Mutation analysis of dihydrofolate reductase (dfr) and dihydropteroate synthase (sul) genes in trimethoprim-sulfamethoxazole-resistant Escherichia coli from urinary tract infections
,
Abstract
Background and Objectives: Escherichia coli is the leading cause of urinary tract infections (UTIs) and has shown increasing resistance to trimethoprim–sulfamethoxazole (TMP-SMX). Resistance to TMP-SMX is commonly associated with the acquisition of resistance genes such as dfr and sul, often mediated by horizontal gene transfer. This study aimed to characterize the presence of dfr (dfrA1, dfrA5, dfrA7/17) and sul (sul1 and sul2) genes and to describe sequence variations in selected E. coli isolates.
Materials and Methods: Urine culture samples were obtained from 678 patients suspected of having UTIs, and 21 samples yielded positive culture results. This study further analyzed 11 E. coli isolates, consisting of eight TMP-SMX-resistant isolates, two susceptible isolates, and one E. coli ATCC 25922 strain as a control. The clinical isolates were provided by the Clinical Microbiology Laboratory, Faculty of Medicine, Universitas Indonesia. PCR amplification and agarose gel electrophoresis were used to identify dfr and sul genes, followed by Sanger sequencing of selected amplicons. Sequence data were analyzed with BioEdit software and aligned against reference sequences from NCBI.
Results: The distribution of resistance genes varies among resistant isolates. Some isolates carry the dfrA5 gene, while dfrA1 was detected in one isolate. The sul2 gene was present in a number of resistant isolates, while sul1 was identified but showed no differences from the reference sequence. Further sequence analysis revealed amino acid changes in the dfrA1, dfrA5, and sul2 genes, although these changes were not consistently found in all isolates.
Conclusion: This study characterizes the distribution of dfr and sul genes and their sequence variations in a limited number of E. coli isolates. The results suggest that TMP-SMX-resistant isolates may contain diverse genetic determinants associated with resistance. Nevertheless, the limited isolate number restricts the broader interpretation of these findings. Larger-scale studies, supported by functional analyses, are required to determine the contribution of these genes and their sequence variations to antibiotic resistance.
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2. Surgers L, Boyd A, Girard PM, Arlet G, Decré D. Biofilm Formation by ESBL-Producing Strains of Escherichia coli and Klebsiella pneumoniae. Int J Med Microbiol 2019; 309: 13-18.
3. Bedenić B, Sardelić S, Bogdanić M, Zarfel G, Beader N, Šuto S, et al. Klebsiella pneumoniae carbapenemase (KPC) in Urinary Infection Isolates. Arch Microbiol 2021; 203: 1825-1831.
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5. Barnawi Y, Alghamdi A, Ibrahim A, Al-Anazi L, Alhumaida G, Alotaibi R, et al. Prevalence of Urinary Tract Infections in Pregnant Women and Antimicrobial Resistance Patterns in Women in Riyadh, Saudi Arabia: A Retrospective Study. BMC Infect Dis 2024; 24: 502.
6. Sultana KF, Akter A, Saha SR, Ahmed F, Alam S, Jafar T, et al. Bacterial Profile, Antimicrobial Resistance, and Molecular Detection of ESBL and Quinolone Resistance Gene of Uropathogens Causing Urinary Tract Infection in the Southeastern Part of Bangladesh. Braz J Microbiol 2023; 54: 803-815.
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8. Sánchez-Osuna M, Cortés P, Llagostera M, Barbé J, Erill I. Exploration into the Origins and Mobilization of Di-hydrofolate Reductase Genes and the Emergence of Clinical Resistance to Trimethoprim. Microb Genom 2020; 6(11): mgen000440.
9. Krucinska J, Lombardo MN, Erlandsen H, Estrada A, Si D, Viswanathan K, et al. Structure-Guided Functional Studies of Plasmid-Encoded Dihydrofolate Reductases Reveal a Common Mechanism of Trimethoprim Resistance in Gram-Negative Pathogens. Commun Biol 2022; 5: 459.
