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To ensure drinking-water safety, it is necessary to understand the factors that regulate harmful cyanobacterial blooms (HCBs) and the toxins they produce. One controlling factor might be any relationship between fungi and the cyanobacteria. To test this possibility, water samples were obtained from Harsha Lake in southwestern Ohio during the 2015, 2016, and 2017 bloom seasons, i.e., late May through September. In each water sample, the concentration of the filamentous fungus

The efforts towards reduction of nutrient contamination of surface waters have greatly gained attention to mitigate increasing incidences of harmful cyanobacterial blooms (CyanoHABs), but little attention has been paid on the roles and importance of cyanobacterial N2-fixation and phosphorus (P) scavenging pathways during cyanoHABs. Meta-transcriptomic analyses revealed that expressions of genes involved in N2-fixation (nifDKH) and P-scavenging were significantly upregulated during the bloom compared to pre-bloom in Harsha Lake. The activities of N2-fixation occurred during early summer after a late spring phytoplankton bloom, and were associated with high phosphorus and low nitrogen. The highly active cyanobacterial N2-fixers were dominated by Nostoc and Anabaena. Following the activities of N2-fixation and production of new nitrogen, an early summer Microcystis-dominated bloom, a shift of dominance from Nostoc and Anabaena to Microcystis and an increase of microcystin and saxitoxin occurred. By contrast, P-scavenging activities dominated also by Nostoc and Anabaena were associated with low P and the Microcystis bloom. This information can be used to aid in the understanding the impact that nitrogen and phosphorus have on the early summer CyanoHAB and the functional activities of Nostoc- and Anabaena-dominated or Microcystis-dominated communities, and aid in making management decisions related to harmful algal blooms.

Water-based opportunistic pathogens (OPs) are a leading cause of drinking-water-related disease outbreaks, especially in developed countries such as the United States (US). Physicochemical water quality parameters, especially disinfectant residuals, control the (re)growth, presence, colonization, and concentrations of OPs in drinking water distribution systems (DWDSs), while the relationship between OPs and those parameters remain unclear. This study aimed to quantify how physicochemical parameters, mainly monochloramine residual concentration, hydraulic residence time (HRT), and seasonality, affected the occurrence and concentrations of four common OPs (Legionella, Mycobacterium, Pseudomonas, and Vermamoeba vermiformis) in four full-scale DWDSs in the US. Legionella as a dominant OP occurred in 93.8% of the 64 sampling events and had a mean density of 4.27 × 105 genome copies per liter. Legionella positively correlated with Mycobacterium, Pseudomonas, and total bacteria. Multiple regression with data from the four DWDSs showed that Legionella had significant correlations with total chlorine residual level, free ammonia concentration, and trihalomethane concentration. Therefore, Legionella is a promising indicator of water-based OPs, reflecting microbial water quality in chloraminated DWDSs. The OP concentrations had strong seasonal variations and peaked in winter and/or spring possibly because of reduced water usage (i.e., increased water stagnation or HRT) during cold seasons. The OP concentrations generally increased with HRT presumably because of disinfectant residual decay, indicating the importance of well-maintaining disinfectant residuals in DWDSs for OP control. The concentrations of Mycobacterium, Pseudomonas, and V. vermiformis were significantly associated with total chlorine residual concentration, free ammonia concentration, and pH and trihalomethane concentration, respectively. Overall, this study demonstrates how the significant spatiotemporal variations of OP concentrations in chloraminated DWDSs correlated with critical physicochemical water quality parameters such as disinfectant residual levels. This work also indicates that Legionella is a promising indicator of OPs and microbial water quality in chloraminated DWDSs.

