Current microbiological challenges in drinking water

Current microbiological challenges in drinking water Ana C. Afonso a,b,c,d , Maria J. Saavedra c , Inˆ es B. Gomes a,b , Manuel Sim˜ oes a,b , Lúcia C. Sim˜ oes d,e,* a LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr Roberto Frias, 4200-465 Porto, Portugal b ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr Roberto Frias, 4200-465 Porto, Portugal c CITAB, Department of Veterinary Sciences, University of Tr´ as-os-Montes e Alto Douro, 5000-801 Vila Real, Portugal d CEB – Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal e LABBELS - Associate Laboratory in Biotechnology and Bioengineering and Microelectromechanical Systems, Braga/Guimar˜ aes, Portugal ARTICLE INFO Editor: Rong Chen Keywords: Biofilms Disinfection by-products Distribution systems Public health Waterborne pathogens Water treatment ABSTRACT Drinking water (DW) is paramount to human health, serving as a cornerstone of public health worldwide. However, DW is not a sterile product and can harbor a large diversity of microorganisms, including pathogens. This comprehensive review addresses the critical importance of DW for human health and the ongoing challenges posed by microbial pathogens and biofilms in water distribution systems. It further analyzes the growing challenges driven by contemporary factors such as antimicrobial resistance, biofilms, climate change, and micropollutants. Biofilms, in particular, remain underestimated in conventional water treatment processes despite their significant contribution to microbial contamination, the formation of disinfection by-products, and disinfectant resistance. Emerging challenges, such as climate change and micropollutants, have become significant concerns due to their profound impact on microbial communities and their role in shaping biofilm formation. This review highlights the key microbiological threats to DW distribution systems, focusing on the role of biofilms and the emerging challenges posed by climate change. It explores the factors promoting biofilm development, including water composition, pipe materials, and treatment strategies. It also explores the limitations of current water treatment strategies, which often fail to address biofilms effectively, and highlights the need to integrate microbiological considerations into water quality management. This review aims to underscore the urgent need to reassess water treatment and management practices to address current microbiological challenges and ensure the delivery of safe and sustainable DW. 1. Introduction Water, the essence of life, is a fundamental resource indispensable for human survival and well-being [1]. However, its contamination by microbial pathogens presents a persistent and frightening challenge to public health worldwide [2]. Within the vast array of pathogens that inhabit water sources, emerging and reemerging pathogens stand out as particularly concerning entities [3]. These pathogens, spanning bacteria, viruses, fungi, and parasites, have the potential to incite widespread illness and outbreaks if not effectively monitored and controlled [3]. Notifiable diseases associated with waterborne transmission, underscore the critical importance of robust disease surveillance and intervention strategies [4]. Drinking water distribution systems (DWDS), while designed to deliver potable water, often harbor complex microbial communities, namely biofilms [5–7]. These biofilms, consisting of microbial cells embedded in a self-produced extracellular matrix, adhere to pipe surfaces and significantly impact water quality. Some negative effects on DWDS include biocorrosion and increased tolerance to disinfection [8]. A potential consequence of the interplay between these issues is the formation of disinfection by-products (DBPs). Specifically, corrosion products from the piping network and organic matter, including biofilm molecules, can significantly affect the effectiveness of disinfectants and promote the formation of DBPs [9–11]. While evidence of this relationship is still emerging, current models predicting the formation of DBPs during water treatment often underestimate the role of biofilms [12,13]. Adding to these challenges, climate change exacerbates microbial * Corresponding author at: CEB – Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal. E-mail address: [email protected] (L.C. Sim˜ oes). Contents lists available at ScienceDirect Journal of Water Process Engineering journal homepage: www.elsevier.com/locate/jwpe https://doi.org/10.1016/j.jwpe.2025.107614 Received 6 February 2025; Received in revised form 28 March 2025; Accepted 29 March 2025 Journal of Water Process Engineering 72 (2025) 107614 2214-7144/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). proliferation in aquatic environments. Rising temperatures, altered precipitation patterns, and extreme weather events create favorable conditions for biofilm formation and the dissemination of antimicrobial resistance (AMR) [14]. Also, micropollutants have increasingly become a major concern in DWDS. These trace contaminants, originating from diverse sources, persist through conventional treatment processes and can significantly impact biofilm formation, alter microbial diversity, and facilitate the spread of antimicrobial resistance genes [15,16]. Among them, microplastics serve as novel surfaces for microbial colonization, further promoting biofilm development and potentially harboring opportunistic pathogens [17]. These interrelated issues demand a deeper understanding of the environmental and infrastructural factors driving microbial dynamics in DWDS. This review addresses the multifaceted challenges associated with microbiological stability and safety in DW, with a particular focus on the role of biofilms. It explores biofilm formation and persistence, their contributions to biocorrosion and DBP production, and their function as reservoirs for opportunistic pathogens. Recognizing the complexity of these issues, the review emphasizes the need for interdisciplinary collaboration and adaptive management strategies to safeguard water quality and public health amid evolving environmental pressures. By synthesizing current knowledge and identifying research gaps, this review seeks to inspire innovative approaches and technological advancements to ensure the sustainable provision of safe DW for all. 2. Drinking water and public health 2.1. Definition, quality criteria, and treatment Water is the essential component of life, covering over two-thirds of the Earth’s surface. However, <3 % of this water is freshwater, and only about 1 % of it is accessible for use [18]. Given the growing demand for water and its limited availability, sustainable water use has become critical to ensure access to safe drinking water (DW) for present and future generations [19]. The United Nations recognizes access to safe