WO2017116695A1 - Assembly and method for field filtration of water samples - Google Patents

Abstract

The present disclosure provides an assembly for collecting a viable microorganism present in water discharged from a distribution pipe. The assembly includes a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway and a static mixer disposed between the first opening and the filter housing. The filter is configured to retain microorganism from a water sample. The filter housing and the static mixer are operatively connected to form a liquid flow path. The first opening is configured connect with a distribution pipe or an adaptor configured to operatively connect the distribution pipe to the first opening. A method of using the assembly is also provided.

Description

ASSEMBLY AND METHOD FOR FIELD FILTRATION OF WATER SAMPLES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/272,578, filed December 29, 2015, the disclosure of which is incorporated by reference in its entirety herein.

BACKGROUND

[0002] Testing aqueous samples for the presence of microorganisms (e.g., bacteria, viruses, fungi, spores, etc.) and/or other analytes of interest (e.g., toxins, allergens, hormones, etc.) can be important in a variety of applications, including food and water safety, infectious disease diagnostics, and environmental surveillance. For example, comestible samples, such as beverages, process water, and/or public water consumed by the general population may contain or acquire microorganisms that can flourish or grow as a function of the environment in which they are located. This growth may lead to the proliferation of pathogenic organisms, which may produce toxins or multiply to infective doses. By way of further example, a variety of analytical methods can be performed on samples of non-comestible samples (e.g., groundwater, urine, etc.) to determine if a sample contains a particular analyte. For example, groundwater can be tested for a microorganism or a chemical toxin; and urine can be tested for a variety of diagnostic indicators to enable a diagnosis (e.g., diabetes, pregnancy, etc.).

[0003] Microbiological quality of water supplies and recreational water is typically assessed by detection and enumeration of coliforms, fecal coliforms, and Escherichia coli. The traditional methods use most-probable-number (MPN) using serial dilutions within replicate tubes incubated with selective media or a membrane filtration method where a known amount of water is filtered through a membrane and the membrane is placed on media selective for the bacterial group of interest. The regulations for drinking water require detection of 1 cfu/100 ml and the samples are typically brought to the lab and processed. Although large samples of water are typically available for processing, it is difficult to transport large volumes of water to the lab and process them. Also, as the limit of detection is 1 cfu, it is difficult to detect it rapidly without any enrichment. This invention describes filtration devices that can be adapted to filter large samples directly from the water source such as faucets. Large samples (1 to 25) liters can be easily processed in the field and the devices can be brought back to the lab for further processing. SUMMARY

[0004] The present disclosure generally relates to an assembly and method for filtering a large-volume (e.g., > 100 mL, > 200 mL, > 500 mL, > 1000 mL) liquid sample in a non- laboratory situation. In particular, the present disclosure relates to filtering a large-volume water sample as the water sample is discharged from a water-distribution pipe and detecting whether the filter retains a viable microorganism. The inventive method includes neutralization of an antimicrobial agent in the water as the water is being filtered. The method further comprises detecting the viable microorganism or a component thereof.

[0005] In one aspect, the present disclosure provides a method for detecting a

microorganism present in water discharged from a distribution pipe. The method can comprise connecting components of an assembly to an outlet of a water distribution pipe; the assembly having a first opening, a second opening, and components that include a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway, and a static mixer disposed between the first opening and the filter housing. The filter is configured to retain a target microorganism from a water sample. After the components are connected, the assembly forms a liquid flow path that extends from a first end proximate the outlet to a second end. After the components are connected, the assembly forms a liquid flow path that extends from a first end that receives water from the distribution pipe to a second end from which water received from the distribution pipe exits the assembly. After the assembly is connected to the outlet, the static mixer is disposed between the outlet and the filter housing. After connecting the components to form the assembly, the method further comprises flowing a water sample from the distribution pipe through the filter; collecting a viable target microorganism or a component thereof, if present, from the filter housing; and detecting the viable target microorganism or the component thereof. In any embodiment of the above method, connecting components of the assembly to the outlet can comprise connecting to the outlet an assembly that further comprises a pressure governor disposed between the first opening and the filter housing, wherein, after the assembly is connected to the outlet, the pressure governor and the static mixer are disposed between the outlet and the filter housing. In any embodiment of the above methods, connecting components of the assembly to the outlet can comprise connecting two or more preassembled components of the assembly to the outlet.

[0006] In any of the above embodiments, flowing a water sample from the distribution pipe through the filter can comprise flowing a water sample having a volume in the range 100 mL to 25 L, inclusive, through the filter. In any of the above embodiments of the method, collecting a viable target microorganism or a component thereof from the filter housing can comprise removing the filter from the filter housing. In any of the above embodiments of the method, collecting a microorganism, if present in the sample, from the filter housing further can comprise contacting the filter with a cell-lysis reagent. In any of the above embodiments, the method further can comprise treating the filtrand with a process that renders a nucleic acid from nonliving microorganisms incapable of being replicated in a nucleic acid amplification process.

[0007] In another aspect, the present disclosure provides an assembly for collecting a viable microorganism present in water discharged from a distribution pipe. The assembly can comprise a conduit having a first opening and a second opening. The conduit can comprise a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway and a static mixer disposed between the first opening and the filter housing. The filter can be configured to retain microorganism from a water sample. The filter housing and the static mixer can be operatively connected to form a liquid flow path that extends from the first opening to the second opening. The first opening can be configured connect with a distribution pipe or an adaptor configured to operatively connect the distribution pipe to the first opening. The first opening or the adaptor can be configured to receive a water sample from the distribution pipe as water is flowing through the distribution pipe.

[0008] In any of the above embodiments of the assembly, the assembly further can comprise a pressure governor disposed between the first opening and the filter housing, wherein the filter housing, the pressure governor, and the static mixer are coupled together to form a liquid flow path that extends from the first opening to the second opening. In any of the above embodiments of the assembly, the static mixer comprises a venturi pump. In any of the above embodiments of the assembly, the filter can comprise a membrane filter or a hollow-fiber filter. In any of the above embodiments of the assembly, the assembly further can comprise an adaptor configured to operatively connect the distribution pipe to the first opening.

[0009] In yet another aspect, the present disclosure provides a kit. The kit can comprise a filter housing comprising a passageway extending therethrough, a static mixer, and a filter dimensioned to be held in the passageway of the filter housing so that water moving through the passageway passes through the filter. In any embodiment, the kit can be provided with the filter disposed in the passageway of the filter housing such that water moving through the passageway passes through the filter. In any of the above embodiments of the kit, the kit further can comprise a pressure governor.

[0010] In any of the above embodiments, the kit further can comprise a first connector, wherein the first connector is adapted to operatively connect the pressure governor to the filter housing or to operatively connect the static mixer to the filter housing. In any of the above embodiments, the kit further can comprise a second connector that is adapted to operatively connect the pressure governor to the static mixer. In any of the above embodiments, the kit further can comprise a reagent for detecting a viable microorganism. The reagent can be selected from the group consisting of a polynucleotide primer, a polynucleotide probe, a viability dye, a DNA polymerase, luciferin, luciferase, and a nutrient for culturing a microorganism. In any of the above embodiments, the kit further can comprise a container holding an antimicrobial neutralizer. The container can be adapted to be operatively connected to the static mixer.

