Chemicals and reagents
The antibiotics, chemicals and reagents used in this study were purchased from Sigma (St. Louis, MO, USA) unless indicated. Stock solutions of florfenicol (FFL), sulfamethoxazole (SMX), tetracycline (TET) and trimethoprim (TMP) were prepared in dimethyl sulfoxide (DMSO). Marbofloxacin (MBF) and streptomycin (STR) were prepared in sterile distilled water. Besides, a stock solution of tylosin (TYL) was made in 50% ethanol. The stock preparations were diluted in sterile distilled water or appropriate broth medium. The concentrations of DMSO and ethanol in the final diluents never exceeded 0.1% (v/v).
S. Typhimurium strains and culture conditions
S. Typhimurium strains (LVPP-STI2 and LVPP-STI15) isolated from swine [33] and S. Typhimurium ATCC14028 were cultured in Luria-Bertani (LB) - agar (Difco, MD, USA) at 37 °C. Before assays, the bacteria were grown overnight in LB-broth at 37 °C in a shaking incubator. Our previous study revealed that the parent S. Typhimurium LVPP-STI15 possesses a single gyrA mutation resulting in amino acid substitution, Asp87Tyr, whereas no mutations were identified in the parent S. Typhimurium LVPP-STI2 [33]. In addition, our recent studies confirmed the invasive, quorum sensing and virulence potentials of these strains [34, 35].
In vitro static tylosin therapy
S. Typhimurium strains were cultured in 10 mL Mueller–Hinton broth (MHB) containing 3 μg/mL of tylosin and incubated at 37 °C in a shaking incubator. The concentration of tylosin was determined based on the average results of the maximum plasma concentration of tylosin in swine from previous studies [36,37,38]. Twenty microliters of samples were taken at 0, 1, 2, 4, 6, 8, 12 and 24 h from the time of incubation and cultured in LB-agar plates. The experiment was conducted in duplicate in three separate experiments.
Tylosin therapy using the in vitro dynamic model
A previously described in vitro dynamic model which contains a dilution compartment containing fresh Muller-Hinton broth II (MHB II), a central compartment with either a bacterial culture alone (control growth experiment) or a bacterial culture in medium containing tylosin (killing-regrowth experiments), and an elimination compartment containing waste broth and bacteria was used to expose S. Typhimurium strains to tylosin [39]. A magnetic stirrer was placed in both flasks in the central compartment. Peristaltic pumps (Masterflex, Cole-Parmer, USA) circulated in one direction, from the dilution compartment to the central compartment and from the central compartment to the elimination compartment, at a flow rate of 8.1 mL/h that corresponds to average half-life of tylosin in swine [36, 37]. The system was placed in an incubator at 37 °C. Six hundred microliter of an overnight culture of S. Typhimurium was inoculated into the central compartment. After 2 h of incubation, the bacterial cultures reached 108 CFU/mL (6 × 109 CFU per 60 mL central compartment). Tylosin (3 μg/mL) was injected into one of the units in the central compartment. Then, 20 μL aliquot samples were collected from each compartment in the central unit at 0, 1, 2. 4, 8, 12, and 24 h. Samples were cultured on LB-agar plates and incubated at 37 °C for 24 h. The experiment was conducted in triplicate for each isolate.
