Legionella monitoring results by water quality characteristics in a large public water system.

Legionella, the causative agent of Legionnaires' disease, is an emerging concern for water utilities. Passaic Valley Water Commission (PVWC) is a public drinking water supplier, which provides treated surface water to approximately 800,000 customers in New Jersey. To evaluate the occurrence of Legionella in the PVWC distribution system, swab, first draw, and flushed cold water samples were collected from total coliform sites (n = 58) during a summer and winter sampling event. Endpoint PCR detection methods were combined with culture for Legionella detection. Among 58 total coliform sites during the summer, 17.2% (10/58) of first draw samples were positive for 16S and mip Legionella DNA markers and 15.5% (9/58) in flushed samples. Across both summer and winter sampling, a total of four out of 58 sites had low-level culture detection of Legionella spp. (0.5-1.6 CFU/mL) among first draw samples. Only one site had both a first and flush draw detection (8.5 CFU/mL and 1.1 CFU/mL) for an estimated culture detection frequency of 0% in the summer and 1.7% in the winter among flushed draw samples. No L. pneumophila was detected by culture. Legionella DNA detection was significantly greater in the summer than in the winter, and detection was greater in samples collected from areas treated with phosphate. No statistical difference was found between first draw and flush sample detection. Total organic carbon, copper, and nitrate were significantly associated with Legionella DNA detection.

In Vitro and In Silico Analysis of the Inhibitory Activity of EGCG-Stearate against Herpes Simplex Virus-2.

About half a billion people worldwide are infected with herpes simplex virus-2 (HSV-2). Prolonged treatment with acyclovir (ACV) and its analogs leads to the development of resistant strains. The aim of this study was to investigate the antiviral potential of epigallocatechin gallate (EGCG) from Camellia sinensis and a stable analog EGCG-stearate (EGCG-S) against HSV-2 in cultured Vero cells. Cell viability and cell proliferation assays were used to determine the non-cytotoxic concentrations on cultured Vero cells. HSV-2 with a green fluorescent protein (GFP) fusion protein of VP26 virions were treated with non-cytotoxic concentrations of EGCG and EGCG-S. The effects on infectivity and mechanisms were determined by plaque assay, attachment and penetration assays, confocal microscopy, qPCR, and in silico modeling analysis. Our results demonstrate that treatment of HSV-2 virions with EGCG and EGCG-S at a concentration of 75 µM showed greater than 99.9% inhibition by inhibiting the attachment of HSV-2 virions to host cells. The bioinformatic analysis indicated high binding affinity of EGCG-S for glycoprotein D; thus EGCG-S may block fusion of HSV-2 and the cell membrane, preventing entry of HSV-2 into the cell.

In Vitro Analysis of the Antioxidant and Antiviral Activity of Embelin against Herpes Simplex Virus-1.

Herpes simplex virus-1 (HSV-1) causes a wide range of infections from mild to life-threatening in the human population. There are effective treatments for HSV-1 infections that are limited due HSV-1 latency and development of resistance to current therapeutics. The goal of this study was to investigate the antioxidant and antiviral effects of embelin on HSV-1 in cultured Vero cells. Oxidative stress was verified by an extensive production of a reactive oxygen species (ROS) H2O2. Vero cells were infected with a recombinant strain of HSV-1 and antiviral assays, time course attachment, penetration, and post penetration assays, confocal microscopy, qPCR, and antioxidant assays were conducted. Our results lead to the conclusion that embelin is noncytotoxic at concentrations tested ranging from 20 to 70 µM. Treatment of HSV-1 virions with embelin resulted in 98.7-100% inhibition and affected the early stage of HSV-1 infection of Vero cells, by inhibiting the attachment and penetration of HSV-1 virions to host cells. Treatment of virions with concentrations of embelin ranging from 35 to 60 µM significantly reduced the production of H2O2. In conclusion, embelin reduces oxidative damage caused by HSV-1 infection and is an effective antiviral to reduce the infection of HSV-1 in cultured Vero cells. Further studies are needed to explore the possibility of embelin as a medicinal agent.