[0011] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0012] As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, an apparatus comprising "a" filter can be interpreted to mean that the apparatus can comprise "one or more" filters.

[0013] The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0014] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0015] A "static mixer", as used herein, refers to a device for continuous mixing/blending of fluid (e.g., liquid) materials. The static mixer uses the energy of a first fluid stream flowing through the mixer to draw in a second fluid stream into the static mixer and to mix the first and second fluid streams.

[0016] The features and advantages of the present invention will be understood upon consideration of the detailed description of the preferred embodiment as well as the appended claims. These and other features and advantages of the invention may be described below in connection with various illustrative embodiments of the invention.

[0017] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The figures and the detailed description which follow more particularly exemplify illustrative embodiments. Other features, objects and advantages will become apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is a schematic side view of one embodiment of an assembly for collecting a viable microorganism present in water discharged from a distribution pipe according to the present disclosure.

[0019] FIG. 2 is a schematic exploded side view of the assembly of FIG. 1. [0020] FIG. 3 is a block diagram of one embodiment of a method for detecting a microorganism present in water discharged from a distribution pipe according to the present disclosure.

[0021] FIG. 4 is a schematic side view of the assembly of FIG. 1 operatively connected to a water distribution pipe.

DETAILED DESCRIPTION

[0022] Before any embodiments of the present disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms "connected" and "coupled" and variations thereof are used broadly and encompass both direct and indirect connections and couplings. Further, "connected" and "coupled" are not restricted to physical or mechanical connections or couplings. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. Furthermore, terms such as "front," "rear," "top," "bottom," and the like are only used to describe elements as they relate to one another, but are in no way meant to recite specific orientations of the apparatus, to indicate or imply necessary or required orientations of the apparatus, or to specify how the invention described herein will be used, mounted, displayed, or positioned in use.

[0023] Water distribution systems are used to transport water (e.g., municipal potable water) from a treatment facility to an end user. The treatment facilities typically treat the water with a disinfectant (e.g., chlorine) to reduce the number of disease-causing microorganisms (e.g., coliform bacteria such as E. coli). Samples of the water are tested periodically to enumerate the quantity of coliform bacteria in order to ensure the integrity and safety of the water in the distribution system. Thus, coliform bacteria represent one type of "target" microorganism with respect to water supplies.

[0024] In the samples that are desired to be tested for a target microorganism (e.g., coliform bacteria), the target microorganism can be present in the sample at a low concentration. For example, regulations for water safety testing can require that testing devices be able to detect 1 colony-forming unit (cfu) of a bacterium of interest in 100 mL of water. Such a low concentration can be difficult or impossible to detect in a reasonable amount of time, much less in a "rapid" time frame, which is described in greater detail below. In order to decrease detection time, in some cases, the sample may need to be concentrated into a smaller volume. That is, in some cases, in order to reach an appropriate concentration of an analyte of interest so as to achieve a detection threshold of an analytical technique in a shorter amount of time, the sample may need to be concentrated by several orders of magnitude.

[0025] The present disclosure generally relates to an assembly and methods for detecting the presence or absence of a target microorganism in a sample, particularly, in water samples, and more particularly, in water samples that are obtained and tested in an environment other than a laboratory. Furthermore, the present disclosure generally relates to systems and methods for rapidly detecting the target microorganism or an analyte associated therewith. In some embodiments, the analyte is selected for detecting (e.g., the presence or absence of) Escherichia coli or other coliforms, for example, in a water sample. Detection of microorganisms (or other analytes) of interest in a water sample can be difficult, because of the low concentration of these microorganisms. As a result of the low concentration, detection in existing systems and methods can be very slow, because the microorganism(s) need to be grown (or the analyte concentration needs to be increased) to a detectable level, which can take time. The present inventors, however, have invented systems and methods for decreasing the time needed to detect a target microorganism in a water sample, and particularly, a dilute water sample, in an environment other than a laboratory.

[0026] Methods of the present disclosure employ an assembly that is adapted to collect target microorganisms by filtration and inactivate antimicrobial reagents in a water sample during the collection process. Methods of the present disclosure subsequently employ rapid methods known in the art to detect an analyte associated with the target microorganism.

[0027] Methods of the present disclosure generally include providing any embodiment of the assembly described herein, moving a liquid sample through the filter unit so that a portion of the liquid passes through the filter while inactivating an antimicrobial reagent present in the water sample, and analyzing the liquid lysate to detect an analyte associated with a target microorganism

[0028] Samples comprising larger volumes of liquid can be difficult to analyze because any target microorganisms present in this larger volume may not be easily or accurately detected (e.g., by imaging or optically interrogating) at least partly because the microorganism(s), if present, may exist in a relatively low concentration in these larger volumes, and/or because the larger volume may not be suitably positioned (e.g., in an analytical instrument) for detecting the microorganisms or an analyte associated therewith. [0029] For example, a large dilute aqueous sample can be filtered to retain on the filter of the filter unit the microorganisms, if present, by size, charge and/or affinity. Optionally, a diluent (e.g., a buffer) can be contacted with the filter, and microorganisms, if present in the sample. In such embodiments, the filtrand on the filter plus any additional diluent that is added can form the "sample concentrate", cells (e.g., microorganisms) in the sample concentrate can be disrupted (e.g., by "bead-beating"), and the resulting cell lysate can be analyzed. The analysis can detect the presence/absence of an analyte that is associated with (e.g., uniquely associated with) the target microorganism.

[0030] Assemblies of the present disclosure can include parts configured to facilitate the processes of inactivating an antimicrobial reagent and filtering the sample. In addition, the systems and methods of the present disclosure allow for concentration of a large volume sample down to a very small volume, for example, from about 1 L down to about 250 to 1000 microliters. In any embodiment, the cells collected by filtration can be disrupted (e.g., chemically and/or physically) to form a cell lysate. After disruption of the cells, the cell lysate optionally can be concentrated using the commercially available devices such as Centricon (Millipore) centrifugal devices.

[0031] Particularly, in any embodiment, method of the present disclosure can include performing a concentration step comprising filtering an original sample using a filter housing comprising a filter that is configured to retain one or more target microorganisms to form a filtrand on one side of the filter; optionally adding one or more diluents to the filtrand and using the filtrand and any added diluents to form a sample concentrate; and analyzing a portion of the sample concentrate to detect an analyte. The analyte may be associated with (e.g., uniquely associated with) a target microorganism.

[0032] In some existing systems and methods, portions of the sample can become irreversibly trapped in the filter during filtration. Trapping can be overcome using isoporous filters, however, filtration through isoporous filters can be slow, and the pores of the isoporous filter can be easily and rapidly plugged during filtration. Other existing systems and methods rely on the elution of analytes from the filter and subsequent detection of the eluted analytes. However, an advantage of methods of the present disclosure is that elution of target microorganisms from the filter membrane is not necessary in order to detect the analytes associated with the target microorganism as described herein.

[0033] In some embodiments, rapid detection can refer to detection in no greater than 8 hours; in some embodiments, no greater than 6 hours; in some embodiments, no greater than 5 hours; in some embodiments, no greater than 4 hours; in some embodiments, no greater than 3 hours; in some embodiments, no greater than 2 hours; and, in some embodiments, no greater than 1 hour.. The detection time, however, can be dependent upon the type and quantity of microorganisms being detected and/or on the detection technology used in the method.

