Sains Malaysiana 55(6)(2026): 1100-1110

http://doi.org/10.17576/jsm-2026-5506-14

 

Penyebaran Klonal dan Pemindahan Gen Melalui Plasmid Menyumbang kepada Rintangan Pelbagai Antibiotik dalam Pencilan Salmonella Typhimurium daripada Ayam Mentah yang Dibeli Secara Dalam Talian

(Clonal Dissemination and Plasmid - Mediated Gene Transfer Contribute to Multidrug Resistance in Salmonella Typhimurium Isolates from Online-Purchased Raw Chicken)

 

ALIA SYAFIEQAH ZULKIFLI^1,2,3, SAHILAH ABD MUTALIB^1,2, MUHAMAD FIRDAUS SYAHMI SAM-ON^1,2 & NURUL AQILAH MOHD ZAINI^1,2,*

 

¹Jabatan Sains Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 46300 UKM Bangi, Selangor, Malaysia

²Pusat Inovasi Teknologi Manisan (MANIS), Universiti Kebangsaan Malaysia, 46300 UKM Bangi, Selangor, Malaysia

³Jabatan Perkhidmatan Makanan dan Pemakanan, Fakulti Perikanan dan Sains Makanan, Universiti Malaysia Terengganu, 21300 Kuala Terengganu, Terengganu, Malaysia

 

Diserahkan: 9 Mac 2026/Diterima: 12 Jun 2026

 

Abstrak

Salmonella Typhimurium rintang pelbagai antibiotik (MDR) merupakan patogen utama penyakit bawaan makanan yang memberi implikasi yang signifikan terhadap kesihatan awam. Kajian ini menilai 48 pencilan S. Typhimurium daripada ayam mentah yang dibeli secara dalam talian daripada tiga jenama komersial yang diproses di pusat penyembelihan berbeza (Kedah, Selangor dan Negeri Sembilan) bagi mengkaji dinamik penyebaran gen MDR melalui hubungan antara penyebaran klonal dan pemindahan gen mendatar (HGT). Analisis kerintangan antibiotik menggunakan kaedah cakera resapan, ujian konjugasi melalui pemindahan gen rintangan, serta analisis hubungan genetik menggunakan ERIC-PCR telah dijalankan. Hasil kajian menunjukkan kadar rintangan tinggi terhadap eritromisin (100%), tetrasiklin (85 %) dan ampisilin (79 %) dengan indeks Rintangan Pelbagai Antibiotik (MAR) antara 0.25 hingga 1.0, menandakan tahap pencemaran yang tinggi.  Ujian konjugasi terhadap pencilan terpilih mendapati dua daripada tiga pencilan MDR mampu memindahkan fenotip rintangan kepada E. coli DH5α dengan kecekapan tertinggi 1.1 × 10⁻², sekali gus menyokong peranan HGT dalam penyebaran MDR.  Analisis ERIC-PCR menghasilkan lapan kluster utama dan satu asingan tunggal (DI = 0.80), menunjukkan kepelbagaian genetik yang baik dalam kalangan pencilan. Gabungan analisis dendogram ERIC-PCR dan profil antibiogram menunjukkan bahawa penyebaran MDR dalam populasi S. Typhimurium berkemungkinan dipengaruhi oleh kedua-dua mekanisme iaitu penyebaran klonal dan HGT. Secara keseluruhan, keputusan ini menekankan keperluan pemantauan bersepadu dalam rantaian bekalan ayam bagi mengawal penyebaran  MDR S. Typhimurium, sejajar dengan peningkatan pembelian ayam mentah secara dalam talian di Malaysia yang berpotensi menjadi laluan penyebaran strain MDR kepada pengguna.

Kata kunci: Ayam mentah dalam talian; pemindahan gen mendatar (HGT); penyebaran klonal; rintangan pelbagai antibiotik (MDR); Salmonella Typhimurium

 

