Free-living amoebae (FLA) are phagocytic protists that play crucial roles in microbial communities as significant microbial grazers. However, our current knowledge of their diversity, ecology, and population genetic structures is marginal due to the shallow and biased sampling of ecosystems and the use of few, poorly resolving molecular markers. Thirty-two FLA were isolated from soil and water samples collected across representative ecosystems of the State of Morelos in Central Mexico, including the drinking water distribution system (DWDS) from the state capital. We classified our isolates as members of Acanthamoeba, Vermamoeba, Naegleria, and Tetramitus by 18S ribosomal DNA (rDNA) sequencing. Vermamoeba isolates were recovered exclusively from the DWDS samples. In contrast, Naegleria strains displayed a broad distribution in soil and water samples across the natural ecosystems. We used a combination of phylogenetic and population genetic analyses of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (COI) sequences from our isolates and a comprehensive set of reference sequences to analyze the currently known diversity of Naegleria spp. Significant associations were uncovered between the most prevalent lineages of Naegleria and Vermamoeba and broad ecological and geographical variables at regional and global levels. The population structure and cryptic diversity within the Naegleria galeacystis-Naegleria americana and Vermamoeba vermiformis species complexes were thoroughly analyzed. Our results prove that the genus Vermamoeba, which was previously thought to consist of only one species, actually encompasses at least seven widely distributed species, as indicated by consistent evidence from Bayesian phylogenetics, two species-delimitation programs, and population genetics analyses. IMPORTANCE Our study sheds new light on the population genetic structure of V. vermiformis and diverse Naegleria species. Using improved molecular markers and advanced analytical approaches, we discovered that N. americana, previously considered a single species, actually contains multiple distinct lineages, as revealed by COI sequencing. These lineages are highly differentiated, with little gene flow between them. Our findings demonstrate that the genus Vermamoeba holds multiple cryptic species, requiring a significant taxonomic revision in light of multilocus sequence analyses. These results advance our understanding of the ecology, molecular systematics, and biogeography of these genera and species complexes at both regional and global scales. This study has significant implications for diagnosing amoebal infections and evaluating health risks associated with FLA in domestic and recreational waters.

An experimental drinking water distribution system (DWDS) was used to evaluate the evolution of particle size distribution (PSD) and basic quality parameters of ultrafiltered water with or without pre-ozonation. An ultrafiltration (UF) module was set up, associated with a pre-ozonation system (3.7 g O3 /m3 ). The permeate was circulated in the DWDS (300 m; 0.9 m/s) with 0.4 mg/L of chlorine, and the analysis of the PSD was performed using a β-variable mathematical model. A better control of membrane fouling was obtained with pre-ozonation, and PSD was necessary to observe water quality differences between permeates and in the DWDS. A decrease in particle concentration of 1.8 logarithms was obtained with the application of UF membranes, while a decrease of only 1.2 logarithms was obtained with pre-ozonation. The system without pre-ozonation showed a higher efficiency at removing smaller particles (around 2 μm), with the absence of particles larger than 23 μm during both stages. The PSD revealed a worsening of water quality in the DWDS with an increase of particles smaller than 5 μm during the application of UF membranes, while with pre-ozonation, all particle sizes analyzed increased their concentration. PRACTITIONER POINTS: Pre-ozonation led to a better control of membrane fouling, but a worsening of permeate quality according to particle size distribution. Pre-ozonation does not improve the turbidity, dissolved organic carbon or UV254 removal capacity of ultrafiltration during drinking water treatment. Particles size distribution reveals the deterioration of water quality in a drinking water distribution system better than turbidity or DOC. Ozone prior to ultrafiltration membranes led to a worsening of permeate quality, more significant in the drinking water distribution system.