Cyanobacterial harmful algal blooms (CyanoHABs) have been found to transmit from N2 fixer-dominated to non-N2 fixer-dominated in many freshwater environments when the supply of N decreases. To elucidate the mechanisms underlying such "counter-intuitive" CyanoHAB species succession, metatranscriptomes (biotic data) and water quality-related variables (abiotic data) were analyzed weekly during a bloom season in Harsha Lake, a multipurpose lake that serves as a drinking water source and recreational ground. Our results showed that CyanoHABs in Harsha Lake started with N2-fixing Anabaena in June (ANA stage) when N was high, and transitioned to non-N2-fixing Microcystis- and Planktothrix-dominated in July (MIC-PLA stage) when N became limited (low TN/TP). Meanwhile, the concentrations of cyanotoxins, i.e., microcystins were significantly higher in the MIC-PLA stage. Water quality results revealed that N species (i.e., TN, TN/TP) and water temperature were significantly correlated with cyanobacterial biomass. Expression levels of several C- and N-processing-related cyanobacterial genes were highly predictive of the biomass of their species. More importantly, the biomasses of Microcystis and Planktothrix were also significantly associated with expressions of microbial genes (mostly from heterotrophic bacteria) related to processing organic substrates (alkaline phosphatase, peptidase, carbohydrate-active enzymes) and cyanophage genes. Collectively, our results suggest that besides environmental conditions and inherent traits of specific cyanobacterial species, the development and succession of CyanoHABs are regulated by co-occurring microorganisms. Specifically, the co-occurring microorganisms can alleviate the nutrient limitation of cyanobacteria by remineralizing organic compounds.

To better understand public health implications of waterfowl as reservoirs for zoonotic sources of Campylobacter in recreational waters, we developed a Gallus gallus (chick) model of infection to assess the pathogenicity of environmental isolates of Campylobacter. This method involved exposure of 1-day-old chicks through ingestion of water, the natural route of infection. Viable Campylobacter from laboratory-infected animals were monitored by using a modified non-invasive sampling of fresh chick excreta followed by a passive polycarbonate-filter migration culture assay. The method was used to evaluate the infectivities of three laboratory strains of Campylobacter spp. (Campylobacter coli, Campylobacter jejuni, and Campylobacter lari), three clinical isolates of C. jejuni, and four environmental Campylobacter spp. isolated from California gulls (Larus californicus). The results revealed that chicks were successfully infected with all laboratory and clinical isolates of Campylobacter spp. through ingestion of Campylobacter-spiked water, with infection rates ranging from 90% in a dose-dependent manner. More importantly, exposure of chicks with Campylobacter spp. isolated from Gallus gallus excreta also resulted in successful establishment of infection (≤90%). Each monitored Campylobacter spp. contained ≥7.5 × 104 CFU⋅g–1 of feces 7 days post-exposure. These results suggest that a G. gallus model can be used to assess infectivity of Campylobacter isolates, including gull and human clinical isolates. Use of an avian animal model can be applied to assess the importance of birds, such as the G. gallus, as potential contributors of waterborne-associated outbreaks of campylobacteriosis.

Public concern over cyanobacterial blooms has increased due to their higher frequency of occurrence and their potential ecological and health impacts. Detection of microcystin (MC) producers (MCPs) using qPCR and RT-qPCR allows for the rapid identification of blooms by combining specificity and sensitivity with a relatively high throughput capability. Investigation of MCP population composition (correlation, dominance), toxin gene expression, and relationship to MC concentration was conducted using a panel of qPCR assays targeting mcyA, E and G on weekly and daily water samples collected from an Ohio inland lake. Further, these data were used to develop early warning thresholds for prediction of MC concentrations exceeding the US EPA Health Advisory cutoff value (>0.3μg L(−1)) using receiver operating characteristic curves and tobit regression. MCP Microcystis genomic copy number made up approximately 35.1% of the total Microcystis spp. and was the dominant toxic subpopulation of MCPs and remained a stable proportion of the total (30-40%) regardless of sampling dates and sites. The expressed MCPs were 0.2% of the extant genomic copy numbers, while toxic Microcystis had higher expressed proportion (0.5%) than that of toxic Planktothrix (0.04%). Microcystis toxin genes increased in June and July, but decreased in August and September along with similar trends of cell replication. Quantities of both RT-qPCR and qPCR followed the same trend and were highly correlated with MC-ADDA, while RT-qPCR not only reflected the active toxin genes or toxic species, but also indicated the beginning and ending of toxin production. A one-week early warning of MC with a cutoff value (>0.3μg L(−1)) was also made based on signaling of qPCR and RT-qPCR using receiver operating characteristic curves. Together, this study reports the use of qPCR and RT-qPCR as an early warning system (up to 7 days prior to) of extant and MC producing potentials (false positive rates ~70% for LC-MS/MS or ELISA) during a toxic algal bloom, with a significant predictive power of 50%−60% and 30%−40%, respectively.

