B per 2

Omono River virus (OmRV; isolate AK4; Isawa et al., 2011 ▸ ) was isolated from C6/36 Aedes albopictus mosquito cells. Purification was performed at 4°C. The cells were pelleted by centrifugation at 10 000g for 15 min and discarded. The remaining culture fluid was concentrated using a centrifugal filtration tube (Vivaspin 20, Sartorius Stedim) at 6000g. The sample was layered onto a bed of 30% sucrose in TNE buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA pH 7.5) and was pelleted by ultracentrifugation at 28 000 rev min⁻¹ (∼140 000g) for 3 h (SW 40 Ti, Beckman Coulter). The pellet was resuspended in 1 ml TNE buffer, applied onto a 12 ml preformed continuous 5–50% sucrose gradient in TNE buffer and ultracentrifugated at 18 000 rev min⁻¹ (∼58 000g) for 18 h (SW 40 Ti, Beckman Coulter). The virus-containing fraction was identified by measuring the absorbance at 280 nm and by SDS–PAGE and was subsequently incubated with an equal volume of detergent (B-PER II, Thermo Scientific) for 30 min with orbital rotation at room temperature. The sample was filtered through a 0.10 µm membrane (Acrodisc 32 mm, Pall Corporation) and ultracentrifuged at 28 000 rev min⁻¹ (∼140 000g) for 3 h (SW 40 Ti, Beckman Coulter). The pellet was resuspended in 1 ml injection buffer (100 mM ammonium acetate pH 7.5) and then repeatedly dialyzed against the same injection buffer.

A modified procedure of Carba NP using blood cultures (bcCarba NP) was developed that did not require an additional incubation in brain heart infusion (BHI) broth as previously described for this assay (2 (link), 6 (link)). For bcCarba NP, 2 × 1 ml blood culture fluid was transferred to 1.5-ml reaction tubes and mixed with 200 μl of saponin 5% (Sigma-Aldrich Chemie, Munich, Germany) to lyse the blood cells. After incubating at room temperature for 5 min, the samples were centrifuged and supernatants discarded (all centrifugation steps were undertaken at 13,000 × g for 1 min). The bacterial pellets were washed with 1 ml distilled water and 1 ml PBS. Subsequently, 50 μl NaCl 0.85% and 50 μl bacterial protein extraction reagent (B-PERII; Thermo Fisher Scientific, Duisburg, Germany) were added to lyse the bacterial cells. After 30 min of incubation at room temperature, 100 μl test solution without imipenem (negative control) was added to the first reaction tube and 100 μl with imipenem (positive control) was added to the other reaction tube. During incubation at 37°C for a maximum of 2 h, any color change of the positive control from red to orange or yellow, while the negative control remained red, was interpreted as positive. Test solutions were prepared according to the methods described by Nordmann et al. (2 (link)).

Antibiotic resistance against carbapenem was determined using the Carba NP assay. The CRE+ (resistant) strain hydrolyzed the antibiotics and reduced the buffer pH, changing the color of the solution from red to yellow. For each bacterial strain, two reactions were carried out. One of the reactions contained 6 mg/mL imipenem (USP) in the reaction buffer, and the other one did not. The reaction buffer comprised of 0.5% (w/v) red phenol and 10 mM zinc sulfate (buffered to pH 7.8 by adding 0.1 N NaOH). To perform Carba NP, a bacterial colony was first added to a 10-μL lysis buffer (10% B-PERII, Thermo Scientific) that contained 4 μL of magnetic particles. The sample droplet was then mixed by moving through the mixing module back and forth to promote the lysis. After mixing, the sample droplet was merged with a 10-μL reaction buffer droplet, and the merged reaction droplet was mixed again. The final reaction droplet was incubated for 30 min before observation.

