Bacterial strains and growth conditions

S. aureus RN4220 and its isogenic ΔmscL mutant (kindly provided by Dr. Jan M. van Dijl, University Medical Center Groningen, The Netherlands) were grown on blood agar plates at 37 °C for 24 h. A single colony was used to inoculate 10 mL of Tryptone Soya Broth (TSB; OXOID, Basingstoke, UK), incubated at 37 °C for 24 h. This preculture was then diluted 1:20 in 100 mL of fresh TSB and incubated at 37 °C for 16 h.

Cultures were harvested by centrifugation (5000 × g) and washed twice in phosphate buffered saline (PBS: 5 mM K2HPO4, 5 mM KH2PO4, 0.15 M NaCl, pH 7.0). Staphylococci were subsequently resuspended in PBS and sonicated (3 × 10 s, 30 W) in an ice-water bath (Vibra Cell Model 375, Sonics and Materials Inc., Danbury, CT, USA). The bacterial suspension was diluted in PBS to a concentration of 108 mL−1 as determined in a Bürker-Türk counting chamber. Absence of mscL genes in the mutant strain was verified by DNA sequencing using Illumina MiSeq as previously described49 (Supplementary Fig. 1).

Materials preparation and characterization

Polystyrene from 12-well-culture-plates (Greiner Bio-One, Frickenhausen, Germany) and gold-coated (10 nm thickness) glass slides (DLRI, St. Charles, MO, USA) were used as received after extensive rinsing with demineralized water. Borosilicate glass (Menzel-Gläser, Menzel GmbH&Co KG, Braunschweig, Germany) was cleaned with 2% Extran, 5 min sonication with 2% RBS35, methanol, and demineralized water. For creating polymer-brush-like surfaces, clean glass slides were exposed to a solution of 0.5 g L−1 Pluronic F-127 (PEO99PPO65PEO99, molecular weight 12600; Sigma-Aldrich, St. Louis, MO, USA) in demineralized water for 20 min. Gentle rinsing with demineralized water removed non-attached Pluronic F-127. Coupons of 1 cm2 were prepared to fit into 12-well plates.

Surface roughnesses were measured with an atomic force microscope (AFM; BioScope Catalyst, Bruker, Camarillo, CA, USA), using ScanAsyst-air tips (tip curvature radius 2 nm; Bruker) to scan areas of 50 × 50 μm at a rate of 1 Hz. Contact angles were measured with water, formamide and methylene iodide, using the sessile drop technique and a home-made contour monitor. Contact angles with these liquids, having different surface tensions and polarities, allowed calculation of the total surface free energy (γtot), together with its acid-base (γAB) component, which in turn can be divided into electron-donating (γ) and -accepting (γ+) parameters, and the Lifshitz–Van der Waals (γLW) component50. Surface roughness and contact angles were measured in triplicate on three different substratum surfaces.

Contact angles on bacterial lawns and surface free energies

Hydrophobicity of bacterial cell surfaces was determined through contact angle measurements with different liquids on staphylococcal lawns using the sessile drop technique and a home-made contour monitor. Staphylococci were deposited on 0.45-μm pore-size HA membrane filters (Millipore Corporation, Bedford, MA, USA) using negative pressure, and filters were subsequently dried until reaching constant, so-called “plateau” water contact angles, representing bacterial cell surfaces without “free”, but with “bound” water. Contact angles were measured with water, formamide and methylene iodide on six bacterial lawns from three different bacterial cultures, from which surface free energy components and parameters were then calculated as described in the above section.

Zeta potentials

To measure bacterial zeta potentials, bacteria were resuspended in 10 mM potassium phosphate buffer at different pH values (pH 2, 3, 4, 5, 7). Using the Helmholtz–Smoluchowski equation51, zeta potentials were derived from electrophoretic mobilities obtained with particulate microelectrophoresis (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Experiments were performed in triplicate with different bacterial cultures.

