Disinfection of water containing natural organic matter by using ozone-initiated radical reactions

Ozone is widely used to disinfect drinking water and wastewater due to its strong biocidal oxidizing properties. Recently, it was reported that hydroxyl radicals ((.)OH), resulting from ozone decomposition, play a significant role in microbial inactivation when Bacillus subtilis endospores were used as the test microorganisms in pH controlled distilled water. However, it is not yet known how natural organic matter (NOM), which is ubiquitous in sources of drinking water, affects this process of disinfection by ozone-initiated radical reactions. Two types of water matrix were considered for this study. One is water containing humic acid, which is commercially available. The other is water from the Han River. This study reported that hydroxyl radicals, initiated by the ozone chain reaction, were significantly effective at B. subtilis endospore inactivation in water containing NOM, as well as in pH-controlled distilled water. The type of NOM and the pH have a considerable effect on the percentage of disinfection by hydroxyl radicals, which ranged from 20 to 50%. In addition, the theoretical T value of hydroxyl radicals for 2-log B. subtilis removal was estimated to be about 2.4 x 10(4) times smaller than that of ozone, assuming that there is no synergistic activity between ozone and hydroxyl radicals.     

Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log₁₀ units) with LP-UV (20 mJ/cm²) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log₁₀ unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C·t value (product of the disinfectant concentration and contact time) of 60 mg·min·liter⁻¹, frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C·t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4°C to 25°C) and a decrease in pH result in better inactivation.     

Effects of Source Water Quality on Chlorine Inactivation of Adenovirus,Coxsackievirus, Echovirus,and Murine Norovirus

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15°C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration × contact time (CT) values (mg·min/liter) required to achieve 2-, 3-, and 4-log₁₀ reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log₁₀ CT values at 5°C ranging from ≤0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log₁₀ CT values at 5°C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15°C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA''s Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.Disinfection processes are critical for the reduction of infectious virus concentrations in source water, because viruses are less efficiently removed by primary treatment of drinking water (e.g., coagulation and filtration) than are other pathogen types of concern (e.g., bacteria and protozoa). Over the years, many disinfection studies have focused on the inactivation of viruses in purified and buffered, demand-free, reagent-grade water (RGW). However, relatively few investigators have examined the impact of water quality during the disinfection process, even though water quality has been found to be a significant factor for inactivation of viruses.Several researchers found that the inactivation rate of poliovirus by free chlorine increased as the ionic concentration of water increased. In one study, poliovirus 1 was inactivated three times faster in boric acid buffer than in purified water (3). In addition, several investigators found that when the ionic content of buffered water was raised by the addition of NaCl or KCl, poliovirus 1 was inactivated two to four times faster than in the buffered water alone (2, 16, 17). In another study, poliovirus 1 was inactivated 10 times more rapidly in drinking water than in purified water (4).Studies conducted with natural waters have demonstrated both increased and decreased disinfection efficacy of chlorine in these waters compared to purified or buffered waters. In a study comparing chlorine disinfection in purified water and Potomac estuarine water, coxsackievirus A9 was inactivated more rapidly in the source water. The remaining study viruses (coxsackievirus B1, echovirus 7, adenovirus 3, poliovirus 1, and reovirus 3) were all inactivated more slowly in the source water (13). Bacteriophage MS2 was inactivated more slowly by free chlorine in two types of surface water than in buffered, demand-free water. However, there was no difference between the inactivation rates of this virus in the buffered water and groundwater (10). In another study, both feline calicivirus and adenovirus 40 were inactivated more slowly in treated groundwater than in buffered, demand-free water (21).The United States Environmental Protection Agency''s (USEPA) Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources (Guidance Manual) recommends disinfectant concentration × contact time (CT) values of 4, 6, and 8 to achieve 2-, 3-, and 4-log₁₀ inactivation, respectively, with chlorine at 5°C and pH 6 to 9 (23). These CT values, which incorporate a safety factor of 3, were obtained from inactivation experiments conducted with monodispersed hepatitis A virus (HAV) in buffered, demand-free water. As water quality can significantly affect the disinfection efficacy of chlorine, it is unclear whether these CT value recommendations are sufficient for inactivation of viruses in source water. More information is needed to systematically examine the role of water quality in chlorine disinfection of viruses.The objective of the present study was to examine the disinfection efficacy of free chlorine on selected viruses from USEPA''s Contaminant Candidate List (CCL) (22) in one untreated and two partially treated source waters from distinct geographical regions. By comparing the efficacy of chlorine disinfection in the source water types to disinfection in buffered, chlorine-demand-free RGW (7), the impact of water quality could be examined. The four representative CCL viruses selected for this study included human adenovirus 2 (HAdV2), echovirus 1 (E1), coxsackievirus B5 (CVB5), and murine norovirus (MNV), a surrogate for human norovirus (22). The viruses were selected because they were previously found to be the least effectively inactivated viruses of their type in RGW (6). Disinfection experiments were carried out in duplicate in pH 7 and 8 source water at 5 and 15°C using 0.2 and 1 mg/liter free chlorine. Inactivation curves were plotted using Microsoft Excel, and CT values were calculated using the efficiency factor Hom (EFH) model (9).     

