Azithromycin

Azithromycin (Sigma-Aldrich, Stockholm, Sweden) was dissolved in dimethyl sulfoxide (DMSO) and HBECs were exposed to Azithromycin 24h prior to infection with rhinovirus and continuous throughout the experiment.

All chemicals except ZnO nanoparticles, azithromycin and Fe(VI) were purchased from the Merck Company. ZnO nanoparticles were purchased from Sigma-Aldrich Company. Based on the information provided by this company, the size of the ZnO nanoparticles was <50 nm. Analytical grade azithromycin was purchased from Sigma, Malaysia. In this study, Fe(VI) was prepared by the electrochemical method according to the instructions described in [33 (link)]. A digital pH meter, the AZ-86502 (Taiwan) was used to measure the pH. The power of UV radiation was measured using a 340A UV meter (Taiwan). Equation (8) was used to determine the efficiency of antibiotic removal from the water in all experiments. The amount of antibiotic was determined by a COD test. The COD was measured by APHA standard method [34 ].
$RE=\frac{C_1-C_2}{C_1}\times 100$

The protocol was adapted from our previous publications (Beckett et al., 2013; Hodge et al., 2010, 2011; Mukaro et al., 2013). Female BALB/c mice (10 weeks old; CS and CS + Azi: n = 5 mice/group) were exposed to mainstream CS from nine research cigarettes (1R6F reference cigarette, University of Kentucky, KY, USA) for 1 hr, twice daily, 5 days/week, for 12 weeks. The protocol utilized the inExpose system and whole‐body chambers (Scireq Inc, QC, Canada), control mice (n = 5) were exposed to fresh air in an adjacent inExpose chamber. inExpose puff profile was configured to deliver 2 L/min fresh air, with an ISO standard CS puff once every 30 s. Cigarettes were burnt to 3 mm from the tipping paper. During weeks 7–12, the CS + Azi cohort were exposed to 0.2 mg kg day⁻¹ of azithromycin via nebulization [azithromycin (Sigma Aldrich, MO, USA) dissolved in pH 6.0 PBS] into the inExpose whole‐body chamber once per day following their second CS session.
Animals were housed within a specific pathogen‐free barrier facility, in separate cages for each cohort, with a temperature range 18–24°C, 12:12 hr light‐dark cycle, and few regular chow ad‐libitum. The experiments were approved by SAHMRI Animal Ethics Committee (SAM361) and conducted within the National Health and Medical Research Council of Australia guidelines on animal experimentation.

Peptides HHC-10 (KRWWKWIRW-NH₂) [37 (link)], 1002 (VQRWLIVWRIRK-NH₂) [38 (link)], 1018 (VRLIVAVRIWRR-NH₂) [39 (link)] and the D-enantiomer DJK-5 (VQWRAIRVRVIR-NH₂) [25 (link)] were synthesized by CPC Scientific using solid-phase 9-flurenylmethoxy carbonyl (Fmoc) chemistry and purified to >95% purity using reverse-phase high-performance liquid chromatography (HPLC). The lyophilized peptides were resuspended in endotoxin-free water. The antibiotics gentamicin, ciprofloxacin, meropenem, erythromycin, clindamycin, vancomycin, azithromycin, and colistin were purchased from Sigma-Aldrich at a United States Pharmacopeia (USP) Reference Standard grade. erythromycin and azithromycin were initially dissolved in 70% ethanol, while all other antibiotics were dissolved in endotoxin-free water (E-Toxate, Sigma-Aldrich). Antibiotics and peptides were further diluted into saline (Sigma-Aldrich) for in vivo application.

The non-encapsulated S. pneumoniae TIGR4Δcps used as model organism was cultivated in the chemically-defined medium RPMI_modi 1640 (HyClone) [9 ] and grown on Columbia blood agar plates (Oxoid) in the presence of the appropriate antibiotic (150 µg ml⁻¹ kanamycin). The cultivation was performed as described previously [9 ]. At a mid-exponential phase OD₆₀₀ of 0.5 the bacterial cells were treated with either 0.005 µg ml⁻¹ (0.5 × minimal inhibitor concentration (MIC)) cefotaxime (Sigma-Aldrich), 0.064 µg ml⁻¹ (2 × MIC) azithromycin (Sigma-Aldrich), 0.8 µg ml⁻¹ (2 × MIC) moxifloxacin (Sigma-Aldrich), 2 µg ml⁻¹ teixobactin-Arg10 (cooperation with University of Lincoln, School of Life Sciences [31 (link), 32 ]), and combination of 0.5 × MIC cefotaxime and 2 × MIC azithromycin for 90 min. For the control, bacterial cells were cultivated without antibiotic. We obtained five independent biological replicates for the metabolome analysis with exception of teixobactin analog (four independent biological replicates). Extra- and intracellular metabolome samples were taken at 15, 30, 60, and 90 min (t₁₅, t₃₀, t₆₀, and t₉₀) after the addition of each antimicrobial compound.

Antibiotic stocks were prepared in the following solvents: 10 mg/mL of ceftriaxone (USP), doxycycline (Fisher scientific, D9891-1G), gentamicin (Fisher scientific, 1405-41-0), ampicillin (Fisher scientific, 69-52-3), and azlocillin (Flightpath) in sterile cell culture grade water, whereas 5 mg/mL of azithromycin (Sigma, PZ0007-5MG) was dissolved in sterile cell culture grade DMSO. Antibiotics were then aliquoted and stored at either −80 °C or −20 °C until further use. On the day of assay, these stock concentrations were diluted to 1 mg/mL and 100 μg/mL in the respected bacterial culture media for MIC and cell-free, liquid culture MBC assays. For the DH82 cell-based MBC assay, antibiotics were diluted in EMEM media supplemented with 15% heat-inactivated FBS.