BIOCONTROL OF SALMONELLA TYPHIMURIUM IN FRESH SLICED FRUITS USING PHAGES

Öz

Foodborne diseases continue to be a major public health problem worldwide. Pathogenic Salmonella serotypes are the leading cause of microbial food poisoning. This study aimed to inhibit Salmonella Typhimurium with phages as biological agents in fruits. RFLP analysis of Salmonella Typhimurium phages isolated from wastewater, raw milk and chicken meat samples was performed with EcoRV and XbaI enzymes. The latent periods of these phages, whose single-stage growth curves were obtained, were found to be short (5-15 minutes), and their burst size was found to be in the range of 25-111 PFU/cell. The lytic effect of phages against different types of bacteria was examined, but no lytic effect was detected against Gram-positive and Gram-negative bacteria other than Salmonella. With the cocktail prepared from phages, the number of S. Typhimurium in melon decreased by 2 log CFU/g at MOI 1000 and 10000; In pineapple samples, a 1 log CFU/g decrease was detected at MOI 10 and 100. It has been revealed that the prepared phage cocktail may be among the strategies that can be used for Salmonella control in the food industry.

Anahtar Kelimeler

• Bacteriophage
• Biocontrol
• Phage therapy
• Salmonella Typhimurium

TAZE DİLİMLENMİŞ MEYVELERDE SALMONELLA TYPHIMURIUM’UN FAJLARLA BİYOKONTROLÜ

Öz

Gıda kaynaklı hastalıklar dünya çapında halk sağlığı açısından büyük bir sorun olarak varlığını sürdürmektedir. Mikrobiyel kaynaklı gıda zehirlenmelerinin başında ise patojenik Salmonella serotipleri gelmektedir. Bu çalışmada dilimlenmiş meyvelerde de rastlanan Salmonella Typhimurium’un fajlar ile inhibisyonu hedeflenmiştir. Bunun için atık su, çiğ süt ve tavuk eti örneklerinden izole edilen Salmonella Typhimurium fajlarının EcoRV ve XbaI enzimleri ile RFLP analizi yapılmış 9 fajdan 4’ünün genomik olarak birbirinden farklı olduğu tespit edilmiştir. Tek aşamalı gelişme eğrileri çıkarılan bu fajların latent dönemleri kısa (5-15 dk), patlama büyüklükleri ise 25-111 PFU/hücre aralığında bulunmuştur. Fajların farklı cins bakterilere karşı litik etkisi incelenmiş fakat Salmonella dışındaki Gram pozitif ve Gram negatif bakterilere karşı litik etkisi saptanmamıştır. Fajlardan hazırlanan kokteyl ile kavunda S. Typhimurium sayısında MOI 1000 ve 10000 değerlerinde 2 log KOB/g azalış; ananas örneklerinde ise MOI 10 ve 100 değerlerinde 1 log KOB/g azalış tespit edilmiştir. Hazırlanan faj kokteylinin gıda endüstrisinde Salmonella kontrolü için kullanılabilecek stratejiler arasında olabileceği ortaya konmuştur.