High accuracy operon prediction method based on STRING database scores

We present a simple and highly accurate computational method for operon prediction, based on intergenic distances and functional relationships between the protein products of contiguous genes, as defined by STRING database (Jensen,L.J., Kuhn,M., Stark,M., Chaffron,S., Creevey,C., Muller,J., Doerks,T., Julien,P., Roth,A., Simonovic,M. et al. (2009) STRING 8–a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res., 37, D412–D416). These two parameters were used to train a neural network on a subset of experimentally characterized Escherichia coli and Bacillus subtilis operons. Our predictive model was successfully tested on the set of experimentally defined operons in E. coli and B. subtilis, with accuracies of 94.6 and 93.3%, respectively. As far as we know, these are the highest accuracies ever obtained for predicting bacterial operons. Furthermore, in order to evaluate the predictable accuracy of our model when using an organism''s data set for the training procedure, and a different organism''s data set for testing, we repeated the E. coli operon prediction analysis using a neural network trained with B. subtilis data, and a B. subtilis analysis using a neural network trained with E. coli data. Even for these cases, the accuracies reached with our method were outstandingly high, 91.5 and 93%, respectively. These results show the potential use of our method for accurately predicting the operons of any other organism. Our operon predictions for fully-sequenced genomes are available at http://operons.ibt.unam.mx/OperonPredictor/.     

Biogenic Silver for Disinfection of Water Contaminated with Viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag⁰. The antiviral action of biogenic Ag⁰ was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter⁻¹ biogenic Ag⁰ caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg⁰) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter⁻¹ ionic Ag⁺ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag⁰ were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag⁰ was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag⁰ m⁻² on the filter (135 mg biogenic Ag⁰ kg⁻¹ filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag⁰ and its promising opportunities for continuous water disinfection.At least 1 billion people do not have access to safe drinking water, according to the WHO (41). Contamination of drinking water and the subsequent outbreak of waterborne diseases are the leading cause of death in many developing nations. Moreover, the spectrum and incidence of some infectious diseases are increasing worldwide (40). Among them, the transmission of waterborne human noroviruses is considered to be the major cause of acute nonbacterial gastroenteritis (22). Numerous outbreaks of norovirus-associated gastroenteritis have been linked with ingestion of contaminated drinking water, in developed countries also (6; M. Kukkula, L. Maunula, E. Silvennoinen, and C. H. von Bonsdorff, presented at the International Workshop on Human Caliciviruses, Atlanta, GA, 29 to 31 March 1999). Therefore, the development of innovative drinking water quality control strategies is of the utmost importance in this decade.Recent interest has arisen in the use of nanotechnology for water disinfection (20). In particular the formation of by-products by conventional disinfection techniques (e.g., chlorination), has encouraged researchers to explore the antimicrobial activity of several nanomaterials, such as silver (18, 31). Silver-containing nanoparticles have previously been demonstrated to be effective against bacteria and viral particles (10, 28, 34). Several mechanisms of the antiviral activity have been ascribed to (chemically produced) zerovalent silver nanoparticles (nAg⁰) but still remain not fully understood. On the one hand, nAg⁰ can release Ag⁺ ions, which interact with thiol groups in proteins and interfere with DNA replication (11, 21, 24). On the other hand, the adhesion of nAg⁰ as such is responsible for the inactivation of HIV-1 virions (10).Previous studies showed that chemically produced nAg⁰ were unstable in solution and would easily aggregate with average particle sizes of <40 nm or at high concentrations (23). As a consequence, the specific surface of the nanomaterial decreases. Moreover, there is a need for environmentally friendly approaches to production of nanoparticles. To cope with these demands, biological processes have been developed using microorganisms. Microbial approaches to obtain nanoscale Ag⁰ have been demonstrated for the bacterium Pseudomonas stutzeri AG259 (17) and for fungi, e.g., Verticillium sp. (26), Phoma sp. (5), Fusarium sp. (2, 16), and Aspergillus sp. (12, 29, 39). However, these enzymatic reduction processes are slow and yield low concentrations of silver. Moreover, if the nanoparticles are produced intracellularly, specific treatments (e.g., heat treatment at 600°C for 6 h) are necessary to make the nanoparticles accessible for antibacterial or antiviral applications (39).Recently, lactic acid bacteria have been used as reducing agents for the fast, nonenzymatic, and extracellular production of nanoscale-sized Ag⁰ particles (33). The bacterial cell wall hereby serves as a microscale carrier matrix for the nanoparticles. The unique association of the nanoparticles with the (dead) bacterial carrier matrix, called biogenic Ag⁰, prevents them from aggregating and makes the association promising for disinfection technologies. In the case of virus inactivation, smaller nanoparticles are known to be more efficient due to a more effective binding to the glycoproteins of the viral envelope (10, 28). For biogenic Ag⁰ production using lactic acid bacteria, it was demonstrated that different particle sizes could be obtained, depending on the species used (33). Production by Lactobacillus fermentum resulted in the smallest average diameter and a narrow size distribution, potentially favorable for antimicrobial applications (33).The objective of the present study was to examine the inactivation of a bacteriophage (UZ1), isolated from hospital sewage, by biogenic Ag⁰. This DNA phage, a T7-like coliphage of the genus Podovirida (order Caudovirales) (38), is infective for Enterobacter aerogenes BE1, a species belonging to the normal digestive microbiota (30). The virucidal action of biogenic Ag⁰ was evaluated in drinking water and compared with the use of ionic Ag⁺ and chemically produced nAg⁰. To test the antiviral activity of biogenic Ag⁰ against noroviruses as well, the murine norovirus 1 (MNV-1) was used as a surrogate organism for human noroviruses (43). Finally, continuous disinfection by the biogenic nanoparticles was evaluated in a flowthrough system with a coated cartridge filter. To our knowledge, this is the first report to demonstrate the antiviral effect of extracellular biogenic Ag⁰.     

