Computational modeling of a Novel CTX-M β-lactamase Escherichia coliisolated from Yogyakarta, Indonesia Using I-TASSER and SWISS-MODEL
##plugins.themes.bootstrap3.article.sidebar##
##plugins.themes.bootstrap3.article.main##
Anggiresti Kinasih
https://orcid.org/0000-0002-5606-0577
Endah Retnaningrum
https://orcid.org/0000-0001-7936-4920
Abstract
Extended-spectrum β-lactamases (ESBLs), especially those of the CTX-M family, significantly contribute to antibiotic resistance in Escherichia coli. While CTX-M variants are well-characterized in clinical settings, their structural features in foodborne E. coli remain underexplored, particularly in countries like Indonesia. This study aimed to predict and validate the three-dimensional (3D) structure of a novel CTX-M β-lactamase protein from an E. coli isolate (D4DU2-ESBL-EC) culture collection at Laboratory of Microbiology, Universitas Gadjah Mada (UGM). This study reports a novel CTX-M variant from E. coli D4DU2, a locally strain originated from raw chicken meat in Yogyakarta traditional market that has been shown to adapt and thrive in Sleman Regency, Indonesia. Based on phylogenetic and structural analyses, E. coli D4DU2 evolutionary link to Indian CTX-M-15, with distinct mutations at W253L, P254A amino acid near the active site. The 3D structure of D4DU2 CTX-M β-lactamase was predicted using I-TASSER and validated using PROCHECK. Ramachandran analysis showed 78.4% of residues in the most favored regions, 20.5% in allowed regions, and only 1.2% in disallowed regions, confirming high stereochemical quality. These findings highlight potential local adaptation and emergence of a unique ESBL-producing E. coli strain. This study provides a reliable model for CTX-M β-lactamase from foodborne E. coli in Indonesia, supporting One Health-based surveillance and intervention strategies. Overall, this study demonstrates that I-TASSER, complemented by PROCHECK validation, offers a valuable in silico approach for the structural characterization of ESBL proteins from foodborne bacterial isolates in resource-limited contexts
Downloads
##plugins.themes.bootstrap3.article.details##
Copyright (c) 2026 Anggiresti Kinasih, Endah Retnaningrum
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
References
Bonten M, Johnson JR, Biggelaar AHJ., Georgalis L., Geurtsen J, de Palacios PI, Gravenstein S, Verstraeten T, Hermans P, Poolman JT. Epidemiology of Escherichia coli Bacteremia: A Systeamtic Literature Review. Clinical Infectious Diseases, 2021; 72(7): 1211-1219. https://doi.org/10.1093/cid/ciaa210
Golden CE, Rothrock Jr MJ, Mishra A. Mapping Foodborn Pathogen Contamination throughout The Conventional and Alternative Poultry Supply Chains. Poultry Science, 2021; 100: 1-17. https://doi.org/10.1016/j.psj.2021.101157
Wilcox CH, Sandilands V, Mayasari N, Asmara IY, Anang A. A literature review of broiler chicken welfare, husbandry, and assessment. World’s Poultry Science Journal, 2023; 80(1), 3–32. https://doi.org/10.1080/00439339.2023.2264824
Leahy E, Mutua F, Grace D, Lambertini E and Thomas LF. Foodborne zoonoses control in lowand middle-income countries: Identifying aspects of interventions relevant to traditional markets which act as hurdles when mitigating disease transmission. Front. Sustain. Food Syst. 2022; 6:913560. doi: 10.3389/fsufs.2022.913560
Kuan NL, Chen YP, Shien JH, Yeh KS. Characteristics of the extended spectrum-β-lactamase-producing Escherichia coli isolated from diseased livestock and poultry in Taiwan. Nature, 2024; 14: 1-8. DOI: https://doi.org/10.1038/s41598-024-80943-9
Lin X, and Kuck U. Cephalosporins as key lead generation beta-lactam antibiotics. Applied Microbiology and Biotechnology, 2022; 106: 8007-8020. DOI: https://doi.org/10.1007/s00253-022-12272-8
Saliu EM, Vahjen W, and Zentek J. Types and prevalence of extended–spectrum beta–lactamase producing Enterobacteriaceae in poultry, Animal Health Research Reviews, 2017; 18(1): 46-57. DOI: 10.1017/S1466252317000020
