Chlorine and Monochloramine Disinfection of Legionella pneumophila Colonizing Copper and Polyvinyl Chloride Drinking Water Biofilms.

Building water systems promote the regrowth and survival of opportunistic pathogens, such as Legionella pneumophila, especially within biofilms, where most drinking water microbes reside. However, compared to their planktonic form, disinfection efficacy for the biofilm-associated forms of water-based pathogens is unclear. The aim of this study was to determine the effectiveness of free chlorine and monochloramine in the inactivation of biofilm-associated L. pneumophila strain Philadelphia-1 serogroup 1 (LpP1s1). Mature (1.5- to 2-year-old) drinking water biofilms were developed on copper (Cu) and polyvinyl chloride (PVC) slides within biofilm annular reactors, then colonized with LpP1s1 at approximately 4 log10 CFU cm-2 and exposed to 2 mg liter-1 of free chlorine or monochloramine. Ct (disinfectant concentration × time, expressed as mg min liter-1) inactivation values for 2-, 3-, and 4-log10 reductions of planktonic and biofilm LpP1s1 were determined. For planktonic LpP1s1, free chlorine was more effective at inactivation than was monochloramine treatment, and for biofilm-associated LpP1s1, monochloramine was more effective on Cu biofilms while free chlorine was more effective on PVC biofilms. In contrast to monochloramine, free chlorine treatment of Cu and PVC biofilms, negatively impacted LpP1s1 16S rRNA gene transcript levels and may act synergistically with Cu surfaces to further reduce transcript levels. Moreover, LpP1s1 cells shed from biofilms into the bulk water were more resistant to disinfection than were prepared planktonic LpP1s1 cells. Results from this study indicate that biofilm association, disinfectant type, and substratum play an important role in the survival of Legionella pneumophila in building water systems.IMPORTANCE Microbial regrowth within building water systems are promoted by water stagnation, low disinfectant residual, high surface-to-volume ratio, amenable growth temperatures, and colonization of drinking water biofilms. Moreover, biofilms provide protection from environmental stresses, access to higher levels of nutrients, and opportunities for symbiotic interactions with other microbes. Disinfectant efficacy information is historically based on inactivation of pathogens in their planktonic, free-floating forms. However, due to the ecological importance of drinking water biofilms for pathogen survival, this study evaluated the efficacy of two common disinfectants, free chlorine and monochloramine, on Legionella pneumophila colonizing mature, drinking water biofilms established on copper and PVC surfaces. Results showed that inactivation was dependent on the disinfectant type and biofilm substratum. Overall, this, and other related research, will provide a better understanding of Legionella ecological stability and survival and aid policy makers in the management of exposure risks to water-based pathogens within building water systems.

Reconsider the burn: The transient effect of a chlorine burn on controlling opportunistic pathogens in a full-scale chloraminated engineered water system.