Anahtar Kelimeler

• Bakteriyofaj
• Biyokontrol
• Faj terapi
• Salmonella Typhimurium

Kaynakça

1. Abdelsattar, A. S., Safwat, A., Nofal, R., Elsayed, A., Makky, S., El-Shibiny, A. (2021). Isolation and characterization of bacteriophage ZCSE6 against Salmonella spp.: Phage application in milk. Biologics, 1(2), 164-176.https://doi.org/10.3390/ biologics1020010.
2. Acar Soykut, E. (2007). Streptococcus thermophilus ve Lactobacillus bulgaricus virülent fajlarının replikasyon parametreleri, kapsid protein profilleri ve restriksiyon endonükleaz analizleri esas alınarak tanımlanmaları ve sınıflandırılmaları, Ankara Üniversitesi, Fen Bilimleri Enstitüsü, Gıda Mühendisliği Anabilim Dalı Doktora Tezi, Ankara, Türkiye, 188 s.
3. Bao, H., Zhang, H., Wang, R. (2011). Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum. Poultry Science, 90(10), 2370-2377. https://doi.org/10.3382/ ps.2011-01496.
4. Bielke, L., Higgins, S., Donoghue, A., Donoghue, D., Hargis, B. M. (2007). Salmonella host range of bacteriophages that infect multiple genera. Poultry Science, 86(12), 2536-2540. https://doi.org/ 10.3382/ps.2007-00250.
5. Byun, K. H., Han, S. H., Choi, M. W., Park, S. H., Ha, S. D. (2022). Isolation, characterization, and application of bacteriophages to reduce and inhibit Listeria monocytogenes in celery and enoki mushroom. Food Control, 135, 108826. https://doi.org/10.1016/j.foodcont.2022.108826.
6. Cao, S., Yang, W., Zhu, X., Liu, C., Lu, J., Si, Z., Pei, L., Zhang, L., Hu, W., Li, Y., Wang, Z., Pang, Z., Xue, X., Li, Y. (2022). Isolation and identification of the broad-spectrum high-efficiency phage vB_SalP_LDW16 and its therapeutic application in chickens. BMC Veterinary Research, 18(1), 386. https://doi.org/10.1186/s12917-022-03490-3.
7. Chevallereau, A., Pons, B. J., van Houte, S., Westra, E. R. (2022). Interactions between bacterial and phage communities in natural environments. Nature Reviews Microbiology, 20(1), 49-62.
8. Endersen, L., Coffey, A. (2020). The use of bacteriophages for food safety. Current Opinion in Food Science, 36, 1-8. https://doi.org/ 10.1016/j.cofs.2020.10.006.

