0.2 μm cellulose nitrate filter

C. indologenes #3362 culture was grown aerobically at 37 °C in LB broth supplemented with 16 μg/mL meropenem overnight with agitation. Intact cells were removed through centrifugation at 7000 × g for 30 min. The supernatant was filtered through a 0.2 μM cellulose nitrate filter (Sartorius). The filtrate was ultracentrifuged at 150,000 x g for 3 hours to harvest the OMVs, which are then dissolved in phosphate buffered saline (PBS). Resuspended OMVs were plated on LB agar to confirm the complete removal of intact cells. Isolated OMV samples were inspected under TEM.

About 100 mL of water sample was passed through a 0.2 μm cellulose nitrate filter (Sartorius Stedim Biotech GmbH, Goettingen, Germany) and then the filter was placed in an upright position on modified mTEC agar media (BD Difco, New Jersey, USA). The culture plate was incubated at 37°C for 2 hours and then at 44°C overnight to allow growth of thermotolerant E. coli. mTEC medium contains a chromogen (5-bromo-6-chloro-3-indolyl-β-D-glucuronide), which is catabolized by E. coli producing β-D-glucuronidase to glucuronic acid and produces a red- or magenta-coloured compound. Two E. coli isolates were selected from each water sample and sub-cultured on MacConkey agar (BD Difco, New Jersey, USA) and incubated at 37°C for overnight. The presumptive colonies were identified according to their colony characters, microscopical examination using Gram staining, motility test, and biochemical reactions (oxidase, catalase, indole, lactose fermentation, methyl-red, citrate-utilization, H₂S, Voges-Proskauer, and urease tests) as described previously [41 ]. All E. coli isolates were stored in Tryptone soya broth (Oxoid Limited, Hampshire, England) with 30% glycerol (Sigma-Aldrich, Darmstadt, Germany) and kept at -80°C for future use.

A volume of 0.25–0.5 L of lake water was filtered through a 0.2 μm cellulose nitrate filter (Sartorius, Göttingen, Germany) to concentrate cellular organisms on the filter. Filters were stored at -20°C prior to DNA isolation. DNA isolation was performed using a phenol-chloroform based method, which was modified from Fuller et al. (2003) (link). Briefly, 2 mL of lysis buffer (0.75 M sucrose, 0.4 M NaCl, 50 mM Tris, 20 mM EDTA, pH 9.0) and 20 μL lysozyme (50 mg/mL) were added to the filter and incubated while rotating for 30 min at 37°C, followed by heating to 55°C for 10 min. After addition of 2 mL phenol/chloroform/isoamylalcohol (25:24:1 v/v/v) samples were spun at 3,310 g for 5 min at 4°C. Subsequently, the upper phase was separated and mixed with 2 mL of chloroform and spun at 4,000 g for 5 min at 4°C. DNA was then precipitated with two volumes of ethanol and 0.1 volume of sodium-acetate 3 M, pH 5.2 for a minimum of 1 h at -20°C. The samples were centrifuged for 45 min at 4,000 g at 4°C, and the resulting pellet was washed with 0.5 mL 80% ethanol. DNA was re-suspended in 50–100 μL Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and quantified with a NanoVue Plus spectrophotometer (GE Healthcare, Little Chalfont, United Kingdom). The 260 nm/280 nm ratios of DNA samples ranged from 1.68 to 2.11. Isolated DNA was aliquoted and stored at -20°C until further analysis.