Abstract

Multidrug-resistant (MDR) Salmonella Typhimurium is a major foodborne pathogen with significant public health implications. This study evaluated 48 S. Typhimurium isolates from raw chicken purchased online from three commercial brands processed at different slaughterhouses (Kedah, Selangor, and Negeri Sembilan) to explore the dynamics of MDR gene dissemination through clonal spread and horizontal gene transfer (HGT). Antimicrobial susceptibility was tested using the disk diffusion method, conjugation assays were performed to evaluate resistance gene transfer, and genetic relatedness was analysed using ERIC-PCR. Results showed high resistance rates to erythromycin (100%), tetracycline (85%), and ampicillin (79%), with a Multiple Antibiotic Resistance (MAR) index ranging from 0.25 to 1.0, indicating high contamination levels. Conjugation assays showed that two out of three selected MDR isolates were able to transfer resistance phenotypes to E. coli DH5α, with the highest transfer efficiency of 1.1 × 10⁻², supporting the role of HGT in MDR dissemination. ERIC-PCR analysis (DI = 0.80) showed good genetic diversity with eight major clusters and one singleton. The combined ERIC-PCR dendrogram and antibiogram profiling indicated that MDR spread in the S. Typhimurium population is likely driven by both clonal expansion and HGT. Overall, these findings highlight the need for integrated monitoring along the poultry supply chain to control the dissemination of MDR S. Typhimurium, particularly as online chicken purchases in Malaysia increase, which may serve as a potential route for MDR strain transmission to consumers.

Keywords: Clonal dissemination; horizontal gene transfer (HGT); multidrug resistance (MDR); online raw chicken; Salmonella Typhimurium

 

RUJUKAN

Alderliesten, J.B., Duxbury, S.J., Zwart, M.P., De Visser, J.A.G., Stegeman, A. & Fischer, E.A. 2020. Effect of donor-recipient relatedness on the plasmid conjugation frequency: A meta-analysis. BMC Microbiology 20(1): 135.

Alsultan, A. & Elhadi, N. 2022. Evaluation of ERIC-PCR method for determining genetic diversity among Escherichia coli isolated from human and retail imported frozen shrimp and beef. International Journal of Food Contamination 9(1): 12.

Anjay, A.K., Agarwal, R., Ramees, T., Dubal, Z., Kaushik, P., Kumar, M.S., Dudhe, N., Milton, A., Abhishek, B. & Shagufta, B. 2015. Molecular typing of Salmonella Typhimurium and S. Enteritidis serovars from diverse origin by ERIC-PCR. J. Pure Appl. Microbiol. 9: 2627-2634.

Bauer, A., Kirby, W., Sherris, J.C. & Turck, M. 1966. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45(4_ts): 493-496.

Bilung, L.M., Radzi, E.S., Tahar, A.S., Zulkharnain, A., Ngui, R. & Apun, K. 2025. BOX-PCR and ERIC-PCR evaluation for genotyping shiga toxin-producing Escherichia coli and Salmonella enterica serovar Typhimurium in raw milk. Foods and Raw Materials 13(2): 264-275.

Clinical and Laboratory Standards Institute (CLSI). 2020. Performance Standards for Antimicrobial Susceptibility Testing. Edisi ke-30. CLSI Supplement M100. Wayne, PA: CLSI.

Deli, R.A. & Adzitey, F. 2017. Prevalence and antibiotic resistance of Salmonella serovars isolated from spent hens and its environmental samples in Penang and Kedah, Malaysia. Journal of Tropical Agriculture and Food Science 45: 37-50.

Fendri, I., Ben Hassena, A., Grosset, N., Barkallah, M., Khannous, L., Chuat, V., Gautier, M. & Gdoura, R. 2013. Genetic diversity of food-isolated Salmonella strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). PLoS ONE 8(12): e81315.

Gaissmaier, M.S., Laganenka, L., Herzog, M.K-M., Bakkeren, E. & Hardt, W-D. 2022. The mobilizable plasmid P3 of Salmonella enterica serovar Typhimurium SL1344 depends on the P2 plasmid for conjugative transfer into a broad range of bacteria in vitro and in vivo. Journal of Bacteriology 204(12): e00347-322.

Gerner‐Smidt, P., Trees, E., Carleton, H., Katz, L., den Bakker, H. & Deng, X. 2019. Molecular source tracking and molecular subtyping. Dlm. Food Microbiology: Fundamentals and Frontiers, disunting oleh Doyle, M.P., Diez-Gonzalez, F. & Hill, C. Washington: ASM Press. hlm. 971-988.

Gillespie, I.A., O’Brien, S., Adak, G., Ward, L. & Smith, H. 2005. Foodborne general outbreaks of Salmonella Enteritidis phage type 4 infection, England and Wales, 1992-2002: Where are the risks? Epidemiology & Infection 133(5): 795-801.

Hallman, W.K., Senger-Mersich, A. & Godwin, S.L. 2015. Online purveyors of raw meat, poultry, and seafood products: Delivery policies and available consumer food safety information. Food Protection Trends 35(2): 80-88.

