Abstract

Over the past decade, the occurrence of milk-borne infections caused by Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) has adversely affected consumer health and the milk industry. We aimed to detect and genotype the strains of E. coli and S. Typhimurium isolated from cow and goat milks using two genotyping tools, BOX-PCR and ERIC-PCR. A total of 200 cow and goat milk samples were collected from the dairy farms in Southern Sarawak, Malaysia. First, E. coli and Salmonella spp. detected in the samples were characterized using PCRs to identify pathogenic strains, STEC and S. Typhimurium. Next, the bacterial strains were genotyped using ERIC-PCR and BOX-PCR to determine their genetic relatedness. Out of 200 raw milk samples, 46.5% tested positive for non-STEC, 39.5% showed the presence of S. Typhimurium, and 11% were positive for STEC. The two genotyping tools showed different discrimination indexes, with BOX-PCR exhibiting a higher index mean (0.991) compared to ERIC-PCR (0.937). This suggested that BOX-PCR had better discriminatory power for genotyping the bacteria. Our study provides information on the safety of milk sourced from dairy farms, underscoring the importance of regular inspections and surveillance at the farm level to minimize the risk of E. coli and Salmonella outbreaks from milk consumption.

Keywords

Food safety, epidemiology, public health, Escherichia coli, Salmonella spp., milk-born infections, genotyping

FUNDING

This project was funded by the Ministry of Higher Education in Malaysia (MOHE) under the Research Acculturation Collaboration Effort (RACE Grant Scheme, RACE/b(3)/1095/2013(3)).

