Antimicrobial Resistance (AMR) menjadi isu global akibat meningkatnya kegagalan pengobatan dan penyebaran Extended-Spectrum Beta-Lactamase Producing Escherichia coli (ESBL-Ec) yang banyak ditemukan di lingkungan perairan seperti sungai. Sungai menjadi jalur utama bagi masuknya berbagai limbah, yang dapat membawa ESBL-Ec ke lingkungan. Untuk itu, analisis gen penanda spesifik diperlukan untuk mengidentifikasi sumber kontaminasi ESBL-Ec dan memahami jalur kontaminasi di perairan. Penelitian ini bertujuan untuk menganalisis keberadaan gen penanda ESBL-Ec di air sungai serta jalur kontaminasinya menuju lingkungan dan potensi paparannya terhadap manusia. Studi dilakukan di dua lokasi, yakni Sungai Ciliwung (DKI Jakarta dan Jawa Barat) dan Sungai Brangbiji (Kabupaten Sumbawa, NTB), menggunakan pendekatan Microbial Source Tracking (MST) berbasis metode Polymerase Chain Reaction (PCR) terhadap empat kelompok gen penanda sumber pencemar, yaitu manusia (H8, H12), sapi (Co2, Co3), ayam (Ch7, Ch9, Ch12, Ch13), dan air limbah (W_nqrC, W_clsA_2). Hasil penelitian menunjukkan bahwa di Sungai Ciliwung, keberadaan gen penanda tertinggi adalah air limbah dan ayam, ditunjukkan oleh seluruh isolat (n = 80) yang terdeteksi pada kedua kelompok gen tersebut. Gen penanda manusia dan sapi juga menunjukkan deteksi tinggi, dengan 79 dari 80 isolat menunjukkan keberadaan kedua kelompok gen ini. Di Sungai Brangbiji, seluruh isolat (n = 6) terdeteksi terhadap keempat kelompok gen penanda. Jalur kontaminasi utama di kedua sungai umumnya berasal dari pembuangan limbah domestik dan peternakan yang tidak melalui pengolahan, serta kontribusi dari efluen IPAL. Limbah ini masuk ke badan sungai melalui drainase terbuka, aliran permukaan, atau aktivitas langsung di sekitar bantaran sungai. Temuan ini menunjukkan bahwa sanitasi yang buruk dan pengelolaan limbah yang tidak memadai dapat meningkatkan risiko paparan ESBL- Ec terhadap manusia, baik secara langsung maupun tidak langsung melalui lingkungan. Berdasarkan hasil tersebut, metode MST berbasis PCR terbukti dapat memberikan gambaran mengenai kemungkinan sumber kontaminasi di perairan serta memetakan jalur kontaminasi ESBL-Ec yang berkaitan dengan aktivitas manusia dan hewan di lingkungan sekitar.

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Antimicrobial Resistance (AMR) has become a global concern due to the increasing incidence of treatment failure and the spread of Extended-Spectrum Beta- Lactamase Producing Escherichia coli (ESBL-Ec), which is commonly found in aquatic environments such as rivers. Rivers serve as major pathways for the entry of various waste streams into the environment, potentially carrying ESBL-Ec. Therefore, the analysis of specific marker genes is necessary to identify the sources of ESBL-Ec contamination and to understand its contamination pathways in aquatic systems. This study aims to analyze the presence of ESBL-Ec marker genes in river water, trace their contamination pathways into the environment, and assess the potential exposure risk to humans. The study was conducted in two locations, namely the Ciliwung River (Jakarta and West Java) and the Brangbiji River (Sumbawa Regency, West Nusa Tenggara), using a Microbial Source Tracking (MST) approach based on Polymerase Chain Reaction (PCR) targeting four groups of specific marker genes including human (H8, H12), cattle (Co2, Co3), chicken (Ch7, Ch9, Ch12, Ch13), and human wastewater (W_nqrC, W_clsA_2). The results showed that in the Ciliwung River, the highest prevalence of marker genes was observed for wastewater and chicken, with both gene groups detected in all isolates (n = 80). Human and cattle markers were also highly prevalent, found in 79 out of 80 isolates. In the Brangbiji River, marker genes from all four source categories were detected in all isolates (n = 6).The main contamination pathways in both rivers generally originated from unprocessed domestic and livestock waste discharges, along with contributions from wastewater treatment plant effluents. These wastes entered the river bodies through open drains, surface runoff, or direct activities near the riverbanks. These findings indicate that poor sanitation and inadequate waste management can elevate the risk of human exposure to ESBL-Ec, either directly or indirectly through environmental contact. Based on these results, the PCR-based MST approach demonstrates its capability to provide an overview of potential contamination sources in aquatic environments and to map the contamination pathways of ESBL-Ec linked to human and animal activities in the surrounding area.