Occurrence revisited: Mycobacterium avium and Mycobacterium intracellulare in potable water in the USA.

Nontuberculous mycobacterium (NTM) infections are increasing in the USA and have a high cost burden associated with treatment. Thus, it is necessary to understand what changes could be contributing to this increase in NTM disease rate. Water samples from 40 sites were collected from around the USA. They represented three water types: groundwater disinfected with chlorine and surface water disinfected with chlorine or monochloramine. Two methods, culture and qPCR, were used to measure M. avium and M. intracellulare. Heterotrophic bacteria and NTM counts were also measured. M. avium and M. intracellulare were molecularly detected in 25% (73/292) and 35% (102/292) of samples. The mean concentrations of M. avium and M. intracellulare were 2.8 × 103 and 4.0 × 103 genomic units (GU) L-1. The Northeast sites had the highest sample positively rate for both M. avium and M. intracellulare. The highest NTM counts and M. avium concentrations were observed in the surface water treated with chloramine. Geographic location and source water/disinfectant type were observed to significantly influence M. avium and M. intracellulare occurrence rates. These studies can help improve public health risk management by balancing disinfectant treatments and diverse microbial loads in drinking water. KEY POINTS: • M. avium (MA) culture rate increased significantly: 1% (1999) to 13%. • Culture versus qPCR method: 13% vs 31% for MA and 6% vs 35% for MI. • The results of each method type tell two different stories of MA and MI occurrence.

Phase transitions in evolutionary dynamics.

Sharp changes in state, such as transitions from survival to extinction, are hallmarks of evolutionary dynamics in biological systems. These transitions can be explored using the techniques of statistical physics and the physics of nonlinear and complex systems. For example, a survival-to-extinction transition can be characterized as a non-equilibrium phase transition to an absorbing state. Here, we review the literature on phase transitions in evolutionary dynamics. We discuss directed percolation transitions in cellular automata and evolutionary models, and models that diverge from the directed percolation universality class. We explore in detail an example of an absorbing phase transition in an agent-based model of evolutionary dynamics, including previously unpublished data demonstrating similarity to, but also divergence from, directed percolation, as well as evidence for phase transition behavior at multiple levels of the model system's evolutionary structure. We discuss phase transition models of the error catastrophe in RNA virus dynamics and phase transition models for transition from chemistry to biochemistry, i.e., the origin of life. We conclude with a review of phase transition dynamics in models of natural selection, discuss the possible role of phase transitions in unraveling fundamental unresolved questions regarding multilevel selection and the major evolutionary transitions, and assess the future outlook for phase transitions in the investigation of evolutionary dynamics.

Chloramine Concentrations within Distribution Systems and Their Effect on Heterotrophic Bacteria, Mycobacterial Species, and Disinfection Byproducts.

Chloramine is a secondary disinfectant used to maintain microbial control throughout public water distribution systems. This study investigated the relationship between chloramine concentration, heterotrophic bacteria, and specific Mycobacterium species. Sixty-four water samples were collected at four locations within the utility's distribution network on four occasions. Water samples were analyzed for total chlorine and monochloramine. Traditional culture methods were applied for heterotrophic bacteria and nontuberculous mycobacteria (NTM), and specific quantitative polymerase chain reaction (qPCR) assays were used to detect and quantify Mycobacterium avium, M. intracellulare, and M. abscessus. Total chlorine and monochloramine concentrations decreased between the distribution entry point (4.7 mg/L and 3.4 mg/L as Cl2, respectively) to the maximum residence time location (1.7 mg/L and 1.1 mg/L as Cl2, respectively). Results showed that heterotrophic bacteria and NTM counts increased by two logs as the water reached the average residence time (ART) location. Microbiological detection frequencies among all samples were: 86% NTMs, 66% heterotrophic bacteria, 64% M. abscessus, 48% M. intracellulare, and 2% M. avium. This study shows that heterotrophic bacteria and NTM are weakly correlated with disinfectant residual concentration, R2=0.18 and R2=0.04, respectively. Considering that specific NTMs have significant human health effects, these data fill a critical knowledge gap regarding chloramine's impact on heterotrophic bacteria and Mycobacterial species survival within public drinking water distribution systems.

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.

Phase transition behaviour in yeast and bacterial populations under stress.

