Molecular Epidemiological Surveillance of blaCTX-M in Escherichia coli from Indonesian Environmental Sources Based on Public Genomic Data
DOI:
https://doi.org/10.22437/e-sehad.v6i1.58451Keywords:
blaCTX-M, Escherichia coli, environment, antimicrobial resistance, genomic surveillanceAbstract
ABSTRACTBackground: Antimicrobial resistance mediated by the blaCTX-M gene in Escherichia coli represents a major global public health threat, while molecular epidemiological surveillance data from environmental sources in Indonesia remain highly limited. This is study aimed to analyze the molecular epidemiology of the blaCTX-M gene in environmental Escherichia coli isolates from Indonesia using a genomic surveillance approach based on publicly available genome data and Linux-based bioinformatics pipeline.
Method: This quantitative descriptive study employed a comparative genomics and phylogenomic analysis design based on an in silico approach. The study included 86 Escherichia coli genome assemblies retrieved from the EnteroBase database using the selection criteria of Environment as the source niche and Indonesia as the country of origin. Data analysis was performed using QUAST, Prokka, AMRFinderPlus, ABRicate, MLST, Snippy, IQ-TREE, and FastTree on the Ubuntu 22.04 LTS operating system.
Result: All isolates met the assembly quality criteria, and the analytical pipeline was validated using both positive and negative controls. Most isolates originated from water and river samples, with the highest number collected in 2019. The blaCTX-M subtypes were predominantly assigned to the CTX-M-1 group (88,2%), particularly CTX-M-15 and CTX-M-55, while resistome profiling revealed a multidrug-resistant pattern. Sequence type ST2 and ST471 were the most prevalent, and although the genomic population was highly heterogeneous, four clonal clusters were identified.
Conclusion: These findings highlight the need for sustained deposition of environmental Escherichia coli genome data from Indonesia with more balanced temporal and geographic representation to strengthen the national One Health-based genomic surveillance system for antimicrobial resistance.
References
Naghavi M, Vollset SE, Ikuta KS, Swetschinski LR, Gray AP, Wool EE, et al. Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet. 2024;404(10459):1199-1226. https://doi.org/10.1016/S0140-6736(24)01867-1
Walsh TR, Gales AC, Laxminarayan R, Dodd PC. Antimicrobial resistance: addressing a global threat to humanity. PLoS Med. 2023;20(7):e1004264. https://doi.org/10.1371/journal.pmed.1004264
IHME. Antimicrobial resistance (AMR) [Internet]. Seattle (WA): Institute for Health Metrics and Evaluation; 2025 [cited 2026 May 30]. Available from: https://www.healthdata.org/research-analysis/health-topics/antimicrobial-resistance-amr
Sintondji K, Fabiyi K, Hougbenou J, Koudokpon H, Lègba B, Amoussou H, et al. Prevalence and characterization of ESBL-producing Escherichia coli in healthy pregnant women and hospital environments in Benin: an approach based on Tricycle. Front Public Health. 2023;11:1227000. https://doi.org/10.3389/fpubh.2023.1227000
Di Marcantonio L, Chiatamone Ranieri S, Toro M, Marchegiano A, Cito F, Sulli N, et al. Comprehensive regional study of ESBL Escherichia coli: genomic insights into antimicrobial resistance and inter-source dissemination of ESBL genes. Front Microbiol. 2025;16:1595652. https://doi.org/10.3389/fmicb.2025.1595652
Faridah HD, Wibisono FM, Wibisono FJ, Nisa N, Fatimah F, Effendi MH, et al. Prevalence of the blaCTX-M and blaTEM genes among extended-spectrum beta-lactamase-producing Escherichia coli isolated from broiler chickens in Indonesia. J Vet Res. 2023;67(2):179. https://doi.org/10.2478/jvetres-2023-0025
Bevan ER, Jones AM, Hawkey PM. Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype. J Antimicrob Chemother. 2017;72(8):2145-2155. https://doi.org/10.1093/jac/dkx146
