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

Abstract

The coexistence of pharmaceuticals and microorganisms in source separated urine poses a risk for the development of antimicrobial resistance (AMR), especially when urine-based fertilizers 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 (bla _{CTX - 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 (bla _{CTX - 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 min of UV/H₂O₂ treatment at pH 10.8, offering a low-energy alternative for sanitizing source-separated urine.

Keywords: fertilizer; hygienisation; microbial risk; pathogens; safe nutrient recycling; source separation; wastewater.

Figure 1

Inactivation of pathogens in KOH alkalized human urine (pH 10.8) exposed to treatments of UV, UV/H₂O₂, and H₂O₂, and control for β lactamase producing E. coli (A), and Vancomycin resistant E. faecium (B). UV irradiation was done using 65 W low pressure high output mercury lamps emitting light radiation at 185 and 254 nm. Experiments involving H₂O₂ treatment were dosed with 1.25 g H₂O₂ L⁻¹. Hollow markers show plate count results that are below the detection limit. Inactivation kinetics were predicted using Equations 1, 2, represented by broken lines, E. coli (Blue) and E. faecium (orange). Shaded regions represent prediction interval for inactivation models for treatments of UV (blue) and UV/H₂O₂ (orange). The shaded area shows the 95% prediction interval derived from the fitted model and reflects uncertainty in parameter estimates, not experimental variation from replicated samples.

Figure 2

Degradation of ARGs in KOH alkalized urine subjected to treatments of UV, UV/H₂O₂, H₂O₂ and control for bla_{CTX − M} gene at pH 10.8 (A) and pH 7.0 and 12.5 (B), and van-A gene at pH 10.8 (C) and at pH 7.0 and 12.5 (D). Degradation kinetics are represented by broken lines with shaded regions showing prediction intervals for UV + H₂O₂ (orange) and UV (blue) treatment, respectively. UV irradiation is done using 65 W low pressure high output mercury lamps emitting photons at 185 and 254 nm. Samples involving H₂O₂ treatment were dosed with 1.25 g H₂O₂ L⁻¹. Standard deviations are color coded inaccordance with the treatment type.