[0034] The term "analyte" is generally used to refer to a substance or biomolecule to be detected (e.g., by a laboratory or field test). A sample can be tested for the presence and/or quantity of particular analytes that are associated with (e.g., uniquely associated with) a target microorganism or group of microorganisms. Examples of analytes can include, but are not limited to, biomolecules (e.g., polypeptides, polynucleotides, polysaccharides, and the like) or small molecules associated with a target microorganism.

[0035] A variety of testing methods can be used to identify or quantitate an analyte of interest, including, but not limited to, biochemical assays (e.g. immunoassay, nucleic acid amplification), or a combination thereof. In some embodiments, analytes of interest can be detected genetically; immunologically; colorimetrically; fluorimetrically; lumimetrically; by detecting, for example, a polynucleotide or antigen released from a cell in the sample; by detecting light that is indicative of the analyte associated with the target microorganism; by detecting light by absorbance, reflectance, fluorescence, or combinations thereof; or combinations thereof.

[0036] Specific examples of testing methods that can be used include, but are not limited to, enzyme assays, antigen-antibody interactions, molecular sensors (affinity binding), thermal analysis, spectroscopy (e.g., mass spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, infrared (IR) spectroscopy, x-ray spectroscopy, attenuated total reflectance spectroscopy, Fourier transform spectroscopy, gamma-ray spectroscopy, etc.), spectrophotometry (e.g., absorbance, reflectance, fluorescence, luminescence (e.g., detection of ATP bioluminescence), colorimetric detection etc.), electrochemical analysis, genetic techniques (e.g., polymerase chain reaction (PCR), transcription mediated amplification (TMA), hybridization protection assay (HPA), DNA or RNA molecular recognition assays, etc.), adenosine triphosphate (ATP) detection assays, immunological assays (e.g., enzyme- linked immunosorbent assay (ELISA)), or a combination thereof.

[0037] The term "microorganism" is generally used to refer to any prokaryotic or eukaryotic microscopic organism that can be captured by a filter, including without limitation, one or more of bacteria (e.g., motile or vegetative, Gram positive or Gram negative), viruses (e.g.,

Norovirus, Norwalk virus, Rotavirus, Adenovirus, DNA viruses, RNA viruses, enveloped, non- enveloped, human immunodeficiency virus (HIV), human Papillomavirus (HPV), etc.), bacterial spores or endospores, algae, fungi (e.g., yeast, filamentous fungi, fungal spores), prions, mycoplasmas, and protozoa. In some cases, the microorganisms of particular interest are those that are pathogenic, and the term "pathogen" is used to refer to any pathogenic microorganism. Examples of pathogens can include, but are not limited to, members of the family Enter obacteriaceae, or members of the family Micrococaceae, or the genera

Staphylococcus spp., Streptococcus, spp., Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp., Shigella spp., Yersinia spp., Enterobacter spp., Escherichia spp., Bacillus spp., Listeria spp., Campylobacter spp., Acinetobacter spp., Vibrio spp., Clostridium spp., and Corynebacterium spp. Particular examples of pathogens can include, but are not limited to, Escherichia coli including enterohemorrhagic E. co/z e.g., serotype 0157:H7, 0129:H11;

Pseudomonas aeruginosa; Bacillus cereus; Bacillus anthracis; Salmonella enteritidis;

Salmonella enterica serotype Typhimurium; Listeria monocytogenes; Clostridium botulinum; Clostridium perfringens; Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; Campylobacter jejuni; Yersinia enterocolitica; Vibrio vulnificus; Clostridium difficile;

vancomycin-resistant Enterococcus; Cronobacter sakazakii; and coliforms. Environmental factors that may affect the growth of a microorganism can include the presence or absence of nutrients, pH, moisture content, oxidation-reduction potential, antimicrobial compounds, temperature, atmospheric gas composition and biological structures or barriers

[0038] The term "biomolecule" is generally used to refer to a molecule, or a derivative thereof, that occurs in or is formed by an organism. For example, a biomolecule can include, but is not limited to, at least one of an amino acid, a nucleic acid, a polypeptide, a protein, a polynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide, and combinations thereof. Specific examples of biomolecules can include, but are not limited to, a metabolite (e.g., staphylococcal enterotoxin), an allergen (e.g., peanut allergen(s ), egg allergen(s ), pollens, dust mites, molds, danders, or proteins inherent therein, etc.), a hormone, a toxin (e.g., Bacillus diarrheal toxin, aflatoxin, Clostridium difficile toxin etc.), RNA (e.g., mR A, total RNA, tR A, etc.), DNA (e.g., plasmid DNA, plant DNA, etc.), a tagged protein, an antibody, an antigen, ATP, and combinations thereof.

[0039] The term "filtering" is generally used to refer to the process of separating matter by size, charge and/or function. For example, filtering can include separating soluble matter and a solvent (e.g., diluent) from insoluble matter, or filtering can include separating soluble matter, a solvent and relatively small insoluble matter from relatively large insoluble matter. As a result, a liquid composition can be "pre-filtered" to obtain a sample that is to be analyzed using the methods of the present disclosure. A variety of filtration methods can be used, including, but not limited to, passing the water sample from the water distribution pipe through a filter, other suitable filtration methods, and combinations thereof.

[0040] A "filter" is generally used to describe a device used to separate the soluble matter (or soluble matter and relatively small (e.g., <10 μπι diameter, <5 μπι diameter, <1 μπι diameter, <0.5 μπι diameter, <0.2 μπι diameter) insoluble matter) and solvent from the insoluble matter (or relatively large insoluble matter) in a liquid composition and/or to filter a sample during sample concentration. Examples of filters can include, but are not limited to, a woven or non- woven mesh (e.g., a wire mesh, a cloth mesh, a plastic mesh, etc.), a woven or non-woven polymeric web (e.g., comprising polymeric fibers laid down in a uniform or nonuniform process, which can be calendered), a surface filter, a membrane (e.g., a ceramic membrane (e.g., ceramic aluminum oxide membrane filters available under the trade designation

ANOPORE from Whatman Inc., Florham Park, NJ), a polycarbonate membrane (e.g., track- etched polycarbonate membrane filters available under the trade designation NUCLEOPORE from Whatman, Inc.)), a polyester membrane (e.g., comprising track-etched polyester, etc.), a sieve, a frit, filter paper, foam, etc., and combinations thereof.

[0041] In any embodiment, the filter can be configured to separate a target microorganism from a sample, for example, by size, charge, and/or affinity. For example, in some

embodiments, the filter can be configured to retain a target microorganism, such that a filtrand retained on the filter comprises the target microorganism.

[0042] In any embodiment, the filter can be configured to retain at least 30% of the target microorganisms in a sample; in any embodiment, at least 50%; in any embodiment, at least 80%; in any embodiment, at least 85%; in any embodiment, at least 90%; and in any embodiment, at least 95% of the target microorganisms in the sample.

