Multiplex PCR-based Molecular Characterization of Fecal-Derived Multidrug-Resistant Escherichia coli Isolates

Öz

Antimicrobial resistance (AMR) in Escherichia coli presents a serious public health threat, particularly when resistant strains spread through contaminated food and water. This study characterized fecal-derived multidrug-resistant (MDR) E. coli isolates using multiplex polymerase chain reaction (PCR) to detect extended-spectrum β-lactamase (ESBL) gene variants (blaTEM, blaOXA, blaSHV, and blaCTX-M), sulfonamide resistance genes (sul1, sul2, and sul3), and integron-associated integrase genes (int1, int2, and int3). We analyzed 62 uspA-positive E. coli isolates. Results showed that 82.25% of isolates carried none of the targeted bla genes. The blaCTX-M and blaOXA genes were present in 8.06% and 9.67% of isolates, respectively. Co-occurrence of bla and sul genes appeared in 12.91% of isolates. Co-occurrence of bla and int genes occurred in 12.90%. Only 6.45% of isolates carried at least one gene from all three categories, indicating potential MDR genotypes. These findings highlight the importance of molecular surveillance in tracking resistance genes outside clinical settings. They support the need for integrated One Health strategies to combat AMR.

Anahtar Kelimeler

• Escherichia coli
• multiplex PCR
• ESBL
• sulfonamide resistance
• integrons
• fecal isolates

Destekleyen Kurum

N/A

Etik Beyan

No ethical approval required.

Teşekkür

This study is a part of Dogan SARIGUL's master thesis.

Gaita Kaynaklı Çoklu İlaç Dirençli Escherichia coli İzolatlarının Multipleks PZR ile Moleküler Karakterizasyonu

Öz

Escherichia coli'de antimikrobiyal direnç (AMR), özellikle dirençli suşlar kontamine gıda ve su yoluyla yayıldığında ciddi bir halk sağlığı tehdidi oluşturmaktadır. Bu çalışmada, gaita kaynaklı çoklu ilaç dirençli (MDR) E. coli izolatları, genişlemiş spektrumlu beta-laktamaz (GSBL) gen varyantlarını (blaTEM, blaOXA, blaSHV ve blaCTX-M), sülfonamid direnç genlerini (sul1, sul2 ve sul3) ve integron ilişkili integraz genlerini (int1, int2 ve int3) tespit etmek amacıyla multipleks polimeraz zincir reaksiyonu (PZR) kullanılarak karakterize edilmiştir. uspA-pozitif 62 E. coli izolatı analiz edilmiştir. Sonuçlar, izolatların %82,25'inin hedeflenen bla genlerinin hiçbirini taşımadığını göstermiştir. blaCTX-M ve blaOXA genleri sırasıyla izolatların %8,06 ve %9,67'sinde tespit edilmiştir. bla ve sul genlerinin birlikte bulunma oranı izolatların %12,91'inde görülmüştür. bla ve int genlerinin birlikte bulunma oranı ise %12,90 olarak gerçekleşmiştir. İzolatların yalnızca %6,45'i her üç kategoriden en az bir gen taşımış olup bu durum potansiyel MDR genotiplerine işaret etmektedir. Bu bulgular, direnç genlerinin klinik ortamlar dışında izlenmesinde moleküler sürveyansın önemini vurgulamaktadır. Ayrıca, AMR ile mücadelede entegre Tek Sağlık stratejilerine duyulan ihtiyacı desteklemektedir.

Anahtar Kelimeler

• Escherichia coli
• multipleks PZR
• GSBL
• sülfonamid direnci
• integronlar
• gaita izolatları

Destekleyen Kurum

YOK

Etik Beyan

Etik Kurul Onayı gerekmemektedir.

Teşekkür

Bu çalışma Doğan SARIGÜL'ün yüksek lisans tezinin bir bölümünü kapsamaktadır.