9. Islam, M. S., Zhou, Y., Liang, L., Nime, I., Liu, K., Yan, T., Wang, X., Li, J. (2019). Application of a phage cocktail for control of Salmonella in foods and reducing biofilms. Viruses, 11(9), 841. https://doi.org/10.3390/v11090841.
10. Guang-Han, O., Leang-Chung, C., Vellasamy, K. M., Mariappan, V., Li-Yen, C., Vadivelu, J. (2016). Experimental phage therapy for Burkholderia pseudomallei infection. PLoS One, 11(7), e0158213. https://doi.org/10.1371/journal.pone.0158213.
11. Guo, Y., Li, J., Islam, M. S., Yan, T., Zhou, Y., Liang, L., Connerton., I. F., Deng, K., Li, J. (2021). Application of a novel phage vB_SalS-LPSTLL for the biological control of Salmonella in foods. Food Research International, 147, 110492. https://doi.org/10.1016/j.foodres.2021.110492.
12. Halkman, A. K. (2019). Gıda Mikrobiyolojisi. Editör: A. Kadir Halkman. Başak Matbaacılık ve Tanıtım Hizmetleri Ltd, Ankara, 648 s. ISBN: 978-605-245-683-5; www.mikrobiyoloji.org
13. Huang, C., Virk, S. M., Shi, J., Zhou, Y., Willias, S. P., Morsy, M. K., Abdelnabby, H. E., Liu, J., Wang, X., Li, J. (2018a). Isolation, characterization, and application of bacteriophage LPSE1 against Salmonella enterica in ready to eat (RTE) foods. Frontiers in Microbiology, 9, 1046. https://doi.org/10.3389/fmicb.2018.01046.
14. Huang, C., Shi, J., Ma, W., Li, Z., Wang, J., Li, J., Wang, X. (2018b). Isolation, characterization, and application of a novel specific Salmonella bacteriophage in different food matrices. Food Research International, 111, 631-641. https://doi.org/10.1016/j.foodres.2018.05.071.
15. Khan, M. A. S., Rahman, S. R. (2022). Use of phages to treat antimicrobial-resistant Salmonella infections in poultry. Veterinary Sciences, 9(8), 438. https://doi.org/10.3390/vetsci9080438.
16. Kim, S. H., Park, J. H., Lee, B. K., Kwon, H. J., Shin, J. H., Kim, J., Kim, S. (2012). Complete genome sequence of Salmonella bacteriophage SS3e. Journal of Virology, 86(18). https://doi.org/10.1128/jvi.01550-12
17. Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E., Johnson, R. P. (2009). Enumeration of bacteriophages by double agar overlay plaque assay. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, 69-76.
18. Lee, S., Kim, M. G., Lee, H. S., Heo, S., Kwon, M., Kim, G. B. (2017). Isolation and characterization of listeria phages for control of growth of Listeria monocytogenes in milk. Korean Journal for Food Science of Animal Resources, 37(2), 320–328. https://doi.org/10.5851/ kosfa.2017.37.2.320.
19. Li, F., Tian, F., Nazir, A., Sui, S., Li, M., Cheng, D., Nong., S., Ali, A., Kakar, M.U., Li, L., Feng, Q., Tong, Y. (2022). Isolation and genomic characterization of a novel Autographiviridae bacteriophage IME184 with lytic activity against Klebsiella pneumoniae. Virus Research, 319, 198873. https://doi.org/10.1016/j.virusres.2022.198873.
20. Li, P., Zhang, X., Xie, X., Tu, Z., Gu, J., Zhang, A. (2020). Characterization and whole-genome sequencing of broad-host-range Salmonella-specific bacteriophages for bio-control. Microbial Pathogenesis, 143, 104119. https://doi.org/ 10.1016/j.micpath.2020.104119
21. López-Cuevas, O., Castro-del Campo, N., León-Félix, J., González-Robles, A., Chaidez, C. (2011). Characterization of bacteriophages with a lytic effect on various Salmonella serotypes and Escherichia coli O157: H7. Canadian Journal of Microbiology, 57(12), 1042-1051. https://doi.org/ 10.1139/w11-099.
22. Lu, M., Liu, H., Lu, H., Liu, R., Liu, X. (2020). Characterization and genome analysis of a novel Salmonella phage vB_SenS_SE1. Current Microbiology, 77, 1308-1315, https://doi.org/ 10.1007/s00284-020-01879-7
23. Maal, K. B., Delfan, A. S., Salmanizadeh, S. (2015). Isolation and identification of two novel Escherichia coli bacteriophages and their application in wastewater treatment and coliform's phage therapy. Jundishapur Journal of Microbiology, 8 (3). https://doi.org/10.5812/jjm.14945.
24. Makalatia, K., Kakabadze, E., Bakuradze, N., Grdzelishvili, N., Stamp, B., Herman, E., Tapinos, A., Coffey, A., Lee, D., Papadopoulos, N.G., Robertson, D.L., Chanishvili, N., Megremis, S. (2021). Investigation of Salmonella phage–bacteria infection profiles: network structure reveals a gradient of target-range from generalist to specialist phage clones in nested subsets. Viruses, 13(7), 1261. https://doi.org/ 10.3390/v13071261.