DW as a human right [1], and the World Health Organization (WHO) emphasizes the importance of maintaining high standards for DW quality to safeguard public health [20]. By 2020, 5.8 billion people were using “safely managed” DW services, yet 771 million people still lacked basic services, particularly in low-income or informal urban settlements [2]. Addressing these disparities is central to the WHO/UNICEF Joint Monitoring Program’s Sustainable Development Goal 6, which aims to ensure the availability and sustainable management of water and sanitation for all by 2030 [21]. Safe DW is generally defined as water that does not pose significant health risks over a lifetime of consumption [22]. WHO guidelines set comprehensive quality criteria across chemical, microbiological, and physical parameters to ensure the safety of DW [20]. Chemical criteria limit contaminants such as heavy metals, pesticides, and organic pollutants, while microbiological criteria focus on preventing pathogens that can cause waterborne diseases. Physical parameters, including colour, taste, and turbidity, ensure that water is aesthetically acceptable. The WHO guidelines also stress the importance of monitoring and surveillance to maintain high standards of water quality and prevent risks to public health [20]. DW treatment plants (DWTPs) and DWDS function collaboratively to ensure the chemical and biological safety of DW, safeguarding public health [23]. DWTPs utilize a multi-barrier approach to remove contaminants and inactivate pathogens through a sequence of physical, chemical, and biological processes [23]. The treatment process typically begins with coagulation and flocculation, where chemical coagulants such as aluminum sulfate or ferric chloride are added to destabilize suspended particles and microorganisms, forming larger aggregates called flocs [24]. These flocs are then removed in the sedimentation stage, where gravitational settling eliminates a significant portion of particulate matter, including microbial contaminants [24]. Filtration follows, using materials like sand, activated carbon, or membranes to trap remaining particles and microorganisms, further enhancing water clarity and safety [25]. The final and most critical step is disinfection, which employs chemical agents such as chlorine, chloramines, or ozone, as well as non-chemical methods like ultraviolet (UV) light or advanced oxidation processes (AOPs), to inactivate bacteria, viruses, and protozoa [26]. Once treated, water enters the DWDS, where maintaining its chemical and biological integrity is vital to delivering safe water to consumers. Residual disinfectants, such as free chlorine or monochloramine, are maintained throughout the distribution system to prevent microbial regrowth and protect against contamination [27]. However, while water treatment processes are effective in ensuring the safety and quality of DW, several limitations and drawbacks challenge their efficacy and sustainability. Table 1 highlights the challenges and limitations at each stage of water treatment and distribution. These limitations highlight the need for continuous innovation in water treatment technologies, coupled with effective monitoring and maintenance strategies, to address the growing challenges for water safety and quality. 2.2. Challenges of microbial etiology in DW Each year, 1.5 million people worldwide die from waterborne diseases, with the majority being children who die due to dehydration caused by diarrhea [2]. According to the WHO, approximately 1.7 billion cases of diarrhea occur in children annually, being the second leading cause of death in children under 5 years of age [2]. Microorganisms are normally present in DWDS at relatively high concentrations (10 3 to 10 6 cells/mL) but without compromising public health [28]. This safety is ensured by a combination of water treatment processes, stringent regulatory oversight, the predominantly nonpathogenic nature of these microorganisms, competitive microbial ecology that inhibits pathogen growth, and the robustness of the human immune system [20,29–31]. Nevertheless, DWDS must be microbiologically safe and stable. In other words, a microbiologically stable DWDS must have a constant concentration and bacterial community composition, from the point of DW production to the tap [28]. However, unwanted bacterial growth inside the pipes can be responsible for adverse changes in DW and DWDS quality. The presence of some microorganisms may be responsible for some deterioration of the organoleptic properties of the water [28], which represents the main consumer complaint to DWDS companies [32]. The water distribution system itself can also be affected by the presence of bacteria, for example, the presence of biofilms in pipe walls can accelerate its corrosion [33]. In addition, biofilms can also harbor pathogenic microorganisms that can pose a danger to human health [8]. The quality of water is inherently shaped by its geographic and geological context, including factors like temperature, precipitation, leaching, and the release of elements from the crust of Earth [34]. Nevertheless, human activities have a significant impact on surface water bodies globally [34]. In rural regions of developing nations, individuals facing economic challenges frequently rely on untreated surface water for bathing, laundry, cooking, and occasionally even drinking [34,35]. Similarly, in both developed and developing countries, surface waters serve purposes such as recreational swimming, fishing, and irrigation [34,36]. Waterborne diseases can be caused by a variety of pathogens including protozoa, parasites, bacteria, and viruses [37,38]. Due to the impracticality of testing for all pathogens in all locations, monitoring efforts for polluted water often prioritize the detection of specific indicator organisms that signal the potential presence of pathogens [38,39]. One of the most employed indicators is faecal coliform bacteria [39]. While most faecal coliform bacteria are not inherently harmful, they are typically associated with human and animal waste. Furthermore, elevated levels of these bacteria often indicate the presence of harmful pathogens [20,40]. Numerous countries have acknowledged the correlation between faecal coliform bacteria and health risks, leading them to develop guidelines dictating permissible A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 2 levels of these bacteria in water sources. Evaluating the degree of pathogen pollution requires a comparison of the levels of faecal coliform bacteria against established safety benchmarks [34]. The WHO has established these benchmarks, drawing upon water quality standards from 17 countries (Table 2). However, throughout the different phases of DW systems, complex microbial communities thrive, engaging in intricate networks of interactions [41]. Additionally, as previously mentioned, existing