Kaynakça

1. [1] Sarowska, J., Futoma-Koloch, B., Jama-Kmiecik, A., Frej-Madrzak, M., Ksiazczyk, M., Bugla-Ploskonska, G., et al., Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports, Gut Pathogens, 11, 10, 2019.
2. [2] Pokharel, P., Habouria, H., Bessaiah, H., Dozois, C.M., Serine Protease Autotransporters of the Enterobacteriaceae (SPATEs): Out and About and Chopping It Up, Microorganisms, 7(12), 594, 2019.
3. [3] Paterson, D.L., Bonomo, R.A., Extended-spectrum beta-lactamases: a clinical update, Clinical Microbiology Reviews, 18(4), 657-686, 2005.
4. [4] Husna, A., Rahman, M.M., Badruzzaman, A.T.M., Sikder, M.H., Islam, M.R., Rahman, M.T., et al., Extended-Spectrum β-Lactamases (ESBL): Challenges and Opportunities, Biomedicines, 11(11), 2937, 2023.
5. [5] Baran, A., Kwiatkowska, A., Potocki, L., Antibiotics and Bacterial Resistance-A Short Story of an Endless Arms Race, International Journal of Molecular Sciences, 24(6), 5777, 2023.
6. [6] Deng, Y., Bao, X., Ji, L., Chen, L., Liu, J., Miao, J., et al., Resistance integrons: class 1, 2 and 3 integrons, Annals of Clinical Microbiology and Antimicrobials, 14, 45, 2015.
7. [7] Partridge, S.R., Kwong, S.M., Firth, N., Jensen, S.O., Mobile Genetic Elements Associated with Antimicrobial Resistance, Clinical Microbiology Reviews, 31(4), e00088-17, 2018.
8. [8] Tängdén, T., Cars, O., Melhus, A., Löwdin, E., Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers, Antimicrobial Agents and Chemotherapy, 54(9), 3564-3568, 2010.

