Fluorescent differentiation of ECM components using luminescent oligothiophenes

Two LCOs, h-HTAA and h-FTAA (Figure 1a), selected from our library of synthesised LCO molecules for their amyloid sensitivity, were screened for their suitability as optotracers of biofilm ECM components on an isogenic collection of S. Enteritidis based on the wild-type (wt) strain 3934 (Supplementary Table 1). To facilitate analysis of surface-bound biofilm formed at the air-liquid interface, bacteria were grown in wells with inclined square glass coverslips (Figure 1b). After gentle removal of the coverslips, LCOs were first applied directly onto the surfaces, which then were prepared for microscopic analysis. Fluorescence microscopy of the biofilms demonstrated distinct labelling, suggesting that h-HTAA (green) and h-FTAA (red) fluorescence signals can complement phase contrast when visualising biofilm morphology (Figure 1c and Supplementary Figure 1). In contrast, no fluorescent signals were identified from a ΔcsgD mutant strain unable to produce curli and cellulose (Figure 1d). Individual contribution of the two ECM elements to the positive LCO-biofilm staining was analysed using ΔbcsA (curli+ cellulose−) and ΔcsgA (curli− cellulose+) mutant strains. Phase contrast microscopy of the cellulose-deficient mutant (strain ΔbcsA) showed similar morphology to the wt, and distinct fluorescence signals from both LCOs (Figure 1e). A thin and brittle bacterial layer, typically formed in the absence of curli,22 was observed in the ΔcsgA mutant strain and LCO staining revealed distinct fluorescence that was more pronounced in areas with higher cell density (Figure 1f).

Figure 1 LCO staining patterns distinguish Salmonella biofilms. (a) Structure of h-HTAA and h-FTAA. (b) Schematic of the incline glass coverslip setup enabling microscopic analysis of biofilm at air–liquid interface after removal of coverslips. (c–f) Fluorescence confocal microscopy using indicated excitation and emission wavelengths (left) and transmission confocal microscopy (right) of h-HTAA- and h-FTAA-stained biofilms from strains 3934 (c) wt, (d) ΔcsgD, (e) ΔbcsA and (f) ΔcsgA with indicated curli and cellulose phenotypes. Single optical sections are shown. Scale bar=50 μm. Full size image

Growth curves of the wt strain in the absence and presence of LCOs revealed a common generation time of 23±1 min, indicating that neither h-HTAA nor h-FTAA exert bacteriostatic or bactericidal effects (Figure 2a). This corroborated previous demonstrations of a non-toxic nature of the oligothiophene family on eukaryotic cells in vitro and in intravital mouse models.23,24 We therefore tested LCOs in live cultures, to monitor the extracellular appearance of curli fibres and cellulose during growth. A custom-designed, small-volume 96-well assay (Supplementary Figure 2) enabled analysis of pellicle and surface-attached biofilm at selected time points using spectrophotometric recordings in a standard plate reader. Fluorescence analysis of wt cultures in the presence of h-HTAA or h-FTAA (Figure 2b) showed a comparable increase of biofilm growth to parallel CV assays (Figure 2c). We next addressed if LCOs can distinguish between different biofilm phenotypes by analysing wt and mutant biofilm formation in the small-volume 96-well assay. Fluorometric read-outs (excitation wavelength (λ Ex ) of 405 nm and emission wavelength (λ Em ) of 556 nm) taken at set time points from a continuous culture grown in the presence of h-HTAA or h-FTAA, revealed that the wt and ΔbcsA (curli+ cellulose−) strains produced high quantities of biofilm (Figure 2d,e). In contrast, ΔcsgA (curli− cellulose+) and ΔcsgD (curli− cellulose−) mutants produced only low amounts. These results supported the microscopy-based demonstration of the ΔbcsA mutant as a substantial biofilm producer (Figure 1e). This result differed however from traditional CV assay results, which showed poor biofilm formation by all three mutants (Figure 2f). LCO-based detection appears to differentiate between biofilm phenotypes, with substantially improved sensitivity in biofilms with curli as the major ECM component.