[0043] Additional examples of suitable filters are described in co-pending PCT Publication No. W02011/156251 (Rajagopal, et al.), which claims priority to US Patent Application No. 61/352,229; PCT Publication No. W02011/156258 (Mach et al.), which claims priority to US Patent Application No. 61/352,205; PCT Publication No. W02011/152967 (Zhou), which claims priority to US Patent Application Nos. 61/350,147 and 61/351,441; and PCT Publication No. W02011/153085 (Zhou), which claims priority to US Patent Application Nos. 61/350, 154 and 61/351,447, all of which are incorporated herein by reference in their entirety.

[0044] In some embodiments, the term "filtrand" is generally used to describe the solid remaining after a liquid source (e.g., water to be tested) has been filtered to separate insoluble matter from soluble matter. Such a filtrand can be diluted or resuspended to form a sample concentrate to be further processed in a method of the present disclosure. The filtrand may be present on one surface or side of the filter, and/or may have penetrated at least partially into the depth of the filter.

[0045] "Bead beating" is a term used herein to describe a method of extracting

polynucleotides (e.g., DNA) from cells (e.g., microbial cells). A method of obtaining microbial DNA from soil samples is described by Yeates et al. ("Methods for microbial extraction from soil for PCR amplification"; 1998; C. Yeates, M R. Gillings, A D. Davidson, N. Altavilla, and D.A. Veal; Biological Procedures Online; Vol. 1, pp. 40-47) and is incorporated herein by reference in its entirety. In addition, Lavender et al. ("A cross comparison of QPCR to agar- based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs"; 2009; J.S. Lavender and J.L. Kinzelman; Water Research; Vol. 43, pp 4967-4979) and Haugland et al. ("Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis"; 2005; R.A. Haugland, S.C.

Siefring, L.J. Wymer, K.P. Brenner, and A.P. Dufour; Water Research; Vol. 39, pp 559-568), both of which are incorporated herein by reference in their entireties, describe methods of extracting microbial DNA from microbes captured on membrane filters.

[0046] Bead beating methods typically include placing cells (e.g., microorganism cells) into a container with a buffer and a plurality of particles (e.g., glass beads) and subjecting the container to agitation forces (e.g., in a bead beater such as, for example, a Mini-Beadbeater-16 available from BioSpec Products, Bartlesville, OK) sufficient to cause disruption of the cells in the container.

[0047] The terms "hydrophobic" and "hydrophilic" are generally used as commonly understood in the art. Thus, a "hydrophobic" material has relatively little or no affinity for water or aqueous media, while a "hydrophilic" material has relatively strong affinity for water or aqueous media. The required levels of hydrophobicity or hydrophilicity may vary depending on the nature of the sample, but may be readily adjusted based on simple empirical observations of a liquid sample when applied to various hydrophobic or hydrophilic surfaces.

[0048] Filters used in an assembly of the present disclosure can include membrane filters.

Suitable membranes may be characterized as porous membranes or as nanofiber membranes. Nanofiber filter membranes can have the fiber diameter less than 5 μπι such as, for example, less than 1 μπι. Nanofiber membranes may be prepared from, for example, polyacrylonitrile, polyvinylidene fluoride, a cellulose ester, polyvinyl acetate, polyvinyl alcohol, polyvinyl butyral, and/or combinations thereof.

[0049] Certain TIPS polyolefin membranes can be prepared so that they possess a single, homogeneous zone of membrane structure, each zone having a different pore microstructure. In other cases, a TIPS membrane may be prepared as a multi-zone membrane that includes two or more zones, each zone having a different pore microstructure. A multi-zone TIPS membrane may contain distinct zones or, alternatively, may possess a transition zone between two otherwise distinct zones.

[0050] Exemplary membranes include filter membranes that are described in, for example, in U.S. Patent No. 4,539,256; U.S. Patent No. 4,726,989; U.S. Patent No. 4,867,881; U.S. Patent No. 5,220,594; U.S. Patent No. 5,260,360; PCT Publication No. WO2010/078234; PCT Publication No. WO2010/071764; PCT Publication No. WO2011/152967; and PCT

Publication No. WO2011/151085. [0051] Turning to the drawings, FIGS. 1 and 2 show various schematic views of one embodiment of an assembly 10 used in a method according to the present disclosure. The assembly forms a conduit that has a first opening 2 through which water enters the assembly and a second opening 4 and includes the following components described below.

[0052] Proximate the second end 4, the assembly 10 comprises a filter housing 40. The filter housing 40 has passageway (not shown in FIGS. 1 and 2) extending therethrough from a first aperture 41 (FIG. 2) through which water enters the passageway to a second aperture 42 (FIG. 2) through which water exits the passageway. The filter housing 40 further comprises a filter (not shown in FIGS. 1 and 2) disposed in the passageway such that a water sample moving along the passageway through the filter housing passes through the filter. The filter is configured to retain a target microorganism from a water sample passing therethrough.

[0053] A person having ordinary skill in the art will know that "configured to retain a target microorganism" means the filter can have one or more of a variety of properties that cause it to retain the target microorganism. For example, the filter may have a pore size (e.g., 0.45 μπι, 0.2 μπι) that is smaller than an average dimension (e.g., diameter) of the target microorganism.

Alternatively, or additionally, the filter may have surface molecules that selectively or nonselectively interact (e.g., via ionic charge, hydrophilic interaction, specific binding pair (e.g., antigen/antibody) interaction, or the like) to cause retention of the target microorganism on or in the filter as a water sample containing the target microorganism passes therethrough.

[0054] Suitable filter housings that contain a filter (or are be configured to place a filter therein) are commercially available. Nonlimiting examples of suitable filter housing with filters therein include a BioASSURE™ filter unit with a 0.2 um 50 mm polyethersulfone filter (available from 3M Company, St. Paul, MN), a Millex-GP filter unit with a 0.22 μπι polyethersulfone filter (available from EMD Millipore Corporation, Billerica, MA), a Sterivex- GP filter unit with a 0.22 μπι polyethersulfone filter (available from EMD Millipore

Corporation), and a ElutraCon 50 Hollow Fiber Filter (available from Elutrasep, Woodstock, GA).

[0055] Referring back to FIGS. 1 and 2, the assembly 10 further comprises a static mixer 30 disposed between the first opening 2 and the filter housing 40.

[0056] The static mixer 30 is operatively connected (e.g., via connector tubing 22) to a source 50 (e.g., a reservoir) of an antimicrobial neutralizer (e.g., a container holding a liquid antimicrobial neutralizer). The antimicrobial neutralizer is present in the source 50 at a concentration that, when diluted with the water sample in the static mixer, attains a concentration effective to inactivate residual antimicrobial in the water from a water disinfection process. Thus, in any embodiment, the assembly 10 further comprises a source 50 of antimicrobial neutralizer operatively connected to the static mixer. In an embodiment (not shown in FIGS. 1 and 2), the static mixer may include the first opening.

[0057] An "antimicrobial neutralizer", according to the present disclosure, refers to a chemical that inhibits the antimicrobial activity associated with an antimicrobial reagent, or a derivative thereof, that is used to disinfect water. Nonlimiting examples of antimicrobial reagents used to disinfect water include sodium hypochlorite, calcium hypochlorite, gaseous chlorine, and chlorine dioxide. Suitable antimicrobial neutralizers according to the present disclosure include, for example, sodium thiosulfate, sodium sulfite, ascorbic acid, sodium ascorbate, and mixtures thereof.