Inhibition of Biofilm Formation by the Synergistic Action of EGCG-S and Antibiotics.

Biofilm, a stress-induced physiological state, is an established means of antimicrobial tolerance. A perpetual increase in multidrug resistant (MDR) infections associated with high mortality and morbidity have been observed in healthcare settings. Multiple studies have indicated that the use of natural products can prevent bacterial growth. Recent studies in the field have identified that epigallocatechin gallate (EGCG), a green tea polyphenol, could disrupt bacterial biofilms. A modified lipid-soluble EGCG, epigallocatechin-3-gallate-stearate (EGCG-S), has enhanced the beneficial properties of green tea. This study focuses on utilizing EGCG-S as a novel synergistic agent with antibiotics to prevent or control biofilm. Different formulations of EGCG-S and selected antibiotics were used to study their combinatorial effects on biofilms produced by five potential pathogenic bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Mycobacterium smegmatis. The crystal violet (CV) assay and the sensitive fluorescence-based resazurin biofilm viability assay were used to assess the biofilm production. Our results identified optimal formulation for each bacterium, effectively inhibiting biofilm formation to an extent of 95-99%. Colony-forming unit (CFU) and cell viability analyses showed a decrease of viable bacteria. These results depict the potential of EGCG-S as a synergistic agent with antibiotics and as an anti-biofilm agent.

Prediction of recreational water safety using Escherichia coli as an indicator: case study of the Passaic and Pompton rivers, New Jersey.

As contact with high concentrations of pathogens in a waterbody can cause waterborne diseases, Escherichia coli is commonly used as an indicator of water quality in routine public health monitoring of recreational freshwater ecosystems. However, traditional processes of detection and enumeration of pathogen indicators can be costly and are not time-sensitive enough to alarm recreational users. The predictive models developed to produce real-time predictions also have various methodological challenges, including arbitrary selection of explanatory variables, deterministic statistical approach, and heavy reliance on correlation instead of the more rigorous multivariate regression analyses, among others. The objective of this study is to address these challenges and develop a cost-effective and timely alternative for estimating pathogen indicators using real-time water quality and quantity data. As a case study we use New Jersey, where pathogens represent the most common cause of impairment for water quality, and Passaic and Pompton rivers, which are among the largest in the state and the country. We used Membrane Filtration Method and mColiblue24 media to enumerate Escherichia coli in a total of 69 water samples collected from April to November 2016 from the two rivers. We also collected data on environmental variables concurrently and performed stepwise and logistic regression analyses to address the said methodological challenges and determine the variables significantly predicting whether or not the Escherichia coli count was above prescribed levels for recreation activities. The results show that source water, higher specific conductance, lower pH, and cumulative rainfall for the 72 h antecedent the sampling significantly impacted the density of Escherichia coli. In addition to using the Bagging technique to validate the results, we also assessed Whole Model Tests, R2, Entropy R2, and Misclassification Rates. This approach improves the prediction of bacteria counts and their use in informing the potential safety/hazard of that waterbody for recreational activities.

Green Tea Polyphenol Epigallocatechin-3-Gallate-Stearate Inhibits the Growth of Streptococcus mutans: A Promising New Approach in Caries Prevention.