Nitrification is a serious water-quality issue in chloraminated engineered water systems (EWSs). Nitrification is often remediated by a chlorine burn (i.e., a free­chlorine conversion), a short-term switch from chloramination to chlorination in EWSs. Opportunistic pathogens (OPs) are the dominant infectious agents in EWSs. However, the responses of OPs to a chlorine burn are unknown. This study for the first time assessed how a chlorine burn affected OPs in a full-scale EWS. We determined the impact of a 1.5-month chlorine burn on four dominant OPs (Legionella, Mycobacterium, Pseudomonas, and Vermamoeba vermiformis) in a representative full-scale chloraminated EWS in the United States. Legionella and Mycobacterium were the most abundant OPs. In the water main, the summed concentration of the four OPs during the chlorine burn [3.27 ± 1.58 log10(GCN·L-1); GCN: genome or gene copy number] was lower (p ≤ 0.001) than before the burn [4.83 ± 0.50 log10(GCN·L-1)]. After the burn, the summed concentration increased to 4.27 ± 0.68 log10(GCN·L-1), comparable to before the burn (p > 0.05), indicating a transient effect of the chlorine burn in the water main. At the residential sites, the summed concentrations of the four OPs were comparable (p > 0.05) at 5.50 ± 0.84, 5.27 ± 1.44, and 5.08 ± 0.71 log10(GCN·L-1) before, during, and after the chlorine burn, respectively. Therefore, the chlorine burn was less effective in suppressing OP (re)growth in the premise plumbing. The low effectiveness might be due to more significant water stagnation and disinfectant residual decay in the premise plumbing. Indeed, for the entire sampling period, the total chlorine residual concentration in the premise plumbing (1.8 mg Cl2·L-1) was lower than in the water main (2.4 mg Cl2·L-1). Consequently, for the entire sampling period, the summed concentration of the four OPs in the premise plumbing [5.26 ± 1.08 log10(GCN·L-1)] was significantly higher (p < 0.001) than in the water main [4.04 ± 1.25 log10(GCN·L-1)]. In addition, the chlorine burn substantially increased the levels of disinfection by-products (DBPs) in the water main. Altogether, a chlorine burn is transient or even ineffective in suppressing OP (re)growth but raises DBP concentrations in chloraminated EWSs. Therefore, the practice of chlorine burns to control nitrification should be optimized, reconsidered, or even replaced.

Spatial and Temporal Variability of Saxitoxin-Producing Cyanobacteria in U.S. Urban Lakes.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season.

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled "Treated," 0.15 g of glucose was added, and nothing was added to the container labeled "Control." After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the "Treated" container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

Legionella pneumophila transcriptional response following exposure to CuO nanoparticles.

Copper ions are an effective antimicrobial agent used to control Legionnaires' disease and Pontiac fever arising from institutional drinking water systems. Here, we present data on an alternative bactericidal agent, copper oxide nanoparticles (CuO-NPs), and its efficacy on Legionella pneumophila. In broth cultures, the CuO-NPs caused growth inhibition, which appeared to be concentration and exposure time dependent. The transcriptomic response of L. pneumophila to CuO-NP exposure was investigated by using a whole-genome microarray. The expression of genes involved in metabolism, transcription, translation, DNA replication and repair, and unknown/hypothetical proteins was significantly affected by exposure to CuO-NPs. In addition, expression of 21 virulence genes was also affected by exposure to CuO-NP and further evaluated by quantitative reverse transcription-PCR (qRT-PCR). Some virulence gene responses occurred immediately and transiently after addition of CuO-NPs to the cells and faded rapidly (icmV, icmW, lepA), while expression of other genes increased within 6 h (ceg29, legLC8, legP, lem19, lem24, lpg1689, and rtxA), 12 h (cegC1, dotA, enhC, htpX, icmE, pvcA, and sidF), and 24 h (legP, lem19, and ceg19), but for most of the genes tested, expression was reduced after 24 h of exposure. Genes like ceg29 and rtxA appeared to be the most responsive to CuO-NP exposures and along with other genes identified in this study may prove useful to monitor and manage the impact of drinking water disinfection on L. pneumophila.

Metagenomic mapping of cyanobacteria and potential cyanotoxin producing taxa in large rivers of the United States.

Cyanobacteria and cyanotoxin producing cyanobacterial blooms are a trending focus of current research. Many studies focus on bloom events in lentic environments such as lakes or ponds. Comparatively few studies have explored lotic environments and fewer still have examined the cyanobacterial communities and potential cyanotoxin producers during ambient, non-bloom conditions. Here we used a metagenomics-based approach to profile non-bloom microbial communities and cyanobacteria in 12 major U.S. rivers at multiple time points during the summer months of 2019. Our data show that U.S. rivers possess microbial communities that are taxonomically rich, yet largely consistent across geographic location and time. Within these communities, cyanobacteria often comprise significant portions and frequently include multiple species with known cyanotoxin producing strains. We further characterized these potential cyanotoxin producing taxa by deep sequencing amplicons of the microcystin E (mcyE) gene. We found that rivers containing the highest levels of potential cyanotoxin producing cyanobacteria consistently possess taxa with the genetic potential for cyanotoxin production and that, among these taxa, the predominant genus of origin for the mcyE gene is Microcystis. Combined, these data provide a unique perspective on cyanobacteria and potential cyanotoxin producing taxa that exist in large rivers across the U.S. and can be used to better understand the ambient conditions that may precede bloom events in lotic freshwater ecosystems.