regulations governing the microbial safety of DWDS primarily target the probability of faecal contamination. These regulations rely on conventional culture-based methodologies to detect and quantify coliforms, particularly Escherichia coli. Despite their centurylong application, these techniques have remained largely unaltered. Critically, these conventional indicators present several limitations: the vast majority of bacteria, over 99 %, evade culturing [42,43]; the emergence of non-faecal pathogens in DW; and the correlation between total coliforms and other pathogenic markers is weak [40,44,45]. The microbiome of DW is influenced by factors like source water quality, treatment processes, flow dynamics, temperature, and the presence of biofilms in the distribution system, among others [46]. Abkar et al. [27] reviewed key studies that elucidate how operational conditions shape microbial communities within DW systems. Lautenschlager et al. [47] provided foundational insights into the effects of stagnation and low flow rates, showing that these conditions promote microbial regrowth and biofilm formation, particularly in dead-end zones of the distribution network. They also observed that prolonged stagnation fosters microbial communities with increased metabolic activity, which can degrade water quality. Prest et al. [28] emphasized the selective pressures exerted by disinfectant residuals, demonstrating how different concentrations of chlorine and chloramine impact microbial diversity, often favoring resistant taxa like Nitrospira and opportunistic pathogens. Fish et al. [48] further explored the effects of temperature increases, linking higher temperatures to accelerated microbial activity, enhanced biofilm growth, and shifts in microbial community composition. The study highlighted how temperature-driven changes can increase the prevalence of Proteobacteria and other biofilm-associated taxa, raising concerns about potential pathogen proliferation. Additionally, Abkar et al. [27] highlighted studies focusing on taxa-specific responses, such as the dominance of Pseudomonas and Acinetobacter under nutrient-rich and low-disinfectant conditions, contributing to infrastructure corrosion and antimicrobial resistance. Collectively, these studies reveal the complex interplay between physical, chemical, and environmental factors in DW systems, stressing the need for dynamic management strategies that address microbial proliferation while maintaining water quality. All of the aforementioned studies underscore the critical importance of investigating the microbiome of DW. Such research not only enhances the understanding of microbial dynamics within DW systems but also holds significant implications for public health by elucidating factors influencing microbial abundance, diversity, and community structure. The microbiome of DW encompasses a diverse array of microorganisms that can significantly influence water quality, treatment processes, and human health. Understanding these changes is crucial for water utilities to ensure safe DW by comprehending their impact on microbiomes and pathogen prevalence. By elucidating the microbial ecology of water systems, one can identify potential sources of contamination, track the spread of pathogens, and develop targeted strategies for water treatment and distribution. Moreover, studying the DW microbiome can also shed light on the ecological relationships within aquatic ecosystems, contributing to broader efforts in environmental conservation and resource management [46]. 3. Waterborne pathogens 3.1. Pathogens of concern in drinking water It is estimated that there are 1407 human-infecting pathogens, with 177 being considered emerging or reemerging [49]. These pathogens encompass bacteria (538 species), viruses (208 types), fungi (317 species), parasitic protozoa (57 species), and helminths (287 species) [50]. Water, wastewater, food, and soils can harbor infectious agents, potentially contaminating DW [50]. Some potentially harmful bacteria Table 1 Limitations and drawbacks of water treatment processes in DWDS, highlighting the challenges associated with each stage of treatment and distribution. Ver este artigo e completar com info de l´ a (Microbial ecology of drinking water from source to tap). Stage Description Limitations and drawbacks References Coagulation and Sedimentation Coagulation involves adding chemical coagulants (e.g., aluminum sulfate) to destabilize and aggregate particles, followed by sedimentation to remove flocs Generates significant quantities of sludge requiring proper disposal, posing environmental and logistical challenges. Effectiveness can be reduced by fluctuations in water chemistry (e.g., pH, temperature) Prest et al. [28] Filtration Filters, such as sand and activated carbon, are used to physically remove remaining particles and microorganisms Sand and activated carbon filters may become clogged or fouled over time, reducing efficiency and requiring frequent maintenance. Membrane filtration is costly and prone to fouling and damage, increasing operational costs Prest et al. [28] Chemical Disinfection Chemical agents like chlorine, chloramines, and ozone are used to kill pathogens and provide residual protection Chlorine has reduced effectiveness against protozoan cysts (Cryptosporidium, Giardia) and reacts with natural organic matter, forming harmful DBPs. Chloramines are less effective as primary disinfectants and produce nitrosamines. Ozone lacks residual activity, making water vulnerable to recontamination and bromate formation Oliveira et al. [26] Non-Chemical Disinfection UV light or AOPs are used to inactivate microorganisms through non-chemical means UV disinfection inactivates microorganisms but does not provide residual protection, leaving water susceptible to regrowth and contamination. AOPs have high energy demands and operational costs, limiting their widespread adoption. Abkar et al. [27] Residual Disinfection Disinfectant (e.g., chlorine) is added to maintain a residual concentration (0.2–2 mg/L, max. 5 mg/L) to inhibit microbial regrowth The effectiveness of residual disinfectants can be influenced by changes in water quality, such as fluctuations in pH or temperature, and interactions with biofilms, reducing their long-term efficacy. Formation of DBPs. Does not fully prevent microbial growth and alters microbial community structure. Abkar et al. [27] Table 2 Classification of pathogen water pollution based on river concentrations of faecal coliform bacteria, utilizing benchmarks derived from water quality standards of 17 countries. Classification for water pollution Faecal coliform (CFU/100 mL) Explanation Low pollution ≤200 Generally suitable for contact (e.g. swimming and bathing) Moderate pollution 200 <x ≤1000 Appropriate only for contact during irrigation and fishing activities Severe pollution >1000 Unsuitable for contact