Bacterial Strains and Culture Conditions
Strains of Salmonella Typhimurium ATCC 19585, S. Typhimurium KCCM 40253, and S. Typhimurium CCARM 8009 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), Korean Culture Center of Microorganism (KCCM; Seoul, Korea), and Culture Collection of Antibiotic Resistant Microbes (CCARM; Seoul, Korea), respectively. The bacterial cells were cultured in trypticase soy broth (TSB; BD, Becton, Dickinson and Co., Sparks, MD, USA) at 37oC for 20 h, centrifuged at 7000 × g for 10 min at 4oC, and washed with phosphate-buffered saline (PBS; pH 7.2). The harvested cells were diluted to 108 CFU/mL for further assays.
In vitro Stepwise Selection Assay
To induce antibiotic-resistant S. Typhimurium ATCC 19585 was exposed with serially increasing ciprofloxacin concentrations according to the serial passage procedure [16]. Salmonella Typhimurium ATCC 19585 was repeatedly cultured in TSB and trypticase soy agar (TSA) containing ciprofloxacin concentrations from 0.0078 to 1 μg/mL. The ciprofloxacin-induced resistant S. Typhimurium ATCC 19585 was stable for more than ten passages in antibiotic-free TSB at 37°C for 20 h prior to use.
Bacteriophage Propagation
Salmonella bacteriophages, P22, P22-B1, PBST-10, PBST-13, PBST-32, and PBST-35, were obtained from ATCC and Bacteriophage Bank at Hankuk University of Foreign Studies (Yongin, Gyeonggi, Korea). All bacteriophages were propagated at 37°C for 20 h in TSB containing S. Typhimurium KCCM 40253. The propagated bacteriophages were collected by centrifuging at 6000 × g for 10 min, filtered through by a 0.2-μm filter to remove bacterial lysates, and further purified using polyethylene glycol (PEG) precipitation assay [17]. The titers of bacteriophage were determined by using a soft-agar overlay method [18]. In brief, the selected bacteriophages were serially (1:10) diluted with PBS and gently mixed with the host cells (107 CFU/mL) in TSB containing 0.5% agar. The mixture was poured onto the pre-warmed base agar and solidified at room temperature, and then incubated at 37°C for 20 h to enumerate the bacteriophages expressed as a plaque-forming unit (PFU).
Lytic Activity of Bacteriophage
Salmonella bacteriophages (P22, P22-B1, PBST-10, PBST-13, PBST-32, and PBST-35) were used to evaluate the lytic activity against STWT, STCIP, STLAB, and STMDR. The selected strains (108 CFU/mL each) were mixed with bacteriophage (1010 PFU/mL each) and incubated at 37oC for 10 min. The incubated cultures were centrifuged at 6,000 × g for 5 min, serially diluted (1:10) with PBS, and plated on TSA using an Autoplate® Spiral Plating System (Spiral Biotech Inc.). The plates were incubated at 37°C for 24-48 h. The lytic activity was expressed as log N/N0; N and N0 denote the counts of bacterial cells treated with and without bacteriophages, respectively.
Fluctuation Assay
The fluctuation assay was used to determine mutant distribution whether STWT, STCIP, STLAB, and STMDR mutants were spontaneous or inducible in the presence of bacteriophages [19]. In brief, STWT, STCIP, STLAB, or STMDR cells (103 CFU/mL) were distributed into 10 test tubes (0.2 mL; Group A) and one test tube (2 mL; Group B). The tubes were incubated at 37oC for 3 h. Group A (1 replicate/tube) and Group B (10 replicates/tube) were plated on TSA with P22 and incubated for 37oC for 24-48 h to enumerate viable cells.
Induction of Bacteriophage-insensitive Salmonella Typhimurium
Bacteriophage P22-insensitive mutant S. Typhimurium ATCC 19585 (STWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (STCIP), S. Typhimurium KCCM 40253 (STLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (STMDR) were isolated using the spot plate assay [20]. P22 (2 × 106 PFU/5 μl) were spotted on 0.5 % soft-agar containing BSSTWT, BSSTCIP, BSSTLAB, and BSSTMDR (107 CFU/mL each) and incubated at 37°C until recovery of host cells within the clear zone, which were assigned as BISTWT, BISTCIP, BISTLAB, and BISTMDR, respectively. The isolated colonies were subcultured in TSB at 37°C for 20 h. The BIST cells were suspended gently in 0.5 % soft-agar plates and poured onto the pre-warmed base agar. The 10-fold diluted P22 (5 μl each) was spotted onto the plates. After 20 h incubation at 37°C, the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR) were tested for the multiple-resistance to P22, P22-B1, PBST-10, PBST-13, PBST-32, and PBST-35.
