Campylobacter species, Salmonella serotypes and ribosomal RNA-based fecal source tracking in the Kokemäki River watershed.

Fecal contamination of surface water compromises the usability of surface water for drinking water production due to an increase in human health risks. In this study, we collected surface water samples for two years from the Kokemäki River (Finland). The downstream river stretch is used for feeding production of artificial ground water for a major drinking water treatment plant. The prevalence of Campylobacter species and Salmonella serotypes together with fecal source identifiers targeting general, human, gull, swine, and ruminant were evaluated at 16 sampling sites throughout the studied watershed. We detected Campylobacter spp. from all 16 sampling sites with Campylobacter jejuni and Campylobacter lari as the most detected species. Salmonella spp. was detected in 10 out of 16 sampling sites, with Salmonella Typhimurium being the most common serovar. Regarding spatial variation in the hygienic quality of surface water, the upstream area (urban proximity) and downstream area (agricultural proximity) had higher microbial loads than the middle section of the study area. Samples taken in fall and spring had higher microbial loads than summer and winter samples. The lower ratio of rRNA to rRNA-gene (rDNA) of studied microbes in the winter than in other seasons may indicate low metabolic activity of bacterial targets during winter. The number of gulls, swine, and cattle in the catchment area concorded with the number of fecal source identifiers in the surface water. Further, the prevalence of gull-specific source identifier agreed with the detection of C. coli, C. lari, and S. Typhimurim, whereas the prevalence of swine- and ruminant-specific source identifiers agreed with the detection of C. jejuni and C. coli. Thus, fecal source identifiers are shown to be important tools for monitoring zoonotic pathogens affecting microbial quality of surface water. Further, variation in fecal loads indicates such variation in health risks related to surface water use.

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.

A comparative analysis employing a gene- and genome-centric metagenomic approach reveals changes in composition, function, and activity in waterworks with different treatment processes and source water in Finland.

The emergence and development of next-generation sequencing technologies (NGS) has made the analysis of the water microbiome in drinking water distribution systems (DWDSs) more accessible and opened new perspectives in microbial ecology studies. The current study focused on the characterization of the water microbiome employing a gene- and genome-centric metagenomic approach to five waterworks in Finland with different raw water sources, treatment methods, and disinfectant. The microbial communities exhibit a distribution pattern of a few dominant taxa and a large representation of low-abundance bacterial species. Changes in the community structure may correspond to the presence or absence and type of disinfectant residual which indicates that these conditions exert selective pressure on the microbial community. The Archaea domain represented a small fraction (up to 2.5%) and seemed to be effectively controlled by the disinfection of water. Their role particularly in non-disinfected DWDS may be more important than previously considered. In general, non-disinfected DWDSs harbor higher microbial richness and maintaining disinfectant residual is significantly important for ensuring low microbial numbers and diversity. Metagenomic binning recovered 139 (138 bacterial and 1 archaeal) metagenome-assembled genomes (MAGs) that had a >50% completeness and <10% contamination consisting of 20 class representatives in 12 phyla. The presence and occurrence of nitrite-oxidizing bacteria (NOB)-like microorganisms have significant implications for nitrogen biotransformation in drinking water systems. The metabolic and functional complexity of the microbiome is evident in DWDSs ecosystems. A comparative analysis found a set of differentially abundant taxonomic groups and functional traits in the active community. The broader set of transcribed genes may indicate an active and diverse community regardless of the treatment methods applied to water. The results indicate a highly dynamic and diverse microbial community and confirm that every DWDS is unique, and the community reflects the selection pressures exerted at the community structure, but also at the levels of functional properties and metabolic potential.

Monitoring groundwater quality with real-time data, stable water isotopes, and microbial community analysis: A comparison with conventional methods.

Groundwater provides much of the world's potable water. Nevertheless, groundwater quality monitoring programmes often rely on a sporadic, slow, and narrowly focused combination of periodic manual sampling and laboratory analyses, such that some water quality deficiencies go undetected, or are detected too late to prevent adverse consequences. In an effort to address this shortcoming, we conducted enhanced monitoring of untreated groundwater quality over 12 months (February 2019-February 2020) in four shallow wells supplying potable water in Finland. We supplemented periodic manual sampling and laboratory analyses with (i) real-time online monitoring of physicochemical and hydrological parameters, (ii) analysis of stable water isotopes from groundwater and nearby surface waters, and (iii) microbial community analysis of groundwater via amplicon sequencing of the 16S rRNA gene and 16S rRNA. We also developed an early warning system (EWS) for detecting water quality anomalies by automating real-time online monitoring data collection, transfer, and analysis - using electrical conductivity (EC) and turbidity as indirect water quality indicators. Real-time online monitoring measurements were largely in fair agreement with periodic manual measurements, demonstrating their usefulness for monitoring water quality; and the findings of conventional monitoring, stable water isotopes, and microbial community analysis revealed indications of surface water intrusion and faecal contamination at some of the studied sites. With further advances in technology and affordability expected into the future, the supplementary methods used here could be more widely implemented to enhance groundwater quality monitoring - by contributing new insights and/or corroborating the findings of conventional analyses.