10. Somorin YM, Weir NJM, Pattison SH, Crockard MA, Hughes CM, Tunney MM, et al. Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection. BMC Microbiol 2022; 22: 144.
11. Wüthrich D, Brilhante M, Hausherr A, Becker J, Meylan M, Perreten V. A Novel Trimethoprim Resistance Gene, dfrA36, Characterized from Escherichia coli from Calves. mSphere 2019; 4(3): 00255-19.
12. Venkatesan M, Fruci M, Verellen LA, Skarina T, Mesa N, Flick R, et al. Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics. Nat Commun 2023; 14: 4031.
13. Catania AM, Stella MC, Cimino F, Zoppi S, Grego E. Sulfonamide Resistance Evaluation in Five Animal Species and First Report of Sul4 in Companion Animals. Vet Microbiol 2024; 296: 110170.
14. Dančová N, Gregová G, Szabóová T. Assessment of Bacterial Contamination and Antimicrobial Resistance of Escherichia coli Isolates from Slovak Dairy Farms. Animals (Basel) 2024; 14: 3095.
15. de los Santos E, Laviña M, Poey ME. Strict Relationship between Class 1 Integrons and Resistance to Sulfamethoxazole in Escherichia coli. Microb Pathog 2021; 161: 105206.
16. Adeniji OO, Elsheikh EAE, Okoh AI. Prevalence of Classes 1 and 2 Integrons in Multidrug-Resistant Acinetobacter baumanni Isolates Recovered from Some Aquatic Environment in South Africa. Sci Rep 2022; 12: 20319.
17. Bobenchik AM, Deak E, Hindler JA, Charlton CL, Humphries RM. Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Enterobacteriaceae with Vitek 2 (2009 FDA) and 2014 CLSI Breakpoints. J Clin Microbiol 2015; 53: 816-823.
18. Bogožalec Košir A, Lužnik D, Tomič V, Milavec M. Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Biosensors (Basel) 2023; 13: 463.
19. Alzayn M, Dulyayangkul P, Satapoomin N, Heesom KJ, Avison MB. OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired β-Lactamases. Antimicrob Agents Chemother 2021; 65(11): e0100421.
20. Mohammadi MM, Bavi O. DNA Sequencing: An Overview of Solid-State and Biological Nanopore-Based Methods. Biophys Rev 2021; 14: 99-110.
21. Racewicz P, Majewski M, Biesiada H, Nowaczewski S, Wilczyński J, Wystalska D, et al. Prevalence and Characterisation of Antimicrobial Resistance Genes and Class 1 and 2 Integrons in Multiresistant Escherichia coli Isolated from Poultry Production. Sci Rep 2022; 12: 6062.
22. Poey ME, de los Santos E, Aznarez D, García-Laviña CX, Laviña M. Genetics of Resistance to Trimethoprim in Cotrimoxazole Resistant Uropathogenic Escherichia coli: Integrons, Transposons, and Single Gene Cassettes. Front Microbiol 2024; 15: 1395953.
23. Sacher-Pirklbauer A, Klein-Jöbstl D, Sofka D, Blanc-Potard AB, Hilbert F. Phylogenetic Groups and Antimicrobial Resistance Genes in Escherichia coli from Different Meat Species. Antibiotics (Basel) 2021; 10: 1543.
24. 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: 1199-1226.
25. Bunduki GK, Heinz E, Phiri VS, Noah P, Feasey N, Musaya J. Virulence Factors and Antimicrobial Resistance of Uropathogenic Escherichia coli (UPEC) Isolated from Urinary Tract Infections: A Systematic Review and Meta-Analysis. BMC Infect Dis 2021; 21: 753.
26. Asmare Z, Erkihun M, Abebe W, Tamrat E. Antimicrobial Resistance and ESBL Production in Uropathogenic Escherichia coli: A Systematic Review and Meta-Analysis in Ethiopia. JAC Antimicrob Resist 2024; 6(3): dlae068.