Adapted from UNEP [34]. A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 3 that could be present in DW include Vibrio cholerae, Salmonella Typhi, Shigella spp., Campylobacter jejuni, E. coli, Yersinia enterolitica, Legionella spp., Aeromonas spp., various species of Mycobacterium (e.g. M. avium, M. tuberculosis, M. fortuitum M. abscessus, M. chelonae, M. kansasii. M. marinum, M. xenopi), and Pseudomonas aeruginosa [51,52]. One important factor for disease development is the minimal infective dose (MID) which varies widely with the type of pathogen [50]. The MID of these waterborne pathogens are presented in Table 3. Some of these previous mentioned bacteria are also included in the list of notifiable infectious diseases, requiring healthcare providers to report cases to public health authorities. Notifiable diseases, particularly those associated with waterborne transmission, pose significant challenges to public health worldwide [53]. These diseases can rapidly spread through contaminated water sources, leading to outbreaks and widespread illness [53]. Examples of such waterborne diseases include cholera caused by V. cholerae, typhoid fever caused by S. typhi, shigellosis caused by Shigella spp., listeriosis caused by Listeria monocytogenes, and campylobacteriosis caused by C. jejuni. Timely reporting of notifiable diseases is vital for effective disease surveillance, enabling public health authorities early detection of outbreaks and implementation of targeted interventions to control transmission [4]. By monitoring and promptly notifying health authorities of suspected or confirmed cases, it becomes possible to implement targeted interventions, such as water treatment and sanitation improvements, to prevent further spread and protect community health [4]. Despite the remarkable success of water treatment and sanitation programs in improving public health, sporadic cases and point-source outbreaks of waterborne diseases continue to occur [54]. According to the Centers for Disease Control and Prevention (CDC), a waterborne disease outbreak (WBDO) is an incident in which two or more epidemiologically-linked persons experience a similar illness after exposure to the same water source and epidemiologic evidence implicates the water as the likely source of the illness [55]. Table 4 shows the WBDO, with bacterial etiology, that occurred in Europe in the period between 2000 and 2023. A search in SCOPUS (November 2024) filtered using the keywords “Waterborne”, “Outbreak” and “Europe” in the title, keywords and abstract; limited to English and the period between 2000 and 2023, displayed 88 document results. Of these, 64 correspond to studies on viruses (17), chemicals (2), climate change (6), parasites (19), other continents (4) and other subjects (study tools, control strategies, projections, non-waterborne, etc.) (29). Only 11 were directly related to waterborne bacterial outbreaks. Of the 11 records found, three were associated with the genus Campylobacter, three with L. pneumophila, one with Salmonella kottbus, one with S. typhi and three with multiple aetiologies. In general, cases of campylobacteriosis and Legionnaires’ disease occurred in milder seasons. Similar to what was previously described for Campylobacter spp., infections tend to occur more frequently in warmer months, which corresponds to the ideal temperature range of 25 to 42 ◦C for Legionella [56]. Studies have observed that temperature, rainfall, and river systems play significant roles in influencing the incidence of legionellosis in various locations [57–59]. In 2006, cases of S. kottbus infections on Gran Canaria (Spain) were documented [60], predominantly affecting children under the age of one, necessitating hospitalization. Instances of outbreaks attributed to this specific Salmonella species are infrequent in the literature, with only five reported outbreaks since 1959 [61–65]. Additionally, the outbreak on Gran Canaria marked the first instance of S. kottbus associated with commercially bottled water in Europe. Also, in non-endemic countries, occurrence of typhoid fever are scarce and typically sporadic [66], resulting in limited public health outcomes. Typhoid fever, a severe systemic illness, is caused by Salmonella enterica subspecies enterica, serovar Typhi [66]. Its transmission usually occurs through faecal contamination of food or water and is prevalent in regions with inadequate sanitation [66]. On April 2022, a significant outbreak of typhoid fever emerged in the Netherlands among asylum seekers housed on a renovated vessel. Ooms et al. [67] reported that the contamination originated from a leakage of the wastewater tank into the DW tank supply. 3.2. Emerging and reemerging pathogens in drinking water Emerging and reemerging pathogens in DW refer to two distinct yet interrelated categories. Emerging pathogens are those that have only recently been identified as waterborne threats, either because they are newly discovered or because advancements in detection methods have revealed their presence and significance. These may include previously unknown microorganisms or pathogens that were not previously recognized as a concern in DW but have gained attention due to their public health implications. In contrast, reemerging pathogens are those that were once considered controlled or of limited concern but have resurged, leading to an increase in infections or an expansion in their geographical distribution [3]. In recent years, waterborne pathogens have come to light, encompassing both known pathogens originating from faecal contamination and novel pathogens arising from environmental reservoirs [54]. Various factors contribute to the resurgence or emergence of these waterborne pathogens, including shifts in human demographics, alterations in human habits, deterioration of public health infrastructures, microbial evolution, and modifications in agricultural methods [68]. For instance, urbanization and population growth can lead to increased contamination of water sources, while changes in agricultural practices such as irrigation techniques can introduce pathogens into water reservoirs. Additionally, climate change may influence the distribution and survival of pathogens in water environments, further complicating efforts to mitigate the risks associated with waterborne diseases [3,69]. The emerging and reemerging pathogenic bacteria highlighted herein (Table 5) have the potential to be transmitted through DW; however, they do not necessarily correlate with the presence of E. coli or other commonly used DW quality indicators. Typically, existing microbiological indicators used to assess DW quality may not reliably detect the presence of these emerging pathogenic bacteria, posing challenges for their identification and monitoring [54]. The species listed in Table 5 stand out for their remarkable proliferation in water systems. Their