Non-equilibrium phase transitions from survival to extinction have recently been observed in computational models of evolutionary dynamics. Dynamical signatures predictive of population collapse have been observed in yeast populations under stress. We experimentally investigate the population response of the budding yeast Saccharomyces cerevisiae to biological stressors (temperature and salt concentration) in order to investigate the system's behaviour in the vicinity of population collapse. While both conditions lead to population decline, the dynamical characteristics of the population response differ significantly depending on the stressor. Under temperature stress, the population undergoes a sharp change with significant fluctuations within a critical temperature range, indicative of a continuous absorbing phase transition. In the case of salt stress, the response is more gradual. A similar range of response is observed with the application of various antibiotics to Escherichia coli, with a variety of patterns of decreased growth in response to antibiotic stress both within and across antibiotic classes and mechanisms of action. These findings have implications for the identification of critical tipping points for populations under environmental stress.

Draft Genome Sequences of Seven Legionella pneumophila Isolates from a Hot Water System of a Large Building.

Public health data show that a significant fraction of the nation's waterborne disease outbreaks are attributable to premise plumbing. We report the draft genome sequences of seven Legionella pneumophila serogroup 1 isolates from hot water lines of a large building. Genomic analysis identified the isolates as belonging to sequence type 1.

Descriptive epidemiology of hepatitis C in individuals referred for specialized HCV care in Newfoundland and Labrador, 1996-2014.

Background: Despite growing awareness of the significant burden of disease caused by hepatitis C virus (HCV) infection worldwide, understanding of the epidemiology and demographic distribution of HCV infection in Canada, specifically in Atlantic Canada, is limited. Currently, data on the demographic and clinical profile of HCV-infected individuals in Newfoundland and Labrador is limited. The aim of this study is to address this knowledge gap. Methods: A retrospective cohort study of HCV-positive individuals referred for specialized care in St. John's, Newfoundland, between 1996 and 2014, was conducted. Descriptive data were obtained through chart review and access to a database consisting of individuals referred for specialized HCV care in St. John's. Results: During the study period, 767 individuals were referred for specialized HCV care, of whom 714 were included in our analysis. These individuals represent 57.5% of HCV-positive cases identified by the province's public health department during the same time frame. HCV infection was more common among men (68.2%) and urban dwellers (74.8%). The majority of cases were HCV genotype 1 (52.1%). Intravenous and intranasal drug use were the most common self-reported risk factors for HCV transmission. High loss-to-follow-up rates were found among those referred from the province's correctional system. Conclusions: This study provides important insights into the demographic and clinical profile of individuals referred for HCV-related care in Newfoundland and Labrador and fills a gap in the current understanding of HCV-positive individuals in this Atlantic province. These findings can help inform future directions for HCV-related health policy, resource allocation, and clinical care initiatives in Newfoundland and Labrador and across Canada.

Multiple phase transitions in an agent-based evolutionary model with neutral fitness.

Null models are crucial for understanding evolutionary processes such as speciation and adaptive radiation. We analyse an agent-based null model, considering a case without selection-neutral evolution-in which organisms are defined only by phenotype. Universal dynamics has previously been demonstrated in a related model on a neutral fitness landscape, showing that this system belongs to the directed percolation (DP) universality class. The traditional null condition of neutral fitness (where fitness is defined as the number of offspring each organism produces) is extended here to include equal probability of death among organisms. We identify two types of phase transition: (i) a non-equilibrium DP transition through generational time (i.e. survival), and (ii) an equilibrium ordinary percolation transition through the phenotype space (based on links between mating organisms). Owing to the dynamical rules of the DP reaction-diffusion process, organisms can only sparsely fill the phenotype space, resulting in significant phenotypic diversity within a cluster of mating organisms. This highlights the necessity of understanding hierarchical evolutionary relationships, rather than merely developing taxonomies based on phenotypic similarity, in order to develop models that can explain phylogenetic patterns found in the fossil record or to make hypotheses for the incomplete fossil record of deep time.

Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169.

We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169T possesses unique virulence genes.

Nationwide reconnaissance of contaminants of emerging concern in source and treated drinking waters of the United States.