Water quality deterioration of drinking water distribution systems (DWDSs) caused by water source switching has been reported previously. However, systematic investigation of the biostability of DWDS under water source switching is limited. Aged pipes, including three commonly used pipe materials dug out from a practical DWDS, were used to systematically investigate the biofilm stability mechanism of DWDS under water source switching to quality-improved water. An increase in adenosine triphosphate (ATP) concentration in the bulk water during the initial stage of the switching period was observed, indicating the risk of biofilm release through aged pipe surfaces after water source switching. Sloughing of biofilms might contribute to temporary instability. From day 35, the ATP concentration in the polyethylene (PE) and plastic stainless steel composite (PS) pipes were maintained at approximately 2.40 and 2.56 ng/L, respectively. In contrast, the ATP concentration in the ductile iron (DI) pipes was higher, at approximately 3.43 ng/L from day 42. The water quality variation could cause areas of the biofilm to slough and reduce the biomass of biofilm, causing partial alteration of the microbial community. 16S rRNA gene amplicon sequencing-based functional prediction revealed that the biofilm could increase the abundance of chlorine-resistant bacteria attributed to the increase in Pseudomonas and Methylobacterium after switching to quality-improved water. Moreover, the profiles of specific pathways linked to human diseases, antibiotic resistance, and antibiotic biosynthesis revealed that the safety of the biofilm could improve after switching to quality-improved water. KEY POINTS: • The PE and PS biofilm showed improved resistance to water quality perturbation. • Greater number of Methylobacterium was found in the biofilm after water source switching. • 3.16S gene-based metagenomics prediction revealed that the safety of the biofilm under water source switching.

Information on the co-occurrence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) among bacterial communities in drinking water distribution systems (DWDSs) is scarce. This study characterized ARGs and MRGs in five well-maintained DWDSs in Finland. The studied DWDSs had different raw water sources and treatment methods. Two of the waterworks employed artificially recharged groundwater (ARGW) and used no disinfection in the treatment process. The other three waterworks (two surface and one groundwater source) used UV light and chlorine during the treatment process. Ten bulk water samples (two from each DWDS) were collected, and environmental DNA was extracted and then sequenced using the Illumina HiSeq platform for high-throughput shotgun metagenome sequencing. A total of 430 ARGs were characterized among all samples with the highest diversity of ARGs identified from samples collected from non-disinfected DWDSs. Furthermore, non-disinfected DWDSs contained the highest diversity of bacterial communities. However, samples from DWDSs using disinfectants contained over double the ratio of ARG reads to 16S rRNA gene reads and most of the MRG (namely mercury and arsenic resistance genes). The total reads and types of ARGs conferring genes associated with antibiotic groups namely multidrug resistance, and bacitracin, beta-lactam, and aminoglycoside and mercury resistance genes increased in waterworks treating surface water with disinfection. The findings of this study contribute toward a comprehensive understanding of ARGs and MRGs in DWDSs. The occurrence of bacteria carrying antibiotic or metal resistance genes in drinking water causes direct exposure to people, and thus, more systematic investigation is needed to decipher the potential effect of these resistomes on human health.

UNASSIGNED: Legionella are Gram-negative bacteria that are ubiquitous in the natural environment. Contaminated water in man-made water systems is a potential source of transmission of legionnaires\' disease. The aim of this study is to explore the prevalence of Legionella pneumophila in the drinking water distribution system (DWDS) of Primorje-Gorski Kotar (PGK) County, Croatia, for the period 2013-2019, coupled with the incidence of legionnaires\' disease. A number of L. pneumophila-positive samples (>100 CFU/L), serogroup distribution and the degree of contamination of specific facilities (health and aged care, tourism, and sports) were assessed. Based on the obtained results, the reasoning for the implementation of a mandatory Legionella environmental surveillance program was assessed.
UNASSIGNED: Sample testing for Legionella was carried out according to the standard method for enumeration of this bacterium. A heterotrophic plate count (HPC) and Pseudomonas aeruginosa number were analysed along with the basic physicochemical indicators of drinking water quality. The research period was divided into two parts, namely, the 2013-2018 period (before implementation of the prevention program, after the outbreak of legionnaires\' disease), and the year 2019 (proactive approach, no disease cases recorded).
UNASSIGNED: During the 7-year observation period in PGK County, an increase in the number of samples tested for Legionella was found. An increase in Legionella-positive samples (particularly pronounced during the warmer part of the year) was recorded, along with a growing trend in the number of reported legionnaires\' disease cases. In addition to hot water systems, the risk of Legionella colonisation also applies to cold water systems. Health and aged care facilities appear to be at highest risk. In addition to the higher proportion of positive samples and a higher degree of microbiological load in these facilities, the highest proportion of L. pneumophila SGs 2-14 was identified. Due to the diagnostic limitations of the applied tests, the number of legionnaires\' disease cases is underdiagnosed.
UNASSIGNED: The introduction of a mandatory preventive approach to monitoring Legionella in DWDS water samples, along with the definition of national criteria for the interpretation of the results will create the preconditions for diagnosis and adequate treatment of larger numbers of legionnaires\' disease cases.