Hashemi, A. & Baghbani‐Arani, F. 2015. The effective differentiation of Salmonella isolates using four PCR‐based typing methods. Journal of Applied Microbiology 118(6): 1530-1540.

Hradecka, H., Karasova, D. & Rychlik, I. 2008. Characterization of Salmonella enterica serovar Typhimurium conjugative plasmids transferring resistance to antibiotics and their interaction with the virulence plasmid. Journal of Antimicrobial Chemotherapy 62(5): 938-941.

Hunter, P.R. & Gaston, M.A. 1988. Numerical index of the discriminatory ability of typing systems: An application of Simpson's index of diversity. Journal of Clinical Microbiology 26(11): 2465-2466.

Ibrahim, H.A. & Abo El-Makarem, H.S. 2021. Bacterial food poisoning in chicken and some chicken products. Alexandria Journal of Veterinary Sciences 69(1): 92.

Innes, G.K., Nachman, K.E., Abraham, A.G., Casey, J.A., Patton, A.N., Price, L.B., Tartof, S.Y. & Davis, M.F. 2021. Contamination of retail meat samples with multidrug-resistant organisms in relation to organic and conventional production and processing: A cross-sectional analysis of data from the United States National Antimicrobial Resistance Monitoring System, 2012-2017. Environmental Health Perspectives 129(5): 057004.

Ismail, Z., Azmi, N.N., Mahyudin, N.A., Omar, W.H.W., Rahman, M.A. & Sapar, M. 2024. Salmonella isolated from raw chicken meats at selected slaughterhouses in Peninsular Malaysia: Antibiotic resistance profiles and biofilm formation on nutrient-limited media. Malaysian Applied Biology 53(2): 55-71.

Khan, J.A., Irfan, A., Soni, S., Maherchandani, S. & Maherchandani, S. 2015. Antibiogram and multiple antibiotic resistance index of Salmonella enterica isolates from poultry. Journal of Pure and Applied Microbiology 9(3): 2495-2500.

Kim, M.B. & Lee, Y.J. 2025. Biosecurity practices for reducing antimicrobial use in commercial broiler farms in Korea. The Journal of Poultry Science 62: 2025001.

Li, W., Bai, X., Sheng, H., Chen, J., Wang, Z., Wang, T. & Yang, B. 2022. Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut.  Food Research International 157: 111263.

Mohapatra, B.R., Broersma, K. & Mazumder, A. 2007. Comparison of five rep-PCR genomic fingerprinting methods for differentiation of fecal Escherichia coli 237 from humans, poultry, and wild birds. FEMS Microbiology Letters 277(1): 98-106.

Mooijman, K.A., Pielaat, A. & Kuijpers, A.F. 2019. Validation of EN ISO 6579-1-microbiology of the food chain-horizontal method for the detection, enumeration and serotyping of Salmonella-Part 1 detection of Salmonella spp. International Journal of Food Microbiology 288: 3-12.

Murray, C.J., Vos, T., Lozano, R., Naghavi, M., Flaxman, A.D., Michaud, C., Ezzati, M., Shibuya, K., Salomon, J.A. & Abdalla, S. 2012. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: A systematic analysis for the Global Burden of Disease Study 2010. The Lancet 380(9859): 2197-2223.

Ou, J.T., Baron, L.S., Dai, X. & Life, C.A. 1990. The virulence plasmids of Salmonella  Typhimurium, Choleraesuis, Dublin, and Enteritidis, and the cryptic plasmids of Salmonella serovars Copenhagen and Sendai belong to the same incompatibility group, but not those of Salmonella serovars Durban, Gallinarum, Give, Infantis and Pullorum. Microbial Pathogenesis 8(2): 101-107.

Phornphisutthimas, S., Thamchaipenet, A. & Panijpan, B. 2007. Conjugation in Escherichia coli: A laboratory exercise. Biochemistry and Molecular Biology Education 35(6): 440-445.

Puangseree, J., Prathan, R., Srisanga, S. & Chuanchuen, R. 2024. Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand. PLoS ONE 19(5): e0304250.

ReAct. 2018. Antibiotic Use in Food Animals: Malaysia Overview.  https://www.reactgroup.org/wpcontent/uploads/2018/11/Antibiotic_Use_in_Food_Animals_Malaysia_Overview_2018web. pdf (Diakses pada 18 September 2025).