When chemical or microbial contaminants are assessed for potential effect or possible regulation in ambient and drinking waters, a critical first step is determining if the contaminants occur and if they are at concentrations that may cause human or ecological health concerns. To this end, source and treated drinking water samples from 29 drinking water treatment plants (DWTPs) were analyzed as part of a two-phase study to determine whether chemical and microbial constituents, many of which are considered contaminants of emerging concern, were detectable in the waters. Of the 84 chemicals monitored in the 9 Phase I DWTPs, 27 were detected at least once in the source water, and 21 were detected at least once in treated drinking water. In Phase II, which was a broader and more comprehensive assessment, 247 chemical and microbial analytes were measured in 25 DWTPs, with 148 detected at least once in the source water, and 121 detected at least once in the treated drinking water. The frequency of detection was often related to the analyte's contaminant class, as pharmaceuticals and anthropogenic waste indicators tended to be infrequently detected and more easily removed during treatment, while per and polyfluoroalkyl substances and inorganic constituents were both more frequently detected and, overall, more resistant to treatment. The data collected as part of this project will be used to help inform evaluation of unregulated contaminants in surface water, groundwater, and drinking water.

Microbial pathogens in source and treated waters from drinking water treatment plants in the United States and implications for human health.

An occurrence survey was conducted on selected pathogens in source and treated drinking water collected from 25 drinking water treatment plants (DWTPs) in the United States. Water samples were analyzed for the protozoa Giardia and Cryptosporidium (EPA Method 1623); the fungi Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus (quantitative PCR [qPCR]); and the bacteria Legionella pneumophila (qPCR), Mycobacterium avium, M. avium subspecies paratuberculosis, and Mycobacterium intracellulare (qPCR and culture). Cryptosporidium and Giardia were detected in 25% and in 46% of the source water samples, respectively (treated waters were not tested). Aspergillus fumigatus was the most commonly detected fungus in source waters (48%) but none of the three fungi were detected in treated water. Legionella pneumophila was detected in 25% of the source water samples but in only 4% of treated water samples. M. avium and M. intracellulare were both detected in 25% of source water, while all three mycobacteria were detected in 36% of treated water samples. Five species of mycobacteria, Mycobacterium mucogenicum, Mycobacterium phocaicum, Mycobacterium triplex, Mycobacterium fortuitum, and Mycobacterium lentiflavum were cultured from treated water samples. Although these DWTPs represent a fraction of those in the U.S., the results suggest that many of these pathogens are widespread in source waters but that treatment is generally effective in reducing them to below detection limits. The one exception is the mycobacteria, which were commonly detected in treated water, even when not detected in source waters.

Increased Frequency of Nontuberculous Mycobacteria Detection at Potable Water Taps within the United States.

Nontuberculous mycobacteria (NTMs) are environmental microorganisms that can cause infections in humans, primarily in the lung and soft tissue. The prevalence of NTM-associated diseases is increasing in the United States. Exposure to NTMs occurs primarily through human interactions with water (especially aerosolized). Potable water from sites across the U.S. was collected to investigate the presence of NTM. Water from 68 taps was sampled 4 times over the course of 2 years. In total, 272 water samples were examined for NTM using a membrane filtration, culture method. Identification of NTM isolates was accomplished by polymerase chain reaction (PCR) amplification of the 16S rRNA and hsp65 genes. NTMs were detected in 78% of the water samples. The NTM species detected most frequently were: Mycobacterium mucogenicum (52%), Mycobacterium avium (30%), and Mycobacterium gordonae (25%). Of the taps that were repeatedly positive for NTMs, the species M. avium, M. mucogenicum, and Mycobacterium abscessus were found to persist most frequently. This study also observed statistically significant higher levels of NTM in chloraminated water than in chlorinated water.

Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

Widespread molecular detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States.

In the United States, 6,868 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009-2010. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to L. pneumophila Sg1 may occur from aerosolization and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect L. pneumophila and the other L. pneumophila Sg1) were determined to be highly sensitive and selective for their respective targets. Over 272 water samples, collected in 2009 and 2010 from 68 public and private water taps across the United States, were analyzed using the two qPCR assays to evaluate the incidence of L. pneumophila Sg1. Nearly half of the taps showed the presence of L. pneumophila Sg1 in one sampling event, and 16% of taps were positive in more than one sampling event. This study is the first United States survey to document the occurrence and colonization of L. pneumophila Sg1 in cold water delivered from point of use taps.

Hepatitis C virus load and expression of a unique subset of cellular genes in circulating lymphoid cells differentiate non-responders from responders to pegylated interferon alpha-ribavirin treatment.