25. Mhone, A. L., Makumi, A., Odaba, J., Guantai, L., Gunathilake, K. D., Loignon, S., Ngugi, C. W., Akhwale, J. K., Moineau, S., Svitek, N. (2022). Salmonella Enteritidis bacteriophages isolated from Kenyan poultry farms demonstrate time-dependent stability in environments mimicking the chicken gastrointestinal tract. Viruses, 14(8), 1788. https://doi.org/10.3390/v14081788.
26. Peng, Q., Yuan, Y. (2018). Characterization of a newly isolated phage infecting pathogenic Escherichia coli and analysis of its mosaic structural genes. Scientific Reports, 8(1), 8086.
27. Petsong, K., Benjakul, S., Chaturongakul, S., Switt, A. I. M., Vongkamjan, K. (2019). Lysis profiles of Salmonella phages on Salmonella isolates from various sources and efficiency of a phage cocktail against S. Enteritidis and S. Typhimurium. Microorganisms, 7(4), 100. https://doi.org/10.3390/microorganisms7040100.
28. Principi, N., Silvestri, E., Esposito, S. (2019). Advantages and limitations of bacteriophages for the treatment of bacterial infections. Frontiers in Pharmacology, 10, 513. https://doi.org/10.3389/ fphar.2019.00513.
29. Shang, Y., Sun, Q., Chen, H., Wu, Q., Chen, M., Yang, S., Du, M., Zha, F., Ye, Q., Zhang, J. (2021). Isolation and characterization of a novel Salmonella phage vB_SalP_TR2. Frontiers in Microbiology, 12, 664810. https://doi.org/ 10.3389/fmicb.2021.664810.
30. Sharma, C. S., Dhakal, J., Nannapaneni, R. (2015). Efficacy of lytic bacteriophage preparation in reducing Salmonella in vitro, on turkey breast cutlets, and on ground turkey. Journal of Food Protection, 78(7), 1357-1362. https://doi.org/ 10.4315/0362-028X.JFP-14-585.
31. Shashidhar, R., Dhokane, V. S., Hajare, S. N., Sharma, A., Bandekar, J. R. (2007). Effectiveness of radiation processing for elimination of Salmonella Typhimurium from minimally processed pineapple (Ananas comosus Merr.). Journal of Food Science, 72(3), M98-M101. https://doi.org/10.1111/j.1750-3841.2007.00300.x.
32. Sritha, K. S., Bhat, S. G. (2018). Genomics of Salmonella phage ΦStp1: candidate bacteriophage for biocontrol. Virus Genes, 54, 311-318. https://doi.org/10.1007/s11262-018-1538-3
33. Sun, X., Wang, J., Dong, M., Zhang, H., Li, L., Wang, L. (2022). Food spoilage, bioactive food fresh-keeping films and functional edible coatings: Research status, existing problems and development trend. Trends in Food Science & Technology, 119, 122-132. https://doi.org/ 10.1016/j.tifs.2021.12.004.
34. Thung, T. Y., Premarathne, J. M. K. J. K., San Chang, W., Loo, Y. Y., Chin, Y. Z., Kuan, C. H., Tan, C. W., Basri, D.F., Radzi, C. W. J. W. M., Radu, S. (2017). Use of a lytic bacteriophage to control Salmonella Enteritidis in retail food. LWT, 78, 222-225. https://doi.org/10.1016/ j.lwt.2016.12.044.
35. Yan, T., Liang, L., Yin, P., Zhou, Y., Mahdy Sharoba, A., Lu, Q., Dong, X., Liu, K., Connerton, I. F., Li, J. (2020). Application of a novel phage LPSEYT for biological control of Salmonella in foods. Microorganisms, 8(3), 400. https://doi.org/10.3390/microorganisms8030400.
36. Wessels, K., Rip, D., Gouws, P. (2021). Salmonella in chicken meat: Consumption, outbreaks, characteristics, current control methods and the potential of bacteriophage use. Foods, 10(8), 1742. https://doi.org/10.3390/foods10081742.
37. Wongsuntornpoj, S., Switt, A. I. M., Bergholz, P., Wiedmann, M., Chaturongakul, S. (2014). Salmonella phages isolated from dairy farms in Thailand show wider host range than a comparable set of phages isolated from US dairy farms. Veterinary Microbiology, 172(1-2), 345-352. https://doi.org/10.1016/j.vetmic.2014.05.023.
38. Woolston, J., Parks, A. R., Abuladze, T., Anderson, B., Li, M., Carter, C., Hanna, L.F., Heyse, S., Charbonneau, D., Sulakvelidze, A. (2013). Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces. Bacteriophage, 3(3), e25697. https://doi.org/10.4161/bact.25697.
39. WHO, (2023). Food Safety Incidents. https://www.who.int/news/item/31-05-2023-infosan-quarterly-summary-2023-1 (Erişim Tarihi: 20.11.2023)