27. Prastiyanto ME, Iswara A, Khairunnisa A, Sofyantoro F, Siregar AR, Mafiroh WU, et al. Prevalence and antimicrobial resistance profiles of multidrug-resistant bacterial isolates from urinary tract infections in Indonesian patients: a cross-sectional study. Clin Infect Pract 2024; 22: 100359.
28. Ambrose SJ, Hall RM. DfrA Trimethoprim Resistance Genes Found in Gram-Negative Bacteria: Compilation and Unambiguous Numbering. J Antimicrob Chemother 2021; 76: 2748-2756.
29. Wang K, Xu J, Lu X, Yin P, Chen L, Zhao Z, et al. Two Novel Trimethoprim Resistance Genes, dfra50 and dfra51, Identified in Phage-Plasmids. Antimicrob Agents Chemother 2025; 69(7): e0169524.
30. Jiang H, Ran M, Wang X, Chen Q, Wang J, Ruan Z, et al. Prevalence and Characterization of Class I Integrons in Multidrug-Resistant Escherichia coli Isolates from Humans and Food-Producing Animals in Zhejiang Province, China. BMC Microbiol 2025; 25: 76.
31. Solis MN, Loaiza K, Torres-Elizalde L, Mina I, Šefcová MA, Larrea-Álvarez M. Detecting Class 1 Integrons and Their Variable Regions in Escherichia coli Whole-Genome Sequences Reported from Andean Community Countries. Antibiotics (Basel) 2024; 13: 394.
32. Chin JJ, Lee HM, Lee SY, Lee YY, Chew CH. High Carriage of tetA, sul1, sul2 and blaTEM Resistance Genes among the Multidrug-resistant Uropathogenic Escherichia coli (UPEC) Strains from Malaysian Patients. Trop Life Sci Res 2024; 35: 211-225.
33. Liu M, Liu J, Ma J, Li W, Zhao X, Jia W, et al. Antimicrobial Resistance and Molecular Characterization of Gene Cassettes from Class 1 Integrons in Carbapenem-Resistant Escherichia coli strains. Microb Pathog 2022; 170: 105669.
34. Alzaidi E, Alshammari M, AlKhulaifi M, Alrashidi B, Alhetheel A, Aljaaidi R, et al. Detection of integrons in Escherichia coli associated with urinary tract infections in Riyadh, Saudi Arabia. Int J Microbiol 2025; 2025: 1013136.
35. Florensa AF, Kaas RS, Clausen PTLC, 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: 000748.
36. Nasrollahian S, Graham JP, Halaji M. A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli. Front Cell Infect Microbiol 2024; 14: 1387497.
2. Surgers L, Boyd A, Girard PM, Arlet G, Decré D. Biofilm Formation by ESBL-Producing Strains of Escherichia coli and Klebsiella pneumoniae. Int J Med Microbiol 2019; 309: 13-18.
3. Bedenić B, Sardelić S, Bogdanić M, Zarfel G, Beader N, Šuto S, et al. Klebsiella pneumoniae carbapenemase (KPC) in Urinary Infection Isolates. Arch Microbiol 2021; 203: 1825-1831.
4. Ullah H, Bashir K, Idrees M, Ullah A, Hassan N, Khan S, et al. Phylogenetic analysis and antimicrobial susceptibility Profile of Uropathogens. PLoS One 2022; 17(1): e0262952.
5. Barnawi Y, Alghamdi A, Ibrahim A, Al-Anazi L, Alhumaida G, Alotaibi R, et al. Prevalence of Urinary Tract Infections in Pregnant Women and Antimicrobial Resistance Patterns in Women in Riyadh, Saudi Arabia: A Retrospective Study. BMC Infect Dis 2024; 24: 502.
6. Sultana KF, Akter A, Saha SR, Ahmed F, Alam S, Jafar T, et al. Bacterial Profile, Antimicrobial Resistance, and Molecular Detection of ESBL and Quinolone Resistance Gene of Uropathogens Causing Urinary Tract Infection in the Southeastern Part of Bangladesh. Braz J Microbiol 2023; 54: 803-815.