Effects of temperature and light exposure on the decay characteristics of fecal indicators, norovirus, and Legionella in mesocosms simulating subarctic river water.

Knowledge of the decay characteristics of health-related microbes in surface waters is important for modeling the transportation of waterborne pathogens and for assessing their public health risks. Although water temperature and light exposure are major factors determining the decay characteristics of enteric microbes in surface waters, such effects have not been well studied in subarctic surface waters. This study comprehensively evaluated the effect of temperature and light on the decay characteristics of health-related microbes [Escherichia coli, enterococci, microbial source tracking markers (GenBac3 & HF183 assays), coliphages (F-specific and somatic), noroviruses GII and Legionella spp.] under simulated subarctic river water conditions. The experiments were conducted in four different laboratory settings (4 °C/dark, 15 °C/dark, 15 °C/light, and 22 °C/light). The T90 values (time required for a 90 % reduction in the population of a target) of all targets were higher under cold and dark (2.6-51.3 days depending upon targets) than under warm and light conditions (0.6-3.5 days). Under 4 °C/dark (simulated winter) water conditions, F-specific coliphages had 27.2 times higher, and coliform bacteria had 3.3 times higher T90 value than under 22 °C/light (simulated summer) water conditions. Bacterial molecular markers also displayed high variation in T90 values, with the greatest difference between 4 °C/dark and 22 °C/light recorded for HF183 DNA (20.6 times) and the lowest difference for EC23S857 RNA (6.6 times). E. coli, intestinal enterococci, and somatic coliphages were relatively more sensitive to light than water temperature, but F-specific coliphages, norovirus, and all bacterial rDNA and rRNA markers were relatively more sensitive to temperature than light exposure. Due to the slow microbial decay in winter under subarctic conditions, the microbial quality of river water might remain low for a long time after a sewage spill. This increased risk associated with fecal pollution during winter may deserve more attention, especially when river waters are used for drinking water production.

Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland.

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area. Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered. Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Food-Grade Bacteria Combat Pathogens by Blocking AHL-Mediated Quorum Sensing and Biofilm Formation.

Disrupting bacterial quorum sensing (QS) signaling is a promising strategy to combat pathogenic biofilms without the development of antibiotic resistance. Here, we report that food-associated bacteria can interfere with the biofilm formation of a Gram-negative pathogenic bacterium by targeting its AHL (acyl-homoserine lactone) QS system. This was demonstrated by screening metabolic end-products of different lactobacilli and propionibacteria using Gram-negative and biofilm-forming Chromobacterium violaceum as the QS reporter and our anti-QS microscale screening platform with necessary modifications. The method was optimized in terms of the inoculation technique and the concentrations of D-glucose and L-tryptophan, two key factors controlling the synthesis of violacein, a purple pigment indicating the activation of the QS system in C. violaceum. These improvements resulted in ca. 16-times higher violacein yields and enabled revealing anti-QS effects of Lactobacillus acidophilus, Lentilactobacillus kefiri, Lacticaseibacillus rhamnosus and Propionibacterium freudenreichii, including new cheese-associated strains. Our findings also suggest that acetate and propionate excreted by these species are the main factors that interrupt the QS-mediated signaling and subsequent biofilm growth without affecting the cell viability of the C. violaceum reporter. Thus, the present study reports a revised anti-QS screening method to accurately define new bacteria with an ability to combat pathogens in a safe and sustainable way.

Surface Water Intrusion, Land Use Impacts, and Bacterial Community Composition in Shallow Groundwater Wells Supplying Potable Water in Sparsely Populated Areas of a Boreal Region.