Based on investigations of liver biopsy material, certain cellular genes have been implicated as correlates of success or failure to interferon alpha-ribavirin (IFN/RBV) therapy against hepatitis C. The current study aimed at determining whether expression of host genes thought to be relevant to HCV replication in the liver would be correlated with HCV infection status in peripheral blood mononuclear cells (PBMCs) and also with patient responsiveness to IFN/RBV treatment. Therefore, PBMCs from patients with chronic hepatitis C responding (n = 35) or not (n = 49) to IFN/RBV and from healthy controls (n = 15) were evaluated for HCV RNA load and cellular gene expression. Non-responders had 3- to 10-fold higher basal levels of interleukin (IL)-8, IFN-stimulated gene 15 (ISG15), 2',5'-oligoadenylate synthetase (OAS), and Toll-like receptors (TLR)-4, -5, and -7 compared to responders. Non-responders with similar post-treatment follow-ups as responders persistently expressed 6- to 20-fold greater levels of IL-8, ISG15, and OAS after therapy. Higher expression of IFN-α, IFN-γ, and IFN-λ was found in PBMCs of individuals achieving sustained virological response, either before or after therapy. Pre-treatment HCV RNA loads in PBMCs of non-responders were significantly higher (P = 0.016) than those of responders. In conclusion, the data indicate that immune cells of responders and non-responders to IFN/RBV therapy exhibited significantly different virological and host gene expression profiles. Elevated baseline HCV loads and TLR-4, -5, and -7 levels, and persistently high levels of IL-8, ISG15, and OAS were correlated with IFN non-responsiveness. The results warrant further investigations on the utilization of PBMCs for predicting success or failure to IFN-based therapies.

Detection of Mycobacterium avium subsp. paratuberculosis in drinking water and biofilms by quantitative PCR.

It has been suggested that Mycobacterium avium subspecies paratuberculosis has a role in Crohn's disease. The organism may be acquired but is difficult to culture from the environment. We describe a quantitative PCR (qPCR) method to detect M. avium subsp. paratuberculosis in drinking water and the results of its application to drinking water and faucet biofilm samples collected in the United States.

Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection.

BACKGROUND & AIMS: Infection of the lymphatic system by hepatitis C virus (HCV) appears to be an intrinsic characteristic of chronic hepatitis C (CHC) and low-level (occult) HCV infection, but the subsets of immune cells involved were not defined. The aim of this study was to characterize HCV replication status and to assess virus compartmentalization in CD4+ and CD8+ T lymphocytes, B cells, and monocytes in CHC, and silent infection persisting after resolution of hepatitis C. METHODS: Immune cell subtypes isolated from 7 patients with CHC and 7 individuals with occult infection were analyzed for HCV-RNA-positive and -negative strands and, in selected cases, nonstructural protein 5A display and HCV variants. RESULTS: All subtypes of immune cells investigated support HCV replication in both forms of infection, although significant differences were found between patients, and virus loads in the cells were greater in CHC than in occult infection. Although HCV RNA occurred at a comparable frequency in all cell subtypes in CHC, monocytes contained the greatest loads. In contrast, B cells tended to carry the highest virus quantities in occult infection, whereas monocytes appeared to be the least frequently infected. Detection of HCV nonstructural protein 5A and HCV variants that were not found in plasma confirmed virus replication in different immune cell types. CONCLUSIONS: This work documents that the immune system supports HCV replication regardless of clinical appearance of infection and identifies immune cells that are reservoirs of HCV in symptomatic and occult infections.

A critical evaluation of a flow cytometer used for detecting enterococci in recreational waters.

The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the

Watershed management and organizational dynamics: nationwide findings and regional variation.

Recent attention has focused on resource management initiatives at the watershed scale with emphasis on collaborative, locally driven, and decentralized institutional arrangements. Existing literature on limited selections of well-established watershed-based organizations has provided valuable insights. The current research extends this focus by including a broad survey of watershed organizations from across the United States as a means to estimate a national portrait. Organizational characteristics include year of formation, membership size and composition, budget, guiding principles, and mechanisms of decision-making. These characteristics and the issue concerns of organizations are expected to vary with respect to location. Because this research focuses on organizations that are place based and stakeholder driven, the forces driving them are expected to differ across regions of the country. On this basis of location, we suggest basic elements for a regional assessment of watershed organizations to channel future research and to better approximate the organizational dynamics, issue concerns, and information needs unique to organizations across the country. At the broadest level, the identification of regional patterns or organizational similarities may facilitate the linkage among organizations to coordinate their actions at the much broader river basin or ecosystem scale.