Swinnex filter holder

In order to determine community composition for the inoculum communities, 30 mL of the inoculum communities were pushed through Swinnex Filter holders (13 mm, Millipore) containing 13 mm 0.22 µm filters (autoclaved, Durapore membrane PVDF, Millipore) connected to Luer-Lok syringes (BD). Filter paper was removed from the holder, cut into 4–6 smaller pieces, submerged in 125 µL QE buffer with 1% Ready-Lyse Lysozyme (Epicenter, Quick Extract Kit) in eppendorf tubes, and shook at 400 rpm overnight at room temperature. The tubes were spun down at 1700 rpm for 5 min the second day and the supernatant was stored at −20 °C till future use.
The composition for communities growing in SSM were determined at early stationary phase. For each community, 200 µL of sample was taken and filtered through MultiScreen HTS GV filter plates (0.22 µm, sterile, PVDF membrane, Millipore) by spinning the plates for 5 min at 3000 rpm. Each well was incubated overnight in 100 µL QE buffer with 1% Ready-Lyse Lysozyme (Epicenter) on a tabletop shaker at 400 rpm. DNA extract was collected by spinning the plates for 5 min at 3000 rpm and obtaining the flow through.

All fluid samples were filtered using 13 mm diameter SPI–pore polycarbonate track etch filters (SPI supplies, West Chester Pennsylvania, USA), held in 13 mm Swinnex® filter holders (Millipore, Billerica, Massachusetts, USA) attached to syringes with Luer-Lok® couplings to prevent sample leaks. In a Class II Biosafety Cabinet, bacterial suspensions were made form growth on agar plates. Approximately two loop-fulls of bacteria were suspended in 1 ml PBS. If the suspension was too turbid a 10X dilution was made. The filter was first wetted by passing 2 ml of PBS through the apparatus. Then 0.2 ml of the bacterial suspension was applied to the filter with a 1 ml syringe, followed by three consecutive 2 ml washes of PBS using a 2 ml syringe. Finally 2 ml of 4% glutaraldehyde was applied with a 2 ml syringe. After one hour wait, the filter was washed with 2 ml 50% ethanol, 2 ml 70% ethanol, 2 ml 85% ethanol, 2 ml 95% ethanol, and then 2 ml of 100% ethanol and finally air dried. Syringes were either operated by hand, or with a Legato 200 syringe pump (KD Scientific, Holliston, Massachusetts, USA).

Isopore track-etched PC membranes with 5 and 8 μm pore size and 13 mm polypropylene Swinnex filter holders were purchased from the Merck Millipore (MA, USA) and Sterlitech (WA, USA), respectively. Before the dead-end MFP, either PC or SiN membranes were mounted between two pieces of a 13 mm Swinnex filter holder, in which the top piece was connected directly to a syringe. To test the stability of the delivery setup and prime the microporous filters, 70% ethanol and sterilized DI water followed by PBS was flushed through a 3 ml syringe mounted on the syringe pump at the flow rate of 1 ml min⁻¹. Next, the cell suspension was pumped through the microporous filter membranes by a syringe pump (KDS Legato, Sigma Aldrich Inc, MO, USA), and treated cells were collected in an Eppendorf tube for further assays.

In order to verify and to visualize the impact of AMPs on the bacterial membrane, electron microscopy was conducted. Therefore, bacterial suspensions of E. coli and S. aureus with a concentration of 2 × 10⁵–8 × 10⁵ CFU/mL were incubated with 128 μg/mL of one AMP (LF11-322-C₈) and PMB in a microplate over night at 37 °C. After overnight culture, the bacteria were prepared for SEM. To achieve this, the bacteria suspensions were filtered through a sterile filter (Swinnex filter holders, Merck Millipore, Burlington, MA, USA). The filters with the collected bacteria were than washed with PBS (10 mM, pH 7.2) and fixed with a 2.5% glutaraldehyde solution (Carl Roth, Karlsruhe, Deutschland). The fixed specimens were gradually dehydrated by increasing ethanol solutions (10% to 100%) and sputtered with gold (Q150R ES Sputter Coater, Quorum Technologies Ltd., East Sussex, UK) prior to examination with a scanning electron microscope (FlexSEM 1000, Hitachi, Tokyo, Japan).

Aliquots of bacterial cultures as well as abiotic controls (not inoculated with Lysinibacillus sp. TchIII 20n38 cells) were filtered through a 0.2 μm GTTP isopore polycarbonate filters using a Swinnex filter holder (Merck Millipore, Darmstadt, Germany). Filters were then air-dried, mounted on aluminum supports with carbon tape, and coated with carbon (7–8 nm thickness), gold (7 nm thickness), or platinum (5 nm thickness). SEM observations were performed using two different instruments; a Hitachi SU 3500 SEM installed at the electron microscopy platform of the Muséum National d’Histoire Naturelle (Paris, France), and a Zeiss Ultra 55 field emission gun SEM equipped with a Brucker EDS QUANTAX detector (Brucker Corporation, Houston, TX, United States) installed at IMPMC (Sorbonne Université, Paris, France). For observations using the Hitachi SU 3500 instrument observations were made in secondary electron mode with an acceleration voltage of 15 kV. SEM-FEG images were acquired in secondary electron mode using with the Zeiss Ultra 55 instrument with an in column detector (InLens) at 2 to 5 kV and a working distance of 3 mm. Energy dispersive X-ray spectroscopy (EDX) analyses were performed at 15 kV and a working distance of 7.5 mm after calibration with reference copper.

For SEM observations, about 100 µL of suspension of each sample were filtered through a 0.2 µm GTTP polycarbonate filter in a Swinnex filter holder (Merck Millipore, Darmstadt, Germany). Dried filters were carbon coated and analyzed using the Zeiss Ultra 55 SEM operating at IMPMC (Paris, France) and equipped with a field emission gun and a Brucker EDS QUANTAX detector (Brucker Corporation, Houston, TX, USA). Imaging was performed in secondary electron mode (In Lens detector) at 5 kV and a working distance of 3 mm. Energy dispersive X-ray spectrometry (EDXS) analyses and mapping were performed at 15 kV and a working distance of 7.5 mm after calibration with reference copper.