Streptococcus mutans (S. mutans) is the main etiological bacteria present in the oral cavity that leads to dental caries. All of the S. mutans in the oral cavity form biofilms that adhere to the surfaces of teeth. Dental caries are infections facilitated by the development of biofilm. An esterified derivative of epigallocatechin-3-gallate (EGCG), epigallocatechin-3-gallate-stearate (EGCG-S), was used in this study to assess its ability to inhibit the growth and biofilm formation of S. mutans. The effect of EGCG-S on bacterial growth was evaluated with colony forming units (CFU) and log reduction; biofilm formation was qualitatively determined by Congo red assay, and quantitatively determined by crystal violet assay, fluorescence-based LIVE/DEAD assays to study the cell viability, and scanning electron microscopy (SEM) was used to evaluate the morphological changes. The results indicated that EGCG-S was able to completely inhibit growth and biofilm formation at concentrations of 250 µg/mL. Its effectiveness was also compared with a commonly prescribed mouthwash in the United States, chlorhexidine gluconate. EGCG-S was shown to be equally effective in reducing S. mutans growth as chlorhexidine gluconate. In conclusion, EGCG-S is potentially an anticariogenic agent by reducing bacterial presence in the oral cavity.

Characterization of zinc stress response in Cyanobacterium Synechococcus sp. IU 625.

The ability of cyanobacteria to survive many environmental stress factors is a testament to their resilience in nature. Of these environmental stress factors, overexposure to zinc is important to study since excessive zinc intake can be a severe hazard. Zinc toxicity in freshwater has been demonstrated to affects organisms such as invertebrates, algae and cyanobacteria. Cyanobacteria which possess increased resistance to zinc have been isolated. It is therefore important to elucidate the mechanism of survival and response to determine what factors allow their survival; as well as any remediation implications they may have. To characterize the effects of zinc in freshwater cyanobacteria, we investigated the response of Synechococcus sp. IU 625 (S. IU 625) over 29days to various concentrations (10, 25, and 50mg/L) of ZnCl2. S. IU 625 was shown to be tolerant up to 25mg/L ZnCl2 exposure, with 10mg/L ZnCl2 having no outward physiological change and 50mg/L ZnCl2 proving lethal to the cells. To determine a potential mechanism Inductive Coupled Plasma-Mass Spectrometry (ICP-MS) and RNA-seq analysis were performed on zinc exposed cells. Analysis performed on days 4 and 7 indicated that response is dose-dependent, with 10mg/L ZnCl2 exhibiting nearly all zinc extracellular, corresponding with upregulation of cation transport response. Whereas the 25mg/L ZnCl2 exhibited half of total zinc sequestered by the cells, which corresponds with the upregulation of sequestering proteins such as metallothionein and the downregulation of genes involved with ATP synthesis and phycobilisome assembly. These analyses were combined with growth monitoring, microscopy, quantitative polymerase chain reaction (qPCR) and flow cytometry to present a full spectrum of mechanisms behind zinc response in S. IU 625.

Antiviral activity of theaflavin digallate against herpes simplex virus type 1.

Tea is the second most consumed drink in the world. The beneficial effects of tea have been mostly attributed to its catechin content. Black tea is derived from the leaves of Camellia sinensis plant, and it is rich in theaflavin polyphenols, in particular theaflavin (TF1), theaflavin-3-monogallate (TF2A), theaflavin-3'-monogallate (TF2B), and theaflavin-3,3'-digallate (TF3). Vero and A549 cells were used to evaluate the effect of purified individual black tea theaflavins as anti-herpes simplex virus 1 agents. With the rise of HSV resistant strains, there is a critical need to develop novel antiherpesviral treatments. Results of the cytotoxicity assay tested by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] showed that TF1, TF2, and TF3 are not toxic to Vero and A549 cells at a concentration up to 75 µM. The antiviral activity of the individual theaflavins was tested by plaque reduction assay, MTS assay, flow cytometric analysis and confocal microscopy observations. The results showed that TF1, TF2, and TF3 exhibit potent, dose-dependent anti-HSV-1 effect, with TF3 being the most efficient in both Vero and A549 cells. A concentration of 50 µM TF3 and above was sufficient to inhibit >99% of the production of HSV-1 viral particles. The anti-HSV-1 effect of TF3 is due to a direct effect on the virions, and treating Vero or A549 cells with TF3 for 1h prior to infection, or treating the cells at different times post infection does not inhibit HSV-1 production. TF3 is stable at vaginal pH, indicating its potential to be a promising natural and affordable remedy against herpes simplex viral infections.