Cometabolism of Trihalomethanes by Nitrosomonas europaea

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 μg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants () increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH₃-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (T_c), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.     

Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8T

Leucobacter salsicius M1-8^T is a member of the Microbacteriaceae family within the class Actinomycetales. This strain is a Gram-positive, rod-shaped bacterium and was previously isolated from a Korean fermented food. Most members of the genus Leucobacter are chromate-resistant and this feature could be exploited in biotechnological applications. However, the genus Leucobacter is poorly characterized at the genome level, despite its potential importance. Thus, the present study determined the features of Leucobacter salsicius M1-8^T, as well as its genome sequence and annotation. The genome comprised 3,185,418 bp with a G+C content of 64.5%, which included 2,865 protein-coding genes and 68 RNA genes. This strain possessed two predicted genes associated with chromate resistance, which might facilitate its growth in heavy metal-rich environments.     

Genome sequence of Burkholderia mimosarum strain LMG 23256T,a Mimosa pigra microsymbiont from Anso,Taiwan

Burkholderia mimosarum strain LMG 23256^T is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Mimosa pigra (giant sensitive plant). LMG 23256^T was isolated from a nodule recovered from the roots of the M. pigra growing in Anso, Taiwan. LMG 23256^T is highly effective at fixing nitrogen with M. pigra. Here we describe the features of B. mimosarum strain LMG 23256^T, together with genome sequence information and its annotation. The 8,410,967 bp high-quality-draft genome is arranged into 268 scaffolds of 270 contigs containing 7,800 protein-coding genes and 85 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Serological identification of fast progressors of structural damage with rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) patients with structural progression are in most need of immediate treatment to maintain tissue integrity. The serum protein fingerprint, type I collagen degradation mediated by matrix metalloproteinases (MMP)-cleavage (C1M), is a biomarker of tissue destruction. We investigated whether baseline serum C1M levels could identify structural progressors and if the biomarker levels changed during anti-inflammatory treatment with tocilizumab (TCZ).

Methods

The LITHE-biomarker study ({"type":"clinical-trial","attrs":{"text":"NCT00106535","term_id":"NCT00106535"}}NCT00106535, n = 585) was a one-year phase III, double-blind, placebo (PBO)-controlled, parallel group study of TCZ 4 or 8 mg/kg every four weeks, in RA patients on stable doses of methotrexate (MTX). Spearman''s ranked correlation was used to assess the correlation between baseline C1M levels and structural progression at baseline and at weeks 24 and 52. Multivariate regression was performed for delta structural progression. Change in C1M levels were studied as a function of time and treatment.