Microbial adhesion to hydrocarbons (kinetic MATH assay)

The combined effects of surface hydrophobicity and charge on staphylococcal adhesion to a hydrophobic ligand were determined, as previously described52. Briefly, staphylococci were resuspended in 3 mL of 10 mM potassium phosphate buffer containing 1:20 (v/v) hexadecane at different pH values (pH 2, 3, 4, 5, 7) to an optical density at 600 nm between 0.4 and 0.6 (initial absorbance at time zero [A0]), as spectrophotometrically measured (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY, USA). The suspension was vortexed for 10 s and allowed to settle for 10 min, and the optical density was measured again (absorbance at time t [At]). This procedure was repeated for five more times to enable calculation of an initial rate of bacterial removal from the aqueous phase defined as

$${\mathrm{Rate}}\,{\mathrm{of}}\,{\mathrm{initial}}\,{\mathrm{removal}} = \mathop {{\lim }}\limits_{t \to 0} \frac{{\mathrm{d}}}{{{\mathrm{d}}t}}\log \left( {\frac{{A_t}}{{A_0}} \times 100} \right)$$

(1)

where t is the vortexing time. The experiment was performed in triplicate with different bacterial cultures.

Atomic force microscopy

Single-bacterial contact AFM probes were prepared by immobilizing bacteria on NP-O10 tip-less cantilevers (Bruker), as described previously53. Briefly, cantilevers were calibrated by the thermal tuning method displaying spring constants in range 0.03–0.12 N m−1 and mounted on a micromanipulator (Narishige International, Tokyo, Japan) under microscopic observation (Leica DMIL, Wetzlar, Germany). The cantilever apex was then dipped into a droplet of 0.01% poly-l-lysine (molecular weight 70,000–150,000, Sigma-Aldrich) for 1 min, dried in air for 2 min and dipped into a bacterial suspension droplet (3 × 106 mL−1 in 10 mM potassium phosphate buffer, pH 7.0) for 2 min. Imaging a calibration grid (HS-20MG BudgetSensors, Innovative Solutions Bulgaria Ltd., Sofia, Bulgaria) with the bacterial probe confirmed single-bacterial contact with the substratum surface54, and probes yielding double contour lines were discarded (which seldom or never happened).

AFM force measurements were done on a BioScope Catalyst AFM (Bruker), at room temperature in 10 mM potassium phosphate buffer. Force–distance curves were obtained under a loading force 3 nN at approach and retraction velocity 2 μm s−1, and taken prior to and after 10 s bond-maturation. To verify that measurements did not disrupt bacterial integrity, five force–distance curves at a loading force of 3 nN and 0 s contact time were acquired on clean glass at the onset and end of each experiment. When adhesion forces differed more than 1 nN from the onset to the end of an experiment, the probe and its last data set obtained were discarded and the probe was replaced by a new one. For each strain, AFM measurements were performed with at least three probes prepared from three separate bacterial cultures. With each probe, at least three different spots on each substratum surface were measured, recording five force–distance curves on each spot for each contact time.

Calcein uptake

For measuring the gating of mechanosensitive channels, bacterial uptake of the fluorescent dye calcein (Sigma-Aldrich) was monitored. First, bacteria were allowed to sediment from a suspension in PBS (108 mL−1) onto the different substratum surfaces for 90 min, after which PBS was removed and gentle rinsing with PBS was applied to remove non-adhering bacteria. Subsequently, calcein was added in a concentration of 4 mM for 15 min, followed by fixation with 4% formaldehyde (VWR International, Breda, Netherlands) for 15 min to prevent removal of intracellular calcein. Extensive rinsing in ultrapure water removed the excess extracellular calcein.

As a control, planktonic bacteria were used. Calcein (4 mM) was added to a staphylococcal suspension for 15 min, followed by fixation with 4% formaldehyde for 15 min. Excess extracellular calcein was removed by filtration with 0.45-μm pore-size HA membrane filters (Millipore Corporation). Bacteria were then collected from the filters by vortexing and resuspended in PBS.