Physiological effects of nickel chloride on the freshwater cyanobacterium Synechococcus sp. IU 625.

Harmful algal blooms (HABs) are a serious environmental problem globally. The ability of cyanobacteria, one of the major causative agents of HABs, to grow in heavy metal polluted areas is proving a challenge to environmental restoration initiatives. Some cyanobacteria secrete toxins, such as microcystin, that are potentially dangerous to animals and humans. In this study, the physiology of a cyanobacterium was assessed to nickel chloride exposure. Cell growths were monitored throughout the study with various nickel chloride concentrations (0, 10, 25 or 50 mg/L). Morphological abnormalities were observed with microscopic image analyses. Inductively coupled plasma mass spectrometry (ICP-MS) was carried out to trace the distribution of nickel during the growth period. This study provides insight on potential nickel response mechanisms in freshwater cyanobacteria, which may lead to effective HAB prevention strategy development.

Inhibition of herpes simplex virus type 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate.

Green tea polyphenol epigallocatechin gallate (EGCG) is a strong antioxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG, palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for herpes simplex virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of herpes simplex virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 µM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.

Adaption of Synechococcus sp. IU 625 to growth in the presence of mercuric chloride.

Resistance to heavy metals is important for the survival of bacteria in contaminated environments. In this study, we show that the unicellular cyanobacterial species Synechococcus sp. IU 625 adapts to growth in the presence of mercuric chloride, recovering from pigmentation and morphological defects. Cells accumulate mercury within 2 h of growth and by 3 days, the total mercury concentration is significantly reduced, with all remaining mercury associated with the cells. This suggests that Synechococcus sp. IU 625 can convert mercury to a volatile form.

Temperature-induced activation of freshwater Cyanophage AS-1 prophage.

Synechococcus sp. IU 625 is one of the freshwater cyanobacteria responsible for harmful algal blooms (HAB). Cyanophages can serve as natural control agents and may be responsible for algal bloom prevention and disappearance. Cyanophage AS-1, which infects Synechococcus sp. IU 625 (Anacystis nidulans) and Synechococcus cedrorum, plays an important role in the environment, significantly altering the numbers of its hosts. Since seasonal (temperature-dependent) lytic induction of cyanobacterial prophage has been proposed to affect seawater algal blooms, we investigated if the AS-1 lytic cycle could be induced by a shift to high temperature. Our hypothesis was confirmed, as more phages were released at 35°C than at 24°C, with maximal induction observed with a shift from 24 to 35°C. Furthermore, transmission electron microscopy (TEM) images provide direct evidence of lysogenic to lytic conversion with temperature shift. Thus, temperature is an important inducer for AS-1 conversion from lysogenic to lytic cycle and could have applications in terms of modulating cyanobacterial populations in freshwater aquatic environments. The study gives insight into the effect of climate change on the interaction between cyanophage and cyanobacteria in freshwater ecosystems.

Induction of temperate cyanophage AS-1 by heavy metal--copper.

BACKGROUND: It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle. RESULTS: In this study, the possible temperate A. nidulans was treated with different concentrations of heavy metal-copper. CuSO4 with concentrations of 3.1 x 10(-3) M, 3.1 x 10(-4) M, 3.1 x 10(-5) M and 3.1 x 10(-6) M were used to detect the induction of AS-1 from A. nidulans. The population of the host, unicellular cyanobacteria Anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units (PFU) by a direct plating method. The ratio of AS-1 release from A. nidulans was also determined. From these results it appears that AS-1 lysogenic phage can be induced by copper at concentrations from 3.1 x 10(-6) M to 3.1 x 10(-4) M. Maximal phage induction occurred at 6 hours after addition of copper, with an optimal concentration of 3.1 x 10(-6) M. CONCLUSION: Cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems.