Amikacin

J774A.1 cells were purchased from the Procell Life Science & Technology Co., Ltd. Cells were routinely cultured in Dulbecco's modified Eagle medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) at 37°C in a 5% CO₂ atmosphere. Cells were seeded in 12-well plates at a density of 1 × 10⁶ cells per well and co-cultured with S. Typhimurium at the multiplicity of infection (MOI) of 10:1. Cells were washed with PBS; and fresh medium containing amikacin (100 μg/ml, MilliporeSigma, Burlington, MA, USA) was added to kill the extracellular bacteria for 2 h. Afterwards, infected cells were washed and subsequently cultured in fresh medium containing amikacin (10 μg/ml, MilliporeSigma) to limit extracellular replication of bacteria. Proteins were extracted at 8 hpi using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology).

The Salmonella suspension was prepared as described above. Following 1 h of incubation with bacteria, cells were washed 3 times with PBS and then placed in fresh medium containing 10% (v/v) heat-inactivated FBS and 100 μg/ml amikacin (MilliporeSigma). After 2 h, the cell culture medium was replaced with fresh medium containing 10% (v/v) heat-inactivated FBS and 10 μg/ml amikacin (MilliporeSigma).

The bacteria reaching the late‐logarithmic phase were washed three times with phosphate buffer saline (PBS) and then quantified using a spectrophotometer to determine optical density at 600 nm and calculate the multiplicity of infection (MOI). Caco-2 and HeLa cells were infected with bacteria at MOI of 100 in DMEM-10% FBS (v/v). One hour later, the cells were washed twice with warm PBS, and the medium was replaced with DMEM-10% FBS (v/v) containing 100 µg/mL amikacin (MilliporeSigma, Burlington, MA, USA) to prevent the growth of extracellular bacteria for 2 h. Afterward, the infected cells were washed and subsequently incubated with DMEM-FBS (10%) and 10 µg/mL amikacin. Cells were subsequently treated with DMSO (MilliporeSigma) vehicle, 5 µM MG123 (Selleck Chemicals, Houston, TX, USA) or 100 nM bafilomycin A1 (Baf A1; MCE) when appropriate.

Cells were seeded in 12-well plates (5 × 10⁵ cells/well) and co-cultured with S. Typhimurium at a multiplicity of infection (MOI) of 100:1. At 1 hpi, cells were washed with PBS three times and incubated further for 2 h with a fresh medium containing amikacin (100 µg/ml, MilliporeSigma, Burlington, MA, USA) to kill the extracellular bacteria. Afterward, cells were washed again, and a fresh medium containing amikacin (10 µg/ml, MilliporeSigma) was added to limit extracellular replication of bacteria. For inhibition of PDK1 activation, cells were pretreated with AR-12 (1 μM; S1106, Selleck, USA) 1 h before infection. For inhibition of RSK phosphorylation, cells were pretreated with BI-D1870 (10 μM; HY-10510, MCE) 1 h before infection. Cells were pretreated with Bafilomycin A1 (100 nM; ab120497, Abcam) 2 h before infection to interfere with autophagy. Proteins were extracted using a radioimmunoprecipitation assay (RIPA, Beyotime Biotechnology) buffer containing protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology).

dTHP-1 cells (5 × 10⁵/mL) were infected for 3 h with Mabs at the multiplicity of infection (MOI) of 10 at 37 °C with 5% CO₂, in absence of antibiotics. Afterwards, extracellular mycobacteria were killed by 1 h incubation with 250 µg/mL Amikacin (Merck Life Science S.r.l, Milano, Italy) and cells were treated for 18 h with empty liposomes, lipo-RIF, polymer-coated lipo-RIF or free RIF. Rifampicin, where present, was used at 96 µM, which is the most effective concentration on the basis of our previous investigation [29 (link)]. Finally, intracellular bacterial growth was assessed by Colony-forming unit (CFU) assay; cells were lysed with 1% deoxycholate (Merck Life Science S.r.l, Milano, Italy), samples diluted in PBS–Tween 80 (0.01%) and CFU quantified by plating bacilli in triplicate on 7H10 supplemented with OADC.

MIC testing was conducted using the doubling dilution technique [54 (link)]. In brief, bacterial overnight cultures grown in LB broth were diluted to 10⁶ CFU/mL and 5 μL aliquots were spotted onto Muller–Hinton agar plates (BD Difco, Franklin Lakes, NZ, USA) containing amikacin (Merck, Auckland, New Zealand), tobramycin (Mylan New Zealand Ltd., Auckland, New Zealand) or gentamicin (Pfizer New Zealand Ltd., Auckland, New Zealand). Control agar plates had no antibiotic supplementation. Plates were incubated overnight at 37 °C. The lowest antibiotic concentration that inhibited growth was taken as the MIC. Bacteria were categorised into resistant and sensitive phenotypes following CLSI guidelines [55 ]. Isolates categorized as having intermediate resistance were treated as meeting the threshold level of resistance. Tobramycin and gentamicin had resistance breakpoints of 8 μg/mL and amikacin had a breakpoint of 32 μg/mL.