Results

At baseline, C1M was significantly correlated to C-reactive protein (P <0.0001), visual analog scale pain (P <0.0001), disease activity score28-erythrocyte sedimentation rate (DAS28-ESR) (P <0.0001), joint space narrowing (JSN) (P = 0.0056) and modified total Sharp score (mTSS) (P = 0.0006). Baseline C1M was significantly correlated with delta-JSN at Week 24 (R² = 0.09, P = 0.0001) and at Week 52 (R² = 0.27, P <0.0001), and with delta-mTSS at 24 weeks (R² = 0.006, P = 0.0015) and strongly at 52 weeks (R² = 0.013, P <0.0001) in the PBO group. C1M levels were dose-dependently reduced in the TCZ + MTX group.

Conclusions

Baseline C1M levels correlated with worsening joint structure over one year. Serum C1M levels may enable identification of those RA patients that are in most need of aggressive treatment

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00106535","term_id":"NCT00106535"}}NCT00106535     

Complete genome sequence of Mesorhizobium opportunistum type strain WSM2075T

Mesorhizobium opportunistum strain WSM2075^T was isolated in Western Australia in 2000 from root nodules of the pasture legume Biserrula pelecinus that had been inoculated with M. ciceri bv. biserrulae WSM1271. WSM2075^T is an aerobic, motile, Gram negative, non-spore-forming rod that has gained the ability to nodulate B. pelecinus but is completely ineffective in N₂ fixation with this host. This report reveals that the genome of M. opportunistum strain WSM2075^T contains a chromosome of size 6,884,444 bp, encoding 6,685 protein-coding genes and 62 RNA-only encoding genes. The genome contains no plasmids, but does harbor a 455.7 kb genomic island from Mesorhizobium ciceri bv. biserrulae WSM1271 that has been integrated into a phenylalanine-tRNA gene.     

Monochloramine Disinfection Kinetics of Nitrosomonas europaea by Propidium Monoazide Quantitative PCR and Live/Dead BacLight Methods