Phase-contrast and fluorescent images were acquired with a fluorescence microscope (Leica, Wetzlar, Germany) on both adhering and planktonic bacteria, to evaluate total number of adhering bacteria or in suspension and the percentage of bacteria that displayed calcein uptake. The percentage of bacteria that displayed fluorescence in suspension or adhering to a surface was taken as a measure for calcein uptake, according to a protocol previously described for gating in mechanosensitive channels reconstituted in liposomes33 and validated here for use in bacteria (Table 3). For validation, channels were blocked by 15-min exposure to 4% formaldehyde34 or blocking was induced by 15-min exposure to 100 μM gadolinium55 before calcein exposure.

The increase in the percentage fluorescent staphylococci (Y) as a function of their adhesion forces (F) on the different substratum surfaces was fitted to an exponential function to yield a plateau level of the percentage of fluorescent staphylococci and a critical adhesion force according to

$$Y = Y_0 + \left( {Y_{{\mathrm{plateau}}} – Y_0} \right)\left( {1 – e^{ – \frac{F}{{F_{\mathrm{c}}}}}} \right)$$

(2)

in which Y0 is the percentage fluorescent staphylococci in suspension (zero adhesion force) and Fc is the critical adhesion force. Yplateau is the plateau level of the percentage fluorescent staphylococci reached.

To allow more accurate fitting, the plateau level was mathematically confined to the maximum percentage of adhering, fluorescent staphylococci observed for each strain. Experiments were performed in triplicate with different bacterial cultures.

Dihydrostreptomycin susceptibility and uptake

First, MIC and MBC of the staphylococci for dihydrostreptomycin were determined. To this end, bacterial cultures (105 mL−1 in TSB) were dispensed in a 96-well microtiter plate with dihydrostreptomycin sesquisulfate (Sigma-Aldrich) in TSB with known concentrations and applying a step factor dilution of 2 starting from 512 μg mL−1. Incubation was done at 37 °C for 24 h. After incubation, MIC was taken as the lowest antibiotic concentration not generating visible turbidity. Then, 10 μL of bacterial suspensions of each well showing no turbidity, were plated on TSB agar plates and incubated at 37 °C for 24 h. The MBC was taken as the lowest concentration at which no colonies were visible on the plate. Experiments were performed in triplicate with different bacterial cultures.

To evaluate the differential uptake of dihydrostreptomycin through mechanosensitive channels15 in adhering and planktonic staphylococci, bacterial suspensions (108 mL−1 in PBS) were allowed to sediment on different surfaces for 90 min or maintained planktonically in suspension. After subsequent exposure to dihydrostreptomycin (512 μg mL−1) in PBS for 120 min, bacteria were collected by 1 min sonication in a water bath and serially diluted in PBS. Exposure to PBS was applied as a control. Next, 10-μL aliquots were plated on TSB agar plates and incubated for 16 h at 37 °C. The number of colonies formed on the plates was manually counted. Experiments were performed in triplicate with different bacterial cultures.

The reduction in the number of CFUs per mL as a function of their adhesion forces, F, on the different substratum surfaces was fitted to an exponential function to yield a plateau level of the reduction in the number of CFUs per mL and a critical adhesion force, similar as done for the percentage fluorescent staphylococci (Eq. (2)). To allow more accurate fitting, the plateau level was mathematically confined to the maximum percentage of reduction in the number of CFUs per mL observed for each strain. Experiments were performed in triplicate with different bacterial cultures.

Statistical analysis

GraphPad Prism, version 8.4.3 (San Diego, CA, USA) was employed for statistical analysis. Data were tested for normal distribution with Shapiro–Wilk normality test. If data were normally distributed, one-way analyses of variance (ANOVA) with Tukey’s HSD post hoc test or a two-tailed Student’s t-test were employed. When data were not normally distributed, Kruskal–Wallis test with Dunns’ approximation replaced ANOVA. Comparisons of dihydrostreptomycin killing across substratum surfaces between the parent strain and its mutant were made using one-way ANOVA test with Sidak’s multiple comparison adjustment (four comparisons). p < 0.05 was used as significance for all tests.

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