7. Chowdhury SS, Tahsin P, Xu Y, Mosaddek ASM, Muhamadali H, Goodacre R. Trends in Antimicrobial Resistance of Uropathogens Isolated from Urinary Tract Infections in a Tertiary Care Hospital in Dhaka, Bangladesh. Antibiotics (Basel) 2024; 13: 925.
8. Sánchez-Osuna M, Cortés P, Llagostera M, Barbé J, Erill I. Exploration into the Origins and Mobilization of Di-hydrofolate Reductase Genes and the Emergence of Clinical Resistance to Trimethoprim. Microb Genom 2020; 6(11): mgen000440.
9. Krucinska J, Lombardo MN, Erlandsen H, Estrada A, Si D, Viswanathan K, et al. Structure-Guided Functional Studies of Plasmid-Encoded Dihydrofolate Reductases Reveal a Common Mechanism of Trimethoprim Resistance in Gram-Negative Pathogens. Commun Biol 2022; 5: 459.
10. Somorin YM, Weir NJM, Pattison SH, Crockard MA, Hughes CM, Tunney MM, et al. Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection. BMC Microbiol 2022; 22: 144.
11. Wüthrich D, Brilhante M, Hausherr A, Becker J, Meylan M, Perreten V. A Novel Trimethoprim Resistance Gene, dfrA36, Characterized from Escherichia coli from Calves. mSphere 2019; 4(3): 00255-19.
12. Venkatesan M, Fruci M, Verellen LA, Skarina T, Mesa N, Flick R, et al. Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics. Nat Commun 2023; 14: 4031.
13. Catania AM, Stella MC, Cimino F, Zoppi S, Grego E. Sulfonamide Resistance Evaluation in Five Animal Species and First Report of Sul4 in Companion Animals. Vet Microbiol 2024; 296: 110170.
14. Dančová N, Gregová G, Szabóová T. Assessment of Bacterial Contamination and Antimicrobial Resistance of Escherichia coli Isolates from Slovak Dairy Farms. Animals (Basel) 2024; 14: 3095.
15. de los Santos E, Laviña M, Poey ME. Strict Relationship between Class 1 Integrons and Resistance to Sulfamethoxazole in Escherichia coli. Microb Pathog 2021; 161: 105206.
16. Adeniji OO, Elsheikh EAE, Okoh AI. Prevalence of Classes 1 and 2 Integrons in Multidrug-Resistant Acinetobacter baumanni Isolates Recovered from Some Aquatic Environment in South Africa. Sci Rep 2022; 12: 20319.
17. Bobenchik AM, Deak E, Hindler JA, Charlton CL, Humphries RM. Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Enterobacteriaceae with Vitek 2 (2009 FDA) and 2014 CLSI Breakpoints. J Clin Microbiol 2015; 53: 816-823.
18. Bogožalec Košir A, Lužnik D, Tomič V, Milavec M. Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Biosensors (Basel) 2023; 13: 463.
19. Alzayn M, Dulyayangkul P, Satapoomin N, Heesom KJ, Avison MB. OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired β-Lactamases. Antimicrob Agents Chemother 2021; 65(11): e0100421.
20. Mohammadi MM, Bavi O. DNA Sequencing: An Overview of Solid-State and Biological Nanopore-Based Methods. Biophys Rev 2021; 14: 99-110.
21. Racewicz P, Majewski M, Biesiada H, Nowaczewski S, Wilczyński J, Wystalska D, et al. Prevalence and Characterisation of Antimicrobial Resistance Genes and Class 1 and 2 Integrons in Multiresistant Escherichia coli Isolated from Poultry Production. Sci Rep 2022; 12: 6062.
22. Poey ME, de los Santos E, Aznarez D, García-Laviña CX, Laviña M. Genetics of Resistance to Trimethoprim in Cotrimoxazole Resistant Uropathogenic Escherichia coli: Integrons, Transposons, and Single Gene Cassettes. Front Microbiol 2024; 15: 1395953.
23. Sacher-Pirklbauer A, Klein-Jöbstl D, Sofka D, Blanc-Potard AB, Hilbert F. Phylogenetic Groups and Antimicrobial Resistance Genes in Escherichia coli from Different Meat Species. Antibiotics (Basel) 2021; 10: 1543.