[0058] In any embodiment, the static mixer 30 can be a venturi-type static mixer that draws the antimicrobial neutralizer from the source 50 due to negative pressure created by water flow through the assembly 10 from the first opening 2 to the second opening 4.

[0059] The static mixer 30 can be regulated to provide a substantially-constant

predetermined concentration of antimicrobial neutralizer in the sample flowing through the filter housing 40. The residual chlorine (typically, 0.2 to 0.4 ppm in U.S. drinking water) in the distribution system will inactivate microorganisms and the residual chlorine should be neutralized to recover and detect all viable microorganisms present in water. The normal protocol for existing methods of testing potable water is to add 0.1% sodium thiosulfate to the sample collection bottle to neutralize chlorine before testing the sample. However, this is not feasible for direct filtration of samples, (especially, potable water samples) with the existing filtration devices. The static mixer of the present disclosure blends the neutralizer solution with the water sample and, thus, neutralizes the antimicrobial agent causing the water flowing through the filter to be substantially free of the antimicrobial reagent used to disinfect the water during routine water treatment. This prevents a viable target microorganism retained by the filter during the filtering process from being killed after the target microorganism is retained on the filter and, thus, provides an accurate estimate of the viable microorganisms that are collected during the sampling process.

[0060] Optionally, the assembly 10 further comprises a connector 35 (e.g. a piece of tubing or a plastic or metal pipe) that operatively connects the static mixer 30 to another component (e.g., the filter housing 40 via the first aperture 41 of the filter housing) of the assembly 10. "Operatively connect", as used herein refers to a connection that facilitates passage of a water sample via the connector from the first component of the assembly to the second component of the assembly without substantial loss of volume or pressure due to the connector. The connector 35 can operatively connect to one or more of the components by friction fit or by complementarily-threaded ends, for example. [0061] Optionally, an assembly 10 of the present disclosure further comprises a pressure governor 20 disposed between the first opening 2 and the filter housing 40. The pressure governor 20 is disposed in the assembly 10 between the first opening 2 and the filter housing 40. The static mixer 30 may be disposed between the pressure governor 20 and the filter housing 40, as shown in the embodiment of FIGS. 1 and 2 or, in an alternative embodiment (not shown), the pressure governor may be disposed between the static mixer and the filter housing.

[0062] In a first instance, the pressure governor 20 functions to determine a maximum pressure that a water sample will flow through the filter housing 40. This first effect protects the filter from excessive water pressure that might otherwise disintegrate the filter and negatively impact its ability to retain the target microorganism. In addition, the pressure governor 20 may also function to provide a more-constant flow rate of water through the assembly than may otherwise occur in a system that might be subject to pressure variations. This second effect can help maintain a constant flow rate through the assembly, thereby enabling the operator to estimate the volume of sample tested by the amount of time the water is flowing through the assembly.

[0063] Nonlimiting examples of suitable pressure governors for use in an assembly and method of the present disclosure include a plastic water pressure regulator, a brass water pressure regulator (model no. 40055), and a brass water pressure regulator with gauge (model no. 40064); each available from Cameo Manufacturing, Inc. (Greensboro, NC).

[0064] Optionally, the assembly 10 further comprises a connector 25 (e.g. a piece of tubing or a plastic or metal pipe) that operatively connects the pressure governor 20 to another component (e.g., the static mixer 30) of the assembly 10. The connector 25 can operatively connect to one or more of the components by friction fit or by complementarily-threaded ends, for example.

[0065] In any embodiment, the assembly of the present disclosure further comprises an adaptor (e.g., a piece of conformable tubing, not shown) adapted to operatively connect the first opening to a water distribution pipe or a terminal structure (e.g., a faucet, a spigot, a fire hydrant) of a water distribution pipe.

[0066] In any embodiment, any one or all of the components of the assembly may be designed for a single use and then discarded. Preferably, the components are sterilized or disinfected prior to use so that any microorganisms detected in the method are microorganisms that were originally present in the water sample. In some embodiments, one or more of the components (e.g., the filter housing) may be designed for reuse. In these embodiments, it may be preferred to use components that can withstand repeated cycles of sterilization or disinfection without deterioration. [0067] The assembly of the present disclosure is used in a method of detecting a microorganism present in water discharged from a distribution pipe. FIG. 3 shows a block diagram of one embodiment of one embodiment of a method 200 according to the present disclosure.

[0068] The method 200 comprises the step 82 of connecting components of an assembly to an outlet of a water distribution pipe. The assembly can be any embodiment of the assembly described herein for collecting a viable microorganism present in water discharged from a distribution pipe. In any embodiment, connecting components of the assembly to the outlet can comprise connecting a preassembled assembly (i.e., an assembly comprising the filter housing, the static mixer, and the pressure governor) to the outlet. Alternatively, in any embodiment, connecting components of the assembly to the outlet of the water distribution pipe can comprise connecting an individual component (e.g., the pressure governor) of the assembly or a subassembly (e.g., a preassembled subassembly comprising the pressure governor and the static mixer) to the outlet of the water distribution pipe and, subsequently, connecting the remaining component(s) to form the assembly. FIG. 4 shows a schematic side view of the assembly of

FIG. 1 connected to an outlet of a water distribution pipe.

[0069] A "water distribution pipe", as used herein, refers to a conduit through which potable water is delivered from a water treatment facility to an end-user. As used herein, the water distribution pipe includes elements (e.g., faucet, valve, spigot, a fire hydrant) that are present at the terminus (e.g., the terminus of a pipe) of a water distribution system. The water treatment facility may be a municipal treatment or a private treatment facility. The purpose of the treatment (e.g., filtration and/or chemical treatment) is to remove contaminants and disinfect the water being delivered through the distribution pipe.

[0070] After connecting the components, the method 200 further comprises the step 84 of flowing a water sample from the distribution pipe through the filter. Flowing a water sample from the distribution pipe through the filter can comprise flowing a predetermined volume of a water sample from the distribution pipe through the filter. The predetermined volume may be a minimum volume (e.g., at least 100 mL, at least 500 mL, or at least 1000 mL), a prescribed nominal volume (e.g., 50 mL, 100 mL, 200 mL, 250 mL, 500 mL, 100 mL, 15000 mL, 2000 mL, 5000 ml, or 10000 mL), or a volume range (100-200 mL, 250-500 mL, 500-1000 mL,

1000-10,000 mL).

[0071] In any embodiment, flowing a water sample from the distribution pipe through the filter further can comprise opening a valve to cause the water the flow out of the distribution pipe and into the assembly. Advantageously, it is contemplated that the assembly may be adapted to receive the water as it is flowing out of the distribution pipe. Thus, the water need not be collected at one location and flowed through the assembly at a different location (e.g., a laboratory).

[0072] In any embodiment, flowing a water sample from the distribution pipe through the filter further can comprise measuring the water that has flowed through the assembly. This can be done, for example, by using a flow meter or by collecting the water that has passed through the assembly and measuring the volume (e.g., by collecting the water using a graduated cylinder a graduated container, or the like.

