Introduction
As a reservoir of domestic, livestock, medical and industrial wastes, wastewater accounts for significant interactions among animals, plants, humans and the environment. The interconnection among humans, animals, plants and their shared environment is a significant component of the concept of One Health. According to the US Centers for Disease Control and Prevention and the One Health Commission, ‘One Health is defined as a collaborative, multisectoral, and transdisciplinary approach – working at the local, regional, national, and global levels – with the goal of achieving optimal health outcomes recognizing the interconnection between people, animals, plants, and their shared environment’ (Mackenzie & Jeggo Reference Mackenzie and Jeggo2019). It focuses on the consequences of all actions at the animal–human–ecosystems interface, and wastewater is a common endpoint to many such interactions. Antimicrobial resistance (AMR) is one of these consequences, that it represents a significant policy and public health threat (Mackenzie & Jeggo Reference Mackenzie and Jeggo2019).
Wastewater treatment (WWT) plays an often-underappreciated role in the daily lives of people across the globe. It is designed to remove physical, chemical and biological wastes from wastewater. The wastewater entering a WWT plant (WWTP; i.e., the influent) is systematically treated to ensure the downstream aquatic ecosystems are not burdened by these harmful wastes when the treated wastewater leaving the WWTP (i.e., the effluent) is discharged into them. Vital to the process of municipal WWT is disinfection, which inactivates pathogens and reduces the likelihood of the spread of waterborne diseases. The disinfection of wastewater in a WWTP is achieved chemically through the use of chlorine, chlorine dioxide and peracetic acid, as well as other technologies such as ultraviolet light and ozonation. The combination of its low cost and ease of use make chlorination preeminent in this area (Collivignarelli et al. Reference Collivignarelli, Abba, Benigna, Sorlini and Torretta2018, Gelete et al. Reference Gelete, Gokcekus, Ozsahin, Uzun and Gichamo2020).
The biocidal mode of action of chlorine involves compromising the membrane permeability of bacteria, which results in the leakage of intracellular constituents that include nucleic acids (Jin et al. Reference Jin, Liu, Wang, Yang, Liu and Yin2020, Adefisoye & Olaniran, Reference Adefisoye and Olaniran2022). The mode of action of alternative disinfection technologies such as ozonation and peracetic acid is similar; they are strong oxidizers and damage the cell membranes, leading to the leakage of intracellular constituents (Collivignarelli et al. Reference Collivignarelli, Abba, Benigna, Sorlini and Torretta2018).
Despite its preeminent use in WWT, the very mode of action of chlorine facilitates the evolution and propagation of AMR. Disinfectants can be considered micropollutants of concern, as their continuous presence at sublethal concentrations enables the selection of resistant phenotypes of bacteria, especially in a complex environment such as wastewater (Mutuku et al. Reference Mutuku, Gazdag and Melegh2022). AMR is the ability of a microorganism to withstand the curative and therapeutic effects of previously effective drugs, rendering existing antimicrobial interventions ineffective (Tang et al. Reference Tang, Millar and Moore2023, Ahmed et al. Reference Ahmed, Hussein, Qurbani, Ibrahim, Fareeq, Mahmood and Mohamed2024). The leakage of intracellular constituents during disinfection increases the availability of all genetic material, including any AMR genes (ARGs), which, being freely available, are taken up by other bacteria in a process called ‘natural transformation’. This involves the uptake of naked DNA by a microorganism, which is usually released from another microorganism (Levy & Marshall Reference Levy and Marshall2004). Newly acquired ARGs are transferred to the next generation by a process called ‘vertical transmission’. Most importantly, the process of disinfection also results in the selection of disinfection-resistant bacteria (DRB; Jin et al. Reference Jin, Liu, Wang, Yang, Liu and Yin2020, Jathar et al. Reference Jathar, Shinde, Dakhni, Fernandes, Jha, Desai and Jobby2021, Adefisoye & Olaniran, Reference Adefisoye and Olaniran2022). Recent studies have identified the role of free chlorine in the dissemination of AMR through natural transformation. Zhang et al. (Reference Zhang, Wang, Lu, Yu, Song, Bond and Guo2021a) demonstrated that chlorine-based disinfectants (CBDs) increased the natural transformation of environmentally available ARGs by a previously susceptible strain of Acinetobacter baylyi ADP1. The observed increase in the rates of transformation of ARGs is a culmination of increases in the levels of reactive oxygen species (ROS), cell membrane damage, ROS-mediated DNA damage and elevated stress responses, all in response to exposure to CBDs (Zhang et al. Reference Zhang, Wang, Lu, Yu, Song, Bond and Guo2021a).