Mutant Frequency Assay
The mutation rates of STWT, STCIP, STLAB, and STMDR cultured in the absence and presence of P22 for 48 h were estimated on the TSA containing bacteriophages (107 PFU/mL). The cultured cells were plated on TSA with and without bacteriophages and incubated at 37oC for 24-48 h. The mutant frequencies were estimated as the proportion of the numbers of surviving colonies on the TSA with and without bacteriophages.
Lysogenic Induction Assay
Lysogenic cells were induced by mitomycin C [18]. In brief, the cultured BSSTWT, BSSTCIP, BSSTLAB, BSSTMDR, BISTWT, BISTCIP, BISTLAB, and BISTMDR were treated with mitomycin C (0.5 μg/mL) at 37oC for 2 h. After incubation, the mixtures were centrifuged at 7000 × g for 5 min and filtered through a 0.2-μm filter. The collected supernatants were spot tested to confirm the lytic bacteriophages against BSSTLAB.
Bacteriophage Adsorption Assay
Bacteriophage adsorption rates were estimated to evaluate the bacteriophage-binding receptors on the bacteriophage-sensitive and bacteriophage-insensitive STWT, STCIP, STLAB, and STMDR. In brief, BSSTWT, BSSTCIP, BSSTLAB, BSSTMDR, BISTWT, BISTCIP, BISTLAB, and BISTMDR (106 CFU/mL) were infected with the bacteriophages (P22, P22-B1, PBST-10, PBST-13, PBST-32, and PBST-35) at MOI of 0.1 and then allowed to adsorb at 37oC for 15 min. After incubation, the cultures were centrifuged at 16,000 × g for 2 min at 4°C. The supernatants were serially diluted and plated to determine unabsorbed bacteriophage titers according to a soft-agar overlay assay. The adsorption percentage was estimated using the equation: Adsorption rate (%) = [(initial phage titer-phage titer in the supernatant) / (initial phage titer)] ×100.
Antibiotic Susceptibility Assay
The antibiotic susceptibilities of BSSTWT, BSSTCIP, BSSTLAB, and BSSTMDR were determined by using an agar disc diffusion assay to compare with those of BISTWT, BISTCIP, BISTLAB, and BISTMDR. The cultured cells (0.5 McFarlan) were spread on Mueller–Hinton agar plate and then allowed to dry for 5 min. The antibiotic discs (Becton, Dickinson and Company, NJ, USA), including ampicillin (10 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 µg), imipenem (10 μg), streptomycin (10 μg), and tetracycline (30 μg) were placed on the surface on Mueller–Hinton agar and incubated at 37°C for 20 h. The diameter of the inhibition zone was measured by using a digital vernier caliper to evaluate the antibiotic susceptibility.
Quantitative RT-PCR Assay
Total RNA was extracted from BSSTWT, BSSTCIP, BSSTLAB, BSSTMDR, BISTWT, BISTCIP, BISTLAB, and BISTMDR according to the protocol of RNeasy Protect Bacteria Mini kit protocol (Qiagen, Hilden, Germany). The pre-cultured cells were mixed with 1 mL of RNA protect Bacteria Reagent to stabilize RNA, and the mixture were centrifuged at 5,000 × g for 10 min. The collected cells were lysed with a lysozyme-containing buffer TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0). The lysate cells were mixed with 95% ethanol to extract RNA through an RNeasy mini column. According to the QuantiTech reverse transcription procedure (Qiagen), cDNA was synthesized. Briefly, the RNA extracts were rinsed with a Wipe buffer to remove genomic DNA and mixed with a master mixture containing reverse transcriptase, RT buffer, and RT primer mix. The mixture was incubated at 42°C for 15 min followed by 95°C for 3 min. For amplification, the PCR mixture (20 μl) containing 10 μl of 2× QuantiTect SYBR Green PCR Master, 2 μl of each primer, and 2 μl of cDNA, and 4 μl of RNase-free water was denatured at 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec, 55°C for 20 sec, and 72°C for 15 sec using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems™, USA). The synthesized oligonucleotide primers used in this study are listed in Table 1. The relative gene expression levels were determined using the comparative method [21].
Statistical Analysis
All analyses were performed in duplicate on three replicates. Data were analyzed using Statistical Analysis System (SAS). The general linear model (GLM) and least significant difference (LSD) procedures were used to determine significant mean differences among treatments at P < 0.05.