Evaluation of lytic bacteriophages for control of multidrug-resistant Salmonella Typhimurium

Background The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium. Methods The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009. Results P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction). Conclusion The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.


Background
Over the last few decades, the repeated misuse and overuse of antibiotics has accelerated the emergence of antibiotic-resistant bacteria, leading to serious clinical and public health problems [1,2]. The rapid spread of antibiotic resistance has left the development of new antibiotics due to the long and expensive clinical testing [3]. The antibiotic resistance of Salmonella species is mainly acquired by the production of antibiotic-degrading enzymes, alteration in membrane permeability, and activation of multidrug efflux pumps [4]. Recently, the prevalence of multidrug-resistant (MDR) Salmonella serotypes has increased the failure in antibiotic treatments [5][6][7]. The MDR Salmonella infection has become a global public health concern due to the annual increase in morbidity and mortality rates [8]. The therapeutic limitation of current antibiotics has added to the difficulty in treating multidrug-resistant bacterial infections. Hence, the development of alternative therapeutic treatments over antibiotics is essential for controlling multidrug-resistant bacteria.

Annals of Clinical Microbiology and Antimicrobials
Bacteriophage has received much attention as a possible alternative due to the specificity and self-replicating property with no adverse effects on beneficial microflora and human cells [9]. The specificity to target bacteria is attributed to the binding ability of bacteriophages to host cell surface receptors such as flagella, capsule, slime layer, lipopolysaccharides, and outer membrane proteins, resulting in the lysis of bacteriophage-infected bacteria expressed as lytic activity [10,11]. The bacteriophagebinding receptors on the host cell surface can be altered though the modification of outer membrane components [12][13][14]. However, there is relatively little knowledge on the interaction between bacteriophages and multidrugresistant bacteria in terms of the alteration in host cell surface receptors. Therefore, the bacteriophages are not directly applicable to multidrug-resistant bacteria. For the successful application of bacteriophage, this study was aimed to evaluate the lytic activity of potential Salmonella bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) against Salmonella enterica serovar Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009, having different antibiotic resistance profiles.

Bacterial strains and culture conditions
Strains of S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009 were purchased from Korean Culture Center of Microorganism (KCCM, Seoul, Korea), American Type Culture Collection (ATCC, Manassas, VA, USA), and Culture Collection of Antibiotic Resistant Microbes (CCARM, Seoul, Korea), respectively. Escherichia coli ATCC 25922 was used as control strain to evaluate the antibiotic susceptibility. All strains were cultured in trypticase soy broth (TSB) (BD, Becton, Dickinson and Co., Sparks, MD, USA) at 37 °C for 20 h. The cultured cells were harvested by centrifugation at 3000×g for 20 min at 4 °C, washed twice with phosphate-buffered saline (PBS, pH 7.2), and diluted to 10 8 CFU/ml prior to use.

Stepwise selection method
The strain of S. Typhimurium ATCC 19585 was exposed to ciprofloxacin to induce antibiotic-resistant isogenic strain according to a serial passage assay [15]. Salmonella Typhimurium ATCC 19585 strain was cultured repeatedly in TSB and TSA by increasing ciprofloxacin concentrations from 0.03 to 1 µg/ml. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 were stable for more than 10 passages in antibiotic-free TSB.
The strain was cultured in ciprofloxacin-free TSB at 37 °C for 20 h prior to use.

Bacteriophage propagation
Salmonella bacteriophages, P22-B1 and P22, were purchased from ATCC and PBST10, PBST13, PBST32, and PBST 35 were obtained from Bacteriophage Bank at Hankuk University of Foreign Studies (Yongin, Gyeonggi, Korea). The bacteriophages were propagated at 37 °C for 24 h in TSB containing S. Typhimurium KCCM 40253. The propagated bacteriophages were centrifuged at 5000×g for 10 min, filtered through a 0.2-μm filter to eliminate bacterial lysates, and further purified using a polyethylene glycol (PEG) precipitation assay. The bacteriophage titers were determined by using a soft-agar overlay method [16]. In brief, the collected bacteriophages were serially (1:10) diluted with PBS and gently mixed with the host cells (10 7 CFU/ml) in TSB (0.5% agar). The mixture was poured over the pre-warmed base agar lawn. The plates were incubated at 37 °C for 24 h to enumerate the bacteriophages expressed as plaque-forming unit (PFU).

Morphological assay
The morphological properties of Salmonella bacteriophages were determined by transmission electron microscope (TEM, LEO 912AB Omega; Carl Zeiss NTS GmbH, Oberkochen, Germany), located at the Korea Basic Science Institute (KBSI; Gangwon, Korea). The bacteriophages were transferred to the surface of carboncoated copper film and negatively stained with 5% aqueous uranyl acetate (pH 4.0). After air-drying, the stained bacteriophages were observed under TEM (120 kV; 125,000× magnification).

Heat and pH stability of bacteriophages
The susceptibility of bacteriophages to heat was evaluated at the range of 30-80 °C for 30 min. For isothermal treatment, 0.1 ml of each Salmonella bacteriophage (5 × 10 5 PFU/ml) was inoculated into a preheated tube containing 9.9 ml of TSB and then treated at each target temperature for 30 min. After heat treatment, each tube was immediately cooled in an icebath. The pH stability of bacteriophages was evaluated in phosphate buffered saline (PBS, pH 7.2) adjusted to pHs ranged from 2 to 12. Each Salmonella bacteriophage (5 × 10 5 PFU/ml) was exposed to different pH levels at 37 °C for 30 min. The pH-treated bacteriophages were immediately diluted with PBS to avoid further inactivation. After heat or pH treatment, viable bacteriophages were enumerated by using a soft-agar overlay assay.