Figure 2 LCO-based fluorometric biofilm quantification in a small-volume 96-well assay. (a) Growth curve, shown as viable counts, of strain 3934 wt cultured in the absence (control) and presence of h-HTAA and h-FTAA. (b and c) End point quantification of biofilm formed by 3934 wt at indicated times based on (b) fluorescence from cultures grown in the presence of h-HTAA and h-FTAA and (c) the crystal violet assay. (d–f) Quantification of biofilm formed at 24 and 48 h by 3934 wt (■), ΔbcsA (), ΔcsgA (▨) and ΔcsgD (□) based on fluorescence from (d) h-HTAA, (e) h-FTAA, and based on the (f) crystal violet assay. Data represent n: 1 of 3 with standard deviations shown. CFU, colony forming units; CV, crystal violet; RFU, relative fluorescence units. Full size image

Non-disruptive analysis of biofilm formation

When studying the kinetics of biofilm formation, longitudinal experiments should ideally be performed with minimum disturbance, such as washing procedures. To test whether LCOs can be used for non-disruptive studies, strains were cultured in h-HTAA-supplemented medium in 96-well plates, and the fluorescence of each well was directly recorded at 0, 24 and 48 h. A 4–6-fold fluorescence increase was observed across all strains (Figure 3a) compared with equivalently washed, end-point experiments (Figure 2d). The signals, however, no longer distinguished between the biofilm phenotypes, suggesting that the complex composition of the LB culture contributes to a high background, emitting in the same wavelength window as h-HTAA. To test this hypothesis, excitation spectra from each 24 h culture were collected. Identical spectra, sharing one characteristic peak, were observed for all strains irrespective of the amounts and phenotypes of the biofilms (Figure 3b). This suggested that h-HTAA binds a biofilm component different from curli and cellulose, which is ubiquitously present in all cultures. While limited in use for the purpose of the present study, h-HTAA still represents an interesting alternative to current biofilm dyes as a general non-bacteriocidal, fluorescence alternative for endpoint studies.

Figure 3 Individual and simultaneous LCO-based quantification of curli and cellulose in longitudinal biofilm cultures. (a) Fluorescence of h-HTAA at 24 and 48 h in non-disrupted liquid cultures of 3934 wt (■), ΔbcsA (), ΔcsgA (▨) and ΔcsgD (□). (b) Spectra of h-HTAA in cultures at 24 h of 3934 wt (△) ΔbcsA (◊), ΔcsgA (□) and ΔcsgD (○), with emission read at 545 nm. (c) Fluorescence of h-FTAA at 24 and 48 h in non-disrupted liquid cultures of 3934 wt (■), ΔbcsA (), ΔcsgA (▨) and ΔcsgD (□). (d) Spectra of h-FTAA at 24 h of 3934 wt (△)ΔbcsA (◊), ΔcsgA (□) and ΔcsgD (○), with emission read at 545 nm. (e) Spectra of h-FTAA mixed with cellulose (5 mg/ml) with emission read at 545 nm. Data represent n: 1 of 3 with standard deviations shown in a and c. RFU, relative fluorescence units. Full size image

The h-FTAA was, however, able to differentiate biofilm phenotypes in the longitudinal assays (Figure 3c). With h-FTAA, the cellulose-deficient mutant ΔbcsA showed significant amounts of biofilm, with a 4–5 fold increase in signal intensity at 24 h, a similar pattern to the washed endpoint experiments (Figure 2e). No further increase was observed, indicating that the bulk of this curli-based biofilm had formed in the first 24 h. Despite high background, spectral analysis showed increased fluorescence intensity from curli-producing wt and ΔbcsA compared with the curli-deficient strains at λ Ex 405 nm, which may represent a signal unique to h-FTAA bound to curli fibres (Figure 3d). A unique excitation peak also appeared at ~480 nm in wt and ΔcsgA (curli− cellulose+) strains suggesting the simultaneous detection of a second target.

Based on the genetic make-up of our Salmonella strains, cellulose, whose biosynthesis is a shared trait in the wt and ΔcsgA (curli− cellulose+) strains appears to be a strong candidate to serve as a second binding target for h-FTAA. To test this hypothesis, the excitation spectra of h-FTAA combined with different concentrations of pure cellulose was obtained. The spectra showed an evident peak with λ max at ~480 nm when emission was recorded at 545 nm (Figure 3e (5 mg/ml) and Supplementary Figure 3 (0.04–2.5 mg/ml). This peak is comparable to that generated by the cellulose-producing Salmonella strains (Figure 3d), and demonstrates the exclusive spectral signature of h-FTAA binding to cellulose. This finding extends the applicability of LCOs from conformation-sensitive spectral probes detecting amyloid protein aggregates15–17 to also include polysaccharides.

