Structural Insights into TEM-1 β-Lactamase-Mediated Ceftriaxone Resistance in Escherichia coli: A Molecular Docking and Toxicity Analysis

Most Farhana Akter¹*, Amena Khatun Manica², Md Abu Bakar Siddique³, Tufael⁴, Md. Robiul Islam¹, Sabiha Nusrat⁵

This study reveals structural causes of Ceftriaxone resistance and guides future β-lactamase inhibitor development and therapeutic strategies. 

Abstract

Background: The escalating resistance of Escherichia coli to Ceftriaxone has become a pressing global concern, undermining the clinical utility of this critical β-lactam antibiotic. Among the underlying mechanisms, the TEM-1 β-lactamase enzyme plays a pivotal role in hydrolyzing β-lactam rings, leading to reduced antibiotic efficacy.

Objective: This study investigated the molecular basis of Ceftriaxone resistance and evaluated structural interactions between TEM-1 and selected β-lactam compounds to guide future therapeutic designs.

Methods: Antibiotic resistance trends (2010–2023) were retrieved from the CDC AR Data Hub and NARMS databases, revealing a steady rise in Ceftriaxone resistance in E. coli. Molecular docking analyses were performed using PyRx with Ceftriaxone, Cefepime, and Avibactam as ligands, and the resulting interactions were visualized through PyMOL and Discovery Studio. Toxicity predictions were conducted using ProTox-II.

Results: Ceftriaxone demonstrated the strongest binding affinity to TEM-1 (-7.8 kcal/mol), forming stable hydrogen bonds with GLU240, GLU104, and SER130. Cefepime exhibited slightly weaker binding (-7.3 kcal/mol) with reduced interaction stability, while Avibactam showed promising π-π and electrostatic interactions, suggesting potential β-lactamase inhibitory effects. Toxicity profiling indicated higher nephrotoxicity and respiratory toxicity risks for Cefepime, highlighting its safety limitations.

Conclusion: Collectively, these findings provide structural and mechanistic insights into TEM-1–mediated resistance and emphasize the need for next-generation β-lactamase inhibitors to sustain the efficacy of β-lactam antibiotics against evolving antimicrobial resistance.

Keywords: Antimicrobial Resistance (AMR), Ceftriaxone Resistance, TEM-1 β-lactamase, Molecular Docking, β-lactam Antibiotics

References

Avery, C., Baker, L., & Jacobs, D. J. (2022). Functional Dynamics of Substrate Recognition in TEM Beta-Lactamase. Entropy, 24(5), 729. https://doi.org/10.3390/e24050729

Birgy, A., Delecourt, M., Geslain, G., Desselas, E., Caseris, M., Magnan, M., Mariani-Kurkdjian, P., Bidet, P., & Bonacorsi, S. (2017). A combination of mecillinam and amoxicillin/clavulanate can restore susceptibility of high-level TEM-1-producing Escherichia coli to mecillinam. Journal of Antimicrobial Chemotherapy, 72(7), 1911–1914. https://doi.org/10.1093/jac/dkx087

Bush, K. (2010). Bench-to-bedside review: The role of β-lactamases in antibiotic-resistant Gram-negative infections. Critical Care, 14(3), 224. https://doi.org/10.1186/cc8892

Bush, K., & Bradford, P. A. (2014). β-Lactamases: Historical Perspectives. In Enzyme-Mediated Resistance to Antibiotics (pp. 65–79). ASM Press. https://doi.org/10.1128/9781555815615.ch6

Carlton, J. M., Sahu, P. K., Wassmer, S. C., Mohanty, S., Kessler, A., Eapen, A., Tomko, S. S., Walton, C., Joshi, P. L., Das, D., Albert, S., Peter, B. K., Pradhan, M. M., Dash, A. P., & Das, A. (2022). The Impact, Emerging Needs, and New Research Questions Arising from 12 Years of the Center for the Study of Complex Malaria in India. The American Journal of Tropical Medicine and Hygiene, 107(4_Suppl), 90–96. https://doi.org/10.4269/ajtmh.21-1277

Clasen, J., Birkegård, A. C., Græsbøll, K., & Folkesson, A. (2019). Evolution of TEM-type extended-spectrum β-lactamases in Escherichia coli by cephalosporins. Journal of Global Antimicrobial Resistance, 19, 32–39. https://doi.org/10.1016/j.jgar.2019.03.010

