Stacy Pfaller

Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms by Quantitative PCR

ABSTRACT It has been suggested that Mycobacterium avium subspecies paratuberculosis has a role in Crohns 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.

Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period

ABSTRACT 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.

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 from29 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.

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.

Epidemiology of nontuberculous mycobacteria isolations among central North Carolina residents, 2006–2010

BACKGROUND Nontuberculous mycobacteria (NTM) are environmental mycobacteria associated with a range of infections. Reports of NTM epidemiology have primarily focused on pulmonary infections and isolations, however extrapulmonary infections of the skin, soft tissues and sterile sites are less frequently described. METHODS We comprehensively reviewed laboratory reports of NTM isolation from North Carolina residents of three counties during 2006-2010. We describe age, gender, and race of patients, and anatomic site of isolation for NTM species. RESULTS Among 1033 patients, overall NTM isolation prevalence was 15.9/100,000 persons (13.7/100,000 excluding Mycobacterium gordonae). Prevalence was similar between genders and increased significantly with age. Extrapulmonary isolations among middle-aged black males and pulmonary isolations among elderly white females were most frequently detected. Most isolations from pulmonary sites and blood cultures were Mycobacterium avium complex; rapidly growing NTM (e.g. Mycobacterium chelonae, Mycobacterium fortuitum) were most often isolated from paranasal sinuses, wounds and skin. CONCLUSIONS We provide the first characterization of NTM isolation prevalence in the Southeastern United States (U.S.). Variation in isolation prevalence among counties and races likely represent differences in detection, demographics and risk factors. Further characterization of NTM epidemiology is increasingly important as percentages of immunocompromised individuals and the elderly increase in the U.S.

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.

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.

The Use of PCR to Detect a Pathogen in Compost

Testing compost for pathogens can be very difficult, time consuming and expensive. A method to purify DNA from compost for amplification by polymerase chain reaction (PCR) was sought. The published techniques used to purify DNA from soils or sediments did not work with compost samples in our laboratory. We have devised an extraction protocol specifically for compost samples. In a series of Escherichia coli-spiked compost samples, ranging from 108 to 103 organisms per gram of compost, the removal of substances that inhibit PCR amplification of DNA was accomplished by extracting the organisms from the compost and treating the extract with base resin. After cell lysis, the collected DNA was further purified on a 1 percent low melt agarose gel. A 260 base-pair region of the lac Z gene, unique to E. coli, was successfully amplified by PCR in the DNA extracted from the gel. The technique was sensitive to the level of 104 E. coli cells per gram of compost.

Resilience of microbial communities in a simulated drinking water distribution system subjected to disturbances: role of conditionally rare taxa and potential implications for antibiotic-resistant bacteria

Many US water utilities using chloramine as their secondary disinfectant have experienced nitrification episodes that detrimentally impact water quality in their distribution systems. A semi-closed pipe-loop chloraminated drinking water distribution system (DWDS) simulator was used to evaluate the biological stability of the system and describe the response of microbial communities in the bulk water (BW) and biofilm (BF) phase to a disturbance caused by changes in the operational parameters. The DWDS simulator was operated through five successive operational schemes, including an episode of nitrification, followed by a ‘chlorine burn’ by switching the disinfectant from chloramine to free chlorine. Community comparisons showed significant differences in the structure based on disinfectant and phase (e.g., BW and BF). Both disturbances created changes in the relative abundances of the core microbiome and some members of the rare biosphere (i.e., conditionally rare taxa); however, the microbial community was resilient and returned to its stable state. Genes associated with multiple antibiotic resistance mechanisms were found to be a component of the core genomes of waterborne isolates. These results provide evidence of variations in the bulk water/biofilm microbial community structure during episodes of disturbance (e.g., disinfectant switching practices, nitrification) and its recovery after disturbance.

Shared Mycobacterium avium Genotypes Observed among Unlinked Clinical and Environmental Isolates

ABSTRACT 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.