The COVID-19 pandemic significantly enhanced the public perception of the need for personal and surface hygiene, and this resulted in a substantial increase in the global use of antimicrobials. This greater use of antimicrobials has increased their discharge into wastewater systems and the environment. However, the levels of antimicrobials in wastewater systems are seldom monitored, even in developed countries. Nevertheless, the bacterial communities in wastewaters are constantly exposed to antimicrobials, and this eventually results in the selection of resistant phenotypes (Van Dijk et al. Reference Van Dijk and Verbrugh2022). This Perspective article aims to identify the One Health and public health policy challenges of the increasing presence of antimicrobials in wastewater systems. Emphasis is placed on CBDs owing to their increased use during the COVID-19 pandemic. Knowledge gaps of significant policy and public health importance to the scientific community are identified after carefully reviewing the existing literature.
Recent trends in the use of disinfectants
The United States Environmental Protection Agency (US EPA) approved many different chemicals for use as disinfectants during the COVID-19 pandemic. These include ethanol, isopropanol, hydrogen peroxide, quaternary ammonium compounds, sodium hypochlorite (NaClO), hypochlorous acid and CBDs (Dewey et al. Reference Dewey, Jones, Keating and Budhathoki-Uprety2021). Due to their availability and ability to inactivate enveloped viruses such as SARS-CoV-2, CBDs were some of the most used disinfectants during the pandemic (La Rosa et al. Reference La Rosa, Bonadonna, Lucenitni, Kenmoe and Suffredini2020). The global use of CBDs was worth approximately USD 7.3 billion in 2024 (Market Research Future 2024), and it is projected to reach USD 10.15 billion by 2034. Municipal WWTPs accounted for 35% of the CBD market revenue (Market Research Future 2024). This rapid growth is also being facilitated by the use of CBDs in healthcare (disinfecting surfaces, instruments and medical waste), food processing (sanitization of equipment, packaging and food contact surfaces), drinking and WWT systems and hospitality (cleaning and maintenance). Thus, the role of WWTPs in the presence, persistence and propagation of AMR needs to be investigated, with the main reasons for this being the presence of antimicrobial-resistant bacteria (ARB) in wastewater systems due to the constant discharge of antimicrobials and ARB, the inefficiency of current disinfection technologies to completely remove ARB and the evolution of AMR due to the very mode of action of disinfection technologies (Novo & Manaia Reference Novo and Manaia2010).
An unintentional and inevitable consequence associated with the increased use of disinfectants is their misuse. More than one in three adults in the USA are likely to be using disinfectants in an unsafe manner (Kuehn Reference Kuehn2020); they may mix multiple chemical products, use higher concentrations than recommended, use them in poorly ventilated areas and with improper protection or use them often on a daily basis for surface cleaning applications (Chang et al. Reference Chang, Schnall, Law, Bronstein, Marraffa and Spiller2020, Kuehn Reference Kuehn2020).
The increased use of disinfectants has meant an increase in their discharge into wastewater and the environment. For example, up to 0.4 mg/L of residual chlorine was detected in lakes in China during the height of the pandemic owing to this increased use of CBDs (Yin et al. Reference Yin, Wang, Zhang and Lei2020). This is 20 times higher than the level of chlorine known to exert acute toxic effects on freshwater organisms (0.019 mg/L), and it is 35 times higher than the level of chlorine known to exert chronic toxic effects on freshwater organisms (0.011 mg/L; US EPA 2014). The presence of residual disinfectants also disrupts the nitrogen cycle in aquatic ecosystems by inactivating the bacteria involved in the continual transformation of nitrogenous compounds (Chu et al. Reference Chu, Fang, Deng and Xu2021). Additionally, residual chlorine reacts with natural organic matter in wastewater to form disinfection byproducts (DBPs), including regulated compounds such as trihalomethanes and haloacetic acids, as well as unregulated but more toxic compounds such as haloacetaldehydes, haloacetonirtiles and halonitromethanes (Zhang et al. Reference Zhang, Zhou, Han, Guo, Wu and Fang2021b). As much as the process of chlorination is known to facilitate the propagation of AMR, the same was observed in the case of regulated DBPs. Bromoacetic acid (BAA), a regulated DBP, facilitates the transformation and uptake of environmental DNA by A. baylyi ADP1 (Mantilla-Calderon et al. Reference Mantilla-Calderon, Plewa, Michoud, Fodelianakis, Daffonchio and Hong2019). This phenomenon is concentration dependent and is attributed to the ability of BAA to induce DNA damage via oxidative stress; the increased transformation rate occurred in response to the DNA damage (Mantilla-Calderon et al. Reference Mantilla-Calderon, Plewa, Michoud, Fodelianakis, Daffonchio and Hong2019). Elevated levels of chlorine in influents have also negatively affected the performance of an aerobic biological sewage treatment process by altering microbial communities in the activated sludge due to the presence of chlorine (Dang et al. Reference Dang, Zhang, Zheng, Meng, Wang and Zhong2023).
