Composition of active bacterial communities and presence of opportunistic pathogens in disinfected and non-disinfected drinking water distribution systems in Finland.

Many factors, including microbiome structure and activity in the drinking water distribution system (DWDS), affect the colonization potential of opportunistic pathogens. The present study aims to describe the dynamics of active bacterial communities in DWDS and identify the factors that shape the community structures and activity in the selected DWDSs. Large-volume drinking water and hot water, biofilm, and water meter deposit samples were collected from five DWDSs. Total nucleic acids were extracted, and RNA was further purified and transcribed into its cDNA from a total of 181 water and biofilm samples originating from the DWDS of two surface water supplies (disinfected with UV and chlorine), two artificially recharged groundwater supplies (non-disinfected), and a groundwater supply (disinfected with UV and chlorine). In chlorinated DWDSs, concentrations of <0.02-0.97 mg/l free chlorine were measured. Bacterial communities in the RNA and DNA fractions were analysed using Illumina MiSeq sequencing with primer pair 341F-785R targeted to the 16S rRNA gene. The sequence libraries were analysed using QIIME pipeline, Program R, and MicrobiomeAnalyst. Not all bacterial cells were active based on their 16S rRNA content, and species richness was lower in the RNA fraction (Chao1 mean value 490) than in the DNA fraction (710). Species richness was higher in the two DWDSs distributing non-disinfected artificial groundwater (Chao1 mean values of 990 and 1 000) as compared to the two disinfected DWDSs using surface water (Chao1 mean values 190 and 460) and disinfected DWDS using ground water as source water (170). The difference in community structures between non-disinfected and disinfected water was clear in the beta-diversity analysis. Distance from the waterworks also affected the beta diversity of community structures, especially in disinfected distribution systems. The two most abundant bacteria in the active part of the community (RNA) and total bacterial community (DNA) belonged to the classes Alphaproteobacteria (RNA 28 %, DNA 44 %) and Gammaproteobacteria (RNA 32 %, DNA 30 %). The third most abundant and active bacteria class was Vampirovibrionia (RNA 15 %), whereas in the total community it was Paceibacteria (DNA 11 %). Class Nitrospiria was more abundant and active in both cold and hot water in DWDS that used chloramine disinfection compared to non-chlorinated or chlorine-using DWDSs. Thirty-eight operational taxonomic units (OTU) of Legionella, 30 of Mycobacterium, and 10 of Pseudomonas were detected among the sequences. The (RT)-qPCR confirmed the presence of opportunistic pathogens in the DWDSs studied as Legionella spp. was detected in 85 % (mean value 4.5 × 104 gene copies/100 ml), Mycobacterium spp. in 95 % (mean value 8.3 × 106 gene copies/100 ml), and Pseudomonas spp. in 78 % (mean value 1.6 × 105 gene copies/100 ml) of the water and biofilm samples. Sampling point inside the system (distance from the waterworks and cold/hot system) affected the active bacterial community composition. Chloramine as a chlorination method resulted in a recognizable community composition, with high abundance of bacteria that benefit from the excess presence of nitrogen. The results presented here confirm that each DWDS is unique and that opportunistic pathogens are present even in conditions when water quality is considered excellent.

Phage Biocontrol of Pseudomonas aeruginosa in Water.

Bacteriophage control of harmful or pathogenic bacteria has aroused growing interest, largely due to the rise of antibiotic resistance. The objective of this study was to test phages as potential agents for the biocontrol of an opportunistic pathogen Pseudomonas aeruginosa in water. Two P. aeruginosa bacteriophages (vB_PaeM_V523 and vB_PaeM_V524) were isolated from wastewater and characterized physically and functionally. Genomic and morphological characterization showed that both were myoviruses within the Pbunavirus genus. Both had a similar latent period (50-55 min) and burst size (124-134 PFU/infected cell), whereas there was variation in the host range. In addition to these environmental phages, a commercial Pseudomonas phage, JG003 (DSM 19870), was also used in the biocontrol experiments. The biocontrol potential of the three phages in water was tested separately and together as a cocktail against two P. aeruginosa strains; PAO1 and the environmental strain 17V1507. With PAO1, all phages initially reduced the numbers of the bacterial host, with phage V523 being the most efficient (>2.4 log10 reduction). For the environmental P. aeruginosa strain (17V1507), only the phage JG003 caused a reduction (1.2 log10) compared to the control. The cocktail of three phages showed a slightly higher decrease in the level of the hosts compared to the use of individual phages. Although no synergistic effect was observed in the host reduction with the use of the phage cocktail, the cocktail-treated hosts did not appear to acquire resistance as rapidly as hosts treated with a single phage. The results of this study provide a significant step in the development of bacteriophage preparations for the control of pathogens and harmful microbes in water environments.

Diverse and active archaea communities occur in non-disinfected drinking water systems-Less activity revealed in disinfected and hot water systems.

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.

Active eukaryotes in drinking water distribution systems of ground and surface waterworks.