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Possible Antagonism between Cladosporium cladosporioides and Microcystis aeruginosa in a Freshwater Lake during Bloom Seasons.

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 Cladosporium cladosporioides was determined by quantitative PCR (qPCR) assay, and Microcystis aeruginosa microcystin-gene transcript copy number (McyG TCN) was quantified by reverse-transcriptase qPCR (RT-qPCR) analyses. The results showed that during each bloom season, the C. cladosporioides concentration and McyG TCN appeared to be interrelated. Therefore, C. cladosporioides concentrations were statistically evaluated via regression on McyG TCN in the water samples for lag times of 1 to 7 days. The regression equation developed to model the relationship demonstrated that a change in the C. cladosporioides concentration resulted in an opposing change in McyG TCN over an approximately 7-day interval. Although the interaction between C. cladosporioides and McyG TCN was observed in each bloom season, the magnitude of each component varied yearly. To better understand this possible interaction, outdoor Cladosporium spore-count data for the Harsha Lake region were obtained for late May through September of each year from the South West Ohio Air Quality Agency. The average Cladosporium spore count in the outdoor air samples was significantly greater in 2016 than in either 2015 or 2017, and the M. aeruginosa McyG TCN was significantly lower in Harsha Lake water samples in 2016 compared to 2015 or 2017. These results suggest that there might be a "balanced antagonism" between C. cladosporioides and M. aeruginosa during the bloom season.

Prophylactic Addition of Glucose Suppresses Cyanobacterial Abundance in Lake Water.

To mitigate harmful cyanobacterial blooms (HCBs), toxic algicides have been used, but alternative methods of HCB prevention are needed. Our goal was to test the prophylactic addition of glucose to inhibit HCB development, using Microcystis and the toxin microcystin as the HCB model. Water samples were collected weekly, from 4 June to 2 July, from Harsha Lake in southwestern Ohio during the 2021 algal bloom season. From each weekly sample, a 25 mL aliquot was frozen for a 16S rRNA gene sequencing analysis. Then, 200 mL of Harsha Lake water was added to each of the three culture flasks, and glucose was added to create concentrations of 0 mM (control), 1.39 mM, or 13.9 mM glucose, respectively. The microcystin concentration in each flask was measured after 1 and 2 weeks of incubation. The results showed an 80 to 90% reduction in microcystin concentrations in glucose-treated water compared to the control. At the end of the second week of incubation, a 25 mL sample was also obtained from each of the culture flasks for molecular analysis, including a 16S rRNA gene sequencing and qPCR-based quantification of Microcystis target genes. Based on these analyses, the glucose-treated water contained significantly lower Microcystis and microcystin producing gene (mcy) copy numbers than the control. The 16S rRNA sequencing analysis also revealed that Cyanobacteria and Proteobacteria were initially the most abundant bacterial phyla in the Harsha Lake water, but as the summer progressed, Cyanobacteria became the dominant phyla. However, in the glucose-treated water, the Cyanobacteria decreased and the Proteobacteria increased in weekly abundance compared to the control. This glucose-induced proteobacterial increase in abundance was driven primarily by increases in two distinct families of Proteobacteria: Devosiaceae and Rhizobiaceae. In conclusion, the prophylactic addition of glucose to Harsha Lake water samples reduced Cyanobacteria's relative abundance, Microcystis numbers and microcystin concentrations and increased the relative abundance of Proteobacteria compared to the control.

Effective Early Treatment of Microcystis Exponential Growth and Microcystin Production with Hydrogen Peroxide and Hydroxyapatite.