Rural communities often rely on groundwater for potable water supply. In this study, untreated groundwater samples from 28 shallow groundwater wells in Finland (<10 m deep and mostly supplying untreated groundwater to <200 users in rural areas) were assessed for physicochemical water quality, stable water isotopes, microbial water quality indicators, host-specific microbial source tracking (MST) markers, and bacterial community composition, activity, and diversity (using amplicon sequencing of the 16S rRNA gene and 16S rRNA). Indications of surface water intrusion were identified in five wells, and these indications were found to be negatively correlated, overall, with bacterial alpha diversity (based on amplicon sequencing of the 16S rRNA gene). High levels of turbidity, heterotrophs, and iron compromised water quality in two wells, with values up to 2.98 nephelometric turbidity units (NTU), 16,000 CFU/ml, and 2,300 µg/liter, respectively. Coliform bacteria and general fecal indicator Bacteroidales bacteria (GenBac3) were detected in 14 and 10 wells, respectively (albeit mostly at low levels), and correlations were identified between microbial, physicochemical, and environmental parameters, which may indicate impacts from nearby land use (e.g., agriculture, surface water, road salt used for deicing). Our results show that although water quality was generally adequate in most of the studied wells, the continued safe use of these wells should not be taken for granted. IMPORTANCE Standard physicochemical water quality analyses and microbial indicator analyses leave much of the (largely uncultured) complexity of groundwater microbial communities unexplored. This study combined these standard methods with additional analyses of stable water isotopes, bacterial community data, and environmental data about the surrounding areas to investigate the associations between physicochemical and microbial properties of 28 shallow groundwater wells in Finland. We detected impaired groundwater quality in some wells, identified potential land use impacts, and revealed indications of surface water intrusion which were negatively correlated with bacterial alpha diversity. The potential influence of surface water intrusion on groundwater wells and their bacterial communities is of particular interest and warrants further investigation because surface water intrusion has previously been linked to groundwater contamination, which is the primary cause of waterborne outbreaks in the Nordic region and one of the major causes in the United States and Canada.

Surfaceome and Exoproteome Dynamics in Dual-Species Pseudomonas aeruginosa and Staphylococcus aureus Biofilms.

Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance, and Persistence.

The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 months by label-free proteomics and compared with cell surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggest that specific changes in mycolic acid synthesis (cord factor), Esx1 secretion, and cell wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting 64× MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria. IMPORTANCE Mycobacteria are naturally resilient, and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance-, and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

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.

Bacterial diversity and predicted enzymatic function in a multipurpose surface water system - from wastewater effluent discharges to drinking water production.

BACKGROUND: Rivers and lakes are used for multiple purposes such as for drinking water (DW) production, recreation, and as recipients of wastewater from various sources. The deterioration of surface water quality with wastewater is well-known, but less is known about the bacterial community dynamics in the affected surface waters. Understanding the bacterial community characteristics -from the source of contamination, through the watershed to the DW production process-may help safeguard human health and the environment. RESULTS: The spatial and seasonal dynamics of bacterial communities, their predicted functions, and potential health-related bacterial (PHRB) reads within the Kokemäenjoki River watershed in southwest Finland were analyzed with the 16S rRNA-gene amplicon sequencing method. Water samples were collected from various sampling points of the watershed, from its major pollution sources (sewage influent and effluent, industrial effluent, mine runoff) and different stages of the DW treatment process (pre-treatment, groundwater observation well, DW production well) by using the river water as raw water with an artificial groundwater recharge (AGR). The beta-diversity analysis revealed that bacterial communities were highly varied among sample groups (R = 0.92, p <  0.001, ANOSIM). The species richness and evenness indices were highest in surface water (Chao1; 920 ± 10) among sample groups and gradually decreased during the DW treatment process (DW production well; Chao1: 320 ± 20). Although the phylum Proteobacteria was omnipresent, its relative abundance was higher in sewage and industrial effluents (66-80%) than in surface water (55%). Phyla Firmicutes and Fusobacteria were only detected in sewage samples. Actinobacteria was more abundant in the surface water (≥13%) than in other groups (≤3%). Acidobacteria was more abundant in the DW treatment process (≥13%) than in others (≤2%). In total, the share of PHRB reads was higher in sewage and surface water than in the DW treatment samples. The seasonal effect in bacterial communities was observed only on surface water samples, with the lowest diversity during summer. CONCLUSIONS: The low bacterial diversity and absence of PHRB read in the DW samples indicate AGR can produce biologically stable and microbiologically safe drinking water. Furthermore, the significantly different bacterial communities at the pollution sources compared to surface water and DW samples highlight the importance of effective wastewater treatment for protecting the environment and human health.

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.

The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland.