Residual manganese(II) in finished water undergoes further oxidation and deposition in drinking water distribution systems (DWDS), and Mn deposits can function as sites for accumulating organic and inorganic pollutants. This study aims to explore how Mn transformation and deposition affect the formation of disinfection byproducts (DBPs) in chlorinated DWDS, and trihalomethanes (THMs) was selected as a representative DBP. In a 100 μg/L Mn system, regulated THMs (chlorinated/bromated-THMs) increased by over 20% higher than Mn-free system after 150-day operation; when 50 μg/L iodide (I-) entered pipe systems after 150 days, iodinated THMs (I-THMs) in 100 μg/L Mn system increased by over 30% compared with Mn-free system. These promotions were attributed primarily to the accumulation of biomolecules and organic substances by tight and hard chlorinated Mn deposits. The residence of inactivated cells and the bridging role of surface Mn(III) in Mn deposits increased the quantity of THM precursors in DWDS. Furthermore, the rapid catalytic oxidation of Mn(II) by preformed Mn oxides (MnOx) inhibited the conversion of free iodine (HOI/OI-) to iodate, resulting in the generation of more I-THMs. This study provides new insights into the DBP risks caused by Mn in DWDS.

Antimicrobial resistance (AMR), as well as the development of biofilms in drinking water distribution systems (DWDSs), have become an increasing concern for public health and management. As bulk water travels from source to tap, it may accumulate contaminants of emerging concern (CECs) such as antibiotics and heavy metals. When these CECs and other selective pressures, such as disinfection, pipe material, temperature, pH, and nutrient availability interact with planktonic cells and, consequently, DWDS biofilms, AMR is promoted. The purpose of this review is to highlight the mechanisms by which AMR develops and is disseminated within DWDS biofilms. First, this review will lay a foundation by describing how DWDS biofilms form, as well as their basic intrinsic and acquired resistance mechanisms. Next, the selective pressures that further induce AMR in DWDS biofilms will be elaborated. Then, the pressures by which antibiotic and heavy metal CECs accumulate in DWDS biofilms, their individual resistance mechanisms, and co-selection are described and discussed. Finally, the known human health risks and current management strategies to mitigate AMR in DWDSs will be presented. Overall, this review provides critical connections between several biotic and abiotic factors that influence and induce AMR in DWDS biofilms. Implications are made regarding the importance of monitoring and managing the development, promotion, and dissemination of AMR in DWDS biofilms.