[0073] After flowing the water sample through the filter, the method 200 further comprises the step 86 of collecting a viable microorganism or a component thereof, if present, from the filter housing. The viable microorganisms can be collected from the filter housing in an intact state or, alternatively, they can be collected from the filter housing by lysing the intact viable microorganisms and collecting the components thereof from the filter housing.

[0074] The viable microorganisms can be collected from the filter by a variety of processes known in the art. For example, the microorganisms can be collected from the filter by back- flushing the filter with a relatively small volume (e.g., < 2 mL, < 1 mL, < 0.5 mL, < 0.25 mL, < 0.2 mL, < 0.1 mL) of eluent. The filter housing may be detached from the assembly and the eluent can be urged through the filter in a direction opposite that through which the water sample was flowed through the filter. Alternatively, or additionally, the filter may be removed from the filter housing and placed in a container (e.g., a microcentrifuge tube) and the tube can be agitated to release the cells from the filter.

[0075] Suitable eluents include, but are not limited to sterile water, a buffer solution, a physiological saline solution. In any embodiment, the eluent further comprises a surfactant capable of facilitating release of the viable microorganisms from the filter.

[0076] Optionally, after releasing the viable microorganisms from the filter into an eluent, the released microorganisms can be concentrated, for example, by centrifuging the eluate and resuspending the pellet in a relatively smaller volume of suspending medium (e.g., a buffer solution) prior to detecting the viable microorganism.

[0077] As for collecting a viable microorganism from a filter by centrifugation, this can be done for example by using a filter housing that is adapted to form a container that can be placed in a centrifuge to recover the filtrand, as described in PCT Publication No. WO 2015/095142, which is incorporated herein by reference in its entirety. The container is used to concentrate the microorganisms, if present, in order to rapidly detect and, optionally, identify them.

[0078] As an alternative to collecting viable microorganisms from the filter, the method contemplates that a component (e.g., a biomolecule such as a protein, a nucleic acid, or an antigen) of the viable microorganism can be collected and analyzed. In some embodiments, the component can be released from the viable cells by contacting the filter, and/or microorganisms released therefrom, with a cell lysis agent. If the filter is contacted with the cell lysis agent, the components of the lysed cells can be recovered by the filter using an eluent as described herein.

[0079] In an embodiment wherein a component of the microorganism is recovered from the filter, the filter housing is configured to be coupled to a lysate collection vessel as described in U. S. Provisional Patent Application Serial No. 62/272,557 filed December 29, 2015, incorporated herein by reference in its entirety. In this embodiment, the filter and the filtrand are disintegrated in a bead-beating process using cell-disruption particles. The disintegrated sample (liquid lysate) then can be moved to a predefined location in the lysate collection vessel where a portion of it can be recovered and analyzed. That is, in some embodiments, a method of the present disclosure can employ a sequential combination of filtration, cell disruption, and centrifugation in order to isolate and detect an analyte indicative of a target microorganism, if present in a sample. Advantageously, the filtrand does not need to be eluted from the filter before any microorganisms present in the filtrand are subjected to cell disruption. This enhances detection of analytes because the method does not require elution of the

microorganisms from the filter in order to detect the microorganisms.

[0080] Referring back to FIG. 3, the method 200 comprises a step 88 of detecting the viable microorganism or the component thereof. The viable microorganism can be detected using any suitable method of detecting viable microorganisms known in the art (e.g., culture methods, ATP bioluminescence methods). In any embodiment, the step 88 of detecting the viable microorganism comprises contacting the filter and/or microorganism eluted therefrom in contact with a nutrient culture medium (e.g., a selective culture medium) in order to promote growth and, optionally, reproduction of the viable microorganism. In any embodiment, the period of contact can be < 1 hour, < 1.5 hours, < 2 hours, < 3 hours, < 4 hours, < 5 hours, < 6 hours, < 8 hours, < 10 hours, or < 12 hours. For rapid methods, preferably, the period of contact is 4 hours or less.

[0081] In any embodiment, the method further may comprise a step of treating the filtrand with a process that renders a nucleic acid from nonliving microorganisms incapable of being replicated in a nucleic acid amplification process. A method of the present disclosure that uses nucleic acid amplification to detect target microorganisms can be adapted to selectively detect live target microorganisms (i.e., acellular nucleic acid and/or nucleic acid from dead microorganisms is not detected in the method). Fittipaldi et al (Journal of Microbiological Methods, 2012, vol. 91, pp. 276-289; which is incorporated herein by reference in its entirety) describe the use of light-reactive viability dyes to suppress amplification of polynucleotides from nonviable microorganisms. Thus, a method of the present disclosure optionally can comprise, before detecting the target microorganisms by nucleic acid amplification, i) contacting the sample filtrand with a light-reactive viability dye capable of suppressing amplification of an acellular polynucleotide or a polynucleotide present in a nonviable microorganism and ii) exposing the contacted filtrand to a suitable source and quantity of electromagnetic energy to suppress amplification of a viability dye-bound polynucleotide.

[0082] The filtrand can be contacted with the viability dye, for example, alternatively, by contacting a small amount of the dye with the filter after the filtrand is collected on the filter, as described in Example 3 of U. S. Provisional Patent Application Serial No. 62/272,557.

[0083] Detecting a component of a viable microorganism can be performed using any suitable method known in the art. As discussed herein, selectively amplifying a polynucleotide from a viable (vs. nonviable) microorganism can be achieved using a viability dye. Selectively detecting other components (e.g. a polypeptide) from viable (vs. nonviable) microorganisms can be achieved using methods that are known in the art. For example, the filtrand may be treated with a protease capable of disintegrating a protein that is otherwise protected from the protease activity when present in a viable cell. The protease is subsequently removed, the cells are lysed, and the intact protein from viable cells is detected.

[0084] In any embodiment, detecting a viable microorganism or a component thereof further comprises enumerating how many target microorganisms, if present, are in the sample.

Quantitative estimates (e.g., plate counts, direct microscopic counts, qPCR) of viable microorganisms can be determined by methods that are known in the art.

[0085] It is contemplated that any embodiment of the assembly of the present disclosure will be used at a variety of non-laboratory locations (i.e., in the "field"). Thus, in yet another aspect, the present disclosure provides a kit for collecting, and optionally detecting, a viable microorganism present in water discharged from a distribution pipe. The kit of the present disclosure includes the components described below in a common package and, optionally, may include in the package instructions for use of each of the components in a method according to the present disclosure.

[0086] A kit of the present disclosure comprises a filter housing comprising a passageway extending therethrough, a filter dimensioned to be held in the passageway of the filter housing so that water moving through the passageway passes through the filter, a pressure governor, and a static mixer.

[0087] In any embodiment, the kit may be provided with the filter disposed in the passageway of the filter housing such that water moving through the passageway passes through the filter.

[0088] The filter housing can be any filter housing as described herein. The filter housing has a first aperture through which a water sample enters the passageway and a second aperture through which water exits the passageway. The first aperture of the filter housing can be configured to be coupled to the pressure governor or the static mixer. Alternatively, the kit further comprises a first connector, the first aperture is configured to operatively connect to the first connector, and the first connector is adapted to operatively connect to the pressure governor or the static mixer.

[0089] In any embodiment, the kit optionally includes a second connector that is adapted to operatively connect the pressure governor to the static mixer.