Dalbanjan, N. P., & Praveen Kumar, S. K. (2024). A Chronicle Review of In-Silico Approaches for Discovering Novel Antimicrobial Agents to Combat Antimicrobial Resistance. Indian Journal of Microbiology, 64(3), 879–893. https://doi.org/10.1007/s12088-024-01355-x

Dhara, L., & Tripathi, A. (2014). Genetic and structural insights into plasmid-mediated extended-spectrum β-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients. International Journal of Antimicrobial Agents, 43(6), 518–526. https://doi.org/10.1016/j.ijantimicag.2014.03.002

Di Bella, S., Sanson, G., Monticelli, J., Zerbato, V., Principe, L., Giuffrè, M., Pipitone, G., & Luzzati, R. (2024). Clostridioides difficile infection: history, epidemiology, risk factors, prevention, clinical manifestations, treatment, and future options. Clinical Microbiology Reviews, 37(2). https://doi.org/10.1128/cmr.00135-23

Díaz, N., Suárez, D., Merz, , Kenneth M., & Sordo, T. L. (2005). Molecular Dynamics Simulations of the TEM-1 β-Lactamase Complexed with Cephalothin. Journal of Medicinal Chemistry, 48(3), 780–791. https://doi.org/10.1021/jm0493663

Farhat, N., Khanam, T., Noor, S., & Khan, A. U. (2024). Structural insight into the binding mode of cefotaxime and meropenem to TEM-1, SHV-1, KPC-2, and Amp-C type beta-lactamases. Cell Biochemistry and Biophysics, 82(2), 1299–1308. https://doi.org/10.1007/s12013-024-01284-y

Fisher, J. F., Meroueh, S. O., & Mobashery, S. (2005). Bacterial Resistance to β-Lactam Antibiotics:  Compelling Opportunism, Compelling Opportunity. Chemical Reviews, 105(2), 395–424. https://doi.org/10.1021/cr030102i

Ghenea, A. E., Zlatian, O. M., Cristea, O. M., Ungureanu, A., Mititelu, R. R., Balasoiu, A. T., Vasile, C. M., Salan, A.-I., Iliuta, D., Popescu, M., Udri?toiu, A.-L., & Balasoiu, M. (2022). TEM,CTX-M,SHV Genes in ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a County Clinical Emergency Hospital Romania-Predominance of CTX-M-15. Antibiotics, 11(4), 503. https://doi.org/10.3390/antibiotics11040503

Huwyler, T., Lenggenhager, L., Abbas, M., Ing Lorenzini, K., Hughes, S., Huttner, B., Karmime, A., Uçkay, I., von Dach, E., Lescuyer, P., Harbarth, S., & Huttner, A. (2017). Cefepime plasma concentrations and clinical toxicity: a retrospective cohort study. Clinical Microbiology and Infection, 23(7), 454–459. https://doi.org/10.1016/j.cmi.2017.01.005

Kather, I., Jakob, R. P., Dobbek, H., & Schmid, F. X. (2008). Increased Folding Stability of TEM-1 β-Lactamase by In Vitro Selection. Journal of Molecular Biology, 383(1), 238–251. https://doi.org/10.1016/j.jmb.2008.07.082

Kluytmans, J., & Struelens, M. (2009). Meticillin resistant Staphylococcus aureus in the hospital. BMJ, 338(feb12 1), b364–b364. https://doi.org/10.1136/bmj.b364

Kolbaba-Kartchner, B., Kazan, I. C., Mills, J. H., & Ozkan, S. B. (2021). The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability. International Journal of Molecular Sciences, 22(6), 2895. https://doi.org/10.3390/ijms22062895

Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A. K. M., Wertheim, H. F. L., Sumpradit, N., Vlieghe, E., Hara, G. L., Gould, I. M., Goossens, H., Greko, C., So, A. D., Bigdeli, M., Tomson, G., Woodhouse, W., Ombaka, E., Peralta, A. Q., Qamar, F. N., Mir, F., … Cars, O. (2013). Antibiotic resistance—the need for global solutions. The Lancet Infectious Diseases, 13(12), 1057–1098. https://doi.org/10.1016/S1473-3099(13)70318-9