Monochloramine disinfection kinetics were determined for the pure-culture ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) by two culture-independent methods, namely, Live/Dead BacLight (LD) and propidium monoazide quantitative PCR (PMA-qPCR). Both methods were first verified with mixtures of heat-killed (nonviable) and non-heat-killed (viable) cells before a series of batch disinfection experiments with stationary-phase cultures (batch grown for 7 days) at pH 8.0, 25°C, and 5, 10, and 20 mg Cl₂/liter monochloramine. Two data sets were generated based on the viability method used, either (i) LD or (ii) PMA-qPCR. These two data sets were used to estimate kinetic parameters for the delayed Chick-Watson disinfection model through a Bayesian analysis implemented in WinBUGS. This analysis provided parameter estimates of 490 mg Cl₂-min/liter for the lag coefficient (b) and 1.6 × 10⁻³ to 4.0 × 10⁻³ liter/mg Cl₂-min for the Chick-Watson disinfection rate constant (k). While estimates of b were similar for both data sets, the LD data set resulted in a greater k estimate than that obtained with the PMA-qPCR data set, implying that the PMA-qPCR viability measure was more conservative than LD. For N. europaea, the lag phase was not previously reported for culture-independent methods and may have implications for nitrification in drinking water distribution systems. This is the first published application of a PMA-qPCR method for disinfection kinetic model parameter estimation as well as its application to N. europaea or monochloramine. Ultimately, this PMA-qPCR method will allow evaluation of monochloramine disinfection kinetics for mixed-culture bacteria in drinking water distribution systems.As a result of stage 1 and stage 2 disinfectant and disinfection by-product rules, chloramination for secondary disinfection in the United States is predicted to increase to 57% of all surface and 7% of all groundwater treatment systems (49). A recent survey reported that 30% of the respondents currently chloraminate to maintain distribution system residual, and other recent surveys suggest that between 8 and 12% of drinking water utilities are contemplating a future switch to chloramination (3, 43).Although chloramines are considered weaker disinfectants than chlorine for suspended bacteria, chloramines are perceived as more effective disinfectants for a biofilm (25, 53). As a result of their lower reactivity, chloramines are believed to penetrate a biofilm further and thereby to more effectively disinfect biofilm bacteria with depth than chlorine (53).Chloramination comes with the risk of distribution system nitrification (2, 21, 22). Based on utility surveys, 30 to 63% of utilities practicing chloramination for secondary disinfection experience nitrification episodes (3, 21, 43, 54). Nitrification in drinking water distribution systems is undesirable and may result in water quality degradation (e.g., disinfectant depletion, coliform occurrences, or nitrite/nitrate formation) and subsequent noncompliance with existing regulations (e.g., surface water treatment rule or total coliform rule) (2). Thus, nitrification control is a major issue in practice and is likely to become increasingly important as chloramination increases.Unfortunately, our understanding of distribution system nitrification and its control is incomplete, which has made this a topic of considerable ongoing research. Recently, Fleming et al. (12) proposed nitrification potential curves as a possible strategy to prevent nitrification in chloraminated drinking water distribution systems. Use of this concept or other modeling approaches inherently requires knowledge of both the growth and disinfection kinetic parameters of nitrifiers, specifically ammonia-oxidizing bacteria (AOB), inhabiting the distribution system.Several chloramine disinfection studies have been reported for nitrifier cultures (2). However, only one study contains a detailed determination of chloramine disinfection kinetics, having investigated the pure-culture AOB Nitrosomonas europaea (33). In contrast to this pure-culture study, AOB are present as mixed cultures in chloraminated drinking water distribution systems, with Nitrosomonas oligotropha rather than N. europaea representing the dominant AOB found (33, 37, 38). Therefore, determination of disinfection kinetics of mixed-culture AOB likely present in chloraminated drinking water (i.e., N. oligotropha) represents a significant knowledge gap in our understanding of nitrification episodes.Disinfection kinetic parameter determination inherently depends on the method used to quantify viable bacteria. In general, there are two classes of viability determinations, i.e., (i) culture-dependent and (ii) culture-independent methods (5, 16, 27). Culture-dependent methods rely on bacterial growth and include plate counts and most-probable-number (MPN) techniques. Culture-independent methods include activity measures (e.g., substrate uptake or oxygen utilization) and other methods that rely on cell membrane integrity as a viability measure. In general, culture-dependent methods result in faster disinfection kinetics than culture-independent methods.As a first step toward gaining more information on AOB disinfection in chloraminated drinking water distribution systems, a culture-independent method with future applicability to mixed-culture AOB was implemented. In the current research, N. europaea was used. Even though N. europaea has not been found to be the dominant AOB in chloraminated systems, its use in the current research provides a comparison to existing literature. The culture-independent method combines the use of propidium monoazide (PMA), which selectively removes DNA from membrane-compromised cells and/or inhibits its amplification by PCR (29-31), with a quantitative PCR (qPCR) method developed for detection of AOB in chloraminated drinking water distribution systems (36). The results using PMA-qPCR were compared with those obtained using another culture-independent membrane integrity-based technique, the Live/Dead BacLight (LD) method. Furthermore, the experimental conditions were selected (pH 8.0 and a chlorine-to-nitrogen mass ratio of 4:1) such that monochloramine was the dominant chloramine species present, and the results are reported as monochloramine disinfection kinetics. The magnitude of the reported disinfection kinetics was closely related to the respective method used for viability determination. For example, in this research a cell was considered viable or nonviable based on the ability of propidium iodide (PI) or PMA to penetrate its membrane and on subsequent processing according to the respective method.LD was previously used to determine detailed N. europaea disinfection kinetics (33) and provides a baseline comparison for the current research. Oldenburg et al. (33) provided a comparison of estimated disinfection kinetic parameters, using both the culture-dependent AOB MPN technique and LD as viability measures. The estimated disinfection kinetic parameters based on the AOB MPN method were 3 orders of magnitude greater than those obtained with the culture-independent LD method, and the lower disinfection kinetics based on LD were more consistent with AOB persistence in chloraminated drinking water distribution systems. Based on this previous research and because the AOB MPN method requires an incubation period of 21 to 30 days, it was not evaluated in the current research (2).Initially, control experiments were conducted with various proportions of heat-killed cells to verify that both the PMA-qPCR and LD methods detected only viable cells. After the control experiments, a series of batch disinfection experiments were conducted where both PMA-qPCR and LD were utilized to quantify viable bacteria, providing two data sets for disinfection kinetic parameter estimation. Ultimately, the PMA-qPCR method used in this research will be applied to mixed-culture AOB typically present in drinking water distribution systems (i.e., N. oligotropha) (36-38).     