Wastewater-based surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used to monitor the population-level prevalence of the COVID-19 disease. In many cases, due to lockdowns or analytical delays, the analysis of wastewater samples might only be possible after prolonged storage. In this study, the effect of storage conditions on the RNA copy numbers of the SARS-CoV-2 virus in wastewater influent was studied and compared to the persistence of norovirus over time at 4 °C, -20 °C, and -75 °C using the reverse-transcription quantitative PCR (RT-qPCR) assays E-Sarbeco, N2, and norovirus GII. For the first time in Finland, the presence of SARS-CoV-2 RNA was tested in 24 h composite influent wastewater samples collected from Viikinmäki wastewater treatment plant, Helsinki, Finland. The detected and quantified SARS-CoV-2 RNA copy numbers of the wastewater sample aliquots taken during 19-20 April 2020 and stored for 29, 64, and 84 days remained surprisingly stable. In the stored samples, the SARS betacoronavirus and SARS-CoV-2 copy numbers, but not the norovirus GII copy numbers, seemed slightly higher when analyzed from the pre-centrifuged pellet-that is, the particulate matter of the influent-as compared with the supernatant (i.e., water fraction) used for ultrafiltration, although the difference was not statistically significant. Furthermore, when wastewater was spiked with SARS-CoV-2, linear decay at 4 °C was observed on the first 28 days, while no decay was visible within 58 days at -20 °C or -75 °C. In conclusion, freezing temperatures should be used for storage when immediate SARS-CoV-2 RNA analysis from the wastewater influent is not possible. Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency.

Bacterial Genes Encoding Resistance Against Antibiotics and Metals in Well-Maintained Drinking Water Distribution Systems in Finland.

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.

Potential and limitations of a pilot-scale drinking water distribution system for bacterial community predictive modelling.

Waterborne disease outbreaks are a persistent and serious threat to public health according to reported incidents across the globe. Online drinking water quality monitoring technologies have evolved substantially and have become more accurate and accessible. However, using online measurements alone is unsuitable for detecting microbial regrowth, potentially including harmful species, ahead of time in the distribution systems. Alternatively, observational data could be collected periodically, e.g. once per week or once per month and it could include a representative set of variables: physicochemical water characteristics, disinfectant concentrations, and bacterial abundances, which would be a valuable source of knowledge for predictive modelling that aims to reveal pathogen-related threats. In this study, we utilised data collected from a pilot-scale drinking water distribution system. A data-driven random forest model was used for predictive modelling and was trained for nowcasting and forecasting abundances of bacterial groups. In all the experiments, we followed the realistic crossline scenario, which means that when training and testing the models the data is collected from different pipelines. In spite of the more accurate results of the nowcasting, the 1-week forecasting still provided accurate predictions of the most abundant bacteria, their rapid increase and decrease. In the future predictive modelling might be used as a tool in designing control measures for opportunistic pathogens which are able to multiply in the favourable conditions in drinking water distribution systems (DWDS). Eventually, the forecasting information will be able to produce practically helpful data for controlling the DWDS regrowth.

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus.

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus.

Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials.

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials-borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene-at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-ß-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points-from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.

Two Drinking Water Outbreaks Caused by Wastewater Intrusion Including Sapovirus in Finland.

Drinking water outbreaks occur worldwide and may be caused by several factors, including raw water contamination, treatment deficiencies, and distribution network failure. This study describes two drinking water outbreaks in Finland in 2016 (outbreak I) and 2018 (outbreak II). Both outbreaks caused approximately 450 illness cases and were due to drinking water pipe breakage and subsequent wastewater intrusion into the distribution system. In both outbreaks, the sapovirus was found in patient samples as the main causative agent. In addition, adenoviruses and Dientamoeba fragilis (outbreak I), and noroviruses, astroviruses, enterotoxigenic and enterohemorragic Escherichia coli (ETEC and EHEC, respectively) and Plesiomonas shigelloides (outbreak II) were detected in patient samples. Water samples were analyzed for the selected pathogens largely based on the results of patient samples. In addition, traditional fecal indicator bacteria and host-specific microbial source tracking (MST) markers (GenBac3 and HF183) were analyzed from water. In drinking water, sapovirus and enteropathogenic E. coli (EPEC) were found in outbreak II. The MST markers proved useful in the detection of contamination and to ensure the success of contaminant removal from the water distribution system. As mitigation actions, boil water advisory, alternative drinking water sources and chlorination were organized to restrict the outbreaks and to clean the contaminated distribution network. This study highlights the emerging role of sapoviruses as a waterborne pathogen and warrants the need for testing of multiple viruses during outbreak investigation.

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.