Mitigating cyanotoxin production is essential to protecting aquatic ecosystems and public health. However, current harmful cyanobacterial bloom (HCB) control strategies have significant shortcomings. Because predicting HCBs is difficult, current HCB control strategies are employed when heavy HCBs have already occurred. Our pilot study developed an effective HCB prediction approach that is employed before exponential cyanobacterial growth and massive cyanotoxin production can occur. We used a quantitative polymerase chain reaction (qPCR) assay targeting the toxin-encoding gene mcyA to signal the timing of treatment. When control measures were applied at an early growth stage or one week before the exponential growth of Microcystis aeruginosa (predicted by qPCR signals), both hydrogen peroxide (H2O2) and the adsorbent hydroxyapatite (HAP) effectively stopped M. aeruginosa growth and microcystin (MC) production. Treatment with either H2O2 (10 mg·L-1) or HAP (40 µm particles at 2.5 g·L-1) significantly reduced both mcyA gene copies and MC levels compared with the control in a dose-dependent manner. While both treatments reduced MC levels similarly, HAP showed a greater ability to reduce mcyA gene abundance. Under laboratory culture conditions, H2O2 and HAP also prevented MC production when applied at the early stages of the bloom when mcyA gene abundance was below 105 copies·mL-1.

Cyanotoxin-encoding genes as powerful predictors of cyanotoxin production during harmful cyanobacterial blooms in an inland freshwater lake: Evaluating a novel early-warning system.

Freshwater harmful cyanobacterial blooms (HCBs) potentially produce excessive cyanotoxins, mainly microcystins (MCs), significantly threatening aquatic ecosystems and public health. Accurately predicting HCBs is thus essential to developing effective HCB mitigation and prevention strategies. We previously developed a novel early-warning system that uses cyanotoxin-encoding genes to predict cyanotoxin production in Harsha Lake, Ohio, USA, in 2015. In this study, we evaluated the efficacy of the early-warning system in forecasting the 2016 HCB in the same lake. We also examined potential HCB drivers and cyanobacterial community composition. Our results revealed that the cyanobacterial community was stable at the phylum level but changed dynamically at the genus level over time. Microcystis and Planktothrix were the major MC-producing genera that thrived in June and July and produced high concentrations of MCs (peak level 10.22 µg·L-1). The abundances of the MC-encoding gene cluster mcy and its transcript levels significantly correlated with total MC concentrations (before the MC concentrations peaked) and accurately predicted MC production as revealed by logistic equations. When the Microcystis-specific gene mcyG reached approximately 1.5 × 103 copies·mL-1 or when its transcript level reached approximately 2.4 copies·mL-1, total MC level exceeded 0.3 µg L-1 (a health advisory limit) approximately one week later (weekly sampling scheme). This study suggested that cyanotoxin-encoding genes are promising predictors of MC production in inland freshwater lakes, such as Harsha Lake. The evaluated early-warning system can be a useful tool to assist lake managers in predicting, mitigating, and/or preventing HCBs.

The Bacterial Community Diversity of Bathroom Hot Tap Water Was Significantly Lower Than That of Cold Tap and Shower Water.