Ruppitsch, W. 2016. Molecular typing of bacteria for epidemiological surveillance and outbreak investigation. Journal of Land Management, Food and Environment 67(4): 199-224.

Sales, A.J., Naebi, S., Nasiri, R. & Bannazadeh-Baghi, H. 2021. The antibiotic resistance pattern and prevalence of Blatem, Blashv, Blactx-M, Blapse-1, Sipb/C, and Cmla/Tetr genes in Salmonella Typhimurium isolated from children with diarrhea in Tabriz, Iran. Int. J. Health Life Sci. 7(4): e118523.

Shafieifini, M., Sun, Y., Staley, Z.R., Riethoven, J.J. & Li, X. 2022. Effects of nutrient level and growth rate on the conjugation process that transfers mobile antibiotic resistance genes in continuous cultures. Applied and Environmental Microbiology 88(19): e01121-22.

Shen, H., Namdarpour, F. & Lin, J. 2022. Investigation of online grocery shopping and delivery preference before, during, and after COVID-19. Transportation Research Interdisciplinary Perspectives 14: 100580.

Singh, S., Yadav, A.S., Singh, S.M. & Bharti, P. 2010. Prevalence of Salmonella in chicken eggs collected from poultry farms and marketing channels and their antimicrobial resistance. Food Research International 43(8): 2027-2030.

Sedrakyan, A., Ktsoyan, Z., Arakelova, K., Gevorgyan, Z., Zakharyan, M., Hakobyan, S., Hovhannisyan, A., Arakelyan, A. & Aminov, R. 2022. Molecular epidemiology and virulence of non-typhoidal Salmonella in Armenia. International Journal of Molecular Sciences 23(16): 9330.

Smadi, H., Sargeant, J.M., Shannon, H.S. & Raina, P. 2012. Growth and inactivation of Salmonellaat low refrigerated storage temperatures and thermal inactivation on raw chicken meat and laboratory media: mixed effect meta-analysis. Journal of Epidemiology and Global Health 2(4): 165-179.

Souza, R.A., Pitondo-Silva, A., Falcão, D.P. & Falcão, J.P. 2010. Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates. Journal of Microbiological Methods 82(2): 141-150.

Sukri, A., Zulfakar, S.S., Taib, I.S.M., Omar, N.F. & Mohamad Zin, N. 2021. The high occurrence of multidrug-resistant Salmonella spp. isolated from raw chicken meat and contact surfaces at wet market in Malaysia. Sains Malaysiana 50(12): 3765-3772.

Thung, T.Y., Mahyudin, N.A., Basri, D.F., Radzi, C.W.M., Nakaguchi, Y., Nishibuchi, M. & Radu, S. 2016. Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia. Poultry Science 95(8): 1888-1893.

Tyrväinen, O. & Karjaluoto, H. 2022. Online grocery shopping before and during COVID-19: A meta-analytical review. Telematics and Informatics 71: 101839.

Utrarachkij, F., Nakajima, C., Siripanichgon, K., Changkaew, K., Thongpanich, Y., Pornraungwong, S., Suthienkul, O. & Suzuki, Y. 2016. Genetic diversity and antimicrobial resistance pattern of Salmonella enterica Enteritidis clinical isolates in Thailand. Journal of Infection and Chemotherapy 22(4): 209-215.

Versalovic, J., Koeuth, T. & Lupski, R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 19(24): 6823-6831.

Wang, X., Biswas, S., Paudyal, N., Pan, H., Li, X., Fang, W. & Yue, M. 2019. Antibiotic resistance in Salmonella Typhimurium isolates recovered from the food chain through national antimicrobial resistance monitoring system between 1996 and 2016. Frontiers in Microbiology 10: 985.

Yang, B., Qu, D., Zhang, X., Shen, J., Cui, S., Shi, Y., Xi, M., Sheng, M., Zhi, S. & Meng, J. 2010. Prevalence and characterization of Salmonella serovars in retail meats of marketplace in Shaanxi, China. International Journal of Food Microbiology 141(1-2): 63-72.

Zhang, Z., Suksawat, F., Zhang, X., Shu, X. & Angkititrakul, S. 2026. Clonal and plasmid-mediated dissemination of multidrug-resistant Salmonella Enteritidis in chicken production, Northeastern Thailand. Pathogens 15(1): 75.

 

*Pengarang untuk surat-menyurat; email: nurulaqilah@ukm.edu.my