Simultaneous inactivation of antibiotic-resistant bacteria and degradation of antibiotic-resistant genes in alkalised human urine

Natnael Girma Demissie^1,2*Annika Christina Nordin¹Prithvi Simha¹Isis Conroy³He Sun¹Anna Schnürer¹Björn Vinnerås¹Adey Desta^2*

• ¹Swedish University of Agricultural Sciences, Uppsala, Sweden
• ²College of Natural Sciences, Addis Ababa University, Addis Ababa, Addis Ababa, Ethiopia
• ³Tampere University of Applied Sciences, Tampere, Pirkanmaa, Finland

The coexistence of pharmaceuticals and microorganisms in source separated urine poses a risk for the development of antimicrobial resistance (AMR), especially when urine-based fertilisers are applied to soils. While prior studies have investigated pathogen inactivation in source-separated wastewater matrices, few have evaluated the simultaneous fate of antibiotic-resistant bacteria (ARBs) and their corresponding resistance genes (ARGs) in real urine matrices, particularly under alkaline conditions. Here, we studied the inactivation of β-lactamase-producing Escherichia coli and vancomycin-resistant Enterococcus faecium and the degradation of their respective ARGs (blaCTX-M and van-A) in alkalized, unhydrolyzed urine (pH 10.8 and 12.5) treated with UV (65 W low pressure dichromatic mercury lamp at 185/254 nm), hydrogen peroxide (1.25 g L -1 H2O2), and their combination (UV/H2O2). UV/H2O2 treatment resulted in >7 log10 inactivation of both ARBs, with inactivation rate constants of -0.058 log10 cfu min -1 (E. coli, UV) and -0.093 log10 cfu min -1 (E. faecium, UV/H₂O₂). In contrast, ARG reduction was limited with UV alone and negligible with H₂O₂ alone. Gene copy reductions of 3 log₁₀ (blaCTX-M, k = -0.055 log₁₀ copies min⁻¹) and 2 log₁₀ (van-A, k = -0.040 log₁₀ copies min⁻¹) were observed under UV/H₂O₂. Notably, brief storage (>3 h) at pH 12.5 achieved similar ARB inactivation and ARG reduction as 80 minutes of UV/H₂O₂ treatment at pH 10.8, offering a low-energy alternative for sanitizing source-separated urine.