Spectral morphotyping of biofilm

More than 90% of S. Typhimurium and S. Enteritidis strains produce a characteristic red, dry and rough colony morphology on solid media, the so-called rdar morphotype.25 This three-dimensional architecture is formed by highly ordered spatial arrangement of cellulose filaments and curli fibre networks.26 We analysed whether LCOs can be used for definitive spectral morphotyping of bacterial colonies. Having ascertained that the strains showed the expected rdar morphotypes on CR plates27 (Figure 4a), we added h-FTAA to re-suspensions of 3-day-old colonies grown on LB agar plates without salt, and performed spectral analysis. Comparison of differently sized colonies at different developmental stages was enabled by normalising each spectrum such that each data point is represented as a percentage of the largest emitted fluorescence of the excitation spectra. A distinct red-shifted cellulose-specific peak was observed at 480 nm in wt and ΔcsgA (curli− cellulose+) colonies, overlapping the signal from h-FTAA binding pure cellulose (Figure 4b). The peak was absent in ΔcsgD and ΔbcsA colonies, both genetically incapable of cellulose production.

Figure 4 LCO-based morphotyping of Salmonella biofilms from agar plates. (a) Morphotypes of strain 3934 wt, ΔcsgD, ΔbcsA and ΔcsgA based on the drop assay on Congo red plates. (b) Normalised spectra of h-FTAA mixed with re-suspended biofilm colonies harvested from indicated strains grown for 48 h on LB agar w/o salt, with emission read at 545 nm. h-FTAA mixed with cellulose and PBS were assayed in parallel for reference. (c) Morphotype of a 3934 wt biofilm colony originating from an individual bacterium on Congo red plates monitored for three consecutive days. (d and e) Spectra of h-FTAA mixed with harvested 3934 wt biofilm colonies at (d) days 2 and 3, and (e) day 1, including cellulose and PBS for reference. Arrows indicate the shift in λ max for h-FTAA in the presence of various amounts of cellulose. n: 1 of 5 in b and d, n: 2 of 5 in e. Scale bars=1 cm. (f) Emission spectra of h-FTAA-supplemented cultures of strain 3934 wt, ΔbcsA, ΔcsgA and ΔcsgD after 24 h incubation, using excitation at 405 nm for curli detection. (g) Same experimental setup as in f using excitation at 500 nm for cellulose detection. Arrows indicate λ max of emission in ΔcsgA and ΔcsgD mutant strains. (h) Normalised fluorescence spectra for cellulose detection from g. Data represent n: 1 of 3. RFU, relative fluorescence units. Full size image

Rdar morphotyping on CR plates requires that a bacterial colony, often initiated from a 25 μl drop of ~105 c.f.u., reach a sufficient size for visual inspection, which usually takes 3 days (Figure 4c). To enable non-biased profiling during biofilm development on LB plates without salt, we analysed daily harvests of individual wt colonies, originating from a single bacterium, for the presence of cellulose. Spectra of colony re-suspensions stained with h-FTAA consistently showed a cellulose peak at 480 nm on day 2, and persisting on day 3 (Figure 4d). The presence of this peak was less consistent on day 1 (Figure 4e). The variability at this early stage reflects a changing ratio of unbound and bound h-FTAA as the amount of cellulose in the colony increases during biofilm maturation. In some day 1 colonies, cellulose was produced which bound all h-FTAA molecules, thus generating the 480 nm peak (Figure 4e, long arrow). In colonies with less cellulose, the higher proportion of unbound h-FTAA was observed as an intermediate degree of red-shifted λ max (Figure 4e, short arrow). The transition of λ max reflects cellulose production during the transition between different bacterial growth stages of colonies growing on solid medium. Such transient events are invisible to eye inspection (Figure 4c).

