Lysozyme solution

Strains were grown overnight on Trypticase Soy Agar (BD Biosciences, Sparks, MD) at 37°C to obtain isolated colonies. Individual colonies were selected to start an overnight culture of Trypticase Soy Broth (BD Biosciences, Sparks, MD). After 12–18 hours of growth, 750 μl of the liquid culture was pelleted and the supernatant was removed. The pelleted cells were stored at -80°C until DNA extraction. To isolate DNA, each pellet was resuspended in 200 μl 1x Phosphate Buffered Saline with 0.2 M EDTA. To lyse the cells, the following were added to each suspension: 12 μl of Lysozyme solution (Sigma, St. Louis, MO), 1 μl RNase (Roche, Mannheim, Germany), 7.5 μl Lysostaphin solution (Sigma, St. Louis, MO), and 7.5 μl Mutanolysin solution (Sigma, St. Louis, MO). The cells were then incubated for 1 hour at 37°C. Forty microliters of Proteinase K (Roche, Mannheim, Germany) was added and the suspension was incubated overnight at 55°C. The following day, a Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) was used to isolate DNA according to the manufacturer’s protocol. The Elution Buffer containing DNA samples was centrifuged for 5 minutes at 8000xg to remove visible debris. The supernatant was transferred into a clean 1.5 mL tube and stored at 4°C until further analysis.

FITC-lysozyme was prepared as described before (Kok et al., 1998 (link)). Briefly, 500 µl of 1 mg/ml FITC (Thermo Scientific, Wilmington, MA, USA) was carefully mixed with 5 ml of 2 mg/ml lysozyme solution (Sigma-Aldrich, St Louis, USA) for 8 h at 4°C in the dark, dialyzed against water and finally lyophilized. Mouse kidney slices were incubated up to 3 h with 1 mg/ml FITC-lysozyme. After 1 and 3 h, the mPCKS were embedded in Tissue-Tek (Sakura, Japan) and snap-frozen in isopentane (−80°C). They were stored at −80°C until 4-μm cryosections were cut perpendicular to the surface of the slices. Cross-sections were allowed to dry on a glass microscope slide and covered with Citifluor (Citifluor, London, UK). Slides were examined using a high-end, fully motorized Zeiss AxioObserver Z1 microscope, and images were acquired using TissueFAXS Image Analysis Software (TissueGnostics, Austria). Images were converted to red/green pseudo-colors using lookup tables in ImageJ 1.47v (http://imagej.nih.gov/ij).

Five micron paraffin-embedded bladder sections were de-paraffinised in xylene/ethanol. Sections were incubated in lysozyme solution (10 mg/ml, Sigma, France) for 15 minutes at 37°C and exposed to 100 μl of universal bacterial 16 S fluorescent rRNA probe (Eub338, GCTGCCTCCCGTAGGAGT-Cy5’, Eurofins, France) at a concentration of 5 ng/μl, in hybridisation buffer (20 mM Tris-HCl, pH7.4, 0.9 M NaCl, 0.01% SDS) at 46°C for 3 hours [57 (link)]. Sections were then incubated in a 48°C prewarmed saline-sodium citrate wash buffer (30 mM sodium citrate, 300 mM sodium chloride, pH7.4, Invitrogen, France) for 20 minutes. To stain polysaccharide-rich content, sections were counterstained with FITC-conjugated wheat germ agglutinin (Invitrogen, France) at a 1:1000 dilution in PBS buffer for 30 min. Slides were mounted with Fluoroshield containing DAPI. Images were acquired using a confocal laser scanning microscope (Zeiss LSM 710) and final images were processed and edited with the ImageJ software.

Bacterial cultures were prepared in triplicate by inoculating pre-warmed MM284 medium with several C. metallidurans CH34 colonies and growing the cells at 30 °C on an orbital shaker at 120 rpm. After 48 h, cultures were diluted with pre-warmed 284 MM medium to an OD₆₀₀ of 0.1 and grown to an OD₆₀₀ of 0.4. At this point, either CuSO₄ was added to a final concentration of 400 µM (copper condition) or a corresponding volume of H₂O (control) was added. After 10 min of exposure, cultures were put on ice and washed twice with a chilled 10 mM MgSO₄ solution at 4 °C. After washing, bacterial pellets were flash frozen and stored at −80 °C until RNA extraction. RNA extraction was performed with the mirVana™ miRNA Isolation kit (Invitrogen™, Carlsbad, CA, USA). Lysis was performed by resuspending the bacterial pellet in 50 µL of a 3 mg/mL lysozyme solution (Sigma Aldrich, Saint Louis, MO, USA) and incubating it at room temperature for 15 min. After lysis, a volume of 500 µL Lysis/Binding buffer was added. The protocol for total RNA extraction was followed without enrichment for small RNAs. Total RNA quality was measured by Agilent 2100 Bioanalyzer using Nano chips. Only samples with a RIN value above 9 were accepted for sequencing.

Pegs with the grown microbiomes were each snapped off from the lid, placed in 1.5 ml tubes containing 180 μl lysozyme solution (20 mg/ml; Sigma, United States) and incubated for 30 min at 37°C. DNA was extracted from the lysate using Purelink Genomic DNA mini Kit (Invitrogen, Waltham, United States) according to the manufacturer’s instructions. DNA was quantified using Qubit 2.0 Fluorimeter and stored at −80°C until subjected to further analysis. Biomass was measured in terms of DNA yield (ng/microbiome).

Sterile paper points were used to collect biofilm samples of the peri-implant sulcus for microbiota analysis as described previously [26 (link)]. The implants were also collected after explantation and their microbiota was likewise analyzed for control purposes. Briefly, biofilm microbes were resuspended in nuclease free water by subjecting paper points (or implants) to a combination of shaking and sonication. The suspension was centrifuged, pellets were stored at − 80 °C and then used for DNA extraction using commercial extraction protocols for genomic DNA (QIAamp Mini Kit, Qiagen, Hilden, Germany). The collected pellets of the supernatant were treated with 180 µL lysozyme solution (20 mg/mL, SIGMA-Aldrich, Taufkirchen, Germany; 20 mM Tris–HCl, pH 8.0, 2 mM EDTA, 1.2% Triton) under shaking at 37 °C for 2:15 h, followed by proteinase K digestion (20 µL proteinase K and 200 µL buffer AL) for 1:15 h under shaking at 56 °C. Finally, the DNA was eluted with 100 µL PCR-clean water and the concentration was quantified using the NanoDrop equipment (PEQLAB, Erlangen, Germany). One empty extraction without any sample material was used a control for background DNA contaminations (contamination control).