Yang JT, Zhang LJ, Lu Y, Zhang RM, Jiang HX. Genomic insights into global blaCTX-M-55-positive Escherichia coli epidemiology and transmission characteristics. Microbiol Spectr. 2023;11(4):e01089-23. https://doi.org/10.1128/spectrum.01089-23
Hidayatullah N, Suandy I, Intanon M, Alter T, Susanti O, Herpianti A, et al. Occurrence and molecular characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in broilers in Indonesia. Antibiotics (Basel). 2025;14(10):1030. https://doi.org/10.3390/antibiotics14101030
Megantara I, Sylviana N, Amira PA, Pradini GW, Krissanti I, Lesmana R. Potential of waterbodies as a reservoir of Escherichia coli pathogens and the spread of antibiotic resistance in the Indonesian aquatic environment. J Water Sanit Hyg Dev. 2023;13(10):776-792. https://doi.org/10.2166/washdev.2023.040
Puspandari N, Pratama R, Goodman R, Suandy I, Efadeswarni E, Febriana T, et al. Genomic investigation of ESBL-producing Escherichia coli reveals likely transmission within and between One Health sectors in Jakarta, Indonesia. Research Square [Preprint]. 2025. https://doi.org/10.21203/rs.3.rs-7825238/v1
Dameanti FNAEP, Yanestria SM, Widodo A, Effendi MH, Plumeriastuti H, Tyasningsih W, et al. Incidence of Escherichia coli producing extended-spectrum beta-lactamase in wastewater of dairy farms in East Java, Indonesia. Biodiversitas. 2023;24(2):1143-1150. https://repository.unair.ac.id/127124/1/Artikel_15_Hani_Plumeriastuti.pdf
Kumavath R, Gupta P, Tatta ER, Mohan MS, Salim SA, Busi S. Unraveling the role of mobile genetic elements in antibiotic resistance transmission and defense strategies in bacteria. Front Syst Biol. 2025;5:1557413. https://doi.org/10.3389/fsysb.2025.1557413
World Health Organization. Global Antibiotic Resistance Surveillance Report 2025: WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) [Internet]. Geneva: World Health Organization; 2025. Available from: https://www.who.int/publications/i/item/9789240116337
Zhou W, Zhang E, Zhou J, He Z, Zhou Y, Han J, et al. Characterization and comparative genomics analysis of IncFII multi-resistance plasmids carrying blaCTX-M and type 1 integrons from Escherichia coli. Front Microbiol. 2021;12:753979. https://doi.org/10.3389/fmicb.2021.753979
Li P, Yan L, Song J, Lin C, Zeng F, Zeng S. Involvement of the blaCTX-M-3 gene in emergence of a peculiar resistance phenotype in Klebsiella pneumoniae. Front Cell Infect Microbiol. 2025;15:1545157. https://doi.org/10.3389/fcimb.2025.1545157
Minja CA, Shirima G, Mshana SE. Conjugative plasmids disseminating ctx-m-15 among humans, animals and the environment in Mwanza, Tanzania: a need to intensify One Health approach. Antibiotics (Basel). 2021;10(7):836. https://doi.org/10.3390/antibiotics10070836
Kementerian Koordinator Bidang Pembangunan Manusia dan Kebudayaan Republik Indonesia. Rencana aksi nasional pengendalian resistensi antimikroba [Internet]. Jakarta: Kemenko PMK; 2022. Available from: https://cdn.who.int/media/docs/default-source/searo/indonesia/20221115_nap-on-amr.pdf
Vanstokstraeten R, Pierard D, Crombé F, De Geyter D, Wybo I, Muyldermans A, et al. Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates. Sci Rep. 2023;13(1):449. https://doi.org/10.1038/s41598-023-27723-z
Horesh G, Blackwell GA, Tonkin-Hill G, Corander J, Heinz E, Thomson NR. A comprehensive and high-quality collection of Escherichia coli genomes and their genes. Microb Genom. 2021;7(2):000499. https://doi.org/10.1099/mgen.0.000499
Murphy R, Palm M, Mustonen V, Warringer J, Farewell A, Parts L, et al. Genomic epidemiology and evolution of Escherichia coli in wild animals in Mexico. mSphere. 2021;6(1):e00738-20. https://doi.org/10.1128/msphere.00738-20
Duggett N, AbuOun M, Stubberfield E, Turner O, Randall L, Horton R, et al. Genomic surveillance of extended-spectrum cephalosporin-resistant Escherichia coli isolated from poultry in the UK from 2016 to 2020. Front Microbiol. 2024;14:1335173. https://doi.org/10.3389/fmicb.2023.1335173
