Kanamycin

Strains and plasmids are described in Table 1. Burkholderia mallei was cultured at 37°C using Brucella medium (BD) supplemented with 5% (vol/vol) glycerol. When indicated, antibiotics were added at the following concentrations: 7.5 μg/mL Polymyxin B (MP Biomedicals), 5 μg/mL kanamycin (MP Biomedicals), 7.5 μg/mL zeocin (Life Technologies). Burkholderia mallei bacteria used to inoculate mice were cultured on agar plates and suspended in PBS to the indicated concentration, as previously reported [41 (link)]. Aliquots of the bacterial suspension were immediately spread onto agar plates to determine the number of colony forming units (CFU) in the inoculum. Escherichia coli was cultured at 37°C using Low Salt Luria Bertani (LSLB) agar (Teknova) supplemented with 15 μg/mL chloramphenicol, 50 μg/mL kanamycin, 100 μg/mL ampicillin (Sigma-Aldrich), or 50 μg/mL zeocin, where applicable. The cell lines A549 (human type II alveolar epithelium; ATCC CCL85) and J774A.1 (murine macrophages; ATCC TIB-67) were cultured as described elsewhere [14 (link), 38 (link)].

Bacteria [E. coli C43 (DE3) or Lemo21 (DE3)] were grown in Luria-Bertani (LB) or 2× YT (for FtsN) medium supplemented with the appropriate antibiotic: ampicillin (100 µg/ml) (from MP Biomedicals), chloramphenicol (30 µg/ml) (from Sigma), or kanamycin (50 µg/ml) (from MP Biomedicals).

Kanamycin was purchased from MP Biomedicals; cefotaxime, cephaloridine, and oxacillin from Sigma-Aldrich; cefazolin from Pharmacia & Upjohn SpA; imipenem from MSD; meropenem from Fresenius Kabi NV/SA; ampicillin from Fisher Scientific; amoxicillin from PanPharma; carbenicillin from Pfizer Italy; piperacillin from Lederle/AHP Pharma; temocillin from Eumedica N.V/S.A; and nitrocefin from Abcam.

HEI-OC1 auditory cells were kindly provided by Dr. D. Lim (House Research Institute, Los Angeles, CA, USA). This conditionally immortalized mouse auditory cell line was established by Kalinec et al. [27 (link)], and is a well-established in vitro system for investigating cellular and molecular mechanisms. Cells were maintained in low-glucose Dulbecco's modified medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; HyClone, UT, USA), 1% penicillin streptomycin, and 1% kanamycin (MP Biomedicals, Ohio, USA) at 33°C in a humidified incubator with 10% CO₂. This culturing medium also served as the control medium in the population doubling experiments. Maintaining HEI-OC1 cells at this temperature and CO₂ concentration is referred to as “permissive conditions” by Kalinec et al. [27 (link)], because lower basal apoptotic rates are present.

Trizma Base, EDTA, Sodium Hydroxide, Sodium dodecyl sulphate, Ethidium bromide, Imidazole, Tween-20, Tween-80, Sodium chloride, Potassium chloride, Glycerol, Acrylamide, bis-acrylamide, Ammonium persulphate, TEMED, Glycine, β-mercaptoethanol, Hydrogen peroxide (H₂O₂), Dithiothreitol (DTT), Horseradish peroxidase (HRP), 5,5′-dithiobis(2-nitrobenzoate) (DTNB), Bovine carbonic anhydrase, and Alpha-lactalbumin were purchased from Sigma Aldrich Pvt. Ltd.
Luria Bertani Medium and Luria Bertani Agar (Difco), PBS tablets (Biobasic), Ni–NTA resin (Qiagen GmbH, Germany), DNA markers (GeneDireX Inc., USA), Precision dual colour Protein marker (Bio-Rad). Ampicillin and Kanamycin were purchased from MP Biomedicals.
Primary (Monoclonal Anti-Prdx6, clone 3A10-2A11 antibody produced in mouse, WH0009588M1) and secondary (Anti-mouse IgG (Fab Specific)-Peroxidase antibody produced in goat, A3682) to detect full length human Prdx6 protein was procured from Sigma-Aldrich.

The ORFs of the AMPs (Table S1), codon-optimized for Lactococcus lactis, were cloned into the pTKR plasmid (Fig. S1), an L. lactis/E. coli shuttle vector developed in the Kearney Lab from the L. lactis plasmid, pT1NX (38 (link)) by adding an E. coli ori site and kanR gene for propagation and selection of E. coli clones. This plasmid includes the P1 acid-inducible promoter and the usp45 signal peptide for transport out of the cell. The ORFs were amplified from a gBlock (IDT) by PCR (1 minute melting at 95°C; 35 cycles of 15 seconds melting at 95°C, 15 seconds annealing at T_m + 3°C, 30 seconds extension at 72°C; and 5 minutes elongation at 72°C) using respective primers and pasted into the pTKR plasmid using restriction enzyme cutsites post agarose gel purification. The recombinant plasmid was electroporated into E. coli 10β cells and plated out on LB agar (Thermo Scientific) plate with kanamycin (MP Biomedicals) to pick successfully cloned colonies. Post-colony-PCR screening, cloned colonies were picked and propagated in LB liquid media with kanamycin (25 µg/mL; Thermo Scientific). The pTKR plamids with AMPs cloned were extracted from the pelleted liquid culture using the Plasmid Extraction Kit (Promega) and electroporated into L. lactis MG1363 (LMBP 3019) cells, followed by erythromycin selection on GM17 plates.

The microdilution protocol was used to assess the resistance of probiotic bacteria to different types of antibiotics [23 (link)] with some modifications according to EFSA Guidance [24 (link)]. The antibiotics ampicillin, chloramphenicol, erythromycin, gentamycin, kanamycin (Sigma Aldrich, St. Louis, MO), and streptomycin (MP Biomedicals, Santa Ana, CA) and the amounts of the active compound were placed into the broth media tube: ampicillin (10 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamycin (10 μg), kanamycin (30 μg), and streptomycin (10 μg). Cultures were inoculated in MRS broth filtered through a 0.22-μm filter and incubating aerobically at 37°C overnight. Furthermore, antibiotic stock solutions were prepared according to the following formula: $\begin{array}{c}\mathrm{W}=\frac{\mathrm{1000}}{\mathrm{P}}\times \mathrm{V}\times \mathrm{C}\end{array}$ where P is potency given by the manufacturer (μg /mg), V is volume required (ml), C is final concentration of solution (multiples of 1000, mg/L), and W is weight of antibiotic (mg) to be dissolved in volume V (mL). The data was expressed after 24 hours as the following formula: $\begin{array}{c}\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\%=\frac{\log \mathrm{N}\mathrm{1}}{\log \mathrm{N}\mathrm{0}}\times \mathrm{100}\\ \end{array}$ $\begin{array}{c}\mathrm{S}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\hspace{0.17em}\hspace{0.17em}\text{of}\hspace{0.17em}\hspace{0.17em}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{t}\mathrm{i}\mathrm{c}=\mathrm{100}-\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\%\\ \end{array}$ log⁡N1 is absorbance of culture 620 nm in MRS broth with different antibiotic types and log⁡N0 is absorbance of culture 620 nm in MRS broth as a control.