Impact/Purpose:

A major limiting factor in assessing the human health risk of microbial pathogens in raw and finished drinking water is the lack of robust, efficient methods for concentrating, identifying and quantifying low levels of bacteria, viruses and protozoa simultaneously, effectively and rapidly. We will develop a microbial isolation and detection protocol capable of qualitative and quantitative identification of waterborne microbial pathogens by combining the latest high-efficiency filtration technology with rapid and sensitive molecular detection techniques including quantitative PCR (qPCR), quantitative reverse transcription-PCR (qRT-PCR), fluorescent in situ hybridization (FISH) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The sensitivity and specificity of the proposed pathogen recovery and detection approach will be directly compared to current USEPA methods via spiking and analysis of raw and finished drinking water samples collected from various water resources and distribution systems. Following method validation, a series of unspiked raw or finished waters (including waters from distribution systems), will be monitored for pathogenic microorganisms to demonstrate the utility of the approach in real world situations.

Description:

The project has been summarized in a series of peer-reviewed published papers as outlined in the Publication section of this report. Pathogens capable of causing waterborne diseases include bacteria, protozoa, and viruses. Fecal indicator bacteria are the primary microorganisms used to evaluate microbial water quality based on the analysis of small volume (100 ml) grab samples. Here a tangential flow, hollow fiber ultrafiltration (UF) method was optimized for the recovery of bacteria, protozoan surrogates, and enteric viruses seeded into 100 L dechlorinated drinking water (DW) and surface water (SW) samples. Recovery efficiency was assessed via culture methods and real-time reverse transcription-PCR (RT-PCR). The combined average recovery efficiencies by culture methods for bacteria (E. coli and E. faecalis), viral surrogates (MS2, PRD1, murine norovirus [MNV-1], and poliovirus), and C. perfringens bacterial spores were 58%, 53%, and 64% in DW (n = 6), respectively. In SW (n = 5), the recovery efficiencies by culture methods for bacteria, viral surrogates, and bacterial spores were 95%, 64%, and 36%, respectively. MNV-1 also was analyzed by real-time RT-PCR. Target MNV-1 RNA was detected in all DW (n = 6) and 3 of 5 SW samples. Following UF optimization, the method was applied for the recovery of enteric viruses from 100 L non-seeded DW (n = 6) and SW (n = 6) collected from two separate drinking water treatment plants (DWTP). Target human enteric viruses were not detected in any DWTP samples. Molecular analyses also included an internal standard RNA (hepatitis G virus) for identification of sample inhibition.

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Commercially available hollow fiber ultrafilter (UF) membranes will be assessed with respect to both microbe collection efficiency and subsequent microbe recoverability using a suite of viruses, bacteria and protozoa. An existing unit for bench-scale testing will be used for these evaluations. The unit is comprised of two parallel channels that allow two membranes to be tested simultaneously. Recovered microorganisms will be enumerated by both the molecular methods outlined above and existing infectivity assays. Following selection of suitable membranes, a scaled-up UF system will be employed to recover spiked microorganisms from large volumes of raw and finished drinking water. As part of the method evaluation, split samples will be analyzed using current USEPA methods for viruses (1MDS filtration and BGM cell culture analysis), protozoa (method 1623) and bacteria (EPA E. coli and enterococci methods 1603 and 1600 respectively). Additional method comparison experiments will be conducted using unaltered natural water samples.

Commercially available hollow fiber ultrafilter (UF) membranes will be assessed with respect to both microbe collection efficiency and subsequent microbe recoverability using a suite of viruses, bacteria and protozoa. An existing unit for bench-scale testing will be used for these evaluations. The unit is comprised of two parallel channels that allow two membranes to be tested simultaneously. Recovered microorganisms will be enumerated by both the molecular methods outlined above and existing infectivity assays. Following selection of suitable membranes, a scaled-up UF system will be employed to recover spiked microorganisms from large volumes of raw and finished drinking water. As part of the method evaluation, split samples will be analyzed using current USEPA methods for viruses (1MDS filtration and BGM cell culture analysis), protozoa (method 1623) and bacteria (EPA E. coli and enterococci methods 1603 and 1600 respectively). Additional method comparison experiments will be conducted using unaltered natural water samples.

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