ability to form biofilms is a critical factor in their resistance to disinfection efforts and environmental stresses, enhancing their persistence in water systems. Climate change, human activities such as urbanization, agricultural runoff, and improper waste management, further contribute to the spread and resilience of these pathogens [70]. The increasing prevalence of these bacteria in water sources underscores the need for improved water quality monitoring, effective disinfection practices, and comprehensive strategies to mitigate their impact on public health [44]. Table 3 MID for harmful waterborne pathogens. Pathogen Minimal infective dose (MID) (CFU/mL) Vibrio cholerae 10 3 Salmonella spp. 10 4 –10 7 Shigella spp. 10 1 –10 2 Campylobacter jejuni ≈500 Escherichia coli 10 6 –10 8 Escherichia coli O157:H7 <100 Yersinia enterocolitica 10 6 Legionella spp. Unknown Aeromonas spp. 10 4 Mycobacterium avium 10 4 –10 7 Pseudomonas aeruginosa Unknown Based on Bitton [50], and Teunis and Figueras [168]. A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 4 4. Biofilms in drinking water systems 4.1. Definition and biofilm-associated problems Biofilms are well-organized structures of microorganisms formed through a sequence of events, starting with the adhesion of planktonic cells to a surface, followed by subsequent replication and production of an extracellular matrix of EPS [71]. However, not all biofilms develop in the same manner, especially within living organisms, in medical and industrial settings, and in nature. In these environments, biofilms can be observed as unattached aggregates [72–74]. A clear example is the formation of clusters of bacteria, commonly called granules, observed in wastewater treatment plants (WWTPs) [75]. Similarly, in marine, lake, and river ecosystems, these biofilms are often termed “marine snow” [76]. Fig. 1 illustrates an adaptation of the biofilm formation model proposed by Sauer et al. [74], where the steps described above are incorporated. The presence of biofilms in DW presents a multitude of challenges, particularly for water disinfection. Bacteria in biofilms are remarkably more resistant to conventional disinfection methods [77]. Assaidi et al. [78] studied the persistence of L. pneumophila in water systems, focusing on the role of biofilms and the effectiveness of sodium chloride disinfection. Assessing various concentrations of sodium chloride on both planktonic and sessile cells, they found that while planktonic cells showed susceptibility to sodium chloride, with survival up to 2 % concentration at 20 ◦C, biofilms were resilient, surviving and regrowing even after exposure to a sodium chloride shock. Interestingly, this resilience was more pronounced in older biofilms compared to the younger ones. Moreover, biofilms serve as reservoirs for various microorganisms, including pathogens, raising concerns about microbial contamination and associated health risks [79]. For instance, research on L. pneumophila biofilms has shown that they protect the colonizer cells from disinfection [5,7,78]. Andreozzi et al. [80] used bacterial cultures and advanced molecular biology techniques to investigate how biofilms protected the replicative form of L. pneumophila, focusing on mip gene expression. The mip gene and its encoded protein, MIP, in Legionella is crucial for understanding its role as a virulence factor, elucidating hostpathogen interactions, developing diagnostic markers, and identifying potential therapeutic targets for Legionnaires’ disease [81]. Results showed an up-regulation of mip gene under nutrient depletion, with biofilms preserving Legionella in the replicative form. Other studies have shown that biofilms in DWDS can also serve as reservoirs for other opportunistic pathogens [7,81]. However, disinfection tolerance and pathogen protection are not the only issues associated with the presence of biofilms in water systems. Biofilms also promote biocorrosion, accelerating the degradation of the piping infrastructure [8]. Biocorrosion, driven by biofilm-forming microbes like bacteria, archaea, and fungi, accelerates pipe material degradation, compromising water quality and infrastructure [82,83]. Microbes such as iron-oxidizing bacteria (IOB), sulfate-reducing bacteria (SRB), and manganese-oxidizing bacteria (MOB) contribute to corrosion by altering local chemistry. IOB oxidize ferrous iron, forming ferric hydroxide deposits that create anaerobic conditions for SRB, which produce corrosive sulfides [84,85]. MOB oxidize manganese, depositing oxides that further corrosion [86]. Biofilms, stabilized by an EPS matrix, trap nutrients and corrosion products, establish redox gradients, and create differential aeration cells, exacerbating corrosion [11,87]. These processes accelerate disinfectant decay, destabilize water quality, and increase harmful DBP production, posing health risks [11]. This topic will be further discussed in Section 5.1, as it is considered one of the emerging key issues associated with biofilm formation. 4.2. Biofilms as a source of waterborne microbiological diseases Biofilms are believed to be the primary source of microorganisms in DWDS, with an estimated 95 % of the total biomass in the water being attached to the pipe walls, while only 5 % is in the water phase [6]. In fact, after treatment procedures like chlorination, bacteria levels in water tanks are at their lowest, but they increase in tap water due to biofilms in plumbing systems [88]. The levels of colony forming units (CFU) vary, ranging from up to 10 6 CFU/mL in groundwater to between 10 2 and 10 5 CFU/mL in household tap water [88,89]. Fig. 2 presents the bacterial concentrations at key stages of the water treatment process. These stages encompass the water source, the treated water immediately post-disinfection within the holding tank, the storage tank before distribution, and finally, the water as it reaches the tap for consumption. Moreover, it provides a breakdown of the predominant and commonly found bacterial classes and genera identified within a household environment across various locations. Each area exhibits distinct microbial compositions, reflecting localized environmental conditions and usage patterns. In fact, bacterial diversity is influenced by factors like disinfectant type, temperature, flow rate, and building materials [90]. For example, in lower parts of plumbing systems, Alphaproteobacteria are dominant over Betaproteobacteria in biofilms, especially in systems with regular water consumption and high flow rates [91]. This dominance was also observed in copper pipes [91], while cross-linked polyethylene (PEX) pipes, particularly in conjunction with hot water, tend to favor Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacilli [89,91,92]. The lack of reporting and surveillance on waterborne outbreaks associated with biofilms represents a significant deficiency