Abstract:

Biofilms are nearly ubiquitous across all environments within the biosphere, with over 80% of surface contaminats in aquatic systems manifesting as biofilm pollutants. Approximately 60%-80% of these biofilms exist as mixed-species biofilms, which may simultaneously harbor antibiotic-resistant bacteria (ARB) and antibiotic-susceptible bacteria (ASB), making them critical reservoirs for antibiotic resistance genes (ARGs) in the environment. Bacteria within biofilms are embedded in a self-produced extracellular polymeric substance (EPS) matrix that facilitates adhesion to surfaces. Composed primarily of polysaccharides and proteins, EPS provides mechanical stability to biofilms, mediates surface attachment, and forms a cohesive three-dimensional polymer network that interconnects and temporarily immobilizes biofilm cells. The growth of biofilm-associated bacteria can be enhanced through EPS production, and biofilms differentiate from their planktonic counterparts by regulating the bacterial growth rates. The stable structure of biofilms offers a protective barrier for embedded bacteria, conferring “emergent properties” such as enhanced rates of antibiotic resistance gene exchange and increased tolerance to environmental stressors, posing significant challenges to public health systems. Furthermore, biofilm formation substantially promotes the horizontal gene transfer (HGT) of ARGs carried by ARB, accelerating their spread. Consequently, disrupting the structural integrity of mixed biofilms and inhibiting ARGs transmission are essential for mitigating biofilm-related contamination. Ultraviolet (UV) irradiation and chlorine disinfection are widely regarded as effective oxidative technologies for eliminating bacterial contamination in public water-treatment systems. However, existing research has predominantly focused on the mechanisms by which environmental factors and oxidative disinfection influence the HGT of resistance plasmids among distinct bacterial species, while studies on ARGs transfer within biofilms under varying stimuli remain limited. Additionally, investigations into oxidative disinfection have revealed that although bacteria undergo oxidative stress responses leading to oxidative damage, the magnitude of oxidative stress exerts differential effects on bacterial survival and behavior. Therefore, it is imperative to examine how intracellular reactive oxygen species (ROSs) levels in biofilms during oxidative disinfection processes influence the HGT of ARGs. Variations in disinfectant dosage are also critical for understanding the impact of oxidative technologies on ARGs transfer within biofilms. Given these gaps, this study emphasizes the need to investigate the effects of varying disinfectant doses on bacterial growth dynamics and oxidative stress responses within mixed biofilms, while elucidating the underlying mechanisms of biofilm removal. Such insights are vital for advancing strategies to control biofilm contamination and curb the spread of antibiotic resistance in aquatic environments. To address this objective, this study employed mixed Escherichia coli biofilms as the research subject and utilized ultraviolet/chlorine (UV/chlorine) combined disinfection as the oxidative treatment to investigate the effects of varying chlorine concentrations on mixed E. coli biofilms. E. coli, one of the primary opportunistic pathogens responsible for biofilm-associated infections, was selected as the model organism. Mixed biofilms were cultivated in a flow reactor using antibiotic-resistant E. coli DH5α (CTX) and antibiotic-sensitive E. coli K12 strains to simulate real-world aquatic environments. During UV/chlorine disinfection, the available chlorine (AC) concentrations were controlled at 0.5, 1, 2, 3, and 5 mg·L⁻¹ in accordance with the Chinese national standard GB 5749—2006. Key parameters were quantified using multiple methodologies: biofilm biomass via crystal violet staining, bacterial viability via the WST-1 assay, EPS content via phenol-sulfuric acid (for polysaccharides) and Lowry methods(for proteins), ARGs conjugative transfer frequency, and intracellular reactive oxygen species (ROS) levels via DCFH-DA fluorescence. Statistical significance was assessed using ANOVA and Bonferroni-corrected Student’s t-test. This study systematically investigated the inhibitory effects and mechanisms of UV/chlorine disinfection at varying chlorine concentrations on mixed E. coli biofilms, focusing on biofilm biomass, metabolic activity, EPS composition, conjugative transfer of ARGs and oxidative stress responses. The results demonstrated that UV/chlorine combined disinfection exhibited superior inhibitory effects on biofilms compared to UV treatment alone. At 24 h, the biomass of mixed biofilms treated with UV/chlorine at 5 mg·L⁻¹ available chlorine (AC) was reduced to 50% of that observed in the untreated control group. Compared to the control, UV/chlorine disinfection with 5 mg·L⁻¹ AC decreased the total bacterial count in biofilms from 5.4×10⁹ CFU·mL⁻¹ to 9.2×10⁸ CFU·mL⁻¹ (representing an 83% reduction) at 24 h. Bacterial growth activity assays within biofilms further revealed that the inhibitory effects on bacterial viability intensified with increasing AC concentrations. Additionally, under UV/chlorine treatment with 5 mg·L⁻¹ AC, the extracellular polymeric substance (EPS) content in mixed biofilms decreased by 45%, from 1 487 μg·mL⁻¹ to 821 μg·mL⁻¹, within 24 h. These findings collectively demonstrate that UV/chlorine disinfection effectively inhibits bacterial proliferation within biofilms and destabilizes their structure. In the untreated control group, as the mixed biofilm formed, the conjugative transfer frequency of antibiotic resistance genes (ARGs) within the biofilm increased 20-fold (from 8.0×10⁻⁷ to 1.6×10⁻⁵) over 24 h. Under UV disinfection alone, the ARGs transfer frequency increased 46-fold (from 8.0×10⁻⁷ to 3.7×10⁻⁵) during the same period. When UV/chlorine disinfection was used with available chlorine (AC) concentrations below 1 mg·L⁻¹, the ARGs transfer frequency in mixed biofilms also increased within 24 h. This may be attributed to membrane permeability changes induced by low-level oxidative stress from sublethal disinfectant concentrations, which enhance ARGs transfer within biofilms. In contrast, UV/chlorine disinfection with AC≥1 mg·L⁻¹ effectively suppressed ARGs dissemination. Particularly at 5 mg·L⁻¹ AC concentration, the UV/chlorine treatment reduced the ARGs transfer frequency by approximately 99.7% (from 1.1×10⁻⁵ to 3.8×10⁻⁸) within 24 h. Oxidative stress analysis revealed that the maximum reactive oxygen species (ROS) levels generated by UV/chlorine at 5 mg·L⁻¹ AC were 3.3 times higher than those produced by UV alone. This synergistic effect was likely due to the co-generation of multiple reactive species by UV/chlorine, which intensified oxidative damage in bacterial cells. These findings demonstrate that high-concentration UV/chlorine disinfection causes severe oxidative damage, leading to reduced bacterial viability and population density in mixed biofilms. Moreover, treatment with higher concentrations disrupted the biofilm structure by decreasing both the quantity and composition of extracellular polymeric substances (EPS), as evidenced by the change in the polysaccharide-to-protein (PS/PN) ratio from 4.4 to 3.5, thereby compromising the biofilm adhesion and structural integrity. In conclusion, this study provides actionable insights for optimizing UV/chlorine disinfection in water treatment systems. By increasing chlorine concentrations (≥1 mg·L⁻¹ AC), the technology can effectively mitigate biofilm contamination, destabilize EPS, and suppress ARGs dissemination. These outcomes advance our understanding of the mechanisms of oxidative disinfection in mixed biofilms and offer practical strategies for enhancing drinking water safety and wastewater management.

