0.45 μm pore size membrane filter

From each sample, multiple volumes were filtered through 0.45 μm pore size membrane filters (Millipore, Amsterdam, the Netherlands). Because samples were analysed as part of different projects, the method used for isolation and enumeration of E. coli varied and entailed either the use of tryptone soya agar (TSA) and tryptone bile agar (TBA), as described in to ISO 9308–1 ‘Rapid test’ [31 ], or alternatively, tryptone bile x-glucuronide agar (TBX) in accordance with ISO 16649–2 [32 ]. In short, filters were incubated on TSA or TBX for 4–5 hours at 36±2°C, and subsequently transferred to TBA or maintained on TBX and incubated for 19–20 hours at 44±0.5°C. Presumptive E. coli identified using the TSA/TBA method (i.e. indole-positive), were additionally confirmed as E. coli by testing for ß-glucuronidase-activity on Brilliance E. coli/coliform agar (BECSA; Oxoid, Badhoevedorp, the Netherlands) or using API20E (Biomerieux). Beta-glucuronidase-positive colonies identified using TBX were additionally confirmed by testing for indole-activity using BBL Dry Slide (BD, Breda, The Netherlands). E. coli concentrations were based on the number of indol-positive (TSA/TBA) or ß-glucuronidase-positive (TBX) colonies and the fraction of these colonies that was confirmed to be E. coli. The concentrations were calculated using Mathematica software 9.0.1 (WolframResearch, Champaign, IL, USA).

The water samples were collected in sterile glass containers, transported to the laboratory at 5 ± 3 °C and processed within the first 24 h. The water samples were diluted and filtered through 0.45 μm pore size membrane filters (Millipore, France), as described in SR EN ISO 9308–2/2014 (for coliform bacteria) and then cultivated on ChromID ESBL agar and ChromID CARBA agar (BioMérieux, France). The resistant colonies developed after cultivation at 37 °C for 24 h in aerobic conditions were subsequently inoculated on the corresponding antibiotic-enriched media for the confirmation of ESBL-(ChromID ESBL) or CP- (ChromID CARBA) producing phenotypes. All resistant strains were identified by MALDI-TOF-MS (Bruker system). In the same time frame with the collection of the water samples (i.e., during a ten-day period prior to the water sampling), Ab and Pa clinical strains were isolated from intra-hospital infections that occurred in the units discharging the wastewater in the sampled WWTPs.
The antibiotic susceptibility profiles of the identified strains (220 Ab and 84 Pa), were determined using the standard disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI guidelines) for 2018 and 2019 [25 , 26 ] (see Additional file 1: Tables S1 and S2).

The influenza viruses A/California/04/09 (pH1N1), A/Puerto Rico/8/34 (H1N1), and A/Hong Kong/1/68 [H3N2 (a reassortant H3N2 virus carrying the HA and NA genes from A/Hong Kong/1/68 and internal genes from A/Puerto Rico/8/34)], were grown in 9 to 10-day-old SPF embryonated chicken eggs (Orient Bio) for 48 h at 37°C. The viruses were harvested from the allantoic fluids by centrifugation at 3,500 × g for 10 min at 4°C and filtration through 0.45 μm pore-size membrane filter (Millipore) and then stored at −80°C until use. All viral experiments were performed under biosafety level 2+ (BSL2+) conditions.

Cells were grown in LB medium until the OD₆₀₀ was 0.5, and EEC at a final concentration of 0.05 mg/mL was added. Water was added for control experiments. Measurement of d-glucose uptake was started by the addition of 1 mM d-glucose containing 6.17KBq/mL of [¹⁴C]-d-glucose (Perkin Elmer, Inc., MA, USA). After incubation for 10, 20, and 30 min at 37 °C, 300 μL aliquots were collected and the reaction was stopped by the addition of 750 μL of ice-cold LB medium containing 140 mM glucose. The cells were collected by filtration through a 0.45-μm pore size membrane filter (Millipore, Ireland), and the filters were then washed with 1 mL of 50 mM ice-cold phosphate buffer (pH6.5). The filters were air-dried, and cell-associated radioactivity was measured in a scintillation counter. All assays were performed in triplicate. Statistical analyses were performed with SPSS software, and between-group differences were detected by T-test.

A 72-hour culture of each isolate was centrifuged at 8,500 g and passaged through a 0.45 μm pore size membrane filter (Millipore) to remove bacteria, and supernatants analyzed for the VacA protein. In brief, 15 μL of sample was mixed with an equal volume of loading buffer (EZ apply, ATTO) and boiled for 3 min. The sample was electrophoresed through a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Migrated proteins then were electrophoretically transferred (0.14 mA for 1 hour) onto a PVDF membrane. After incubation in blocking buffer (5% skim milk in PBS), the PVDF membrane was treated with anti-VacA primary antibody (b-300, sc-25790, Santa Cruz Biotechnology Inc.) at room temperature for 1 hour with gentle shaking. Then membranes were washed with PBS containing 0.1% Tween 20 (T-PBS) and then incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology Inc.) for 1 hour at room temperature. After incubation, the membrane was washed 5 times with T-PBS, and specific bands were detected using the Amersham ECL Plus Western blotting system (GE Healthcare), according to the manufacturer’s protocol.

The PPJ samples were centrifuged at 10,000× g at 4 °C for 10 min in a centrifuge ALC 4239R (ALC, Winchester, VA, USA), the supernatant was filtered by a 0.45 μm pore size membrane filter (Millipore^®, Burlington, MA, USA) and used for total polyphenols, flavonoids and DPPH determination. To evaluate betacyanin and betaxanthin content, 10 g of each PPJ sample were dissolved in 100 mL of deionized water, let to stir at room temperature for 30 min, centrifuged and filtered as previously described.

Betacyanins and betaxanthins were evaluated following the method reported by Ruiz-Gutierrez et al. [22 (link)], with slight modifications. Briefly, 10 g of sample was diluted in 100 mL of deionized water and homogenized with Ultra Turrax T18 equipment (IKA ULTRA-TURRAX^®, Wilmington, NC, USA). After homogenization, the sample was centrifuged at 10,000× g at 4 °C for 10 min in a centrifuge (ALC 4239R) and the obtained supernatant was filtered by a 0.45 μm pore-size membrane filter (Millipore^®, Burlington, MA, USA). The extract was analyzed spectrophotometrically at 536 nm and 481 nm, for betacyanin and betaxanthin, respectively, using the molecular weight (Mw) and molar extinction coefficient (ε) in water of betanin (Mw = 550 g/mol; ε = 60,000 L/mol) and indicaxanthin (Mw = 308 g/mol; ε = 48,000 L/mol). All the measurements were conducted in triplicate and the results were expressed as the mg of betacyanin and betaxanthin in 100 g of PPPF (dry matter).