REFERENCES

1. Pakbin B, Brück WM, Rossen JWA. Virulence factors of enteric pathogenic Escherichia coli: A review. International Journal of Molecular Sciences. 2021;22(18):9922. https://doi.org/10.3390/ijms22189922
2. Oluwarinde BO, Ajose DJ, Abolarinwa TO, Montso PK, Preez ID, Njom HA, et al. Safety properties of Escherichia coli O157:H7 specific bacteriophages: Recent advances for food safety. Foods. 2023;12(21):3989. https://doi.org/10.3390/foods12213989
3. Mkangara M. Prevention and control of human Salmonella enterica infections: An implication in food safety. International Journal of Food Science. 2023;2023:8899596. https://doi.org/10.1155/2023/8899596
4. Zenu F, Bekele T. Major food-borne zoonotic bacterial pathogens of livestock origin: A review. Foods and Raw Materials. 2024;12(1):179–193. https://doi.org/10.21603/2308-4057-2024-1-595
5. Lamichhane B, Mawad AMM, Saleh M, Kelley WG, Harrington II PJ, Lovestad CW, et al. Salmonellosis: An overview of epidemiology, pathogenesis, and innovative approaches to mitigate the antimicrobial resistant infections. Antibiotics. 2024;13(1):76. https://doi.org/10.3390/ANTIBIOTICS13010076
6. Humphreys H, Coleman DC. Contribution of whole-genome sequencing to understanding of the epidemiology and control of meticillin-resistant Staphylococcus aureus. The Journal of Hospital Infection. 2019;102(2):189–199. https://doi.org/10.1016/j.jhin.2019.01.025
7. El-Badawy MF, El-Far SW, Althobaiti SS, Abou-Elazm FI, Shohayeb MM. The first Egyptian report showing the co-existence of blaNDM-25, blaOXA-23, blaOXA-181, and blaGES-1 among carbapenem-resistant K. pneumoniae clinical isolates genotyped by BOX-PCR. Infection and Drug Resistance. 2020;13:1237–1250. https://doi.org/10.2147/IDR.S244064
8. Maurice Bilung L, Sin Chai L, Tahar AS, Ted CK, Apun K. Prevalence, genetic heterogeneity, and antibiotic resistance profile of Listeria spp. and Listeria monocytogenes at farm level: A highlight of ERIC- and BOX-PCR to reveal genetic diversity. BioMed Research International. 2018;2018:3067494. https://doi.org/10.1155/2018/3067494
9. van Kessel JS, Karns JS, Gorski L, Perdue ML. Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR. Journal of Food Protection. 2005;68(12):2707–2712. https://doi.org/10.4315/0362-028X-68.12.2707
10. Nath G, Maurya P, Gulati AK. ERIC PCR and RAPD based fingerprinting of Salmonella Typhi strains isolated over a period of two decades. Infection, Genetics and Evolution. 2010;10(4):530–536. https://doi.org/10.1016/j.meegid.2010.02.004
11. Movahedi M, Zarei O, Hazhirkamal M, Karami P, Shokoohizadeh L, Taheri M. Molecular typing of Escherichia coli strains isolated from urinary tract infection by ERIC-PCR. Gene Reports. 2021;23:101058. https://doi.org/10.1016/j.genrep.2021.101058
12. Bakhshi B, Afshari N, Fallah F. Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks. Brazilian Journal of Microbiology. 2018;49(3):529–533. https://doi.org/10.1016/j.bjm.2017.01.014
13. O’Callaghan TF, Sugrue I, Hill C, Ross RP, Stanton C. Nutritional aspects of raw milk: A beneficial or hazardous food choice. In: Nero LA, De Carvalho AF. Raw milk: Balance between hazards and benefits. Academic Press; 2019. pp. 127–148. https://doi.org/10.1016/B978-0-12-810530-6.00007-9
14. Kapoor S, Goel AD, Jain V. Milk-borne diseases through the lens of one health. Frontiers in Microbiology. 2023;14:1041051. https://doi.org/10.3389/fmicb.2023.1041051
15. Lee E, Radu S, Jambari NN, Abdul-Mutalib NA. Prevalence and antibiogram profiling of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli in raw vegetables, in Malaysia. Biology and Life Sciences Forum. 2021;6(1):44. https://doi.org/10.3390/foods2021-10960
16. Yew CS, Fan CS, Kira R, Tahar AS, Bilung LM. Occurrence of Listeria monocytogenes and Salmonella Typhimurium in fruit juices from local stalls and restaurant in Kuching, Sarawak. Trends in Undergraduate Research. 2018;1(1):a1–7. https://doi.org/10.33736/tur.1133.2018
17. Bilung LM, Tesfamariam F, Andriesse R, San FYK, Ling CY, Tahar AS. Presence of Bacillus cereus from local unhusked (Rough) rice samples in Sarawak, Malaysia. Journal of Sustainability Science and Management. 2018;13(1):181–187.
18. Bilung LM, Ling KK, Apun K, Abdullah MT, Rahman MA, Ming CY, et al. Occurrence of Escherichia coli in wildlife from different habitats of Sarawak, Malaysia. Borneo Journal of Resource Science and Technology. 2014;4(1):19–27. https://doi.org/10.33736/bjrst.240.2014