Microbial drinking water quality in premise plumbing systems (PPSs) strongly affects public health. Bacterial community structure is the essential aspect of microbial water quality. Studies have elucidated the microbial community structure in cold tap water, while the microbial community structures in hot tap and shower water are poorly understood. We sampled cold tap, hot tap, and shower water from a simulated PPS monthly for 16 consecutive months and assessed the bacterial community structures in those samples via high-throughput sequencing of bacterial 16S rRNA genes. The total relative abundance of the top five most abundant phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes) was greater than 90% among the 24 identified phyla. The most abundant families were Burkholderiaceae, Sphingomonadaceae, unclassified Alphaproteobacteria, unclassified Corynebacteriales, and Mycobacteriaceae. A multiple linear regression suggests that the bacterial community diversity increased with water temperature and the age of the simulated PPS, decreased with total chlorine residual concentration, and had a limited seasonal variation. The bacterial community in hot tap water had significantly lower Shannon and Inverse Simpson diversity indices (p < 0.05) and thus a much lower diversity than those in cold tap and shower water. The paradoxical results (i.e., diversity increased with water temperature, but hot tap water bacterial community was less diverse) were presumably because (1) other environmental factors made hot tap water bacterial community less diverse, (2) the diversity of bacterial communities in all types of water samples increased with water temperature, and (3) the first draw samples of hot tap water could have a comparable or even lower temperature than shower water samples and the second draw samples of cold tap water. In both a three-dimensional Non-metric multidimensional scaling ordination plot and a phylogenetic dendrogram, the samples of cold tap and shower water cluster and are separate from hot tap water samples (p < 0.05). In summary, the bacterial community in hot tap water in the simulated PPS had a distinct structure from and a much lower diversity than those in cold tap and shower water.

Co-occurring microorganisms regulate the succession of cyanobacterial harmful algal blooms.

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.

Legionella and other opportunistic pathogens in full-scale chloraminated municipal drinking water distribution systems.

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.

A comprehensive evaluation of monochloramine disinfection on water quality, Legionella and other important microorganisms in a hospital.

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<0.001) after monochloramine treatment. Mycobacterium genus-specific qPCR positivity was reduced from 92% to 65%, but the change was not significant. Heterotrophic bacteria (heterotrophic bacteria plate counts [HPCs]) exhibited large variability which skewed statistical results on a per room basis. However, when all samples were considered, a significant decrease in HPCs was observed after monochloramine addition. Lastly, Pseudomonas aeruginosa and Vermamoeba vermiformis demonstrated large and significant decrease of qPCR signals post-chloramination. General water chemistry parameters including monochloramine residual, nitrate, nitrite, pH, temperature, metals and total trihalomethanes (TTHMs) were also measured. Significant monochloramine residuals were consistently observed at all sampling sites with very little free ammonia present and no water quality indications of nitrification (e.g., pH decrease, elevated nitrite or nitrate). The addition of monochloramine had no obvious impact on metals (lead, copper and iron) and disinfection by-products.

The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3beta-independent manner.

TCF7L2 transcription factor is a downstream effector of the canonical Wnt/beta-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant beta-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell responsiveness to Wnt/beta-catenin signaling. While GSK3beta down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3beta-dependent up-regulation and a GSK3beta-independent modulation of RNA splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7-binding sites. Altogether our data reveal a lithium-induced RNA switch favoring the expression of TCF7L2-short forms, which results in a transcriptional de-repression of lithium target genes negatively regulated by TCF7L2-long forms, like TCF7L2, and thus to an amplification of Wnt-signaling in responsive cells.

Use of qPCR and RT-qPCR for monitoring variations of microcystin producers and as an early warning system to predict toxin production in an Ohio inland lake.

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 reservoir lake. Further, these data were used to develop early warning thresholds for prediction of MC concentrations exceeding the US EPA Health Advisory cutoff value (>0.3 µg L-1) using receiver operating characteristic curves and tobit regression. MCP Microcystis genomic copy number made up approximately 35% of the total Microcystis spp. and was the dominant toxic subpopulation of MCPs. 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 exceedance over the EPA Health Advisory was based on signaling of qPCR and RT-qPCR using receiver operating characteristic curves. This study illustrates the potential use of qPCR or RT-qPCR as an early warning system of extant and MC producing potentials during a toxic algal bloom, with predictive powers of 50%-60% and 30%-40% (p < 0.001), respectively, and false positive rates of about 70% for both LC-MS/MS or ELISA.

Nitrogen-phosphorus-associated metabolic activities during the development of a cyanobacterial bloom revealed by metatranscriptomics.

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.

A Gallus gallus Model for Determining Infectivity of Zoonotic Campylobacter.

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 <10 to >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.