Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia

  • Hadri Latif (Veterinery Public Health and Epidemology Division, School of Veterinary Medicine and Biomedical Sciences (SVMBS), IPB University) ;
  • Debby Fadhilah Pazra (Animal Health Division, Bogor Agricultural Development Polytechnic) ;
  • Chaerul Basri (Veterinery Public Health and Epidemology Division, School of Veterinary Medicine and Biomedical Sciences (SVMBS), IPB University) ;
  • I Wayan Teguh Wibawan (Medical Microbiology Division, School of Veterinary Medicine and Biomedical Sciences (SVMBS), IPB University) ;
  • Puji Rahayu (Quality Control Laboratory and Certification of Animal Products)
  • Received : 2024.01.24
  • Accepted : 2024.04.29
  • Published : 2024.05.31
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Abstract

Importance: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. Objective: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. Methods: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. Results: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. Conclusions and Relevance: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.

Keywords

Acknowledgement

The authors wish to thank the Head of the Tangerang Regency Agriculture and Food Security Service, along with the dedicated staff, for their invaluable support and the facilities provided throughout the research process. The authors also wish to express their appreciation to the Bogor Animal Product Quality Testing and Certification Center Laboratory and its staff for their assistance and the use of laboratory facilities, which contributed significantly to the successful execution of this research.

References

  1. Yao X, Doi Y, Zeng L, Lv L, Liu JH. Carbapenem-resistant and colistin-resistant Escherichia coli co-producing NDM-9 and MCR-1. Lancet Infect Dis. 2016;16(3):288-289. https://doi.org/10.1016/S1473-3099(16)00057-8
  2. He T, Wang R, Liu D, Walsh TR, Zhang R, Lv Y, et al. Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans. Nat Microbiol. 2019;4(9):1450-1456. https://doi.org/10.1038/s41564-019-0445-2
  3. Van Boeckel TP, Pires J, Silvester R, Zhao C, Song J, Criscuolo NG, et al. Global trends in antimicrobial resistance in animals in low- and middle-income countries. Science. 2019;365(6459):1-5. https://doi.org/10.1126/science.aaw1944
  4. Van Cuong N, Nhung NT, Nghia NH, Mai Hoa NT, Trung NV, Thwaites G, et al. Antimicrobial consumption in medicated feeds in vietnamese pig and poultry production. EcoHealth. 2016;13(3):490-498. https://doi.org/10.1007/s10393-016-1130-z
  5. Andersson DI, Hughes D. Microbiological effects of sublethal levels of antibiotics. Nat Rev Microbiol. 2014;12(7):465-478. https://doi.org/10.1038/nrmicro3270
  6. CDC Centers for Disease Control. Antibiotic resistance threats in the United States. US: Centers for Disease Control; 2019, 73-74.
  7. Poirel L, Madec JY, Lupo A, Schink AK, Kieffer N, Nordmann P, et al. Antimicrobial resistance in Escherichia coli. Microbiol Spectr. 2018;6(4):1-27. https://doi.org/10.1128/microbiolspec.ARBA-0026-2017
  8. WHO World Health Organization. GLASS Whole-Genome Sequencing for Surveillance of Antimicrobial Resistance. Geneva: World Health Organization; 2020, p 1-42.
  9. Gong L, Wong CH, Cheng WC, Tjong H, Menghi F, Ngan CY, et al. Picky comprehensively detects high-resolution structural variants in nanopore long reads. Nat Methods. 2018;15(6):455-460. https://doi.org/10.1038/s41592-018-0002-6
  10. Feng Y, Zhang Y, Ying C, Wang D, Du C. Nanopore-based fourth-generation DNA sequencing technology. Genomics Proteomics Bioinformatics. 2015;13(1):4-16. https://doi.org/10.1016/j.gpb.2015.01.009
  11. WHO World Health Organization. WHO Integrated Global Surveillance on ESBL-Producing E. coli Using a "One Health" Approach: Implementation and Opportunities. Geneva: World Health Organization; 2021, p 16-36.
  12. Pazra DF, Latif H, Basri C, Wibawan IW, Rahayu P. Detection of tetracycline resistance genes and their diversity in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Vet World. 2023;16(9):1907-1916. https://doi.org/10.14202/vetworld.2023.1907-1916
  13. Pazra DF, Latif H, Basri C, Wibawan IW. Tetrasiklin Resistance in Escherichia coli Isolated from Pig Farm, Pig Slaughterhouse, and the Environment in Banten Province. J Kedokt Hewan. 2023;17(4):121-126.
  14. CLSI Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. Wayne: Clinical and Laboratory Standards Institute; 2018, p 30-37.
  15. Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data [Internet]. England: Babraham Institute; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Updated 2024. Accessed 2024 April 2.
  16. Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol. 2021;39(11):1348-1365. https://doi.org/10.1038/s41587-021-01108-x
  17. Community G; Galaxy Community. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic Acids Res. 2022;50 W1:W345-W351. https://doi.org/10.1093/nar/gkac247
  18. Seemann T. ABRicate: Mass Screening of Contigs for Antibiotic Resistance Genes [Internet]. California: GitHub; https://github.com/tseemann/abricate. Updated 2022. Accessed 2024 April 2.
  19. Florensa AF, Kaas RS, Clausen PT, Aytan-Aktug D, Aarestrup FM. ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes. Microb Genom. 2022;8(1):000748.
  20. Carattoli A, Hasman H. PlasmidFinder and in silico pmlst: identification and typing of plasmid replicons in whole-genome sequencing (WGS). Methods Mol Biol. 2020;2075:285-294. https://doi.org/10.1007/978-1-4939-9877-7_20
  21. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453-4455. https://doi.org/10.1093/bioinformatics/btz305
  22. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49 W1:W293-W296. https://doi.org/10.1093/nar/gkab301
  23. Peng Z, Hu Z, Li Z, Zhang X, Jia C, Li T, et al. Antimicrobial resistance and population genomics of multidrug-resistant Escherichia coli in pig farms in mainland China. Nat Commun. 2022;13(1):1116.
  24. Carhuaricra D, Duran Gonzales CG, Rodriguez Cueva CL, Ignacion Leon Y, Silvestre Espejo T, Marcelo Monge G, et al. Occurrence and genomic characterization of mcr-1-harboring Escherichia coli isolates from chicken and pig farms in Lima, Peru. Antibiotics (Basel). 2022;11(12):1-10. https://doi.org/10.3390/antibiotics11121781
  25. Zhang P, Shen Z, Zhang C, Song L, Wang B, Shang J, et al. Surveillance of antimicrobial resistance among Escherichia coli from chicken and swine, China, 2008-2015. Vet Microbiol. 2017;203(203):49-55. https://doi.org/10.1016/j.vetmic.2017.02.008
  26. Park J, Gasparrini AJ, Reck MR, Symister CT, Elliott JL, Vogel JP, et al. Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes. Nat Chem Biol. 2017;13(7):730-736. https://doi.org/10.1038/nchembio.2376
  27. WHO World Health Organization. Critically Important Antimicrobials for Human Medicine. 6th Revision. Geneva: World Health Organization; 2018, p 1-45.
  28. Kallau NH, Wibawan IW, Lukman DW, Sudarwanto MB. Analysis of relationship between knowledge and attitudes towards the practice of using antibiotics by pig farms in the city of Kupang, East Nusa Tenggara province [Analisis Hubungan antara Pengetahuan dan Sikap terhadap Praktik Penggunaan Antibiotik oleh Peternakan Babi di Kota Kupang Provinsi Nusa Tenggara Timur]. JSV. 2018;36(2):200-212.
