Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.

PFA-fixed E. coli cells were incubated with 5% skimmed milk (Wako, Tokyo, Japan) in buffer A for 1 h at room temperature.
For primary labelling, the biofilms were incubated with antibodies added to the blocking solution for 1 h at room temperature or overnight at 4C. Antibodies: rabbit anti-Eap antibody developed by Scrum (Tokyo, Japan, 1/200 dilution in the blocking solution) 2  IgG were prepared using a Pierce Fab Preparation Kit (Thermo Fisher Scientific, Tokyo, Japan) and were also used for the primary labelling (1/10 dilution in the blocking solution).
For secondary labelling, biofilms were washed 3-times with PBS, incubated with Fab' fragments bound to 1.4-nm Nanogold and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Nanoprobes, 1/2,000-1/4,000 dilution in the blocking solution), with Fab' fragments bound to 10-nm colloidal gold and Alexa Fluor 488-conjugated goat anti-mouse IgG (Nanoprobes, 1/1,000-1/2,000 dilution in the blocking solution), or with 5-nm colloidal gold-conjugated protein A (EY Laboratories, 1/500 in Can Get Signal A) for 1 h at room temperature, and washed at least 3-times with PBS. After post-fixing with 1% GA, the Nanogold particles were enlarged by gold enhancement using GoldEnhance-EM (Nanoprobes) for 10 min at room temperature, followed by washing with DDW. The biofilms were imaged by ASEM. In some cases a counter-staining step was employed as detailed in the figure legends.
Staphylococcal biofilms were formed directly in an ASEM dish, while E. coli colony biofilms and culture suspensions were placed on an ASEM dish after cultivation. Fixation and staining were performed in the ASEM dishes. The buffer was then exchanged; the biofilms and bacterial cells were immersed in 10 mg/ml D-glucose or ascorbic acid in DDW and observed using the inverted SEM of the ASEM. The acceleration voltage of the SEM was 20 or 30 kV, and backscattered electrons (BSE) from the specimens were recorded by a BSE imaging (BEI) detector to visualise the sample (Fig. 1). The electron dose at the highest magnification of 20,000× was 20 e − /Å 2 , which is less than half the dose permitted in low-dose cryoelectron microscopy aiming at atomic resolution single particle reconstructions.

TEM imaging
S. aureus MR23 biofilm cells were grown in BHIG for 4 h at 37C in 35-mm plastic dishes (Nunc). Biofilms were scraped, collected by centrifugation, and fixed with 2.5% GA in 0.1 M phosphate buffer (PB) (pH 7.4) at room temperature for 1 h and further with 1% osmic acid (OA) in PB at 4C for 1 h. Specimens were dehydrated by treatment with an alcohol gradient series at room temperature, embedded in Epon812, and thin-sectioned using a Leica Ultracut UCT ultramicrotome. Thin sections were stained with uranyl acetate (UA) and lead citrate (LC), and observed with a H7600 TEM (Hitachi, Tokyo, Japan) at 80 kV.
The isolated MV fractions from S. aureus biofilm and planktonic cultures were adsorbed to thin carbon films rendered hydrophilic by glow discharge and supported by copper mesh grids, stained with 2 % UA for 10 sec, air-dried and imaged by TEM.
E. coli colony biofilms with or without curli were observed by negative stain TEM as recently reported 6 .

Conventional SEM imaging
Biofilms formed on a cover slip were fixed in phosphate buffered saline (PBS) containing 2.5% glutaraldehyde at room temperature for 1 h. After washing with double distilled water three times, the specimens were dehydrated through a series of graded ethanol solutions (50%, 70%, 80%, 90%, 95%, and 99.5%), substituted with t-butyl alcohol, freeze-dried, and coated with platinum. The samples were observed using a Keyence VE-9800 SEM (Keyence, Osaka, Japan) as previously reported 7,8 .

Indirect immunofluorescence microscopy
To detect IgG-binding proteins localised on the cell surface, S. aureus cells were cultured in BHIG medium in a glass bottom dish for 3 h at 37C under static conditions. The cells were fixed with 4% PFA and incubated with 4% skimmed milk in PBS with non-specific rabbit IgG (Sigma, 1/1,000 in the blocking solution) for 60 min at room temperature for the primary labelling, and then with Fab' fragments bound to 1.4-nm Nanogold and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Nanoprobes, 1/2,000 dilution in the blocking solution) for 60 min at room temperature for the secondary labelling. In a simple procedure, PFA-fixed cells were incubated with Cy3-conjugated goat anti-rabbit IgG (GE Healthcare, Buckinghamshire, UK, 1/200 dilution in the blocking solution) for 60 min at room temperature. After washing with PBS, the cells were observed using a BZ9000 fluorescence microscope (Keyence).

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
The concentrated supernatant of the biofilm culture and the supernatant and pellet fractions obtained by ultracentrifugation as mentioned above, were analysed by SDS-PAGE, followed by Western blotting. The SDS-PAGE gels were used to transfer the separated proteins to a polyvinyl difluoride (PVDF) membrane with a 0.2 µm pore size (ATTO, Tokyo, Japan). The membrane was incubated in blocking buffer composed of 0.3% bovine serum albumin (BSA), 5% goat serum (Sigma), and Tris buffered saline buffer supplemented with 0.1% (w/v) Tween 20 (TBS-T) for 1 h at room temperature. After washing with TBS-T, the membrane was incubated with the primary antibody (rabbit anti-ClpB IgG or rabbit anti-DnaK IgG) 1/5,000 diluted in CanGet Signal 1 (Toyobo) and with the secondary antibody (HRP-conjugated goat antirabbit IgG, BioRad, Tokyo, Japan) 1/100,000 diluted in CanGet Signal 2 (Toyobo). After washing with TBS-T, the membrane was treated with ECL Plus (GE Healthcare) and the antibodies signals were recoded using a LAS-4000 imager (GE Healthcare).

Swimming assay
Bacterial swimming activity was examined on a soft agar. An overnight culture of E. coli cells grown in LB medium at 30C was 1/100 diluted into YESCA medium, and incubated for a further 4 h at 30C. Aliquots of the culture (2 μl) were spotted onto YESCA soft agar plates containing 0.3% agar, and incubated for 24 h at 30C. The diameter of the colony was recorded.
In parallel, 50 μl of the bacterial culture was placed on the ASEM dish and incubated for 30 min at room temperature. After fixing with 1% GA and 4% PFA, bacteria were labelled with PCG and observed by ASEM to visualise flagella.

Hemolysis assay
Five-microliter aliquots of the MV fraction were spotted on a sheep blood agar plate (Becton Dickinson), and the plate was incubated for 24 h at 37°C. A photo of the plate was then taken using a digital camera.

Coagulase activity assay
Five-microliter aliquots of the MV fraction were spotted on a heat infusion agar plate containing bovine fibrinogen and rabbit serum, and the plate was incubated for 24 h at 37°C. A photo of the plate was then taken using a digital camera. DnaK (e), using the indicated antibodies and colloidal gold-conjugated Spa for primary and secondary labelling, respectively. After the labelling, bacterial cells were further stained with OA and UA. (f)