Determination of MIC in the presence and absence of an efflux pump inhibitor
The minimum inhibitory concentration (MICs) of florfenicol, marbofloxacin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim against S. Typhimurium strains were determined before and after (at 0, 1, 2, 4, 8, 12 and 24 h post-incubation) exposure to the static and dynamic tylosin treatment. The MICs were determined in the presence and absence of an efflux pump inhibitor (Phe-Arg-β-naphthylamide, 40 μg/mL) using the broth microdilution method with an inoculum of approximately 105 CFU/mL. Four colonies isolated from tylosin exposure/time point were assayed and this was repeated for three experiments i.e. a total of at least 12 colonies assayed for a single exposure/time point. The lowest concentrations of antibiotics inhibiting visible bacterial growth after incubation at 37 °C for 18–24 h were considered as MICs. Clinical breakpoints for tetracycline, florfenicol and sulfamethoxazole were: TET ≥ 32 μg/mL, FFL ≥ 16 μg/mL and SMT ≥ 512 μg/mL. Since CLSI breakpoints were not available for marbofloxacin, streptomycin and trimethoprim, the following breakpoints were used: MBF ≥ 1 μg/mL (ciprofloxacin), STR ≥ 64 μg/mL and TMP ≥ 2 μg/mL [40,41,42]. In addition, tylosin is known to be inactive against Gram-negative bacteria and its MIC in Salmonella is ≥1 mg/mL [34].
Growth curves
Based on the results of the MIC assay, the growth curves of S. Typhimurium strains were determined before and after exposure to tylosin for 12 h in the dynamic model. Briefly, S. Typhimurium (105 CFU/mL) was incubated at 37 °C in MH-broth in a shaking incubator. After 0, 1, 2, 4, 8, 12 and 24 h from the time of incubation, 100 μL of the suspension was removed and diluted serially (10-fold). Then, 20 μL of the dilutions were spread plated on LB-agar plates and the CFUs were determined following incubation at 37 °C for 24 h.
Estimation of free radicals
NBT assay was performed to determine the amount of free radicals generated by S. Typhimurium before and after (12 h) exposure to tylosin using a minor modification of a previous method [11]. The quantity of formazan crystals that are produced from water-soluble tetrazolium salt within each bacterial cell is directly proportional to the production of free radicals [43]. Briefly, 4 h cultures of S. Typhimurium were diluted to 106 CFU/mL in LB-broth. Bacteria were centrifuged at 500 xg for 10 min and washed twice in 1× PBS. The pellet was then suspended in 1× PBS (200 μL). Freshly prepared NBT (0.01%) solution was added and incubated at 37 °C for 1 h. Then, it was washed again with 1× PBS and centrifuged at 500 xg for 10 min. The blue, water-insoluble intracellular formazan crystals were dissolved in 60 μL potassium hydroxide solution (2 M, DMSO). Thereafter, the preformed bacterial superoxide anions were quantified using a microplate reader at 630 nm (VersaMax, Molecular Devices, CA, USA). The experiment was conducted in duplicate in three different experiments for each strain.
QRT-PCR analysis of Salmonella efflux pump, the efflux pump global regulators, and outer membrane porins
Quantitative reverse transcription-PCR (qRT-PCR) analysis was performed to determine the impacts of tylosin exposure (12 h) on the gene expression of acrA, acrB, and tolC (encoding the S. Typhimurium AcrAB-TolC efflux pump), marA, soxS, and ramA (encoding their global regulators), and ompC and ompF (encoding outer membrane porins). Total RNA was extracted using TRIzol (Ambion Life Technologies, Carlsbad, CA, USA) and qRT-PCR was conducted similarly to O’Regan et al. [29]. Gene expression levels of acrA, acrB, and tolC, marA, soxS, ramA, ompC and ompF were determined by CFX96 Touch™ real-time PCR detection system (174 Biorad, USA) using IQ™ SYBR® Green Supermix (Biorad, Singapore). The reaction conditions include denaturation at 94 °C for 3 min, followed by 35 cycles of amplification. Each cycle of amplification consists of 1 min at 94 °C, 20–60 s at the appropriate annealing temperature (Additional file 2), and 1 min at 72 °C. The final extension step was at 72 °C for 10 min. The housekeeping gene rrsG was used to normalize gene expression (2-ΔΔCT). The primers used in this experiment are listed in Additional file 2.
Data analysis
Data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA). One-way analyses of variance (ANOVA) followed by Tukey’s HSD test were conducted to compare the mean values among treatment groups. p < 0.05 was considered statistically significant.