Defining optical settings for simultaneous, dual detection of cellulose and curli

The binding of h-FTAA to amyloid curli protein and cellulose suggests its possible application as an optotracer for simultaneous, dual detection of ECM components. We applied the small-volume 96-well assay of defined bacterial cultures to test whether we could identify discrete spectral signatures for each target. To identify the optimum emission wavelength (λ Em ) of h-FTAA bound to curli, we collected the emission spectra with excitation at 405 nm. The three biofilm-forming strains produced red-shifted emission peaks at λ max 550–560 nm, compared with a λ max 525 nm in the non-biofilm forming ΔcsgD mutant (Figure 4f). Increased fluorescence amplitude was only observed in curli-containing biofilms (wt and ΔbcsA), corroborating the finding in Figure 3c. This suggests λ Em 550–560 nm as an optimum range for curli detection. Cellulose does not appear to contribute to the background signal, since the red-shift appearing in the cellulose-expressing ΔcsgA curli mutant was not associated with a fluorescence intensity increase.

To define the optimal emission wavelength for cellulose detection, excitation at 500 nm was used, as this wavelength was observed to maximise the signal-to-background ratio (Figure 3d). Two prominent emission peaks at ~560 nm and ~597 nm were seen for h-FTAA binding to the cellulose-containing biofilm from wt and ΔcsgA (Figure 4g). Strains lacking this ECM component (ΔbcsA and ΔcsgD) showed h-FTAA binding to curli or an unknown component, inferred from the weak λ max signals at 566 and 586 nm that were only noticeable following data normalisation (Figure 4h). Detection above 597 nm in cellulose-producing strains resulted in less background than detection at 560 nm. Ensuring minimal contribution of background, optimal spectral parameters for h-FTAA based cellulose detection were identified as λ Ex 500 nm and λ Em 600 nm. Combining this with spectral parameters optimal for curli (λ Ex 405 nm, λ Em 556 nm) thus enables dual detection of the ECM components.

Real-time detection of curli and cellulose in liquid bacterial cultures

In contrast to traditional fluorophores, fluorescence from the LCOs is modulated by the molecular geometry. As the geometry changes in the bound versus unbound state, fluorescence intensity increases at a given wavelength, showing an ON-like switch as the corresponding binding target appears. We evaluated LCOs as dynamic optotracers of curli and cellulose during biofilm formation, using spectrophotometric recordings of h-FTAA added to bacterial cultures in the small-volume 96-well format. Bacterial GFP expression (plasmid p2777—Supplementary Table 1) enabled simultaneous recording of bacterial growth based on GFP expression (λ Ex 445 nm, λ Em 510), and h-FTAA detection of curli (λ Ex 405 nm, λ Em 556 nm) and cellulose (λ Ex 500 nm, λ Em 600 nm). The appearance of the curli and cellulose spectral signatures coincided with the shift from late logarithmic to early stationary phase at circa 15 h in the wt culture (Figure 5a). Significant signal increase demonstrated pronounced secretion and assembly of both ECM components during early stationary phase. After ~5 h, production ceased and the signals remained at a consistent level throughout late stationary phase. No signal was detected in the curli- and cellulose-deficient strain ΔcsgD-p2777 (Figure 5b).

Figure 5 h-FTAA enables simultaneous, real-time detection of curli and cellulose in liquid Salmonella cultures. (a–d) Combined real-time recording of bacterial growth (left y-axis) measured by intensity of GFP (λ Ex 445 nm, λ Em 510 nm), and h-FTAA staining extracellular curli (λ Ex 405 nm, λ Em 556 nm) and cellulose (λ Ex 500 nm, λ Em 600 nm; right y-axis) in liquid cultures of strains 3934 (a) wt, (b) ΔcsgD, (c) ΔcsgA and (d) ΔbcsA harbouring plasmid p2777. (e) Autofluorescence from LB medium only (□), as well as the combined background fluorescence from biofilm-forming, GFP-expressing bacteria in LB medium (○). (f) Combined real-time recording of bacterial growth, monitored by GFP expression (dashed line, left y-axis) from strain 3934 wt p2777, and appearance of cellulose detected by h-FTAA (right y-axis) in the absence (dotted line) and presence (solid line) of the cellulose-digesting enzyme cellulase. Full size image

In the curli-deficient ΔcsgA-p2777 strain, cellulose was expressed 4 h earlier than the wt culture, during the exponential growth phase (Figure 5c). Multiple comparison analysis (ANOVA) revealed that this difference was statistically significant (Supplementary Figure 4 and Supplementary Table 2). The sharp increase leveled off, and remained constant from early stationary phase onwards. In the curli detection window, a small non-specific signal appeared, which we could ascribe to the broad emission range of h-FTAA bound to cellulose (Figure 4g).