Determination of optimum multiplicity of infection
The lytic ability of Salmonella bacteriophages against S. Typhimurium KCCM 40253 (10 5 CFU/ml) was evaluated at different multiplicity of infections (MOIs) ranging from 0.01 to 10. The host cells infected by each Salmonella bacteriophage were incubated at 37 °C for 6 h. After incubation, the host cells were enumerated using an Autoplate ® Spiral Plating System (Spiral Biotech Inc., Norwood, MA, USA) and a QCount ® Colony Counter (Spiral Biotech Inc.). The reduction rate was estimated by comparing with the number of the control cells treated without bacteriophages.

Bacteriophage spotting assay
The lytic ability of Salmonella bacteriophages against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009 was determined using a spotting test. The serially (1:10) diluted bacteriophages (2 µl each) were spotted onto the soft-agar plates and incubated at 37 °C for 24 h. The formation of clear zone was determined to express the lytic capability.

Lytic activity of bacteriophage
Salmonella bacteriophages were used to evaluate the lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009. The host strains (10 5 CFU/ ml each) were mixed with bacteriophage (10 5 PFU/ml) at MOI of 1 and incubated at 37 °C for 12 h. After 12-h incubation, the cultures were centrifuged at 3000×g for 20 min. The collected cells were serially diluted (1:10) with PBS. The proper dilutions were plated on TSA using an Autoplate ® Spiral Plating System (Spiral Biotech Inc.). After 24-h incubation, the viable cells were enumerated using a QCount ® Colony Counter (Spiral Biotech Inc.). The lytic activity was expressed as log N c /N p ; N c and N p denote the counts of bacterial host cells treated without and with bacteriophages, respectively, after 12-h incubation.

Statistical analysis
All analyses were performed in duplicate on three replicates. Data were analyzed using the Statistical Analysis System software (SAS 9.4; SAS Institute Inc., Cary, NC, USA). The general linear model (GLM) and least significant difference (LSD) procedures were used to compare means at p < 0.05.
The heat sensitivity of Salmonella bacteriophages was evaluated at the temperature ranged from 30 to 80 °C (Fig. 2a). All bacteriophages were stable to heat up to 60 °C, but the numbers of bacteriophages were reduced to below the detection limit at 80 °C. The sensitivity of Salmonella bacteriophages to pH was evaluated at different pH values ranged from 2 to 12 (Fig. 2b). P22-B1, P22, PBST32, and PBST 35 were stable at pHs ranged from 4 to 11, showing no noticeable reduction in the viable bacteriophage counts.
The ability of Salmonella bacteriophages to lyse the host cells (S. Typhimurium KCCM 40253) was evaluated at different MOIs of 0.01, 01, 1, and 10 (Fig. 3). The reduction rates of S. Typhimurium KCCM 40253 were increased with increasing the MOI. The highest reduction rate of host cells was 54% for P22 at MOI of 10, followed by PBST35, PBST13, and PBST32. P22 was most effective on the reduction of host cells, whereas the least lytic activity was observed for P22-B1 at all MOIs.

Discussion
This study demonstrates the lytic activity of selected bacteriophages against multidrug-resistant S. Typhimurium strains, including antibiotic-induced resistant and clinically isolated strains. In order to compare the host specificity depending on the interaction between bacteriophages and host receptors, the isogenic strain was used to induce the antibiotic resistance. The application of bacteriophages can be one possible approach as an alternative agent to control multidrug-resistant S. Typhimurium.
The heat stability of PBST10 and PBST13 was significantly decreased at 70 °C when compared to other bacteriophages (Fig. 2a). PBST10 and PBST13 were relatively more stable than other bacteriophages at pH 3 but less stable at pH above 4 (Fig. 2b). All bacteriophages were highly sensitive to pH 2 and pH 12 (Fig. 2b). This is in good agreement with previous study showing most bacteriophages are highly stable at pHs between 4 and 9 [17,18]. The observations suggest that the adsorption rates of Salmonella bacteriophages tested in this study may not be changed at the broad ranges of temperature and pH, which are the important environmental factors for the lytic activity of bacteriophages [19].
The different lytic activities of P22 and P22-B1 were observed depending on MOI (Fig. 3). The interaction of bacteriophages and host cells plays an important role in practical application. The efficacy of lytic activity is associated with the optimum ratio of bacteriophage to host cells, which leads to the increase in the binding chance of bacteriophages to the host cells [20]. The primary step in bacteriophage infection process is the attachment and adsorption, which contribute to the host specificity [10,11]. Unlike the host cell reduction, the highest phage titers were observed at low MOI (data not shown). The result is due to the lysis-from-without phenomenon that the lysis of host cells can occur without bacteriophage multiplication at high MOI [20,21]. The bacteriophages could effectively self-replicate at the optimum MOI of 1.  The specificity of P22 to antibiotic-sensitive S. Typhimurium ATCC 19585 was not different from that to ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (Fig. 5). The specificity of P22 to S. Typhimurium ATCC 19585 was not changed after the induction of antibiotic resistance. The observation implies that P22 can be used as biocontrol agent against multidrugresistant S. Typhimurium. Bacteriophages have renewed attention to potential alternative over conventional antibiotics [22]. However, the lowest lytic efficacy of bacteriophages against the clinically isolated antibioticresistant S. Typhimurium CCARM 8009 might be due to the alteration in receptors, leading to the decrease in binding affinity [23]. The specificity of bacteriophages against host cells is highly associated with the binding affinity between the receptors in the host and receptorbinding proteins in bacteriophages [10,24]. The alterations in host cell surface receptors are responsible for bacteriophage resistance, resulting in the reduced lytic activity [12,25].