Opportunistic pathogens such as Legionella are of significant public health concern in hospitals. Microbiological and water chemistry parameters in hot water throughout an Ohio hospital were monitored monthly before and after the installation of a monochloramine disinfection system over 16 months. Water samples from fifteen hot water sampling sites as well as the municipal water supply entering the hospital were analyzed using both culture and qPCR assays for specific microbial pathogens including Legionella, Pseudomonas spp., nontuberculous Mycobacteria [NTM], as well as for heterotrophic bacteria. Legionella culture assays decreased from 68% of all sites being positive prior to monochloramine addition to 6% positive after monochloramine addition, and these trends were parallel to qPCR results. Considering all samples, NTMs by culture were significantly reduced from 61% to 14% positivity (p

Aims To examine the occurrence and quantity of potential pathogens and an indicator of microbial contamination in the sediments of municipal drinking water storage tanks (MDWSTs), given the absence of such data across the United States. Methods and Results Sediment samples (87 MDWST) from eighteen locations across ten states of the United States were collected and assayed by qPCR for a range of potential enteric and opportunistic microbial pathogens and a sewage-associated Bacteroides marker. Potential opportunistic pathogens dominated, with the highest detection of occurrence (per cent positive detection; average cell equivalence (CE)) being Mycobacterium spp. (88·9%; 6·7 ± 8·5 × 104 CE g−1), followed by Legionella spp. (66·7%; 5·2 ± 5·9 × 103 CE g−1), Pseudomonas aeruginosa (22·2%; 250 ± 880 CE g−1) and Acanthamoeba spp. (38·9%; 53 ± 70 CE g−1), with no detected Naegleria fowleri. Most enteric pathogens (Campylobacter jejuni, Escherichia coli 0157:H7, Salmonella enterica, Cryptosporidium parvum and Giardia duodenalis) were not detected, except for a trace signal for Campylobacter spp. There was significant correlation between the qPCR signals of Legionella spp. and Acanthamoeba spp. (R2 = 0·61, n = 87, P = 0·0001). Diverse Legionella spp. including Leg. pneumophila, Leg. pneumophila sg1 and Leg. anisa were identified, each of which might cause legionellosis. Conclusions These results imply that potential opportunistic pathogens are common within MDWST sediments and could act as a source of microbial contamination, but need downstream growth to be of potential concern. Significance and Impact of the Study The results imply that opportunistic pathogen risks may need to be managed by regular tank cleaning or other management practices.

The occurrence and densities of opportunistic pathogens (OPs), the microbial community structure, and their associations with sediment elements from eight water storage tanks in Ohio, West Virginia, and Texas were investigated. The elemental composition of sediments was measured through X-ray fluorescence (XRF) spectra. The occurrence and densities of OPs and amoeba hosts (i.e., Legionella spp. and L. pneumophila, Mycobacterium spp., P. aeruginosa, V. vermiformis, Acanthamoeba spp.) were determined using genus- or species-specific qPCR assays. Microbial community analysis was performed using next generation sequencing on the Illumina Miseq platform. Mycobacterium spp. were most frequently detected in the sediments and water samples (88% and 88%), followed by Legionella spp. (50% and 50%), Acanthamoeba spp. (63% and 13%), V. vermiformis (50% and 25%), and P. aeruginosa (0 and 50%) by qPCR method. Comamonadaceae (22.8%), Sphingomonadaceae (10.3%), and Oxalobacteraceae (10.1%) were the most dominant families by sequencing method. Microbial communities in water samples were mostly separated with those in sediment samples, suggesting differences of communities between two matrices even in the same location. There were associations of OPs with microbial communities. Both OPs and microbial community structures were positively associated with some elements (Al and K) in sediments mainly from pipe material corrosions. Opportunistic pathogens presented in both water and sediments, and the latter could act as a reservoir of microbial contamination. There appears to be an association between potential opportunistic pathogens and microbial community structures. These microbial communities may be influenced by constituents within storage tank sediments. The results imply that compositions of microbial community and elements may influence and indicate microbial water quality and pipeline corrosion, and that these constituents may be important for optimal storage tank management within a distribution system.