[0090] In any embodiment, the kit optionally includes an instrument to measure water flow or volume. The instrument can be used to estimate the volume of water sample that has passed through the filter. Suitable instruments include a flow meter, a graduated cylinder, or a graduated beaker, for example.

[0091] In any embodiment, the kit father comprises an eluent for eluting a microorganism from the filter. In any embodiment, the kit further comprises a plurality of particles configured for use in a bead-beating cell lysis procedure.

[0092] In any embodiment, the kit further comprises a reagent for detecting a viable microorganism. The reagent can be selected from the group consisting of a polynucleotide primer, a polynucleotide probe, a viability dye, a DNA polymerase, luciferin, luciferase, an antibody, a PNA, a LNA, an aptamer, and a nutrient for culturing a microorganism.

[0093] The embodiments described above and illustrated in the figures are presented by way of example only and are not intended as a limitation upon the concepts and principles of the present disclosure. As such, it will be appreciated by one having ordinary skill in the art that various changes in the elements and their configuration and arrangement are possible without departing from the spirit and scope of the present disclosure.

[0094] All references and publications cited herein are expressly incorporated herein by reference in their entirety into this disclosure.

[0095] The following embodiments are intended to be illustrative of the present disclosure and not limiting.

EXEMPLARY EMBODIMENTS

[0096] Embodiment A is a method for detecting a microorganism present in water discharged from a distribution pipe, the method comprising:

connecting components of an assembly to an outlet of a water distribution pipe, the assembly having a first opening, a second opening, and components that include:

a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway;

wherein the filter is configured to retain a target

microorganism from a water sample; and

a static mixer disposed between the first opening and the filter housing; wherein, after the components are connected, the assembly forms a liquid flow path that extends from a first end that receives water from the distribution pipe to a second end from which water received from the distribution pipe exits the assembly;

wherein, after the assembly is connected to the outlet, the static mixer is disposed between the outlet and the filter housing;

after connecting the components, flowing a water sample from the distribution pipe through the filter;

collecting a viable target microorganism or a component thereof, if present, from the filter housing; and

detecting the viable target microorganism or the component thereof.

[0097] Embodiment B is the method of Embodiment A, wherein connecting components of the assembly to the outlet comprises connecting to the outlet an assembly that further comprises a pressure governor disposed between the first opening and the filter housing, wherein, after the assembly is connected to the outlet, the pressure governor and the static mixer are disposed between the outlet and the filter housing.

[0098] Embodiment C is the method of Embodiment A or Embodiment B, wherein connecting components of the assembly to the outlet comprises connecting two or more preassembled components of the assembly to the outlet.

[0099] Embodiment D is the method of Embodiment A or Embodiment B, wherein flowing a water sample from the distribution pipe through the filter comprises flowing a water sample having a volume in the range 100 mL to 25 L, inclusive, through the filter.

[00100] Embodiment E is the method of any one of the preceding Embodiments; wherein collecting a viable target microorganism or a component thereof from the filter housing comprises removing the filter from the filter housing.

[00101] Embodiment F is the method of Embodiment E, further comprising subjecting the filter to a bead beating process.

[00102] Embodiment G is the method of any one of Embodiments A through E, wherein collecting a microorganism, if present in the sample, from the filter housing comprises dislodging a microorganism from the filter into a predetermined volume of an elution liquid.

[00103] Embodiment H is the method of Embodiment G, wherein dislodging a

microorganism from the filter into a predetermined volume comprises back-flushing the filter with the predetermined volume of elution liquid.

[00104] Embodiment I is the method of any one of the preceding Embodiments, wherein collecting a microorganism, if present in the sample, from the filter housing further comprises contacting the filter with a cell-lysis reagent. [00105] Embodiment J is the method of any one of Embodiments A through H, further comprising treating the filtrand with a process that renders a nucleic acid from nonliving microorganisms incapable of being replicated in a nucleic acid amplification process.

[00106] Embodiment K is the method of any one of the preceding Embodiments, wherein detecting the viable microorganism or a component thereof comprises detecting a

polynucleotide that indicates a presence of a target microorganism, detecting a polypeptide that indicates a presence of a target microorganism, detecting a cell wall component that indicates a presence of a target microorganism, or detecting an antigen that indicates a presence of a target microorganism.

[00107] Embodiment L is the method of Embodiment E, wherein detecting the viable microorganism or a component thereof comprises contacting the viable microorganism with a nutrient growth medium.

[00108] Embodiment M is the method of any one of the preceding Embodiments, wherein detecting a presence of a target microorganism further includes enumerating how many target microorganisms, if present, are in the sample.

[00109] Embodiment N is an assembly for collecting a viable microorganism present in water discharged from a distribution pipe, the assembly comprising:

a conduit having a first opening and a second opening, the conduit comprising:

a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway;

wherein the filter is configured to retain microorganism from a water sample; and

a static mixer disposed between the first opening and the filter housing;

wherein the filter housing and the static mixer are operatively connected to form a liquid flow path that extends from the first opening to the second opening; wherein the first opening is configured connect with a distribution pipe or an adaptor configured to operatively connect the distribution pipe to the first opening;

wherein the first opening or the adaptor is configured to receive a water sample from the distribution pipe as water is flowing through the distribution pipe.

[00110] Embodiment O is the assembly of Embodiment N, further comprising a pressure governor disposed between the first opening and the filter housing, wherein the filter housing, the pressure governor, and the static mixer are coupled together to form a liquid flow path that extends from the first opening to the second opening.

[00111] Embodiment P is the assembly of Embodiment N or Embodiment O, wherein the static mixer comprises a venturi pump. [00112] Embodiment Q is the assembly of any one of Embodiments N through P, wherein the filter comprises a membrane filter.

[00113] Embodiment R is the assembly of any one of Embodiments N through P, wherein the filter comprises a hollow-fiber filter.

[00114] Embodiment S is the assembly of any one of the Embodiments N through R, further comprising an adaptor configured to operatively connect the distribution pipe to the first opening.

[00115] Embodiment T is the assembly of any one of Embodiments N through S, further comprising a container holding antimicrobial neutralizer, wherein the container is operatively connected to the static mixer.

[00116] Embodiment U is a kit, comprising:

a filter housing comprising a passageway extending therethrough;

a static mixer; and

a filter dimensioned to be held in the passageway of the filter housing so that water moving through the passageway passes through the filter.

[00117] Embodiment V is the kit of Embodiment U, wherein the kit is provided with the filter disposed in the passageway of the filter housing such that water moving through the passageway passes through the filter.

[00118] Embodiment W is the kit of Embodiment U or Embodiment V, further comprising a pressure governor.

[00119] Embodiment X is the kit of any one of Embodiments U through W, further comprising a first connector, wherein the first connector is adapted to operatively connect the pressure governor to the filter housing or to operatively connect the static mixer to the filter housing.

[00120] Embodiment Y is the kit of any one of Embodiments U through X, further comprising a second connector that is adapted to opeatively connect the pressure governor to the static mixer.

[00121] Embodiment Z is the kit of any one of Embodiments U through Y, further comprising an instrument to measure water flow or volume.

[00122] Embodiment AA is the kit of Embodiment Z, wherein the instrument is selected from the group consisting of a flow meter, a graduated cylinder, and a graduated beaker.