9. [9] Hazirolan, G., Mumcuoglu, I., Altan, G., Özmen, B.B., Aksu, N., Karahan, Z.C., Fecal carriage of extended-spectrum beta-lactamase and ampc beta-lactamase-producing Enterobacteriaceae in a Turkish community, Nigerian Journal of Clinical Practice, 21(1), 81-86, 2018.
10. [10] Kaprou, G.D., Bergšpica, I., Alexa, E.A., Alvarez-Ordóñez, A., Prieto, M., Rapid Methods for Antimicrobial Resistance Diagnostics, Antibiotics, 10(2), 209, 2021.
11. [11] Coccaro, N., Tota, G., Anelli, L., Zagaria, A., Specchia, G., Albano, F., Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies, International Journal of Molecular Sciences, 21(9), 3141, 2020.
12. [12] Chen, J., Griffiths, M.W., PCR differentiation of Escherichia coli from other gram- negative bacteria using primers derived from the nucleotide sequences flanking the gene encoding the universal stress protein, Letters in Applied Microbiology, 27(6), 369-371, 1998.
13. [13] Ahmed, S.F., Ali, M.M.M., Mohamed, Z.K., Moussa, T.A., Klena, J.D., Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community, Annals of Clinical Microbiology and Antimicrobials, 13, 22, 2014.
14. [14] Kerrn, M.B., Klemmensen, T., Frimodt-Møller, N., Espersen, F., Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteremia, and distribution of sul genes conferring sulphonamide resistance, Journal of Antimicrobial Chemotherapy, 50(4), 513-516, 2002.
15. [15] Hammerum, A.M., Sandvang, D., Andersen, S.R., Seyfarth, A.M., Porsbo, L.J., Frimodt-Møller, N., et al., Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark, International Journal of Food Microbiology, 106(2), 235-237, 2006.
16. [16] Gündoğdu, A., Long, Y.B., Vollmerhausen, T.L., Katouli, M., Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia, Journal of Medical Microbiology, 60(11), 1633-1642, 2011.
17. [17] Arslan, E., Uçan, U.S., Comparison of Rapid Bacterial DNA Extraction Methods, Selcuk University Journal of Science Faculty, 2(27), 73-76, 2006.
18. [18] Kürekci, C., Aydin, M., Yipel, M., Katouli, M., Gündoğdu, A., Characterization of extended spectrum β-lactamase (ESBL)-producing Escherichia coli in Asi (Orontes) River in Turkey, Journal of Water and Health, 15(5), 788-798, 2017.
19. [19] Düzgün, A.Ö., Okumuş, F., Saral, A., Çiçek, A.Ç., Cinemre, S., Determination of antibiotic resistance genes and virulence factors in Escherichia coli isolated from Turkish patients with urinary tract infection, Revista da Sociedade Brasileira de Medicina Tropical, 52, e20180499, 2019.
20. [20] Abrar, S., Ain, N.U., Liaqat, H., Hussain, S., Rasheed, F., Riaz, S., Distribution of blaCTX-M, blaTEM, blaSHV and blaOXA genes in Extended-spectrum-β-lactamase-producing Clinical isolates: A three-year multi-center study from Lahore, Pakistan, Antimicrobial Resistance and Infection Control, 8, 80, 2019.
21. [21] Yelimlibağ, B., Aydin, M., Characterization of Multiple Resistance Genes of Escherichia coli Strains Isolated from Food and Clinical Samples, Cellular and Molecular Biology, 69(7), 118-126, 2023.
22. [22] Wu, S., Dalsgaard, A., Hammerum, A.M., Porsbo, L.J., Jensen, L.B., Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human, Acta Veterinaria Scandinavica, 52(1), 47, 2010.
23. [23] Urban-Chmiel, R., Marek, A., Stępień-Pyśniak, D., Wieczorek, K., Dec, M., Nowaczek, A., et al., Antibiotic Resistance in Bacteria – A Review, Antibiotics, 11(8), 1079, 2022.
24. [24] Arabi, H., Pakzad, I., Nasrollahi, A., Hosainzadegan, H., Azizi Jalilian, F., Taherikalani, M., et al., Sulfonamide Resistance Genes (sul) in Extended Spectrum Beta Lactamase (ESBL) and Non-ESBL Producing Escherichia coli Isolated from Iranian Hospitals, Jundishapur Journal of Microbiology, 8(7), e19961, 2015.
25. [25] Perewari, D.O., Otokunefor, K., Agbagwa, O.E., Tetracycline-Resistant Genes in Escherichia coli from Clinical and Nonclinical Sources in Rivers State, Nigeria, International Journal of Microbiology, 2022, 9192424, 2022.
26. [26] Trinchera, M., De Gaetano, S., Sole, E., Midiri, A., Silvestro, S., Mancuso, G., et al., Antimicrobials in Livestock Farming and Resistance: Public Health Implications, Antibiotics, 14(6), 606, 2025.
27. [27] Riveros, M., Pons, M.J., Durand, D., Ochoa, T.J., Ruiz, J., Class 1 and 2 Integrons in Escherichia coli Strains Isolated from Diarrhea and Bacteremia in Children Less Than 2 Years of Age from Peru, American Journal of Tropical Medicine and Hygiene, 108(1), 181-186, 2023.
28. [28] Araque, M., Labrador, I., Prevalence of Fecal Carriage of CTX-M-15 Beta- Lactamase-Producing Escherichia coli in Healthy Children from a Rural Andean Village in Venezuela, Osong Public Health and Research Perspectives, 9(1), 9-15, 2018.
29. [29] Lavakhamseh, H., Mohajeri, P., Rouhi, S., Shakib, P., Ramazanzadeh, R., Rasani, A., et al., Multidrug-Resistant Escherichia coli Strains Isolated from Patients Are Associated with Class 1 and 2 Integrons, Chemotherapy, 61(2), 72-76, 2016.
30. [30] Martinson, J.N.V., Walk, S.T., Escherichia coli Residency in the Gut of Healthy Human Adults, EcoSal Plus, 9(1), 2020.
31. [31] Salam, M.A., Al-Amin, M.Y., Salam, M.T., Pawar, J.S., Akhter, N., Rabaan, A.A., et al., Antimicrobial Resistance: A Growing Serious Threat for Global Public Health, Healthcare, 11(13), 1946, 2023.
32. [32] Velazquez-Meza, M.E., Galarde-López, M., Carrillo-Quiróz, B., Alpuche-Aranda, C.M., Antimicrobial resistance: One Health approach, Veterinary World, 15(3), 743-749, 2022.
33. [33] Arnold, K.E., Laing, G., McMahon, B.J., Fanning, S., Stekel, D.J., Pahl, O., et al., The need for One Health systems-thinking approaches to understand multiscale dissemination of antimicrobial resistance, The Lancet Planetary Health, 8(2), e124-e133, 2024.