Yehouenou CL, Bogaerts B, De Keersmaecker SCJ, Roosens NH, Marchal K, Tchiakpe E, et al. Whole-genome sequencing-based antimicrobial resistance characterization and phylogenomic investigation of 19 multidrug-resistant and extended-spectrum beta-lactamase-positive Escherichia coli strains collected from hospital patients in Benin in 2019. Front Microbiol. 2021;12:752883. https://doi.org/10.3389/fmicb.2021.752883
Sugiyono. Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung: Alfabeta; 2013. Available from: https://www.scribd.com/document/671612229/Sugiyono-2013-Metode-Penelitian-Kuantitatif-Kualitatif-dan-R-D-1
Bilal MY, Klutts JS. Molecular epidemiological investigations of localized SARS-CoV-2 outbreaks: utility of public algorithms. Epidemiologia (Basel). 2022;3(3):402-411. https://doi.org/10.3390/epidemiologia3030031
Alizadehmohajer N, Zahedifar S, Sohrabi E, Shaddel Basir S, Nourigheimasi S, Falak R, et al. Using in silico bioinformatics algorithms for the accurate prediction of the impact of spike protein mutations on the pathogenicity, stability, and functionality of the SARS-CoV-2 virus and analysis of potential therapeutic targets. Biochem Genet. 2023;61(2):778-808. https://doi.org/10.1007/s10528-022-10282-9
Tan M, Xia J, Luo H, Meng G, Zhu Z. Applying the digital data and the bioinformatics tools in SARS-CoV-2 research. Comput Struct Biotechnol J. 2023;21:4697-4705. https://doi.org/10.1016/j.csbj.2023.09.044
Kirichenko AD, Poroshina AA, Sherbakov DY, Sadovsky MG, Krutovsky KV. Comparative analysis of alignment-free genome clustering and whole genome alignment-based phylogenomic relationship of coronaviruses. PLoS One. 2022;17(3):e0264640. https://doi.org/10.1371/journal.pone.0264640
van der Putten BC, Huijsmans NA, Mende DR, Schultsz C. Benchmarking the topological accuracy of bacterial phylogenomic workflows using in silico evolution. Microb Genom. 2022;8(3):000799. https://doi.org/10.1099/mgen.0.000799
Olawoye IB, Frost SD, Happi CT. The Bacteria Genome Pipeline (BAGEP): an automated, scalable workflow for bacteria genomes with Snakemake. PeerJ. 2020;8:e10121. http://dx.doi.org/10.7717/peerj.10121
Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-2069. https://doi.org/10.1093/bioinformatics/btu153
Feldgarden M, Brover V, Gonzalez-Escalona N, Frye JG, Haendiges J, Haft DH, et al. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci Rep. 2021;11(1):12728. https://doi.org/10.1038/s41598-021-91456-0
Andersen PS, Stegger M, Aziz M, Contente-Cuomo T, Gibbons HS, Keim P, et al. Complete genome sequence of the epidemic and highly virulent CTX-M-15-producing H30-Rx subclone of Escherichia coli ST131. Genome Announc. 2013;1(6):e00988-13. https://doi.org/10.1128/genomea.00988-13
Campos-Madueno EI, Aldeia C, Perreten V, Sendi P, Moser AI, Endimiani A. Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches. Front Microbiol. 2023;14:1236208. https://doi.org/10.3389/fmicb.2023.1236208
Khetrapal V, Mehershahi KS, Chen SL. Complete genome sequence of the original Escherichia coli isolate, strain NCTC86. Genome Announc. 2017;5(16):e00243-17. https://doi.org/10.1128/genomea.00243-17
Ramadan H, Soliman AM, Hiott LM, Elbediwi M, Woodley TA, Chattaway MA, et al. Emergence of multidrug-resistant Escherichia coli producing CTX-M, MCR-1, and FosA in retail food from Egypt. Front Cell Infect Microbiol. 2021;11:681588. https://doi.org/10.3389/fcimb.2021.681588
Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics. 2013;29(8):1072-1075. https://doi.org/10.1093/bioinformatics/btt086
Kikwai GK, Juma B, Nindo F, Ochieng C, Wamola N, Mbogo K, et al. Genome sequence of Escherichia coli isolated from an adult in Kibera, an urban informal settlement in Nairobi, Kenya. Microbiol Resour Announc. 2022;11(4):e01241-21. https://doi.org/10.1128/mra.01241-21
Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021;38(10):4647-4654. https://doi.org/10.1093/molbev/msab199
