black, green and red dots indicate WT, GFP- and RFP-labelled cells, respectively. three independent experiments, each with an intra-experiment triplicate. **p < 0.01 (Kruskal Wallis test with Benjamini and Hochberg correction for FDR). (C) Flow cytometry analysis of fluorescent variants in biofilm model. Biofilm formation was induced as described in (B). After 72h biofilms were resuspended and sonicated and cell counts were obtained by flow cytometry. Representative histograms are shown.

Figure 2

Schematic representation of interactions identified among pneumococcal strains. For each strain growing in a dual-strain biofilm three possible effects can be observed when its cell counts are compared to those obtained when growing alone (single-strain biofilms depicted on the left): negative (-), if there is a decrease in its cell counts; positive (+), if there is an increase in its cell counts; and neutral (0), if there is no change. Depending on the combination of effects observed for each pair of strains, interactions can be divided into six categories: neutralism (0/0), mutualism (+/+), commensalism (+/0), antagonism (-/+), amensalism (-/0) and competition (-/-) [reviewed in (Faust and Raes, 2012)]. The interaction observed for each pair of strains is indicated below the schematic representation of each interaction.

Figure 3

Dual-strain combinations representative of commensalism (A), competition (B), and amensalism (C). Strains were tested in dual-strain biofilms (one GFP- and one RFP-labelled strain) in a 1:1 ratio. Reverse labelling combinations were also tested as controls. For comparative analysis, single-strain biofilms were grown in parallel. Intra-species interactions were considered to occur when statistically significant differences between a given strain in single and dual-strain biofilms were observed (p<0.05), Mann-Whitney U test corrected with Benjamini and Hochberg method for FDR. (i.) Cell counts. Cell counts of strains in single- and dual-strain biofilms were obtained by flow cytometry, events/mL were calculated and compared. For each strain, one out of three possible outcomes was determined when grown in the presence of another: positive (+), if there was beneficial effect; neutral (0), if there was no effect; and negative (-) if there was harm. Black bars indicate geometric means. Green and red dots correspond to GFP- and RFP-labelled cells, respectively. Graphics represent the ungrouped and grouped analyses for each pair using data from three independent experiments, each with an intra-experiment triplicate. (ii.) Imaging of dual-strain biofilms. (iii.) Imaging of single strain biofilms (iv.). Single-channel images of dual-strain biofilms. All CLSM images are transparent projections of 3D reconstructions from z-sections acquired at 0.81 µm intervals on a Zeiss LSM 880 point scanning confocal microscope using the Airyscan detector, a 20x plan-apochromat 0.8 NA objective (Zeiss) and the 488 nm and 561 nm laser lines.