The cellulose-deficient strain ΔbcsA-p2777, showed a slight signal in the cellulose detection window (Figure 5d), which was identified as GFP ‘bleed-through’ by an excitation scan of a planktonic GFP-producing culture (Figure 5e). Curli production increased only gradually throughout the time course of an experiment, a trend differing from the conventional sigmoidal dynamics of ECM formation (Figure 5a). These data suggest that the absence of cellulose de-regulates the production of curli fibres. The kinetics of immediate LCO fluorescence upon amyloid assembly28 implies that the gradual signal increase directly reflects the appearance of curli in the biofilm. The topic of cellulose- and curli co-regulation in biofilm needs further study, studies in which LCOs can play a major role.

To verify the specificity of h-FTAA as a tool for real-time in situ measurements of assembled biofilm components, we grew the wt-p2777 strain in the presence a cellulose-degrading enzyme. Whereas increased GFP fluorescence indicated bacterial growth was not affected by the addition of cellulase, the h-FTAA signal was suppressed due to the effective elimination of the cellulose polysaccharides (Figure 5f). Taken together, our data show h-FTAA to be able to specifically detect the expression of cellulose and curli simultaneously in a continuously growing culture, opening up many exciting possibilities for further kinetic studies.

Fluorescence imaging of biofilm ECM

Fluorescence-based microscopy techniques are fundamental tools for the better understanding of biological events. As a lab with a strong interest in microscopy, we analysed the use of LCOs for in situ staining of ECM in growing biofilms. Using the inclined coverslip set-up, cultures of wt-p2777 in medium supplemented with h-FTAA-enabled incorporation of the optotracer molecules into biofilm formed at the air–liquid interface of the coverslips. Without any additional treatment, confocal microscopy was performed directly on coverslips removed at 24 h. Large communities of distinct rod-shaped GFP-expressing bacteria were observed, surrounded by dense mesh-like structures visualised by fluorescence from h-FTAA bound to the ECM (Figure 6a and Supplementary Figure 5a,b). The fluorescence of h-FTAA was detectable using standard microscopy settings for Cy3 (λ Ex 559 nm, λ Em 575–675 nm). Higher magnification revealed the spatial relationship more clearly, showing red ECM filling the extracellular spaces between green bacterial cells (Figure 6b). No signs of intracellular h-FTAA binding were observed.

Figure 6 Visualisation of ECM components in biofilms formed by S. Enteritidis and S. Typhimurium in the presence of h-FTAA. (a–c) Fluorescence confocal microscopy of unfixed biofilm formed by S. Enteritidis strain 3934 wt p2777 at (a) lower and (b) higher magnification, as well as (c) S. Typhimurium strain 14028 ssaG:gfp+ during growth on inclined coverslips in medium supplemented with h-FTAA. Bacteria (green) and ECM (red) are detected at indicated wavelengths, representing the microscopes’ pre-defined detection settings for GFP and Cy3. (d–f) Fluorescence confocal microscopy images of 14028 ssaG:gfp+ (green) infected (d) CRL-4031 epithelial cells, and (e) RAW264.7 macrophage cell and in (f) sections of mouse livers stained with h-FTAA (red). Staining with Hoechst 33324 shows nuclei (blue) of each cell type. Single optical sections are shown. Scale bar=10 μm. Full size image

Intracellular cellulose detected by h-FTAA in S. Typhimurium infection

Biofilm is considered an important factor in the pathogenesis of Salmonella infections. Shifting to the common model serovar S. Typhimurium, we first confirmed that its ECM components are also detectable by h-FTAA. Fluorescent spectral profiling of biofilm formed by the S. Typhimurium strain 14028 ssaG::gfp+ (Supplementary Table 1) showed the same distinctive emission spectra as S. Enteritidis, with a signature cellulose peak at 480 nm (Supplementary Figure 6a). h-FTAA also proved useful for kinetic, real-time recordings of curli and cellulose production in growing cultures (Supplementary Figure 6b), and for microscopy-based visualisation of the mesh-like ECM in S. Typhimurium strain 14028 ssaG::gfp+ (Figure 6c). Due to structural similarities, the cellulose polysaccharide can thus be successfully identified by h-FTAA across different S. Enterica serovars.