Ortega-Enríquez JZ, Arenas-Hernández MM, Barrios-Villa E. Draft genome sequence of a triple hybrid Escherichia coli strain isolated from a healthy donor feces. Microbiol Resour Announc. 2024;13(5):e00113-24. https://doi.org/10.1128/mra.00113-24
Aswal M, Singhal N, Kumar M. Genomic analysis of phylogroup D Escherichia coli strains using novel de novo reference-based guided assembly. Sci Data. 2023;10(1):573. https://doi.org/10.1038/s41597-023-02444-0
Coolen JP, den Drijver EP, Verweij JJ, Schildkraut JA, Neveling K, Melchers WJG, et al. Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance. Microb Genom. 2021;7(4):000556. https://doi.org/10.1099/mgen.0.000556
Lipworth S, Crook D, Walker AS, Peto T, Stoesser N. Exploring uncatalogued genetic variation in antimicrobial resistance gene families in Escherichia coli: an observational analysis. Lancet Microbe. 2024;5(11):e1009-e1018. https://doi.org/10.1016/S2666-5247(24)00152-6
Atlaw NA, Keelara S, Correa M, Foster D, Gebreyes W, Aidara-Kane A, et al. Identification of CTX-M-type ESBL-producing Escherichia coli from sheep and their abattoir environment using whole-genome sequencing. Pathogens. 2021;10(11):1480. https://doi.org/10.3390/pathogens10111480
Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol. 2012;50(4):1355-1361. https://doi.org/10.1128/jcm.06094-11
Nakamura A, Takahashi H, Arai M, Tsuchiya T, Wada S, Fujimoto Y, et al. Molecular subtyping for source tracking of Escherichia coli using core genome multilocus sequence typing at a food manufacturing plant. PLoS One. 2021;16(12):e0261352. https://doi.org/10.1371/journal.pone.0261352
Chowdhury PR, Hastak P, DeMaere M, Wyrsch E, Li D, Elankumaran P, et al. Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission. mSystems. 2023;8(5):e01236-22. https://doi.org/10.1128/msystems.01236-22
Lees JA, Kendall M, Parkhill J, Colijn C, Bentley SD, Harris SR. Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation-based study. Wellcome Open Res. 2018;3:33. http://dx.doi.org/10.12688/wellcomeopenres.14265.1
Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37(5):1530-1534. https://doi.org/10.1093/molbev/msaa015
Wong TK, Ly-Trong N, Ren H, Demotte P, Baños H, Roger AJ, et al. IQ-TREE 3: phylogenomic inference software using complex evolutionary models. Mol Biol Evol. 2026;43(5):msag117. https://doi.org/10.1093/molbev/msag117
Cave R, Ter-Stepanyan MM, Mkrtchyan HV. Short- and long-read sequencing reveals the presence and evolution of an IncF plasmid harboring blaCTX-M-15 and blaCTX-M-27 genes in Escherichia coli ST131. Microbiol Spectr. 2023;11(4):e00356-23. https://doi.org/10.1128/spectrum.00356-23
Berwa A, Caspar Y. Easy analysis of bacterial whole-genome sequencing data for clinical microbiologists using open-source Galaxy platform: characterization of ESBL-producing Enterobacterales from bloodstream infections. J Glob Antimicrob Resist. 2024;39:153-158. https://doi.org/10.1016/j.jgar.2024.08.012
Peirano G, Chen L, Nobrega D, Finn TJ, Kreiswirth BN, DeVinney R, et al. Genomic epidemiology of global carbapenemase-producing Escherichia coli, 2015-2017. Emerg Infect Dis. 2022;28(5):924-932. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC9045447/pdf/21-2535.pdf
Maric L, Rupnik M, Janezic S. Diversity of ESBL-producing Escherichia coli in various water and sediment types. PLoS One. 2025;20(12):e0338703. https://doi.org/10.1371/journal.pone.0338703
Richter L, Duvenage S, Du Plessis EM, Msimango T, Dlangalala M, Mathavha MT, et al. Genomic evaluation of multidrug-resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from irrigation water and fresh produce in South Africa: a cross-sectional analysis. Environ Sci Technol. 2024;58(32):14421-14438. https://doi.org/10.1021/acs.est.4c02431
Fagerström A, Mölling P, Khan FA, Sundqvist M, Jass J, Söderquist B. Comparative distribution of extended-spectrum beta-lactamase-producing Escherichia coli from urine infections and environmental waters. PLoS One. 2019;14(11):e0224861. https://doi.org/10.1371/journal.pone.0224861