Aims This study examined the impact of pipe materials and introduced Legionella pneumophila on downstream Leg. pneumophila colonization and microbial community structures under conditions of low flow and low chlorine residual. Methods and results CDC biofilm(™) reactors containing either unplasticized polyvinylchloride (uPVC) or copper (Cu) coupons were used to develop mature biofilms on Norprene(™) tubing effluent lines to simulate possible in-premise biofilm conditions. The microbial communities were characterized through 16S and 18S rRNA gene clone libraries and Leg. pneumophila colonization was determined via specific qPCR assays. The Cu significantly decreased downstream microbial diversity, approximately halved bacterial and eukaryotic abundance, with some groups only detected in uPVC-reactor tubing biofilms. However, some probable amoeba-resisting bacteria (ARB) like Mycobacterium spp. and Rhodobacteraceae were significantly more abundant in the Cu than uPVC-reactor tubing biofilms. In particular, Leg. pneumophila only persisted (postinoculation) within the Cu-reactor tubing biofilms, and the controlled low chlorine residue and water flow conditions led to a general high abundance of possible free-living protozoa in all tubing biofilms. The higher relative abundance of ARB-like sequences from Cu-coupons vs uPVC may have been promoted by amoebal selection and subsequent ARB protection from Cu inhibitory effects. Conclusions Copper pipe and low flow conditions had significant impact on downstream biofilm microbial structures (on plastic pipe) and the ability for Leg. pneumophila colonization post an introduction event. Significance and impact of the study This is the first report that compares the effects of copper and uPVC materials on downstream biofilm communities grown on a third (Norprene(™)) surface material. The downstream biofilms contained a high abundance of free-living amoebae and ARB, which may have been driven by a lack of residual disinfectant and periodic stagnant conditions. Given the prevalence of Cu-piping in buildings, there may be increased risk from drinking water exposures to ARB following growth on pipe/fixture biofilms within premise drinking water systems.

Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied colonization of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila colonized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms.

The goal of this study was to characterize microbial eukaryotes over a 12-month period to provide insight into the occurrence of potential bacterial predators and hosts in premise plumbing. Nearly 6,300 partial 18S rRNA gene sequences from 24 hot (36.9-39.0 °C) and cold (6.8-29.1 °C) drinking water samples were analyzed and classified into major eukaryotic groups. Each major group, consisting of free-living amoebae (FLA)/protozoa, algae, copepods, dinoflagellates, fungi, nematodes, and unique uncultured eukaryotic sequences, showed limited diversity dominated by a few distinct populations, which may be characteristic of oligotrophic environments. Changes in the relative abundance of predators such as nematodes, copepods, and FLA appear to be related to temperature and seasonal changes in water quality. Sequences nearly identical to FLA such as Hartmannella vermiformis, Echinamoeba thermarmum, Pseudoparamoeba pagei, Protacanthamoeba bohemica, Platyamoeba sp., and Vannella sp. were obtained. In addition to FLA, various copepods, rotifers, and nematodes have been reported to internalize viral and bacterial pathogens within drinking water systems thus potentially serving as transport hosts; implications of which are discussed further. Increasing the knowledge of eukaryotic occurrence and their relationship with potential pathogens should aid in assessing microbial risk associated with various eukaryotic organisms in drinking water.