The Pdu MCPs from both S. Typhimurium LT2-WT and LT2-ΔpduA were isolated by detergent treatment and differential centrifugation^{36 (link)}. Briefly, 400 mL cells were harvested and washed twice with 40 mL of buffer A (50 mM Tris-HCl pH 8.0, 500 mM KCl, 12.5 mM MgCl₂, and 1.5% 1,2-PD) and lysed with a mixture of 10 mL of buffer A and 15 mL of the bacteria-specific reagent (BPER-II, Thermo Fisher) supplemented with 5 mM 2-mercaptoethanol (Sigma-Aldrich), 1× protease inhibitor cocktail (PIC; Sigma-Aldrich), 25 mg lysozyme (Sigma-Aldrich), and 2 mg DNase I (Sigma-Aldrich), with 60 rpm shaking at room temperature for half an hour. Subsequently, Pdu MCPs were separated from cell debris by consecutive centrifuge steps at 4 °C (12,000 × g for 5 min twice to pellet cell debris, 20,000 × g for 20 min to pellet Pdu MCPs). The pellet after centrifugation at 20,000 × g was washed with a 10-mL mixture of buffer A and BPER-II (2:3, v/v) containing 1× PIC and then was resuspended in 0.2 mL of buffer B (50 mM Tris-HCl pH 8.0, 50 mM KCl, 5 mM MgCl₂, 1% 1,2-PD) with 1× PIC. Finally, isolated Pdu MCPs were obtained by centrifugation at 12,000 × g (3 × 1 min) to further remove cell debris.

BMCs were isolated from Ec0087, Ec3087, and Ec3090 cells following the procedure described by Sinha et al. [20 (link)], with minor modifications. Cell pellet (~0.7–1.0 g) was re-suspended in ~4 mL of 60% BPER-II (ThermoFisher) in buffer A (50 mM Tris, pH 8.0, 500 mM KCl, 12.5 mM MgSO₄), supplemented with 0.1 mg/mL lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 U/mL DNaseI. Cells were lysed via a French pressure cell at 12,000 psi and centrifuged at 12,000×g for 10 min at 4 °C to remove cell debris. The soluble fraction was then centrifuged at 20,000×g for 20 min at 4 °C to collect the BMCs. The BMCs were washed with 1 mL of 60% BPER-II in buffer A and then harvested again at 20,000×g for 20 min at 4 °C. Crude BMCs were re-suspended in 400 µL of buffer B (50 mM Tris, pH 8.0, 50 mM KCl, 5 mM MgCl₂). The BMC suspension was then centrifuged at 12,000×g for 1 min at 4 °C three times to remove insoluble contaminants. The final purified BMC suspension was stored at 4 °C until use. Total protein content was determined by Bradford assay (Bio-Rad) using BSA as a standard [21 (link)].

Conventional Carba NP was performed by using a modified protocol described by Vasoo et al.^{25 (link)}, who performed the testing in a pair of microcentrifuge tubes. Each tube contained 100 μL of 20 mM bacterial protein extraction reagent (B-PERII, Thermo Scientific, MA, USA). Next, a 1-μL loopful of the bacterial sample was inoculated into the reagent and vortexed for 5 s. This method used the cultures directly instead of using the supernatant of lysed cells, thereby obviating the need for a centrifuge. One hundred microliters of solution A (0.5% w/v phenol red solution (Sigma-Aldrich, MU, Germany) with 10 mM zinc sulfate (Sigma-Aldrich, MU, Germany) buffered to pH 7.8 by adding 0.1 N NaOH) was added to the first tube, and 100 μL of solution A containing 12 mg/mL imipenem/cilastatin (Fresenius Kabi, Bad Homburg, Germany), which contained 6 mg/mL imipenem, was added to the second tube. The protocol used in this study was modified by downscaling the volume of each reagent to 10 μL (90% reduction in volume) and performing the reaction in droplets on the MDM Carba platform. The incubation was performed at room temperature instead of 37 °C.

The Pdu BMCs from both S. Typhimurium LT2-WT and LT2-ΔpduK were isolated by detergent treatment and differential centrifugation^{28 (link)}. Briefly, 400 mL cells grown in MIM (OD₆₀₀ = 1.0–1.2) were harvested and washed with buffer A (50 mM Tris-HCl pH 8.0, 500 mM KCl, 12.5 mM MgCl₂, and 1.5% 1,2-PD), and lysed with the bacteria-specific reagent (BPER-II, ThermoFisher, 78260). Subsequently, Pdu BMCs were separated from cell debris by sequential centrifuge steps (12,000×g for 5 min to pellet cell debris, 20,000×g for 20 min to pellet Pdu BMCs). The isolated Pdu BMCs were washed with a mixture of buffer B (50 mM Tris-HCl pH 8.0, 50 mM KCl, 5 mM MgCl₂, 1% 1,2-PD) and BPER-II, and resuspended in buffer B containing protease inhibitor cocktail (Sigma-Aldrich). Finally, isolated Pdu BMCs were obtained by centrifugation at 12,000×g (3 × 1 min) to further remove cell debris.