in public health monitoring. Recent findings from the CDC underscore the magnitude of this issue, with their report on “Surveillance of Waterborne Disease Outbreaks Associated with Drinking Water” in the United States revealing alarming statistics. Between 2015 and 2020, 87 % of reported outbreaks were linked to biofilms, accounting for 187 cases out of 214. These outbreaks were predominantly attributed to Legionella, comprising 98 % of biofilm-related cases, with isolated instances involving nontuberculous mycobacteria and Pseudomonas spp. [93]. Such data highlights the urgent need for enhanced surveillance and reporting mechanisms globally to effectively address biofilm-associated Table 4 Bacterial-related outbreaks caused by waterborne transmission in Europe between 2000 and 2020. Year Country Pathogenic bacteria Number of cases Source Reference 2000 France Multiple 202 Groundwater system contamination [169] 2002–2003 Sweden Campylobacter jejuni 101 Communal water [170] 2006 Spain Salmonella kottbus 41 Bottled water [60] 2007 Norway Campylobacter spp. 105 Vulnerability of the DW system [171] 2007 Denmark Multiple 140 DW [210] 2010 Belgium Multiple 222 DW [172] 2012 United Kingdom Legionella pneumophila 21 Spa pool device [173] 2014 Portugal Legionella pneumophila 550 Industrial wet cooling systems [174] 2019 Norway Campylobacter jejuni 2000 Municipal DW system [175] 2020–2021 Finland Legionella pneumophila 12 Tap water [176] 2022 Netherlands Salmonella Typhi 72 Wastewater leak in DW tank [67] A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 5 Table 5 Emerging and reemerging waterborne pathogenic bacteria of public health concern. Bacteria Prevalence in water sources Resistance to disinfection Biofilm formation Impact of climate change Human activities impact Emerging or reemerging status Health effects References Acinetobacter spp. 28 % of tested DW samples Resistant to chlorine Biofilm formation enhances resistance to disinfectants and antibiotics Increased survival and dissemination in warmer water Antibiotic overuse, improper waste disposal, untreated wastewater Emerging pathogen due to multidrug resistance (MDR) and survival in diverse environments Nosocomial infections; meningitis, pneumonia, endocarditis, bacteremia, skin and urinary tract infections in immunocompromised patients [99,177–180] Arcobacter spp. 35 % of surface water samples Moderate resistance to chlorine and ozone Biofilm formation enhances resistance to disinfectants and environmental stresses Increased risk of waterborne transmission due to altered water temperatures and precipitation patterns Intensive livestock farming, inadequate wastewater treatment, and agricultural runoff Emerging pathogens due to rising detection in water sources and resistance to common disinfection methods Diarrhea [181–184] Aeromonas hydrophila 18 % of DW samples Resistant to chlorination and oxidative stress Biofilm formation enhances resistance to environmental stresses and disinfectants Increased presence and virulence in warmer waters Agricultural runoff, urbanization, industrial discharges Reemerging pathogen due to persistent water presence and resistance to disinfectants and antibiotics Gastroenteritis [44,185,186] Campylobacter (including C. jejuni, C. coli, and related species) Surface water, groundwater, rural watershed runoff, marine water, rainwater, rivers, and lakes Sensitive to chlorination but persists in biofilms and lownutrient environments Biofilm formation enhances resistance to chlorination and environmental stresses Higher temperatures and intense rainfall increase cases, while heat waves and winter precipitation decrease cases Spread by improper waste management, agricultural runoff, recreational water activities, and occupational exposure in poultry farms Emerging pathogens due to increasing incidence, environmental adaptability, water source presence, and climate and human activity impacts Acute gastroenteritis [69,190,191,187–189,209,211] Helicobacter pylori 30 % of tested DW samples Survives in water for extended periods, resistance to chlorine is uncertain Biofilm formation protects against environmental stresses and disinfection Increased survival in warmer waters due to climate change Improper wastewater management and contaminated water supplies Emerging pathogen due to association with waterborne transmission and resilience in environmental conditions Gastroduodenal disease including chronic active gastritis; peptic and duodenal ulcer disease; gastric cancer [44,192,208] Legionella pneumophila High prevalence in water systems Resistant to standard disinfection, requires thermal treatment Biofilm formation provides protection against disinfectants Increased risk due to higher water temperatures Use of cooling systems, inadequate water system maintenance, urbanization Emerging pathogen due to association with modern water systems and rising incidence Legionnaire’s disease and Pontiac fever [56,191,193,194] Mycobacterium avium complex 40 % of municipal water samples Resistant to chlorine and common disinfectants Biofilm formation provides a protective niche for persistence and growth Enhanced survival in warmer water Urbanization, agricultural runoff, treated water system use Emerging pathogens due to increasing incidence in immunocompromised populations and resilience in treated water systems Pulmonary disease [44,195,196] Listeria monocytogenes 25 % of tested DW sources Resistance to chlorine and other disinfectants Biofilm formation enhances survival and resistance to environmental stresses and disinfectants Increased prevalence due to warmer temperatures and extreme weather events Spread by improper wastewater management, agricultural runoff, and food processing environments Emerging pathogen due to its resilience in various environments, biofilm formation, and increasing detection in water sources Listeriosis, abortion, human meningitis, infection during the perinatal period, granulomatosis infantiseptica, sepsis, diarrhea, pyelitis, and “flu-like” symptoms [197–199] Pseudomonas aeruginosa 42 % of hospital water systems Resistant to chlorine and other disinfectants Biofilm formation enhances resistance to antibiotics and disinfectants Increased prevalence in warmer water and higher rainfall Antibiotic misuse, poor hospital sanitation, improper wastewater treatment Emerging pathogen due to high resistance to antibiotics and disinfectants, association with healthcareassociated infections Infections in immunocompromised patients and patients with underlying diseases (i.e. wound and urinary tract infections and [191,200,201] (continued on next page) A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 6 waterborne illnesses and mitigate public health risks. 