Lecaille, F., Kaleta, J., & Brömme, D. (2002). Human and Parasitic Papain-Like Cysteine Proteases:  Their Role in Physiology and Pathology and Recent Developments in Inhibitor Design. Chemical Reviews, 102(12), 4459–4488. https://doi.org/10.1021/cr0101656

Macy, E., Crawford, W. W., Nguyen, M. T., Adams, J. L., McGlynn, E. A., & McCormick, T. A. (2022). Population-Based Incidence of New Ampicillin, Cephalexin, Cefaclor, and Sulfonamide Antibiotic “Allergies” in Exposed Individuals with and without Preexisting Ampicillin, Cephalexin, or Cefaclor “Allergies.” The Journal of Allergy and Clinical Immunology: In Practice, 10(2), 550–555. https://doi.org/10.1016/j.jaip.2021.10.043

Martin, J. F., Alvarez-Alvarez, R., & Liras, P. (2022). Penicillin-Binding Proteins, β-Lactamases, and β-Lactamase Inhibitors in β-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms. International Journal of Molecular Sciences, 23(10), 5662. https://doi.org/10.3390/ijms23105662

Md Sakil Amin, & Md Jabir Rashid. (2025). Probiotics as Emerging Neurotherapeutics in Spinal Cord Injury: Modulating Inflammation, Infection, and Regeneration. Microbial Bioactives, 8(1), 1–11. https://doi.org/10.25163/microbbioacts.8110290

Nadeem, S. F., Gohar, U. F., Tahir, S. F., Mukhtar, H., Pornpukdeewattana, S., Nukthamna, P., Moula Ali, A. M., Bavisetty, S. C. B., & Massa, S. (2020). Antimicrobial resistance: more than 70 years of war between humans and bacteria. Critical Reviews in Microbiology, 46(5), 578–599. https://doi.org/10.1080/1040841X.2020.1813687

Nørskov-Lauritsen, N., Søndergaard, A., & Lund, M. (2015). TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae. Microbiology, 161(12), 2310–2315. https://doi.org/10.1099/mic.0.000183

Oelschlaeger, P. (2021). β-Lactamases: Sequence, Structure, Function, and Inhibition. Biomolecules, 11(7), 986. https://doi.org/10.3390/biom11070986

Palzkill, T. (2018). Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC β-lactamases. Frontiers in Molecular Biosciences, 5. https://doi.org/10.3389/fmolb.2018.00016

Payne, L. E., Gagnon, D. J., Riker, R. R., Seder, D. B., Glisic, E. K., Morris, J. G., & Fraser, G. L. (2017). Cefepime-induced neurotoxicity: a systematic review. Critical Care, 21(1), 276. https://doi.org/10.1186/s13054-017-1856-1

Pennisi, F., Pinto, A., Ricciardi, G. E., Signorelli, C., & Gianfredi, V. (2025). The Role of Artificial Intelligence and Machine Learning Models in Antimicrobial Stewardship in Public Health: A Narrative Review. Antibiotics, 14(2), 134. https://doi.org/10.3390/antibiotics14020134

Pereira, R. V., Foditsch, C., Siler, J. D., Dulièpre, S. C., Altier, C., Garzon, A., & Warnick, L. D. (2020). Genotypic antimicrobial resistance characterization of E. coli from dairy calves at high risk of respiratory disease administered enrofloxacin or tulathromycin. Scientific Reports, 10(1), 19327. https://doi.org/10.1038/s41598-020-76232-w

Priyamvada, P., Debroy, R., Anbarasu, A., & Ramaiah, S. (2022). A comprehensive review on genomics, systems biology and structural biology approaches for combating antimicrobial resistance in ESKAPE pathogens: computational tools and recent advancements. World Journal of Microbiology and Biotechnology, 38(9), 153. https://doi.org/10.1007/s11274-022-03343-z

R., S., S., A. H., MF, T. A. K., S., M. A. A., & H., R. (2024). A Review on the Use of Third-Generation Cephalosporins on Gram-Positive and Gram-Negative Bacteria Based on its Spectrum of Activity. Recent Trends in Pharmaceutical Sciences and Research, 6(1), 1–7. https://doi.org/10.46610/RTPSR.2024.v06i01.001

