Mixed cellulose ester filter

Binding assays were performed in 20 mM MES pH 5.4, 40 mM Sodium Chloride, 0.01% DDM 0.0005% CHS unless stated otherwise. Five µL of ³H-TAEKDEL or ³H-TAEHDEL (Cambridge peptides, UK) at 20 nM were incubated with 5 µL of Gg KDELR or variants thereof at the desired concentration at 20°C for 10 min. The reaction was then filtered through a 0.22 µm mixed cellulose ester filter (Millipore, USA) using a vacuum manifold. Filters were then washed with 2 × 500 µL buffer. The amount of bound peptide was measured using scintillation counting in Ultima Gold (Perkin Elmer). Experiments were performed a minimum of three times to generate an overall mean and standard deviation. Data was normalised to the maximal binding at pH 5.4 and fit with a four-parameter logistic non-linear regression model.

To check the selectivity of the novel culture-based method, non-motile Klebsiella pneumoniae were mixed with C. jejuni and C. coli. The tested bacterial suspensions (200 μL) were spread on filters (0.45 μm, mixed cellulose ester filter; Merk, Darmstadt, IN, USA) and incubated on mCCDA or Brilliance CampyCount Agar. Petri dishes were incubated bottom-down at 42 °C for 24 ± 4 h under a microaerobic atmosphere. After overnight incubation, the filters were removed, the dishes were inverted, and cultivation continued at 42 °C under a microaerobic atmosphere for another 44 ± 4 h. Finally, bacterial species were identified by PCR and MALDI-TOF/MS. Experiments were performed in triplicate.

In parallel, Campylobacter spp. were isolated from water samples by a novel method without enrichment. In detail, 50 mL water samples were centrifuged at 12,000× g (acceleration time 300 s, deceleration time 40 s; HERMLE Z326K, Wehingen, Germany) for 30 min at 10 °C. Pellets were resuspended in 200 μL of sterile water and spread on a filter (0.45 μm, mixed cellulose ester filter; Merk, Darmstadt, Germany). Filters were placed face-up on top of mCCDA agar and incubated bottom-down for passive filtration at 42 °C under a microaerobic atmosphere. After overnight incubation, the filters were removed and cultivation continued at 42 °C under a microaerobic atmosphere for another 44 ± 4 h. Experiments were performed in triplicate.

Sewage influent samples from 71 cities and 78 wastewater treatment plants (WWTPs) across the United States and one city in Spain were collected during August 2012, January 2013, and May 2013 as part of former surveys. Details on the collection method can be found in (17 (link), 20 (link)). Briefly, 1 liter of single-time point grab or 24-h flow-weighted composite samples were collected and shipped overnight on ice. A total of 25-mL subsamples were filtered through 0.22-μm mixed cellulose ester filters (47-mm diameter; Millipore, Billerica, MA, USA) and stored in 2-mL screw-cap freezer tubes at −80°C. The list of the samples and their associated metadata are reported in Data Set S1.

For fecal samples, bacterial DNA was extracted from approximately 0.2 g of material using QIAmp DNA stool mini kit according to the manufacturer’s instructions (Qiagen, USA). A total of 25 mL for sewage influent and 200 or 400 mL for freshwater samples were filtered onto 0.22-μm mixed cellulose ester filters with a 47 mm diameter (Millipore, USA). DNA from filters was then extracted using the FastDNA spin kit for soil (MP Biomedicals, USA) according to the manufacturer’s instructions. One modification of this protocol was applied: Cells were mechanically lysed using a MiniBeadBeater-8 cell disruptor (BioSpec Products, USA) for 1 min and 2 min at room temperature for sewage and freshwater samples, respectively. DNA was stored at − 20 °C until it was analyzed. DNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

Overnight cultures of donor E. coli MG1655 harboring pKM101, donor E. coli MS411 harboring R1-16, and recipient E. coli MG1655 (WM1652) bacteria were grown at 37°C with shaking at 225 rpm in LB plus antibiotic, diluted in antibiotic-free LB containing DMSO or 150 µM compound, and incubated with shaking at 37°C to reach an OD₆₀₀ of ~0.3. Donor and recipient cells were mixed at a ratio of 1:1 on 0.22-µm-pore-size mixed-cellulose-ester filters (Millipore) on antibiotic-free LB plates for 2 h at 37°C. Filters were aseptically removed from LB plates and incubated in 100 µl antibiotic-free LB to dislodge E. coli from membranes. Serial dilutions were plated on the appropriate antibiotic (or dual-antibiotic) LB plates to determine the number of donor, recipient, and transconjugant cells. Colonies were enumerated after 24 h, and conjugation efficiencies were calculated by dividing the average number of transconjugates by the average number of donors (either pKM101 or R1-16 donor cells). Conjugation efficiency in the presence of compound is expressed as a percentage of conjugation efficiency in DMSO. Data represent six individual experiments for compounds C10 and KSK85 and three individual experiments for GKP42.