19. Tarazi YH, El-Sukhon SN, Ismail ZB, Almestarehieh AA. Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in North Jordan. Veterinary World. 2021;14(10):2827–2832. https://doi.org/10.14202/vetworld.2021.2827-2832
20. Jamshidi A, Ghasemi A, Mohammadi A. The effect of short-time microwave exposures on Salmonella typhimurium inoculated onto chicken drumettes. Iranian Journal of Veterinary Research. 2009;10(4):378–382. https://doi.org/10.22099/IJVR.2009.1730
21. Kuruwita DP, Jiang X, Darby D, Sharp JL, Fraser AM. Persistence of Escherichia coli O157:H7 and Listeria monocytogenes on the exterior of three common food packaging materials. Food Control. 2020;112:107153. https://doi.org/10.1016/j.foodcont.2020.107153
22. Khan MAS, Rahman SR. Use of phages to treat antimicrobial-resistant Salmonella infections in poultry. Veterinary Sciences. 2022;9(8):438. https://doi.org/10.3390/vetsci9080438
23. Singha P, Kaushik G, Hussain CM, Chel A. Food safety issues associated with milk: A review. In: Grumezescu AM, Holban AM. Safety issues in beverage production. Volume 18: the science of beverages. Academic Press; 2020. pp. 399–427. https://doi.org/10.1016/B978-0-12-816679-6.00012-7
24. Fusco V, Chieffi D, Fanelli F, Logrieco AF, Cho G-S, Kabisch J, et al. Microbial quality and safety of milk and milk products in the 21st century. Comprehensive Reviews in Food Science and Food Safety. 2020;19. https://doi.org/10.1111/1541-4337.12568
25. Jenkins C, Bird PK, Wensley A, Wilkinson J, Aird H, Mackintosh A, et al. Outbreak of STEC O157:H7 linked to a milk pasteurisation failure at a dairy farm in England, 2019. Epidemiology and Infection. 2022;150:e114. https://doi.org/10.1017/S0950268822000929
26. Sebastianski M, Bridger NA, Featherstone RM, Robinson JL. Disease outbreaks linked to pasteurized and unpasteurized dairy products in Canada and the United States: A systematic review. Canadian Journal of Public Health. 2022;113:569–578. https://doi.org/10.17269/s41997-022-00614-y
27. Rosario AILS, Castro VS, Santos LF, Lisboa RC, Vallim DC, Silva MCA, et al. Shiga toxin – producing Escherichia coli isolated from pasteurized dairy products from Bahia, Brazil. Journal of Dairy Science. 2021;104(6):6535–6547. https://doi.org/10.3168/jds.2020-19511
28. Quinto EJ, Marín JM, Caro I, Mateo J, Schaffner DW. Modelling growth and decline in a two-species model system: Pathogenic Escherichia coli O157:H7 and psychrotrophic spoilage bacteria in milk. Foods. 2020;9(3):331. https://doi.org/10.3390/foods9030331
29. Gonzalez GMA, Cerqueira MFA. Shiga toxin-producing Escherichia coli in the animal reservoir and food in Brazil. Journal of Applied Microbiology. 2020;128(6):1568–1582. https://doi.org/10.1111/jam.14500
30. Martin A, Beutin L. Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources. International Journal of Food Microbiology. 2011;146(1):99–104. https://doi.org/10.1016/j.ijfoodmicro.2011.01.041
31. Liu B, Furevi A, Perepelov AV, Guo X, Cao H, Wang Q, et al. Structure and genetics of Escherichia coli O antigens. FEMS Microbiology Reviews. 2020;44(6):655–683. https://doi.org/10.1093/femsre/fuz028
32. Hughes AC, Zhang Y, Bai X, Xiong Y, Wang Y, Yang X, et al. Structural and functional characterization of Stx2k, a new subtype of Shiga toxin 2. Microorganisms. 2020;8(1):4. https://doi.org/10.3390/microorganisms8010004
33. Wang F, Deng L, Huang F, Wang Z, Lu Q, Xu C. Flagellar motility is critical for Salmonella enterica serovar typhimurium biofilm development. Frontiers In Microbiology. 2020;11:499150. https://doi.org/10.3389/fmicb.2020.01695
34. Das C, Mokashi C, Mande SS, Saini S. Dynamics and control of flagella assembly in Salmonella tyhimurium. Frontiers in Cellular and Infection Microbiology. 2018;8:252000. https://doi.org/10.3389/fcimb.2018.00036
35. Cesaris L, Gillespie BE, Srinivasan V, Almeida RA, Zecconi A, Oliver SP. Discriminating between strains of Escherichia coli using pulsed-field gel electrophoresis and BOX-PCR. Foodborne Pathogens and Disease. 2007;4(4):473–480. https://doi.org/10.1089/fpd.2007.0038
36. Rasschaert G, Houf K, Imberechts H, Grijspeerdt K, de Zutter L, Heyndrickx M. Comparison of five repetitive-sequence-based PCR typing methods for molecular discrimination of Salmonella enterica isolates. Journal of Clinical Microbiology. 2005;43(8):3615–3623. https://doi.org/10.1128/JCM.43.8.3615-3623.2005