  29. AbuOun M, O'Connor HM, Stubberfield EJ, Nunez-Garcia J, Sayers E, Crook DW, et al. Characterizing antimicrobial resistant Escherichia coli and associated risk factors in a cross-sectional study of pig farms in Great Britain. Front Microbiol. 2020;11(861):861.
  30. Marchant M, Vinue L, Torres C, Moreno MA. Change of integrons over time in Escherichia coli isolates recovered from healthy pigs and chickens. Vet Microbiol. 2013;163(1-2):124-132. https://doi.org/10.1016/j.vetmic.2012.12.011
  31. Zhang WJ, Xu XR, Schwarz S, Wang XM, Dai L, Zheng HJ, et al. Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr. J Antimicrob Chemother. 2014;69(2):385-389. https://doi.org/10.1093/jac/dkt355
  32. Hounmanou YM, Bortolaia V, Dang ST, Truong D, Olsen JE, Dalsgaard A. ESBL and AmpC b-Lactamase encoding genes in E. coli from pig and pig farm workers in Vietnam and their association with mobile genetic elements. Front Microbiol. 2021;12(629139):629139.
  33. Zurfluh K, Nuesch-Inderbinen M, Morach M, Zihler Berner A, Hachler H, Stephan R. Extended-spectrum-β-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam. Appl Environ Microbiol. 2015;81(9):3115-3120. https://doi.org/10.1128/AEM.00258-15
  34. Jacoby GA, Strahilevitz J, Hooper DC. Plasmid-mediated quinolone resistance. Microbiol Spectr. 2014;2(5):1-42. https://doi.org/10.1128/microbiolspec.PLAS-0006-2013
  35. Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013;303(6-7):298-304. https://doi.org/10.1016/j.ijmm.2013.02.001
  36. Burow E, Grobbel M, Tenhagen BA, Simoneit C, Ladwig M, Szabo I, et al. Antimicrobial susceptibility in faecal Escherichia coli from pigs after enrofloxacin administration in an experimental environment. Berl Munch Tierarztl Wochenschr. 2018;131(5-6):170-181.
  37. Wu S, Dalsgaard A, Hammerum AM, Porsbo LJ, Jensen LB. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human. Acta Vet Scand. 2010;52(1):47.
  38. Kikuvi GM, Schwarz S, Ombui JN, Mitema ES, Kehrenberg C. Streptomycin and chloramphenicol resistance genes in Escherichia coli isolates from cattle, pigs, and chicken in Kenya. Microb Drug Resist. 2007;13(1):62-68. https://doi.org/10.1089/mdr.2006.9998
  39. Grave K, Torren-Edo J, Muller A, Greko C, Moulin G, Mackay D, et al.; ESVAC Group. Variations in the sales and sales patterns of veterinary antimicrobial agents in 25 European countries. J Antimicrob Chemother. 2014;69(8):2284-2291. https://doi.org/10.1093/jac/dku106
  40. Huang SY, Zhu XQ, Wang Y, Liu HB, Dai L, He JK, et al. Co-carriage of qnrS1, floR, and bla(CTX-M-14) on a multidrug-resistant plasmid in Escherichia coli isolated from pigs. Foodborne Pathog Dis. 2012;9(10):896-901. https://doi.org/10.1089/fpd.2012.1131
  41. Zhang S, Wen J, Wang Y, Wang M, Jia R, Chen S, et al. Dissemination and prevalence of plasmid-mediated high-level tigecycline resistance gene tet (X4). Front Microbiol. 2022;13(13):969769.
  42. Sun J, Chen C, Cui CY, Zhang Y, Liu X, Cui ZH, et al. Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli. Nat Microbiol. 2019;4(9):1457-1464. https://doi.org/10.1038/s41564-019-0496-4
  43. He F, Shi Q, Fu Y, Xu J, Yu Y, Du X. Tigecycline resistance caused by rpsJ evolution in a 59-year-old male patient infected with KPC-producing Klebsiella pneumoniae during tigecycline treatment. Infect Genet Evol. 2018;66(66):188-191. https://doi.org/10.1016/j.meegid.2018.09.025
  44. Republic of Indonesia. Regulation of the Ministry of Agriculture of the Republic of Indonesia Number 14/Permentan/PK.350/5/2017 concerning Classification of Veterinary Medicines [Peraturan Menteri Pertanian Republik Indonesia Nomor 14/Permentan/PK.350/5/2017 tentang Klasifikasi Obat Hewan]; https://www.fao.org/faolex/results/details/en/c/LEX-FAOC169455/. 2017.
  45. Republic of Indonesia. Decree of the Ministry of Agriculture of the Republic of Indonesia Number 9736/PI.500/F/09/2020 concerning Amendments to Attachment III to Regulation of the Ministry of Agriculture of the Republic of Indonesia Number 14/Permentan/PK.350/5/2017 concerning Classification of Veterinary Medicines [Keputusan Menteri Pertanian Republik Indonesia Nomor 9736/PI.500/F/09/2020 tentang Perubahan Lampiran III Peraturan Menteri Pertanian Republik Indonesia Nomor 14/Permentan/PK.350/5/2017 tentang Klasifikasi Obat Hewan]; 2020.
  46. MOA. Announcement of the Ministry of Agriculture and Rural People's Republic of China No. 193 [Internet]. China: MOA; http://www.moa.gov.cn/ztzl/ncpzxzz/flfg/200709/ t20070919_893091.htm. Updated 2002. Accessed 2023 October 23.
  47. Yang H, Paruch L, Chen X, van Eerde A, Skomedal H, Wang Y, et al. Antibiotic application and resistance in swine production in China: current situation and future perspectives. Front Vet Sci. 2019;6(136):136.
  48. Partridge SR, Kwong SM, Firth N, Jensen SO. Mobile genetic elements associated with antimicrobial resistance. Clin Microbiol Rev. 2018;31(4):1-61. https://doi.org/10.1128/CMR.00088-17
  49. Rozwandowicz M, Brouwer MS, Fischer J, Wagenaar JA, Gonzalez-Zorn B, Guerra B, et al. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J Antimicrob Chemother. 2018;73(5):1121-1137. https://doi.org/10.1093/jac/dkx488
  50. Egervarn M, Borjesson S, Byfors S, Finn M, Kaipe C, Englund S, et al. Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden. Int J Food Microbiol. 2014;171(171):8-14. https://doi.org/10.1016/j.ijfoodmicro.2013.11.005