Genome Sequences of Pelagibaca bermudensis HTCC2601T and Maritimibacter alkaliphilus HTCC2654T,the Type Strains of Two Marine Roseobacter Genera

Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here, we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to-extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.Organisms from the Roseobacter clade of the Alphaproteobacteria are numerically significant in the world''s oceans and have been found in a wide range of habitats (1, 3). Using previously described high-throughput dilution-to-extinction culturing (6, 13), the marine Roseobacter strains Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T were isolated in low-nutrient heterotrophic medium (LNHM) (4) from surface water collected at the Bermuda Atlantic Time-Series Study (BATS) site in the western Sargasso Sea (5, 9). As the type strains for two genera of this globally prolific Roseobacter group, P. bermudensis and M. alkaliphilus were selected for shotgun genome sequencing at the J. Craig Venter Institute through the Moore Foundation Microbial Genome Sequencing Project (http://www.moore.org/microgenome). Draft genomes of P. bermudensis and M. alkaliphilus, with 103 and 46 contigs, respectively, were annotated and analyzed through the Joint Genome Institute IMG/M website (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) (10).The draft genomes of P. bermudensis and M. alkaliphilus comprise 5,425,920 and 4,529,231 bases, 5,522 and 4,764 predicted open reading frames (ORFs), and 66.44% and 64.13% G+C content, respectively. The P. bermudensis genome is predicted to contain 56 tRNA genes, five 5S rRNA genes, four 16S rRNA genes, and five 23S rRNA genes, and that of M. alkaliphilus 49 tRNA genes and one each of the 5S, 16S, and 23S rRNA genes. Both genomes have putative genes for complete glycolysis and Entner-Doudoroff pathways, a complete tricarboxylic acid cycle, and predicted metabolic pathways for the oxidation of C₁ compounds. Both have predicted genes for the synthesis of most essential amino acids and some vitamins and cofactors. Each has putative genes for the utilization of fructose, sucrose, and mannose, confirmed in physiological testing of P. bermudensis (5) but not for M. alkaliphilus (9). P. bermudensis contains a predicted complete RuBisCO complex, unique to the sequenced Roseobacter species (12, 15), a complete assimilatory nitrate reduction pathway, and several type VI secretion genes. M. alkaliphilus is predicted to have complete nitrate reduction pathways to both N₂ and ammonia and most type IV secretion genes. Both are predicted to have complete sec pathways and large numbers of ABC transporters (362 in P. bermudensis and 224 in M. alkaliphilus), similar to other Roseobacter strains (15).M. alkaliphilus was named because of its alkaline growth optimum at pH 10. Na⁺/H⁺ antiporters have been shown to be involved in conferring alkaliphilic phenotypes for a variety of organisms by increasing internal cellular H⁺ concentrations in alkaline conditions where Na⁺ is present (2, 7, 8, 14, 16, 17). As expected, the genome of M. alkaliphilus contains two putative Na⁺/H⁺ antiporters, one homologous to nhaP, important for alkaliphily in several strains (2, 16, 17), and another located adjacent to predicted ABC transporter genes for capsular polysaccharide export.     

An ultrasonic inactivation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> glucose oxidase in aqueous solutions

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37–59°C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constantsk _in, k*_in andk _in(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO^·—DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.     