BACKGROUND: Eukaryotes are ubiquitous in natural environments such as soil and freshwater. Little is known of their presence in drinking water distribution systems (DWDSs) or of the environmental conditions that affect their activity and survival. METHODS: Eukaryotes were characterized by Illumina high-throughput sequencing targeting 18S rRNA gene (DNA) that estimates the total community and the 18S rRNA gene transcript (RNA) that is more representative of the active part of the community. DWDS cold water (N = 124), hot water (N = 40), and biofilm (N = 16) samples were collected from four cities in Finland. The sampled DWDSs were from two waterworks A-B with non-disinfected, recharged groundwater as source water and from three waterworks utilizing chlorinated water (two DWDSs of surface waterworks C-D and one of ground waterworks E). In each DWDS, samples were collected from three locations during four seasons of 1 year. RESULTS: A beta-diversity analysis revealed that the main driver shaping the eukaryotic communities was the DWDS (A-E) (R = 0.73, P < 0.001, ANOSIM). The kingdoms Chloroplastida (green plants and algae), Metazoa (animals: rotifers, nematodes), Fungi (e.g., Cryptomycota), Alveolata (ciliates, dinoflagellates), and Stramenopiles (algae Ochrophyta) were well represented and active-judging based on the rRNA gene transcripts-depending on the surrounding conditions. The unchlorinated cold water of systems (A-B) contained a higher estimated total number of taxa (Chao1, average 380-480) than chlorinated cold water in systems C-E (Chao1 ≤ 210). Within each DWDS, unique eukaryotic communities were identified at different locations as was the case also for cold water, hot water, and biofilms. A season did not have a consistent impact on the eukaryotic community among DWDSs. CONCLUSIONS: This study comprehensively characterized the eukaryotic community members within the DWDS of well-maintained ground and surface waterworks providing good quality water. The study gives an indication that each DWDS houses a unique eukaryotic community, mainly dependent on the raw water source and water treatment processes in place at the corresponding waterworks. In particular, disinfection as well as hot water temperature seemed to represent a strong selection pressure that controlled the number of active eukaryotic species.

Drinking water quality and formation of biofilms in an office building during its first year of operation, a full scale study.

Complex interactions existing between water distribution systems' materials and water can cause a reduction in water quality and unwanted changes in materials, aging or corrosion of materials and formation of biofilms on surfaces. Substances leaching from pipe materials and water fittings, as well as the microbiological quality of water and formation of biofilms were evaluated by applying a Living Lab theme i.e. a research in a real life setting using a full scale system during its first year of operation. The study site was a real office building with one part of the building lined with copper pipes, the other with cross-linked polyethylene (PEX) pipes thus enabling material comparison; also differences within the cold and hot water systems were analysed. It was found that operational conditions, such as flow conditions and temperature affected the amounts of metals leaching from the pipe network. In particular, brass components were considered to be a source of leaching; e. g. the lead concentration was highest during the first few weeks after the commissioning of the pipe network when the water was allowed to stagnate. Assimilable organic carbon (AOC) and microbially available phosphorus (MAP) were found to leach from PEX pipelines with minor effects on biomass of the biofilm. Cultivable and viable biomass (heterotrophic plate count (HPC), and adenosine triphosphate (ATP)) levels in biofilms were higher in the cold than in the hot water system whereas total microbial biomass (total cell count (DAPI)) was similar with both systems. The type of pipeline material was not found to greatly affect the microbial biomass or Alpha-, Beta- and Gammaproteobacteria profiles (16s rRNA gene copies) after the first one year of operation. Also microbiological quality of water was found to deteriorate due to stagnation.

Manual faucets induce more biofilms than electronic faucets.

Electronic faucets (types E1 and E2) and manual (M) faucets were studied for microbial quality, i.e., biomass and pathogenic microbes of biofilms in the faucet aerator, the water, and the outer surface of faucet in a hospital in Finland. Heterotrophic plate count content reflecting culturable microbial biomass and adenosine triphosphate content representing viable microbial biomass were smaller in the biofilms of E1-type electronic faucets than E2-type electronic faucets or M faucets. The likely explanation is the mixing point of cold and hot water (E1 and M: in the faucet; E2: in a separate box 50 cm before the actual faucet part). The highest amounts of Legionella (serogroups 2-15 of Legionella pneumophila) in a water sample (5000 cfu/L) and in biofilm samples (May-June 2008 sampling: 240 cfu/mL; November 2008: 1100 cfu/mL) were found in one E1-type faucet, which was lacking a back pressure valve due to faulty installation. This study reveals that certain types of electronic faucets seem to promote hospital hygiene, as they were associated with less microbial growth in biofilms in the faucet aerator, than some other types of electronic faucets or manual faucets, likely owing to the mixing point of cold and hot water. However, the faucet type had no direct effect on the presence of Legionella spp. Also correct installation is crucial.

Decontamination of a drinking water pipeline system contaminated with adenovirus and Escherichia coli utilizing peracetic acid and chlorine.

A contaminated drinking water distribution network can be responsible for major outbreaks of infections. In this study, two chemical decontaminants, peracetic acid (PAA) and chlorine, were used to test how a laboratory-scale pipeline system can be cleaned after simultaneous contamination with human adenovirus 40 (AdV40) and Escherichia coli. In addition, the effect of the decontaminants on biofilms was followed as heterotrophic plate counts (HPC) and total cell counts (TCC). Real-time quantitative polymerase chain reaction (qPCR) was used to determine AdV40 and plate counting was used to enumerate E. coli. PAA and chlorine proved to be effective decontaminants since they decreased the levels of AdV40 and E. coli to below method detection limits in both water and biofilms. However, without decontamination, AdV40 remained present in the pipelines for up to 4 days. In contrast, the concentration of cultivable E. coli decreased rapidly in the control pipelines, implying that E. coli may be an inadequate indicator for the presence of viral pathogens. Biofilms responded to the decontaminants by decreased HPCs while TCC remained stable. This indicates that the mechanism of pipeline decontamination by chlorine and PAA is inactivation rather than physical removal of microbes.