Xu H, Tan C, Li C, Li J, Han Y, Tang Y, Lei C, Wang H. ESBL-Escherichia coli extracellular vesicles mediate bacterial resistance to β-lactam and mediate horizontal transfer of blaCTX-M-55. International Journal of Antimicrobial Agents, 2024; 63(5): 137-145. DOI: https://doi.org/10.1016/j.ijantimicag.2024.107145
Handayani KS, Setiyono A, Lukman DW, Pisestyani H, Rahayu P. Distribution of extended-spectrum β-lactamase producing Escherichia coli genes in an integrated poultry-fish farming system in Bogor, Indonesia. Vet World. 2024;17(7):1596-1602. Doi:10.14202/vetworld.2024.1596-1602
Ranjan A, Shaik S, Hussain A, Nandanwar N, Semmler TJadhav S, Wieler LH, Ahmed N. Genomic and Functional Portrait of a Highly Virulent, CTX-M-15-Producing H30-Rx Subclone of Escherichia coli Sequence Type 131. Antimicrob Agents Chemother. 2015; 59(10): 6087-6095. https://doi.org/10.1128/aac.01447-15
Ariyanti T, Noor SM, Rachmawati F, Widiyanti PM, Andriani, Sukmawinata E, Pisestyani H, Suhaemi, Mulyati S, Sukatma, Anastasia Y, Purba HHS. Prevalence, antibiotic resistance patterns, and genetic characterization of ESBL-producing Escherichia coli in chicken samples from Tangerang, Indonesia. IOP Conf. Series: Earth and Environmental Science. 2025; 1471: 1-6. Doi:10.1088/1755-1315/1471/1/012062
Wardoyo EH, Sugawara Y, Nakano S, Zuo H, Elahi S, Arai C, Kondo K, Kuntaman K, Sugai M.0.Genomic characterization of extended-spectrum β-lactamase-producing Escherichia coli spread among chickens and healthy residents in Lombok, Indonesia. Appl Environ Microbiol0:e02364-24. https://doi.org/10.1128/aem.02364-24
Aini Eka Puji Dameanti FN, Yanestria SM, Effendi MH, et al. Genotyping of extended-spectrum beta-lactamase-producing Escherichia coli isolated from wastewater of dairy farms in East Java, Indonesia. Vet World. 2025;18(1):162-171. Doi:10.14202/vetworld.2025.162-171
Mardhia M, Liana DF, Mahyarudin M, Ih H. The first report of antibiotic resistance and virulence factor profiles in multidrug-resistant clinical isolates of Klebsiella pneumoniae from Pontianak, Indonesia. Osomh public health and research perspectives. 2025;16(2):160-168. https://doi.org/10.24171/j.phrp.2024.0242
Lengyel, J., Hnath, E., Storms, M. et al. (2014). Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR. J Struct Funct Genomics 15, 117–124. https://doi.org/10.1007/s10969-014-9179-9
Papageorgiou AC, Poudel N, Mattsson J. Protein structure analysis and validation with X-ray crystallography. Protein downstream processing: design, development, and application of high and low-resolution methods. 2021; 2178: 377-404. https://doi.org/10.1007/978-1-0716-0775-6_25
Hu, Y., Cheng K., He, L., Zhang X., Jiang B., Jiang L., Li C., Wang G., Yang Y., Liu M. 2021. NMR-Based Methods for Protein Analysis. Anal.Chem, 93: 1866-1879. https://dx.doi.org/10.1021/acs.analchem.0c03830?ref=pdf
Chang, W. H., Huang, S. H., Lin, H. H., Chung, S. C., & Tu, I. P. (2021). Cryo-EM Analyses Permit Visualization of Structural Polymorphism of Biological Macromolecules. Frontiers in bioinformatics, 1, 788308. https://doi.org/10.3389/fbinf.2021.788308
Zhou, X., Zheng, W., Li, Y., Pearce, R., Zhang, C., Bell, E. W., Zhang, G., & Zhang, Y. (2022). I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nature protocols, 17(10), 2326–2353. https://doi.org/10.1038/s41596-022-00728-0
Carugo O. Identifications of False Positives Amongst Sodium(I) Cations in Protein Three-Dimensional Structures—A Validation Approach Extendible to Any Alkali or Alkaline Earth Cation and to Any Monoatomic Anion. Crystals. 2024; 14(11):918. https://doi.org/10.3390/cryst14110918
Yang J, Zhang Y. Protein Structure and Function Prediction Using I-TASSER. Curr Protoc Bioinformatics. 2015; 52: 5.8.1–5.8.15. Doi:10.1002/0471250953.bi0508s52