24. 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: 1199-1226.
25. Bunduki GK, Heinz E, Phiri VS, Noah P, Feasey N, Musaya J. Virulence Factors and Antimicrobial Resistance of Uropathogenic Escherichia coli (UPEC) Isolated from Urinary Tract Infections: A Systematic Review and Meta-Analysis. BMC Infect Dis 2021; 21: 753.
26. Asmare Z, Erkihun M, Abebe W, Tamrat E. Antimicrobial Resistance and ESBL Production in Uropathogenic Escherichia coli: A Systematic Review and Meta-Analysis in Ethiopia. JAC Antimicrob Resist 2024; 6(3): dlae068.
27. Prastiyanto ME, Iswara A, Khairunnisa A, Sofyantoro F, Siregar AR, Mafiroh WU, et al. Prevalence and antimicrobial resistance profiles of multidrug-resistant bacterial isolates from urinary tract infections in Indonesian patients: a cross-sectional study. Clin Infect Pract 2024; 22: 100359.
28. Ambrose SJ, Hall RM. DfrA Trimethoprim Resistance Genes Found in Gram-Negative Bacteria: Compilation and Unambiguous Numbering. J Antimicrob Chemother 2021; 76: 2748-2756.
29. Wang K, Xu J, Lu X, Yin P, Chen L, Zhao Z, et al. Two Novel Trimethoprim Resistance Genes, dfra50 and dfra51, Identified in Phage-Plasmids. Antimicrob Agents Chemother 2025; 69(7): e0169524.
30. Jiang H, Ran M, Wang X, Chen Q, Wang J, Ruan Z, et al. Prevalence and Characterization of Class I Integrons in Multidrug-Resistant Escherichia coli Isolates from Humans and Food-Producing Animals in Zhejiang Province, China. BMC Microbiol 2025; 25: 76.
31. Solis MN, Loaiza K, Torres-Elizalde L, Mina I, Šefcová MA, Larrea-Álvarez M. Detecting Class 1 Integrons and Their Variable Regions in Escherichia coli Whole-Genome Sequences Reported from Andean Community Countries. Antibiotics (Basel) 2024; 13: 394.
32. Chin JJ, Lee HM, Lee SY, Lee YY, Chew CH. High Carriage of tetA, sul1, sul2 and blaTEM Resistance Genes among the Multidrug-resistant Uropathogenic Escherichia coli (UPEC) Strains from Malaysian Patients. Trop Life Sci Res 2024; 35: 211-225.
33. Liu M, Liu J, Ma J, Li W, Zhao X, Jia W, et al. Antimicrobial Resistance and Molecular Characterization of Gene Cassettes from Class 1 Integrons in Carbapenem-Resistant Escherichia coli strains. Microb Pathog 2022; 170: 105669.
34. Alzaidi E, Alshammari M, AlKhulaifi M, Alrashidi B, Alhetheel A, Aljaaidi R, et al. Detection of integrons in Escherichia coli associated with urinary tract infections in Riyadh, Saudi Arabia. Int J Microbiol 2025; 2025: 1013136.
35. Florensa AF, Kaas RS, Clausen PTLC, 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: 000748.
36. Nasrollahian S, Graham JP, Halaji M. A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli. Front Cell Infect Microbiol 2024; 14: 1387497.
| Files | ||
| Issue | Vol 18 No 4 (2026) | |
| Section | Original Article(s) | |
| Keywords | ||
| Escherichia coli Trimethoprim-sulfamethoxazole Dihydrofolate reductase Dihydropteroate synthase Antibiotic resistance Gene characterization | ||
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How to Cite
1.
Tjampakasari C, Sjatha F, Yasmon A, Syahrurachman A, Guntari N. Mutation analysis of dihydrofolate reductase (dfr) and dihydropteroate synthase (sul) genes in trimethoprim-sulfamethoxazole-resistant Escherichia coli from urinary tract infections. Iran J Microbiol. 2026;18(4):544-552.
