Preparation of hyperimmune serum against multidrug-resistant Escherichia coli isolated from animal products in Bali and Nusa Tenggara, Indonesia: An epidemiological perspective

Background and Aim: The global rise of antimicrobial resistance (AMR), particularly multidrug-resistant Escherichia coli (MDR E. coli), poses a significant threat to public health, animal health, and environmental safety. Traditional antibiotics are increasingly ineffective, necessitating alternative approaches such as immunotherapy. Hyperimmune serum represents a potential solution to combat MDR pathogens by providing immediate passive immunity. The present study aimed to produce and evaluate hyperimmune serum against MDR E. coli isolated from animal products in Bali and Nusa Tenggara, Indonesia, as a novel immunotherapeutic strategy against AMR. Materials and Methods: Five MDR E. coli isolates resistant to eight antibiotics were obtained from the Quality Testing and Certification Center for Animal Products in Bogor, Indonesia. Isolates originated from Bali, West Nusa Tenggara, and East Nusa Tenggara. Bacterial isolates were cultured in Yeast Tris (YT) broth medium supplemented with antibiotics. Bacterial proteins were extracted, solubilized, and emulsified with Freund’s complete and incomplete adjuvants. A 2-year-old horse was immunized with prepared antigens through intramuscular injections over 6 weeks, followed by intravenous administration. Serum was collected pre- and post-immunization. Confirmation of the hyperimmune serum’s specificity and neutralizing capability was performed using an in vitro agar plate test. Results: Successful growth of MDR E. coli isolates was confirmed in antibiotic-supplemented media, whereas no growth was observed in the antibiotic-sensitive native control. Hyperimmune serum obtained post-immunization exhibited strong in vitro specificity, recognized homologous MDR E. coli isolates, and formed significant neutralization zones (5–6 mm) around agar diffusion disks. Pre-immunization serum showed no reactivity, demonstrating effective production of specific antibodies. The neutralization zones were slightly smaller than those achieved with chloramphenicol control, indicating effective but slightly reduced bactericidal activity. Conclusion: The study successfully demonstrated that hyperimmune serum generated from MDR E. coli isolates from animal products has specific neutralizing activity against homologous MDR bacterial strains. These findings support the potential application of hyperimmune sera as an immunotherapeutic tool to mitigate AMR-related infections. Further research is necessary to standardize Immunoglobulin G concentrations, determine optimal immunization protocols, and evaluate clinical efficacy in vivo. The hyperimmune serum preparation represents a promising advancement for tackling MDR bacterial infections and contributes substantially to One Health strategies aimed at reducing the public health threat posed by AMR. Keywords: antimicrobial resistance, hyperimmune serum, immunotherapy, multidrug-resistant Escherichia coli, One Health.