Increased discharge of CBDs into wastewater: data gaps and challenges to One Health
There are critical data gaps pertaining to the presence of elevated levels of CBDs in WWTPs and the challenges they pose to the success of One Health. Those gaps are outlined in the following questions:
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(1) Is the selection of chlorine-resistant bacteria (CRB) being facilitated by elevated levels of chlorine in wastewater influents? To date, only one study has attempted to evaluate the concentration of residual chlorine in the influents of WWTPs: Dang et al. (Reference Dang, Zhang, Zheng, Meng, Wang and Zhong2023) detected residual chlorine concentrations as high as 0.22 mg/L in the influent sewage of WWTPs in Wuhan (China) during February 2020. Hypochlorite is quickly inactivated in the presence of organic matter, which is abundant in wastewater (Ghafoor et al. Reference Ghafoor, Khan, Khan, Ualiyeva and Zaman2021). Detection of measurable concentrations of residual chlorine, considering the short half-life and low persistence of hypochlorite in wastewater, suggests that the levels of CBDs being discharged into wastewater are increasing. This also indicates why it is important to investigate the levels of chlorine in the influents of WWTPs. The exposure to sublethal concentrations of an antimicrobial often results in the selection of resistant phenotypes (Mutuku et al. Reference Mutuku, Gazdag and Melegh2022, Van Dijk et al. Reference Van Dijk and Verbrugh2022). The selection for CRB in WWTP influents renders the disinfection step completely ineffective, compromising the very objectives of WWT. The chlorine disinfection step is known to be one of the main reasons for the selection of CRB in WWTPs (Luo et al. Reference Luo, Wu, Yu, Wang, Chen and Tong2021). This is a major public health policy issue.
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(2) Is the presence of low levels of chlorine in WWTP influents inducing the expression of a viable but non-culturable (VBNC) state in bacteria? Wastewater is a stressful environment for bacteria due to the presence of antibiotics and disinfectants. The constant presence of xenobiotic stressors induces a VBNC state in bacteria – a phenotypic manifestation of bacteria with reduced or halted metabolism (Chebotar et al. Reference Chebotar, Emelyanova, Bocharova, Mayansky, Kopantseva and Mikhailovich2021). Exposure to levels of chlorine as low as 0.5 mg/L has induced a VBNC state in Escherichia coli. An unintended consequence is that cells in the VBNC state overexpress genes that confer resistance to a broad spectrum of antibiotics (Lin et al. Reference Lin, Ye, Chen, Zhang and Yu2017, Ye et al. Reference Ye, Lin, Zhang, Chen and Yu2020). Considering the increase in the use and discharge of antibiotics and CBDs in wastewaters, the formation of VBNC bacteria in wastewater influents is another important issue that needs to be investigated.
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(3) Are increasing levels of chlorine in WWTP influents facilitating the emergence of multidrug-resistant bacteria? Low levels of chlorine damage the cell membrane (Ye et al. Reference Ye, Lin, Zhang, Chen and Yu2020), resulting in the leakage of intracellular constituents. Bacteria harbouring ARGs are frequently discharged into wastewater influents. This is likely to increase the availability of ARGs in wastewater influents. The uptake of freely available ARGs by previously susceptible phenotypes could result in the selection of multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria (Magiorakos et al. Reference Magiorakos, Srinivasan, Carey, Carmeli, Falagas and Giske2012). Klebsiella pneumonia exposed to a minimum inhibitory concentration (MIC; 275 mg/L) and sub-MIC of NaClO for 60 days demonstrated resistance not only to NaClO, but also to the antibiotics erythromycin, polymyxin B, gentamicin, tetracycline and ciprofloxacin (Chen et al. Reference Chen, Zhang, Mao, Wang and Luo2024). Although it would be very unlikely to detect such high concentrations of NaClO in wastewater systems, the constant discharge and presence of CBDs nevertheless are pressing concerns.
Conclusions
The global use of CBDs is very unlikely to decrease owing to their low cost and high availability. This situation leads to an increased discharge of CBDs into wastewater, the consequences of which are not at all clear. The constant presence of sublethal concentrations of CBDs in wastewater eventually facilitates the selection of DRB. We attempt to identify a few data gaps regarding the presence, persistence and propagation of disinfectant resistance in wastewater influents. The selection for DRB in these influents not only defeats the very purpose of WWT, but may also result in the development of multidrug-resistant bacteria. Significant data gaps remain, and the bridging of these is important from a public health and environment standpoint.
Acknowledgements
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Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare none.
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