Key words: UV/chlorine, Escherichia coli mixed biofilm, extracellular polymeric substances, conjugative transfer, oxidative stress

摘要： 细菌生物膜是水体中介质表面污染的主要形式（占比>80%），也是耐药性传播的重要载体。该文从生物膜生物学特性、生物膜内抗生素耐药基因（antibiotic resistance genes，ARGs）接合转移频次以及氧化应激水平等角度探讨了紫外/氯联用消毒方法中不同氯浓度对大肠杆菌（Escherichia coli）混合生物膜的抑制作用及其机制。结果表明，紫外/氯联用消毒能有效抑制大肠杆菌混合生物膜的生物量和生长活力。相较于正常培养至24 h的大肠杆菌混合生物膜，有效氯（Available Chlorine，AC）质量浓度增加至5 mg·L⁻¹时，紫外/氯联用消毒将生物膜内的菌群总数由5.4×10⁹ CFU·mL⁻¹降至9.2×10⁸ CFU·mL⁻¹，降低了83%；并且将大肠杆菌混合生物膜的胞外基质（extracellular polymeric substances，EPS）质量浓度由1 487 μg·mL⁻¹降至821 μg·mL⁻¹。接合转移实验结果表明，相较于常规培养，随着AC浓度的增加，紫外/氯联用消毒能有效降低大肠杆菌混合生物膜内ARGs的接合转移频次，最高时可降低99.7%。此外，含有不同AC浓度的紫外/氯联用消毒均能够造成大肠杆菌混合生物膜氧化应激水平的上升。综上，紫外/氯联用消毒可以通过对大肠杆菌混合生物膜产生氧化损伤从而抑制混合生物膜的菌群数量和EPS浓度，并能够有效抑制大肠杆菌混合生物膜内ARGs的接合转移频次，这为控制和消除水体中的生物膜污染提供了参考。

关键词: 紫外/氯, 大肠杆菌混合生物膜, 胞外基质, 接合转移, 氧化应激