Rajer, F., Allander, L., Karlsson, P. A., & Sandegren, L. (2022). Evolutionary Trajectories toward High-Level β-Lactam/β-Lactamase Inhibitor Resistance in the Presence of Multiple β-Lactamases. Antimicrobial Agents and Chemotherapy, 66(6). https://doi.org/10.1128/aac.00290-22

Ranjbar, R., & Alam, M. (2024). Antimicrobial Resistance Collaborators (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Evidence Based Nursing, 27(1), 16–16. https://doi.org/10.1136/ebnurs-2022-103540

Salam, Md. A., Al-Amin, Md. Y., Salam, M. T., Pawar, J. S., Akhter, N., Rabaan, A. A., & Alqumber, M. A. A. (2023). Antimicrobial Resistance: A Growing Serious Threat for Global Public Health. Healthcare, 11(13), 1946. https://doi.org/10.3390/healthcare11131946

Salleh, M. Z. (2025). Addressing antimicrobial resistance: Structural insights into cefiderocol’s mode of action and emerging resistance mechanisms. Journal of Infection and Public Health, 18(9), 102871. https://doi.org/10.1016/j.jiph.2025.102871

Salverda, M. L. M., De Visser, J. A. G. M., & Barlow, M. (2010). Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance. FEMS Microbiology Reviews, 34(6), 1015–1036. https://doi.org/10.1111/j.1574-6976.2010.00222.x

Shrestha, P., He, S., & Legido-Quigley, H. (2022). Antimicrobial Resistance Research Collaborations in Asia: Challenges and Opportunities to Equitable Partnerships. Antibiotics, 11(6), 755. https://doi.org/10.3390/antibiotics11060755

Siddique. (2025). Targeting p38 MAPK: Molecular Docking and Therapeutic Insights for Alzheimer’s Disease Management. Journal of Primeasia, 6(1), 1–11. https://doi.org/10.25163/primeasia.6110116

Tamma, P. D., Komarow, L., Ge, L., Garcia-Diaz, J., Herc, E. S., Doi, Y., Arias, C. A., Albin, O., Saade, E., Miller, L. G., Jacob, J. T., Satlin, M. J., Krsak, M., Huskins, W. C., Dhar, S., Shelburne, S. A., Hill, C., Baum, K. R., Bhojani, M., … van Duin, D. (2022). Clinical Impact of Ceftriaxone Resistance in Escherichia coli Bloodstream Infections: A Multicenter Prospective Cohort Study. Open Forum Infectious Diseases, 9(11). https://doi.org/10.1093/ofid/ofac572

Vilvanathan, S. (2021). Penicillins, Cephalosporins, and Other β-Lactam Antibiotics. In Introduction to Basics of Pharmacology and Toxicology (pp. 821–834). Springer Nature Singapore. https://doi.org/10.1007/978-981-33-6009-9_54

Wang, H., Feng, Y., & Lu, H. (2022). Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics. Environmental Science & Technology, 56(21), 15074–15083. https://doi.org/10.1021/acs.est.2c00793

Yang, P., Chen, Y., Jiang, S., Shen, P., Lu, X., & Xiao, Y. (2020). Association between the rate of third generation cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae and antibiotic consumption based on 143 Chinese tertiary hospitals data in 2014. European Journal of Clinical Microbiology & Infectious Diseases, 39(8), 1495–1502. https://doi.org/10.1007/s10096-020-03856-1

Zainal-Abidin, R.-A., Afiqah-Aleng, N., Abdullah-Zawawi, M.-R., Harun, S., & Mohamed-Hussein, Z.-A. (2022). Protein–Protein Interaction (PPI) Network of Zebrafish Oestrogen Receptors: A Bioinformatics Workflow. Life, 12(5), 650. https://doi.org/10.3390/life12050650

Zhang, D., Wang, W., Song, C., Huang, T., Chen, H., Liu, Z., Zhou, Y., & Wang, H. (2024). Comparative genomic study of non-typeable Haemophilus influenzae in children with pneumonia and healthy controls. IScience, 27(12), 111330. https://doi.org/10.1016/j.isci.2024.111330