Rashid NNA, Nasir MHM, Hamzah N, Ismail CMKH, Hishamuddin SASN, Suffian IFM, Hamid AAA. Three-Dimensional Structure of Human Epididymis Protein (HE4): A Protein Modelling of an Ovarian Cancer Biomarker Through In Silico Approach. Journal of tropical life science. 2024; 14(2): 331-348. http://dx.doi.org/10.11594/jtls.14.02.13
Reddy B, Fields R. Performance Analysis of Multiple Sequence Alignment Tools. In Proceedings of the 2024 ACM Southeast Conference (ACMSE '24). Association for Computing Machinery. 2024; 2024: 167–174. https://doi.org/10.1145/3603287.3651216
Koichiro Tamura, Glen Stecher, Sudhir Kumar, MEGA11: Molecular Evolutionary Genetics Analysis Version 11, Molecular Biology and Evolution, 2021; 38(7):3022–3027, https://doi.org/10.1093/molbev/msab120
Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870-1874. doi:10.1093/molbev/msw054
Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, et al. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol Biol Evol. 2017;34(12):3299-3302. doi:10.1093/molbev/msx248
Chi, L., Dong, Y., Wang, R. Yang S, Wu L, Xue Y, Chen H. HapNetworkView: a tool for haplotype network exploration and visualization. BMC Genomics. 2025; 26(52):3-6. https://doi.org/10.1186/s12864-025-11206-8
Zheng W, Zhang C, Li Y, Pearce R, Bell EW, Zhang Y. Folding non-homologous proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Rep Methods. 2021;1(3):100014. doi:10.1016/j.crmeth.2021.100014
Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods. 2015;12(1):7-8. doi:10.1038/nmeth.3213
Galdino GT, DesCoteaux T, Teruel N, Najmanovich R. NRGSuite-Qt: A PyMOL plugin for high-throughput virtual screening, molecular docking, normal-mode analysis, the study of molecular interactions and the detection of binding-site similarities. BioRxiv Cold Spring Harbor Laboratory. 2025; 1-13. doi: https://doi.org/10.1101/2025.01.23.634566
Rustagi V, Gupta SRR, Singh A, Singh IK. Beyond trial and error: Leveraging advanced software for Therapeutic discovery. Chemical Biology Letters. 2025; 12(1):1-9. https://doi.org/10.62110/sciencein.cbl.2025.v12.1251
Elmowalid GA, Ahmad AAM, Hassan MN, Abd El-Aziz NK, Abdelwahab AM, Elwan SI. Molecular Detection of New SHV β-lactamase Variants in Clinical Escherichia coli and Klebsiella pneumoniae Isolates from Egypt. Comp Immunol Microbiol Infect Dis. 2018; 60:35-41. doi: 10.1016/j.cimid.2018.09.013.
Shaikh S, Fatima J, Shakil S, Rizvi SM, Kamal MA. Antibiotic resistance and extended spectrum beta-lactamases: Types, epidemiology and treatment. Saudi J Biol Sci. 2015;22(1):90-101. doi: 10.1016/j.sjbs.2014.08.002.
Yu K, Huang Z, Xiao Y, Gao H, Bai X, Wang D. Global spread characteristics of CTX-M-type extended-spectrum β-lactamases: A genomic epidemiology analysis. Drug Resistance Updates. 2024; 73:101036. https://doi.org/10.1016/j.drup.2023.101036
Riccio, M. E., Verschuuren, T., Conzelmann, N., Martak, D., Meunier, A., Salamanca, E., Musicha, P. Household acquisition and transmission of extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae after hospital discharge of ESBL-positive index patients. Clinical Microbiology and Infection. 2021; 27(9), 1322–1329. doi:10.1016/j.cmi.2020.12.024
Shastry B. S. Role of SNP/haplotype map in gene discovery and drug development: An overview. Drug Development Research. 2004; 62(2), 143–150. doi:10.1002/ddr.10377
Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48(1):1-14. doi: 10.1128/AAC.48.1.1-14.2004.
Palzkill T. Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC β-lactamases. Front Mol Biosci. 2018; 23(5):16. doi: 10.3389/fmolb.2018.00016.
Ali A, Danishuddin, Maryam L, Srivastava G, Sharma A et al. Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria. Journal of Biomolecular Structure and Dynamics. 2018; 36 (7): 1806-1821. doi: 10.1080/07391102.2017.1335434
Sariyer E, Saral A. Evaluation of quercetin as a potential β-lactamase CTX-M-15 inhibitor via the molecular docking, dynamics simulations, and MMGBSA. Turk J Chem. 2021; 45(4):1045-1056. doi: 10.3906/kim-2011-52. PMID: 34707432; PMCID: PMC8517493.