4.3. Impact of materials and treatments on biofilm formation Biofilm formation in DW systems is highly variable and influenced by numerous factors, including water composition, materials in use, treatment methods, and the presence of residual disinfectants. The chemical and microbiological composition of water plays a pivotal role; high levels of organic matter, nutrients (e.g., carbon, nitrogen, phosphorus), and ions such as calcium and magnesium often promote biofilm growth, providing substrates for microbial metabolism [6]. In contrast, low-nutrient conditions may limit biofilm density but favor microorganisms adapted to oligotrophic environments [94]. For instance, a study by Dang et al. [95] highlights that nutrient availability not only influences biofilm biomass but also alters microbial community composition, with oligotrophic environments favoring slower-growing, resource-efficient organisms. Organic carbon is often the most critical growth-limiting nutrient due to its role as the primary energy source for heterotrophic bacteria. Bioavailable organic matter (BOM), comprising simple sugars, organic acids, and complex humic substances, represents only a fraction of the dissolved organic carbon (DOC) in water, typically between 1 and 30 % [96]. Even small concentrations of BOM, such as 1 μ g C/L, can support bacterial growth, highlighting the sensitivity of microbial communities to nutrient availability [97]. Additionally, the balance of inorganic nutrients like nitrogen and phosphorus is essential, as deficiencies in these elements can limit bacterial proliferation even in the presence of sufficient organic carbon [98]. These nutrient dynamics shape microbial communities, where competition for substrates influences the diversity and resilience of bacteria in water systems. The materials used in distribution systems also significantly impact biofilm formation. For example, rough or porous surfaces like unlined cast iron facilitate microbial adhesion more than smoother materials such as polyethylene or stainless steel [99]. Polyvinyl chloride (PVC) pipes generally show reduced biofilm formation compared to copper pipes, where ion release may affect microbial communities [100]. However, material choice alone does not determine biofilm growth; factors such as pipe ageing, maintenance, and operational conditions also play critical roles. Poorly maintained systems can exacerbate biofilm formation, leading to aesthetic issues (e.g., discoloration, odor), technical problems (e.g., pipe corrosion), and potential public health risks from waterborne diseases. Studies indicated that high-density polyethylene (HDPE) and unplasticized polyvinyl chloride (PVC-U) have been increasingly preferred in modern networks due to their resistance to corrosion and cost-effectiveness, though biofilm growth on these materials can still occur under certain conditions [101]. Yet, biofilm composition and biomass vary across materials. Despite its smooth surface, research has shown that stainless steel can support significant biofilm growth under low-nutrient conditions, although electropolished surfaces reduce initial bacterial adhesion [102]. Copper pipes, known for releasing antimicrobial ions, typically harbor fewer bacteria, especially when combined with chloramine disinfection [103]. Conversely, plastic materials like PVC-P may exhibit higher biomass and microbial diversity compared to PVC-C, which is more effective in minimizing bacterial accumulation [104]. Treatment methods, such as filtration and coagulation, play a vital role in reducing organic loads, which can help mitigate biofilm formation. However, disinfection methods, such as chlorination, while effective against planktonic microorganisms, can inadvertently alter microbial community structures and promote the selection of resistant species, potentially contributing to biofilm persistence [105]. Advanced disinfection techniques, such as ozonation and UV irradiation, effectively inactivate microorganisms but lack residual disinfection capacity, leaving systems susceptible to biofilm regrowth and recontamination [106]. The choice of disinfection strategy significantly impacts biofilm formation and composition. Conventional disinfectants like chlorine and chloramine have been shown to lower biofilm diversity while selecting Table 5 (continued) Bacteria Prevalence in water sources Resistance to disinfection Biofilm formation Impact of climate change Human activities impact Emerging or reemerging status Health effects References pneumonia); tracheobronchitis in cystic fibrosis patients Stenotrophomonas maltophilia 30 % of hospital water sources Resistant to chlorine and several disinfectants Biofilm formation enhances resistance to disinfectants and environmental stresses Facilitated spread and persistence in changing water availability Antibiotic overuse, inadequate water treatment Emerging pathogen due to increasing prevalence in hospital settings and MDR Nosocomial infections, mainly respiratory tract infections [202–204] Yersinia enterocolitica 27 % of surface water samples Resistant to chlorine, especially in cold water Biofilm formation enhances survival in cold and chlorinated water Increased persistence in cold water due to climate variability Agricultural runoff, untreated wastewater, and improper waste disposal Emerging pathogen due to cold water persistence and moderate resistance to disinfection Gastrointestinal infections [69,191,205–207] A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 7 for opportunistic pathogens, highlighting the importance of maintaining effective residual concentrations to inhibit biofilm-associated risks [26]. However, the inability of current strategies to fully eradicate biofilms underscores the need for innovative approaches. Emerging technologies, including multi-barrier disinfection, bacteriophages, and nanoparticlebased treatments, offer promising solutions to limit biofilm development and control microbial regrowth [26]. Furthermore, the interaction between residual disinfectants and pipe materials, along with the degradation of chloramines, poses additional challenges for biofilm management. Addressing these factors through optimized treatment protocols and advanced materials, such as carbon quantum dots and metal-organic frameworks-based systems, could significantly improve biofilm prevention [107]. Residual disinfectants, particularly chlorine and chloramines, are essential for maintaining microbial control within DWDS. Free chlorine is the most widely used residual disinfectant worldwide, whereas monochloramine is favored in some systems due to its ability to limit disinfection by-product (DBP) formation in high organic load environments [108]. However, countries such as the Netherlands, Germany, Austria, and Switzerland avoid residual disinfectants due to concerns about DBPs, which are increasingly recognized for their potential carcinogenicity [108,109]. Interestingly, research indicates that systems using chlorine residuals, like those in the USA, report ten times more total coliform