The knowledge about the members of active archaea communities in DWDS is limited. The current understanding is based on high-throughput 16S ribosomal RNA gene (DNA-based) amplicon sequencing that reveals the diversity of active, dormant, and dead members of the prokaryote (bacteria, archaea) communities. The sequencing primers optimized for bacteria community analysis may underestimate the share of the archaea community. This study characterized archaea communities at five full-scale drinking water distribution systems (DWDS), representing a variety of drinking water production units (A-E); A&B use artificially recharged non-disinfected groundwater (ARG), the other DWDS\'s supplied water disinfected by using ultraviolet (UV) light and chlorine compounds, C&D were surface waterworks and E was a ground waterworks. For the first time for archaea community analyses, this study employed the archaea-specific high-throughput sequencing primers for 16S ribosomal RNA (rRNA) as a target (reverse-transcribed cDNA; an RNA-based approach) in addition to the previously used 16S rRNA gene target (rDNA; a DNA-based approach) to reveal the active fraction of the archaea present in DWDS. The archaea community structure in varying environmental conditions in the water and biofilm of the five DWDSs were investigated by taking into consideration the system properties (cold or hot water system) and water age (distance from the treatment plants) in samples from each season of one year. The RNA-based archaea amplicon reads were obtained mostly from cold water samples from DWDSs (A-B) distributing water without disinfection where the DNA-based and RNA-based analysis created separate clusters in a weighted beta-diversity analysis. The season and location in DWDS A further affected the diversity of these archaea communities as was seen by different clusters in beta-diversity plots. The recovery of archaea reads was not adequate for analysis in any of the disinfected samples in DWDSs C-E or non-disinfected hot water in DWDSs A-B when utilizing RNA-based template. The metabolically active archaea community of DWDSs thus seemed to be effectively controlled by disinfection of water and in the hot water systems by the temperature. All biofilms regardless of DWDS showed lower species richness values (mainly Nitrososphaeria class) than non-disinfected water from DWDSs A-B where several archaea classes occurred (e.g. Woesearchaeia, Nitrososphaeria, Micrarchaeia, Methanomicrobia, Iairchaeia, Bathyarchaeia) indicating only part of the archaea members were able to survive in biofilms. Thus, Archaea has been shown as a significant part of normal DWDS biota, and their role especially in non-disinfected DWDS may be more important than previously considered.

Perfluoroalkyl substances (PFASs) can occur in water sources, pass through drinking water treatment plants (DWTPs), drinking water distribution systems (DWDSs), to the consumer taps. This investigation was carried out to present the transportation behaviors of 17 PFASs, involving seven DWTPs with different water sources, raw water transportation modes, treatment processes, and DWDS structures in eastern and northern China. The results showed that the long-distance raw water transportation pipelines removed a certain extent of PFASs from raw water, probably due to the accumulation of loose deposits. The long-distance, open-channel South-to-North water diversion increased PFAS contamination risk. In the DWTPs, granular activated carbon (GAC) adsorption and ultraviolet radiation removed less than 25% of PFASs, but ozonation-biological activated carbon (O3-BAC) was superior to GAC alone in removing PFASs. Loose deposits couldsignificantly influence PFAS accumulation and release within branch-structured DWDSs. In loop-structured DWDSs, finished water with different PFAS characteristics could mix along the pipeline, with the corresponding DWTP as the center, ultimately forming a relatively uniform distribution in the entire DWDS.

With the expansion of cities, the deterioration of drinking water quality undergoing complex and long-distance distribution is gaining increasing attention. However, spatial variations between free-living bacteria (FLB) and particle-associated bacteria (PAB) in chlorinated drinking water distribution systems (DWDSs) have not been fully explored, especially in complex water supply areas with multiple interconnected DWDSs. To fill this gap, this study utilized 16S rRNA approaches to characterize the spatial patterns of FLB and PAB in DWDSs with intersection regions. Based on distance-decay analysis, transportation distance is a potential driver of bacterial variation for both FLB (Pearson\'s r = -0.476, p < 0.01) and PAB. (Pearson\'s r = -0.352, p < 0.01). Moreover, the influence of transportation distance was further confirmed by a 1.20-99.45% decline in microbial contribution to the source of FLB and PAB communities in pipe water along the transportation pipelines. Meanwhile, significant difference (PERMANOVA, R2 = 0.14, p < 0.01) was found between FLB and PAB in DWDSs. Average proportions of Pseudomonas spp. were 59.84% and 45.59% for the PAB and intersection regions based on the 16S rRNA results, respectively, suggesting that PAB are potential reservoirs for high-risk bacteria, and a greater microbial risk may exist in intersection regions. In summary, transportation distance and pipeline intersection exerted significant impacts on the FLB and PAB in DWDSs. Therefore, precautionary strategies for controlling microbial risks that consider different microbial components and intersection regions in long-distance and multi-plant DWDSs should be implemented.