UV Light Inactivation of Bacterial Biothreat Agents

Seven species of bacterial biothreat agents were tested for susceptibility to UV light (254 nm). All gram-negative organisms tested required <12 mJ/cm² for a 4-log₁₀ reduction in viability (inactivation). Tailing off of the B. anthracis spore inactivation curves began close to the 2-log₁₀ inactivation point, with a fluence of approximately 40 mJ/cm², and 3-log₁₀ inactivation still was not achieved with a fluence of 120 mJ/cm².The security of our nation''s water supply is a concern for water providers and public health officials. Questions have been asked regarding the possibility of our drinking water becoming contaminated with biothreat agents and the efficacy of current disinfection practices for the reduction in viability (inactivation) of biothreat agents (5, 8, 14). The use of UV irradiation as a supplemental water disinfection practice is increasing for several reasons, among them improving control of protozoa, such as Cryptosporidium spp., and decreasing disinfection by-products created by chemical disinfectants (21). This study employed a bench-scale collimated beam test to determine the UV fluence (dose) required to inactivate seven representative bacterial biothreat agents.Seven species, two isolates each, from the Health and Human Services and U.S Dept. of Agriculture lists of select agents (http://www.selectagents.gov/resources/List%20of%20Select%20Agents%20and%20Toxins_111708.pdf) were used in this study: Bacillus anthracis Ames spores, B. anthracis 34F2 (Sterne) spores, Brucella melitensis ATCC 23456, B. melitensis IL195, Brucella suis KS528, B. suis MO562, Burkholderia mallei M9, B. mallei M13, Burkholderia pseudomallei ATCC 11688, B. pseudomallei CA650, Francisella tularensis LVS, F. tularensis NY98, Yersinia pestis A1122, and Y. pestis Harbin. B. anthracis was grown on soil extract-peptone-beef extract agar (SEA) (1) or in Schaeffer''s sporulation medium (SSM) (10) for 7 days, resulting in >99% spores as determined by phase-contrast microscopy. The cells and spores were then washed by centrifugation (8,000 × g), resuspended in ultrapure water, transferred to centrifuge tubes, treated with 50% ethanol for 1 h at room temperature, and washed five times with sterile ultrapure water before being stored in reverse-osmosis water at −70°C. F. tularensis isolates were grown on cysteine heart agar (1) and all other isolates on Trypticase soy agar with 5% sheep blood (TSA II; Becton Dickinson Microbiology Systems, Sparks, MD) for 24 h before testing. B. anthracis spores were adjusted to 10⁷ CFU/ml and other bacterial suspensions to 10⁸ CFU/ml in Butterfield buffer (3 mM KH₂PO₄, pH 7.2; Becton Dickinson Microbiology Systems), and then isolates were sonicated (40-Hz ultrasonic cleaner; VWR, Suwanee, GA) for 1 min to disperse aggregates. The suspensions were diluted 1:100 in Butterfield buffer for final test concentrations. Five milliliters of each suspension were placed into a small petri dish (50-mm diameter) along with a small sterile stir bar, and the petri dish was placed on a stir plate.UV irradiation was performed by using a collimated beam apparatus (Calgon Carbon, Pittsburgh, PA) equipped with a low-pressure lamp (254 nm) according to the standard method developed by Bolton and Linden (2). The surface of the suspension was placed 5 cm from the end of the collimating tube. The UV intensity was measured with a radiometer at 0.5-cm intervals across the test area and variability compensated for according to the UVCalc software directions (International UV association [http://www.iuva.org]). The fluences (UV doses) were determined using the UVCalc software, and the petri dishes were placed under the beam for at least five time periods to deliver a range of appropriate fluences to the organisms. Each irradiation test was conducted at room temperature (23 ± 2°C) in triplicate and in a random order of fluences. After exposure, 10-fold serial dilutions were performed, and the dilutions were spread plated in triplicate. Plates were placed in a dark incubator within 10 min of plating and incubated at the temperature appropriate for the organism, and the colonies were counted at 24 h and 48 h for B. anthracis and at 3 and 5 days for the remaining organisms. Colonies were counted, and the log₁₀ inactivation at each fluence was determined for each organism. A linear regression of the fluence response data determined the fluence required for 2-, 3-, and 4-log₁₀ inactivation.The UV fluences required for inactivation of each organism are reported in Table ​Table1.1. Little difference in UV susceptibility was seen between the gram-negative organisms. B. suis KS528 and B. melitensis ATCC 23456 required the greatest UV fluence of the gram-negative organisms for 4-log₁₀ inactivation (10.5 and 10.2 mJ/cm², respectively), while the two Y. pestis isolates required the lowest UV fluence (4.1 and 4.9 mJ/cm²) for the same 4-log₁₀ inactivation (Table ​(Table1).1). Generally, the two isolates of each species differed no more than 3 mJ/cm² in the UV fluence required for 4-log₁₀ inactivation.

TABLE 1.

UV fluence required for given log₁₀ inactivation of each organism

Inactivation of Adenoviruses,Enteroviruses, and Murine Norovirus in Water by Free Chlorine and Monochloramine

Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5°C. CT values (disinfectant concentration × time) for 2- to 4-log₁₀ (99 to 99.99%) reductions in virus titers were calculated by using the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg·min/liter (pH 7 and 8) for 4-log₁₀ inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18 mg·min/liter for 2- and 3-log₁₀ reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log₁₀ reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. These data will be useful in drinking water risk assessment studies and disinfection system planning.Disinfection is a critical step in the drinking water treatment process to inactivate infectious viruses because primary treatment is less effective for the removal of viruses. Chlorine and monochloramine are the most widely used disinfectants in the United States (2). Free chlorine is widely used as a primary disinfectant following filtration and also as a secondary disinfectant in distribution systems. Under the Long Term 2 Enhanced Surface Water Treatment Rule (38), monochloramine can also be used as a primary disinfectant, but because it requires longer contact times to achieve the same level of disinfection as free chlorine it is primarily used as a secondary disinfectant to maintain a stable disinfectant residual in the distribution system and minimize disinfection by-product formation and biofilm growth.The efficacy of chlorine disinfection for viruses has been evaluated in numerous studies over the years. Many early studies focused on the disinfection of polioviruses by chlorine (14, 17, 26, 28, 30, 39, 40, 43). Early investigators suggested a number of variables that must be controlled in the disinfection of viruses: contact time, temperature, ionic strength, pH, chlorine concentration, and virus aggregation (29, 30). These researchers concluded that comparisons and general trends of disinfection efficacy can only be discerned for viruses when the same disinfection parameters are applied.Fewer studies have investigated the disinfection efficacy of monochloramine, but monochloramine disinfection has been found to be less effective than free chlorine for viruses. In comparative studies of chlorine and monochloramine disinfection, coxsackievirus B5, adenovirus 2, and adenovirus 41 were found to be inactivated far more readily by chlorine than monochloramine (4, 5, 32). For drinking water treatment systems where monochloramine is used as a secondary disinfectant, it is important to know its efficacy for a wide range of viruses, as infectious viruses may be introduced in the distribution system where only monochloramine is present. In addition, relatively few studies have investigated the efficacy of monochloramine as systematically as free chlorine; frequently only one concentration, pH, or temperature has been investigated. Two notable exceptions were investigations that examined monochloramine disinfection of human adenovirus 2 (HAdV2) and coxsackievirus B5 (CVB5) at multiple pH levels (21, 31).In 2005, the U.S. Environmental Protection Agency (USEPA) published its second candidate contaminant list (CCL2). The CCL2 is comprised of unregulated microbial and chemical contaminants of potential public health concern that are known or anticipated to occur in drinking water systems and includes: echovirus, coxsackievirus, adenovirus, and calicivirus (36). A number of researchers have reported the disinfection efficacy of free chlorine for representatives of the CCL2 viruses (4, 5, 7, 11, 13, 18, 20, 22, 27, 33, 34, 35), but fewer studies have investigated the disinfection efficacy of monochloramine on these viruses (4, 5, 21, 31). In addition, comparison between existing studies of chlorine or monochloramine disinfection is difficult because of differences in the viruses examined, experimental parameters investigated, and analytical methods used.The present study compared the inactivation kinetics for representative CCL2 viruses with levels of free chlorine and monochloramine recommended for drinking water disinfection. Duplicate experiments with both disinfectants were carried out in pH 7 and 8 buffered chlorine-demand-free (CDF) water at 5°C, with eight viruses chosen to represent the CCL2 virus types. Coxsackieviruses B5 and B3 (CVB5 and CVB3) and echoviruses 1 and 11 (E1 and E11) were chosen based on existing data suggesting resistance to free chlorine, disease implications, and likelihood of presence in higher numbers in natural water. Three representative human adenoviruses were studied, including both serotypes of species F HAdV (40 and 41) that cause gastroenteritis and HAdV2, a representative of respiratory HAdV that may be found in water because they are present in fecal excretions (9). Murine norovirus (MNV), phylogenetically similar to human norovirus and the only norovirus that can be propagated in cell culture, was used as a surrogate for human norovirus. Kinetic inactivation curves are presented, and CT values (disinfectant concentration × time, reported in mg·min/liter) were calculated by using the efficiency factor Hom (EFH) model (16).     

Flavonoids: Efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44°C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm²). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k _in, k*_in, and k _in(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01–50 μM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol ? astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO^· and O ₂ ^·? in G6PDH inactivation in solutions.     

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165T

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165^T. The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10⁶ M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.     

Assembly of Collagen Matrices as a Phase Transition Revealed by Structural and Rheologic Studies

We have studied the structural and viscoelastic properties of assembling networks of the extracellular matrix protein type-I collagen by means of phase contrast microscopy and rotating disk rheometry. The initial stage of the assembly is a nucleation process of collagen monomers associating to randomly distributed branched clusters with extensions of several microns. Eventually a sol-gel transition takes place, which is due to the interconnection of these clusters. We analyzed this transition in terms of percolation theory. The viscoelastic parameters (storage modulus G′ and loss modulus G″) were measured as a function of time for five different frequencies ranging from ω = 0.2 rad/s to 6.9 rad/s. We found that at the gel point both G′ and G″ obey a scaling law , with the critical exponent Δ = 0.7 and a critical loss angle being independent of frequency as predicted by percolation theory. Gelation of collagen thus represents a second order phase transition.