failures compared to systems without residuals, such as in the Netherlands, after adjusting for population differences [110]. This suggests that factors beyond disinfection residuals, including infrastructure age and water quality, significantly affect water safety outcomes. Although chlorine generally attenuates bacterial biofilm cell concentrations [111–113], biofilm-bound microorganisms exhibit greater resistance to disinfectants compared to planktonic cells, largely due to the protective EPS matrix. Fish et al. [48] investigated how residual chlorine influences biofilm ecology and overall water quality. The study highlighted that while residual chlorine is effective in controlling planktonic bacteria, it can inadvertently select for chlorine-tolerant species within biofilms, potentially altering microbial community structures and reducing treatment efficacy. The EPS layer not only shields biofilm cells but also plays a role in mechanical stability and influences DBP formation [114–116]. The study of Xu et al. [117] examined the interplay between biofilms and chlorine decay, showing that biofilms not only contribute to the depletion of residual chlorine but also facilitate the formation of DBPs such as trihalomethanes (THMs). Additionally, studies have shown that increasing chlorine concentrations do not necessarily reduce EPS or other biofilm components, meaning risks to water quality persist if biofilms are mobilized [116]. Therefore, biofilm formation in DW systems is shaped by water composition, materials, and treatment methods, highlighting the need for integrated strategies that address microbial dynamics, material interactions, and the limitations of current disinfection approaches. 4.4. Role of metals in biofilm formation In water systems, biofilm formation is not only influenced by organic nutrients, microbial communities, and water composition but also by the accumulation of inorganic deposits such as metals like iron (Fe), manganese (Mn), and aluminum (Al). For example, discolored water events remain a persistent challenge for the DW industry, often leading to customer dissatisfaction [118]. Ginige et al. [119] investigated the contribution of pipe wall biofilms toward Fe and Mn deposition, and discolored water events. Using biofilm reactors and field biofilm monitors designed identically, the study compared Fe and Mn accumulation Fig. 1. Schematic representation of biofilm formation highlighting bacterial aggregation. In the “Aggregation and Attachment” phase, planktonic single cells, autoaggregates, or coaggregates adhere directly to the surface or coadhere with primary colonizers, later participating in the coadhesion process. During the “Growth and Accumulation” phase, active microbial growth and the recruitment of secondary colonizers lead to microcolony formation and EPS excretion. This phase also involves the formation of coaggregation/coadhesion bridges between early colonizers. In the “Disaggregation and Detachment” phase, bacteria can leave the biofilm as aggregates or single cells, depending on the mechanism, and may integrate into other biofilms. Based on Sauer et al. [74]. A.C. Afonso et al. Journal of Water Process Engineering 72 (2025) 107614 8 on glass surfaces in the presence and absence of biofilms. In nonchlorinated reactors, biofilms significantly enhanced Mn deposition, with biofilm-covered surfaces accumulating 33 times more Mn than those exposed to lower Mn concentrations. Conversely, Fe deposition was only about twice as high under high Fe conditions, suggesting that Mn accumulation is more influenced by biofilm activity. Chlorinated reactors exhibited a 95 % reduction in biofilm biomass, yet Mn deposition remained 17 times higher in reactors with elevated Mn concentrations, indicating abiotic deposition mechanisms. Upon chlorination of previously biofilm-rich systems, Fe and Mn concentrations in effluent water increased six-fold, demonstrating that biofilm detachment could lead to metal release and potential water discoloration events. Aluminum, often introduced through water treatment processes or the use of aluminum-based coagulants [120], can also influence biofilm formation. It has been shown to promote microbial adhesion to surfaces and strengthen the structural stability of the biofilm matrix by interacting with EPS and bacterial cell surfaces [121]. Cui et al. [121] examined the effects of low concentrations of residual Al(III) on biofilm formation, exploring mechanisms potentially relevant to biofouling in WTPs and reuse systems. At concentrations below 2.0 mg/L, Al(III) stimulated bacterial growth by approximately 3.7 times and enhanced EPS production, promoting biofilm development. Al(III)-induced flocculation facilitated bacterial aggregation, leading to rapid biofilm formation, with biofilm thickness increasing to 103.0 ±7.1 μ m and 112.0 ±5.7 μ m at 0.6 and 2.0 mg-Al/L, respectively, compared to 61 ±4.24 μ m in control conditions. The zeta potential of bacterial cells decreased with increasing Al(III) concentrations, indicating charge neutralization and reduced electrostatic repulsion, thereby enhancing microbial adhesion. Notably, Al(III) interacted strongly with bacterial outer membranes, causing structural damage, protein leakage, and ATP depletion by up to 55.91 %, leading to a distinct double-layer biofilm structure with inactivated cells and proteins at the bottom and polysaccharides with active cells at the top. The presence of inorganic deposits can also alter the microbial composition within biofilms, promoting the growth of specific metal-tolerant microorganisms that can thrive in the altered chemical environment. Recently, Li et al. [122] investigated the impact of trace metals on water quality and microbial risks by conducting a 168-hour experimental simulation, where biofilms developed on glass beads in tap water supplemented with Fe(III) and Al (III), followed by metagenomic analysis. The study revealed that Fe(III) significantly increased turbidity, while both Fe(III) and Al(III) promoted biofilm growth and enhanced the production of EPS. Bacterial communities, dominated by Pseudomonadota, exhibited adaptive mechanisms in response to metal-induced stress, with Al(III) having a more pronounced effect on bacterial proliferation than Fe(III). The presence of Al(III) also increased the relative abundance of pathogenic bacteria, such as S. enterica and P. aeruginosa, raising concerns about potential health risks. Moreover, Fe(III) facilitated the co-occurrence of antibiotic resistance genes and pathogens, increasing the overall risk of antibioticresistant bacteria in DW systems. These studies collectively underscore the significant role of inorganic deposits in shaping biofilm formation and stability by not only enhancing the accumulation of metals but also contributing to their remobilization. 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