189 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr pISSN 1229-1153 / eISSN 2465-9223 J. Food Hyg. Saf. Vol. 35, No. 2, pp. 189~194 (2020) https://doi.org/10.13103/JFHS.2020.35.2.189 Synergistic Effect of Bacteriophage and Antibiotic against Antibiotic-Resistant Salmonella Typhimurium Kantiya Petsong 1 , Kitiya Vongkamjan 1 , Juhee Ahn 2 * Department of Food Technology, Prince of Songkla University, Hat Yai, Thailand Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon, Korea (Received March 3, 2020/Revised March 25, 2020/Accepted March 26, 2020) ABSTRACT - In this study, we investigated the efficacy of Salmonella phage P22 combined with antibiotics to inhibit antibiotic-resistant S. Typhimurium CCARM 8009. The synergistic effect of phage P22 and antibiotics was evaluated by using disk diffusion and broth dilution assays. The development of Antimicrobial resistance was deter- mined after time-kill assay. The antibiotic susceptibility assay showed the inhibition zone sizes around the antibiotic disks were increased up to 78.8% in the presence of phage (cefotaxime; 13.6%, chloramphenicol; 19.3%, ciprofloxa- cin; 12.7% and erythromycin; 78.8%). The minimum inhibitory concentration values of the combination treatment significantly decreased from 256 to 64 mg/mL for tetracycline, 8 to 4 mg/mL for chloramphenicol, 0.0156 to 0.0078 mg/mL for ciprofloxacin, 128 to 64 mg/mL for erythromycin and 512 to 256 mg/mL for streptomycin. The number of S. Typhimurium CCARM 8009 was approximately 4-log lower than that of the control throughout the combination treatment with phage P22 and ciprofloxacin delete at 37 C for 20 h. The results indicate that the development of anti- microbial resistance in S. Typhimurium could be reduced in the presence of phage treatment. This study provides promising evidence for the phage-antibiotic combination as an effective treatment to control antibiotic-resistant bac- teria. Key words: Bacteriophage, Antimicrobial resistance, Salmonella, Phage-antibiotic combination, Ciprofloxacin Salmonella is one of the major foodborne pathogens that cause public health concern worldwide. The estimated 1.35 million cases associated with Salmonella infections has been reported annually in the U.S. 1) . Among >2500 serovars, Salmonella enterica which presents the majority of Salmonella infections in humans accounted for more than half of the number 2) . In the past several decades, using of antibiotic treatments to control foodborne pathogens in agricultural industry is mis- and over-used, leading to critical problem related antibiotic-resistant development in bacteria 3) . The infections associated with antimicrobial resistance have resulted in the healthcare costs of $20 billion and additional society costs for productivity loss of $35 billion each year in the U.S. 4) . More than 100,000 illnesses associated antibiotic-resistant Salmonella has been reported annually. The study reported that Salmonella isolates conferring resistance to ≥5 antibiotics (i.e. ceftriaxone, ciprofloxacin, streptomycin, tetracycline and erythromycin) accounted for more than 66,000 illnesses from 2009 to 2011 in the U.S 5) . The emergence of multiple antibiotic-resistant Salmonella serovars is responsible for the interruption of antibiotic treatment. The antibiotic-resistant Salmonella infections might increase in mortality rates 2) . The development of effective control method is essential step to solve or decrease the spread of antimicrobial resistance in food production chain. Bacteriophage (phage) is a type of viruses that have the outstanding properties over antibiotic (e.g. specificity to target hosts) 6,7) or chemical agents which have some negative effects on human health 8) . Several phages have been approved as GRAS (Generally Recognized AS Safe) 9) . Phage application is recognized as the potential tool as either an alternative or supplemental to antibiotic treatments 10) . The desirable efficacy of phage-antibiotic combination has been reported and continuously investigated. Synergistic effect of Salmonella phage P22 and ciprofloxacin has been investigated on the inhibition of antibiotic-sensitive S. Typhimurium. The remarkable antimicrobial efficacy of more than 5 log reductions was reported. Moreover, the expression level of genes related to antimicrobial resistance was decreased following the combination treatment 11) . In *Correspondence to: Juhee Ahn, Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon 24341, Korea Tel: +82-33-250-6564; Fax: +82-33-259-5645 E-mail: juheeahn@kangwon.ac.kr