Lb broth

The 11168H strain used in this study is a hypermotile derivative of strain NCTC 11168 [24, 25 (link)]. The strains were stored at −80 °C in Mueller–Hinton (MH) broth (Fluka) supplemented with 15 % glycerol. They were recovered by incubation at 37 °C under microaerophilic conditions in a controlled atmosphere incubator (Don Whitely) or in jars supplemented with a CampyGen gas generating kit (Oxoid) for 24 h. Strains were grown on Columbia blood agar (Oxoid) supplemented with 5 % defribinated horse blood and selective Skirrow supplement (Oxoid).
E. coli XL1 cells were used for molecular cloning, e.g. for the construction of pRRBCD-egfp-lacYA177C and pRRBCD-egfp-araE plasmids. E. coli NEB high-efficiency cells (C2566l) were used for transformation experiments. The strains were grown on Luria–Bertani (LB) agar (Oxoid) supplemented under aerobic conditions at 37 °C. Glycerol stocks of E. coli were made with LB broth (Oxoid) supplemented with 15 % glycerol and stored at −80 °C.

For preparing bacterial lawns, 100 or 300 μL overnight cultures of the selected propagation strain grown in LB (Lysogeny Broth, Merck, Darmstadt, Germany) at 37 °C were mixed with 3.3 or 11 mL of molten overlay agar (LBov; LB broth with 0.6% Agar bacteriological no.1, Oxoid) and spread on 9 or 12 cm LA (LB with 1.2% agar) plates, respectively. After settling for 5 min, lawns were dried in a laminar hood for 35 min and used immediately thereafter. To determine phage titers and host ranges, tenfold serial dilutions (up to 10⁻⁷−10⁻⁹) of the phage stocks in SM buffer (0.1 M NaCl, 8 mM MgSO4.7H2O, 50 mM Tris-HCl, pH 7.5), were prepared and 10 μL aliquots were spotted on bacterial lawns. After incubation, plaques were counted and plaque forming units per ml (pfu/mL) were calculated for each strain. Plaque assays were done for at least two independent replicates and if plaques formed in at least one of the assays, the log pfu/mL was noted. Phage stocks were prepared by plate lysis method [74 ].

The PBS pellets (Density = 1.26 g/cm³; Melting point = 114 °C; Glass transition temperature = −32 °C) were purchased from PTT Public Co. Ltd. in Bangkok, Thailand. Tapioca starch (TPS) powder (Moisture = 11.1%; Bulk density = 0.63 g/cm³; Gelatanization temperature = 51 °C; Viscosity = 5.5 C_p) was bought from PT. Starch Solution Int. in Karawang, Indonesia. Meanwhile, Biomaster-silver (BM) particles were provided by Indochine Bio Plastiques, Senai, Johor, Malaysia. The bacteria strains, Staphylococcus aureus (ATCC 6538P), Escherichia coli (ATCC 11229), and Salmonella Typhimurium (ATCC 14028) (Microbiologics, KWIK-STIKTM 2 pack, St. Cloud, MN, USA) were purchased from the Choice-Care Sdn. Bhd, Malaysia. The culture media including LB agar and LB broth were obtained from Oxoid, Malaysia.

Campylobacter jejuni strains (Table 1) were grown at 37°C in a microaerobic chamber (Don Whitley Scientific, United Kingdom) containing 85% N₂, 10% CO₂ and 5% O₂ either on blood agar (BA) plates containing Columbia agar base (Oxoid, United Kingdom) supplemented with 7% (v/v) horse blood (TCS Microbiology, United Kingdom) and Campylobacter Selective Supplement (Oxoid) or in Brucella broth (Oxoid) with shaking at 75 rpm. C. jejuni strains were grown on BA plates for 24 h prior to use in all assays unless otherwise stated. Escherichia coli XL-2 Blue MRF’ competent cells (Stratagene, United Kingdom) were used for cloning experiments and were grown at 37°C in aerobic conditions either on Luria-Bertani (LB) agar plates (Oxoid) or in LB broth (Oxoid) with shaking at 200 rpm. Antibiotics were added at the following concentrations; ampicillin (100 μg/ml), kanamycin (50 μg/ml), and chloramphenicol (50 μg/ml for E. coli studies or 10 μg/ml for C. jejuni studies). All reagents were obtained from Fisher Scientific (United Kingdom) unless otherwise stated.

The inducibility of colistin resistance was tested in the colistin-susceptible mcr-1 positive E. coli isolate. Briefly, the mcr-1 positive/colistin-susceptible E. coli isolate was cultured overnight on non-selective medium (Columbia blood agar). A bacterial suspension in saline (100 µL) adjusted to turbidity of 0.5 McFarland was inoculated, in triplicate, into 5 mL of LB broth (Luria Bertani, Oxoid, UK) containing colistin at concentrations of 0, 0.064, 0.125, 0.25, 0.5, and 1 mg/L and grown at 37 °C with an agitation of 200 rpm overnight (18 h). The culture grown in the LB broth was centrifuged using 4000 rpm for 10 min, and the minimum inhibitory concentration (MIC) to colistin was determined directly from the bacterial pellet according to the manufacturer’s instructions (Erba Mannheim, Germany).

The MIC and MBC of MDEO for the CRKP strains were determined by broth microdilution assays according to the protocol of the Clinical and Laboratory Standard Institute [12 (link)]. A bacterial suspension of 0.5 McFarland turbidity standard was initially diluted 1000-fold with LB broth (LB, Oxoid). The bacterial suspension was then mixed with MDEO. Essential oil concentrations were prepared from 128 mg/ml to 0.125 mg/ml. An LB broth containing CRKP served as the negative control. The MIC value was defined as the lowest MDEO concentration that resulted in non-significant bacterial growth after overnight incubation. For MBC value determination, cultures without significant bacterial growth were subcultured on MH plates and incubated for 18 h at 37^°C. MBC was defined as the lowest MDEO concentration that eliminated the inoculum growth when subcultured [13 (link)]. Each experiment was performed independently three times.