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      Molecular survey of Legionellaand Naegleria fowleriin private well water and premise plumbing following the 2016 Louisiana flood

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          Abstract

          This is the first community-wide survey of opportunistic pathogens in private wells and premise plumbing following a flooding event.

          Abstract

          Private wells are a critical drinking water source and are susceptible to contamination from flooding. Opportunistic pathogens (OPs), such as Legionella, are an increasing source of drinking water-related outbreaks, but are poorly characterized in private wells. Here we conducted a molecular survey of OPs in private wells and plumbing systems shortly after the 2016 Louisiana flood. Detection frequency of fecal indicators was not notably high (total coliform 24.8% and Escherichia coli3.5% in 113 private wells) ten weeks after flooding. Gene markers of Legionellaspp., L. pneumophila, and Naegleria fowleriwere detected in 77.5%, 15.0%, and 20.0% of a subset of 40 homes that were tested specifically for these OPs, respectively. Legionellaspp. varied from 8.4 gc mL −1to 1.8 × 10 4gc mL −1in first draw and flushed water. Positive detections and levels of Legionellaspp., as well as positive detections of L. pneumophila, were correlated with total bacterial numbers (measured as 16S rRNA gene copy numbers), suggesting that total bacterial numbers could be an indicator of OP occurrence under the conditions of private wells, which usually do not have disinfection treatment installed. Further, Legionellaspp. positivity in first draw water from cold and hot taps was associated with their detection in flushed water, suggesting that the well itself can be a source of OPs. OP detection was not predictable from total coliform, well characteristics, or observable well damage, but was associated with higher metals in flushed water resulting from plumbing corrosion. Given that the majority of Legionnaires' disease cases are sporadic, private wells merit greater attention as a potential source of exposure.

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          Most cited references64

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          Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays.

          Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5'-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5'-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5'-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.
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            Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes.

            There are significantly more microbial cells in drinking water than what can be cultured on synthetic growth media. Nonetheless, cultivation-based heterotrophic plate counts (HPCs) are used worldwide as a general microbial quality parameter in drinking water treatment and distribution. Total bacterial cell concentrations are normally not considered during drinking water treatment as a design, operative or legislative parameters. This is mainly because easy and rapid methods for quantification of total bacterial cell concentrations have, up to now, not been available. As a consequence, the existing lack of data does not allow demonstrating the practical value of this parameter. In this study, we have used fluorescence staining of microbial cells with the nucleic acid stain SYBR((R)) Green I together with quantitative flow cytometry (FCM) to analyse total cell concentrations in water samples from a drinking water pilot plant. The plant treats surface water (Lake Zürich) through sequential ozonation, granular active carbon (GAC) filtration and membrane ultrafiltration (UF). The data were compared with adenosine tri-phosphate (ATP) measurements and conventional HPCs performed on the same water samples. We demonstrated that the impact of all three major treatment steps on the microbiology in the system could accurately be described with total cell counting: (1) ozonation caused chemical destruction of the bacterial cells; (2) GAC filtration facilitated significant regrowth of the microbial community; and (3) membrane UF physically removed the bacterial cells from the water. FCM typically detected 1-2 log units more than HPC, while ATP measurements were prone to interference from extracellular ATP released during the ozonation step in the treatment train. We have shown that total cell concentration measured with FCM is a rapid, easy, sensitive and importantly, a descriptive parameter of several widely applied drinking water treatment processes.
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              Molecular survey of the occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and amoeba hosts in two chloraminated drinking water distribution systems.

              The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.
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                Author and article information

                Contributors
                Journal
                ESWRAR
                Environmental Science: Water Research & Technology
                Environ. Sci.: Water Res. Technol.
                Royal Society of Chemistry (RSC)
                2053-1400
                2053-1419
                July 25 2019
                2019
                : 5
                : 8
                : 1464-1477
                Affiliations
                [1 ]Virginia Tech
                [2 ]Via Civil and Environmental Engineering
                [3 ]Blacksburg
                [4 ]USA
                [5 ]Louisiana State University Health Science Center
                [6 ]New Orleans
                Article
                10.1039/C9EW00109C
                90bde981-32b7-46dd-b8d5-995f0b14b4da
                © 2019

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