Shared Mycobacterium avium Genotypes Observed among Unlinked Clinical and Environmental Isolates

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event

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

Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages

Water, Water Everywhere, but Every Drop Unique: Challenges in the Science to Understand the Role of Contaminants of Emerging Concern in the Management of Drinking Water Supplies.

The protection and management of water resources continues to be challenged by multiple and ongoing factors such as shifts in demographic, social, economic, and public health requirements. Physical limitations placed on access to potable supplies include natural and human-caused factors such as aquifer depletion, aging infrastructure, saltwater intrusion, floods, and drought. These factors, although varying in magnitude, spatial extent, and timing, can exacerbate the potential for contaminants of concern (CECs) to be present in sources of drinking water, infrastructure, premise plumbing and associated tap water. This monograph examines how current and emerging scientific efforts and technologies increase our understanding of the range of CECs and drinking water issues facing current and future populations. It is not intended to be read in one sitting, but is instead a starting point for scientists wanting to learn more about the issues surrounding CECs. This text discusses the topical evolution CECs over time (Section 1), improvements in measuring chemical and microbial CECs, through both analysis of concentration and toxicity (Section 2) and modeling CEC exposure and fate (Section 3), forms of treatment effective at removing chemical and microbial CECs (Section 4), and potential for human health impacts from exposure to CECs (Section 5). The paper concludes with how changes to water quantity, both scarcity and surpluses, could affect water quality (Section 6). Taken together, these sections document the past 25 years of CEC research and the regulatory response to these contaminants, the current work to identify and monitor CECs and mitigate exposure, and the challenges facing the future

Isolation of the Genome Sequence Strain Mycobacterium avium 104 from Multiple Patients over a 17-Year Period

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods

Widespread Molecular Detection of <i>Legionella pneumophila</i> 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 <i>Legionella pneumophila</i> Serogroup (Sg) 1. <i>Legionella</i> spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to <i>L. pneumophila</i> Sg1 may occur from aerosolization and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect <i>L. pneumophila</i> and the other <i>L. pneumophila</i> 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 <i>L. pneumophila</i> Sg1. Nearly half of the taps showed the presence of <i>L. pneumophila</i> 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 <i>L. pneumophila</i> Sg1 in cold water delivered from point of use taps

Water, Water Everywhere, but Every Drop Unique: Challenges in the Science to Understand the Role of Contaminants of Emerging Concern in the Management of Drinking Water Supplies

Abstract The protection and management of water resources continues to be challenged by multiple and ongoing factors such as shifts in demographic, social, economic, and public health requirements. Physical limitations placed on access to potable supplies include natural and human‐caused factors such as aquifer depletion, aging infrastructure, saltwater intrusion, floods, and drought. These factors, although varying in magnitude, spatial extent, and timing, can exacerbate the potential for contaminants of concern (CECs) to be present in sources of drinking water, infrastructure, premise plumbing and associated tap water. This monograph examines how current and emerging scientific efforts and technologies increase our understanding of the range of CECs and drinking water issues facing current and future populations. It is not intended to be read in one sitting, but is instead a starting point for scientists wanting to learn more about the issues surrounding CECs. This text discusses the topical evolution CECs over time (Section 1), improvements in measuring chemical and microbial CECs, through both analysis of concentration and toxicity (Section 2) and modeling CEC exposure and fate (Section 3), forms of treatment effective at removing chemical and microbial CECs (Section 4), and potential for human health impacts from exposure to CECs (Section 5). The paper concludes with how changes to water quantity, both scarcity and surpluses, could affect water quality (Section 6). Taken together, these sections document the past 25 years of CEC research and the regulatory response to these contaminants, the current work to identify and monitor CECs and mitigate exposure, and the challenges facing the future

Association of <i>M. avium</i> spp. isolates with human macrophages.

<p><i>A. Non-opsonic condition.</i> Macrophage monolayers (2.0×10⁵ cells/well) on glass coverslips in triplicate were incubated in the absence of serum with isolates (MOI 25∶1) for 2 h at 37°C, 5% CO₂. MR-specific association was measured in the presence (close bars) of mannan (2.5 mg/ml). <i>B. Opsonic condition.</i> Macrophage monolayers were incubated with isolates as described above and association assessed in the absence (open bars) or presence (close bars) of 10% fresh autologous non-immune serum. Bacteria associated with macrophages were stained with rhodamine-auramine and association assessed by phase- contrast and fluorescence microscopy. >300 cells/coverslip were counted. Shown are cumulative data from N = 3 (mean±SEM) *P<0.05, *P<0.01, ***P<0.0001.</p

Detection of ManLAM on the bacterial surface. A.

<p><i>M. avium</i> spp. isolates (2×10⁶/well) in a 96 well plate (triplicate wells per test group) were blocked with BSA and incubated for 2 h with CS-35 mAb followed by a secondary HRP-Ab. The graph shows cumulative data of N = 3 for selected samples (mean±SEM; *P<0.05). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045411#pone-0045411-t004" target="_blank">table 4</a> for the complete set. <b>B.</b> Live bacilli single cell suspensions (1×10⁶) were blocked with 2% BSA in PBS, washed with PBS followed by staining with anti-LAM CS-35 in 2% BSA in PBS for 20 min at 4C. After further washing, samples were analyzed by flow cytometry. Mean fluorescence intensity (MFI) was measured for each isolate and the average values of 2 samples were obtained. Shown is cumulative data of N = 2 (mean±SEM). *P<0.05.</p