[00123] Embodiment AB is the kit of any one of Embodiments U though AA, further comprising an eluent for eluting a microorganism from the filter.

[00124] Embodiment AC is the kit of any one of Embodiments U though AB, further comprising a plurality of particles configured for use in a bead-beating cell lysis procedure. [00125] Embodiment AD is the kit of any one of Embodiments U though AC, further comprising a reagent for detecting a viable microorganism.

[00126] Embodiment AE is the kit of Embodiment AD, wherein the reagent is selected from the group consisting of a polynucleotide primer, a polynucleotide probe, a peptide nucleic acid, a locked nucleic acid, an aptamer, an antibody, a viability dye, a DNA polymerase, luciferin, luciferase, and a nutrient for culturing a microorganism.

[00127] Embodiment AF is the kit of any one of Embodiments U through AE, further comprising a container holding an antimicrobial neutralizer.

[00128] Embodiment AG is the kit of Embodiment AF, wherein the container is adapted to be operatively connected to the static mixer.

EXAMPLES

[00129] General Methods - Preparation of Bacteria.

[00130] The various bacteria (shown in Table 1) used in the examples were obtained from

ATCC (Manassas, VA).

[00131] Table 1. List of bacteria.

[00132] Pure cultures of the bacterial strains were inoculated into Tryptic Soy Broth (TSB, BD, Franklin Lakes, NJ) and were grown overnight at 37°C. The cultures were diluted serially in Butterfield phosphate buffer (Whatman, Piscataway, NJ) to obtain desired amount of colony forming units (cfu) per ml for spiking into water samples. The bacteria were quantified by plating appropriate dilutions on 3M PETRIFILM E. co/z/Coliform Count Plates (3M Company, St. Paul, MN) according to manufacturer's instruction and incubated overnight at 37°C. The plates were read using 3M PETRIFILM Plate Reader (3M Company) and colony forming units (cfu) were determined.

[00133] Reference Example 1. Detection of bacteria eluted from filter and subjected to PCR detection.

[00134] E. coli was grown over night in TSB at 37° C. The culture was diluted to obtain approximately 10² cfu/ml and 1 ml of the solution was added to 100 ml of sterile water to obtain approximately 100 cfu per 100 mL sample. The solution was filtered through the various filters using a syringe; the filter was attached to the outlet of a Barnstead™ Nanopure™ Life Science UV UF water system (Thermo Scientific, Ashville, NC and washed with about 10 liters of deionized water. The bacteria retained in the filters were eluted by various methods such as back flush, vortexing, back and forth sweeping. The eluted bacteria were further concentrated by centrifugation and DNA was extracted from the pelleted bacteria using QIAamp DNA mini kit (Qiagen, Valencia, CA). The DNA was eluted in 10 μΐ using a MinElute column and PCR reaction was set up using the entire eluted DNA.

[00135] Commercially available reagent for detection of E. coli from Primer Design Ltd,

Southampton, UK (Quantification of E. coli standard kit) was used according to manufacturer's instructions. Ten μΐ of DNA sample was added to 96-well PCR plate containing 10 μΐ of reaction mix (primers, probes, and enzyme mix). Thermal cycling was carried out using ABI 7500 sequence detection system (Life Technologies Corporation, Grand Island, NY) with the following conditions: 2 min at 95° C for denaturation followed by 40 cycles of 20 sec at 95° C and 1 min at 60° C.

[00136] Table 2. Elution of bacteria from filters after filtration and detection by PCR

[00137] Reference Example 2. Detection of bacteria retained filter and subjected to in situ lysis and PCR detection.

[00138] E. coli was grown over night in TSB at 37° C. The culture was diluted to obtain approximately 10² cfu/ml and 1 ml of the solution was added to 100 ml of sterile water to obtain approximately 100 cfu. The solution was filtered through the various filters using a syringe; the filter was attached to the outlet of a Nanopure Life Science UV UF water system (Thermo Scientific Barnstead) and washed with about 10 liters of water. The filters were processed using The PowerWater® Sterivex™ DNA Isolation Kit (MO BIO Laboratories) and DNA was extracted according to manufacturer's instructions. The DNA was eluted in 50 μΐ volume.

[00139] PCR was set up as described in Example 2. 25 μΐ of DNA sample was added to 96- well PCR plate containing 50 μΐ of reaction mix (primers, probes, and enzyme mix).

Thermocycling was carried out using ABI 7500 sequence detection system with the following conditions: 2 min at 95° C for denaturation followed by 40 cycles of 20 sec at 95° C and 1 at 60° C.

[00140] Table 3. Extraction of DNA from filters after filtration and detection by PCR

[00141] Example 1. Direct filtration of tap water and detection by qPCR.

[00142] Filtration of samples:

[00143] Control samples: E. coli was grown over night in TSB at 37° C. The culture was diluted to obtain approximately 10² cfu/ml and 1 ml of the solution was added to 100 ml of sterile water to obtain approximately 100 cfu. A 47 mm track etched 0.4μ polycarbonate Isopore™ Membrane Filter (cat# HTTP04700; EMD Millipore Corporation) was placed in a polypropylene filter holder (Cat # 43303020, Advantec 43303020 polypropylene filter holder for 47-mm membranes, available from Cole-Parmer, item # UX-06623-22; Cole-Parmer, Vernon Hills, IL). The sample containing about 100 cfu of E. coli was filtered through the membrane using a syringe.

[00144] From one set of samples (triplicates) the bacteria retained on the membrane after filtration was quantified using 3M™ PETRIFILM E. co/z/Coliform Count Plates (3M Company). One ml of sterile Butterfield phosphate buffer was pipetted onto E. coli/Coliform Count plate and allowed to hydrate for a minimum of 10 min. The membrane after filtration, was removed and placed on hydrated E. coli/Coliform count plates and incubated at 37°C for 18h. The bacterial colonies on the plate were counted manually and per cent recovery was calculated based on input amount of bacteria. From another set of samples (triplicates), membrane was processed for qPCR as described below. These samples constituted as control samples.

[00145] Non-neutralized samples: For another set of samples (triplicates), the culture was diluted to obtain approximately 10² cfu/ml and 1 ml of the solution was added to 100 ml of sterile water to obtain approximately 100 cfu. A 47 mm track etched 0.4μπι polycarbonate Isopore™ Membrane Filter (cat# HTTP04700; EMD Millipore Corporation) was placed in a polypropylene filter holder (Cat # 43303020, Advantec 43303020 polypropylene filter holder for 47-mm membranes, available from Cole-Parmer, item # UX-06623-22; Cole-Parmer). The sample containing about 100 cfu of E. coli was filtered through the membrane using a syringe.

[00146] A Thermo Scientific™ Nalgene™ Aspirator Vacuum Pump (Cat # 6140-0010; Thermo Scientific) was attached to the sink faucet. The filter holder was attached to the outlet of aspirator vacuum pump and 10 liters of tap water was filtered through the membrane by turning on the faucet. From one set of samples (triplicates), membrane after filtration, was removed and placed on hydrated E. coli/Coliform count plates and incubated at 37°C for 18h for quantitation of bacteria retained on the membrane. The bacterial colonies on the plate were counted manually and per cent recovery was calculated based on input amount of bacteria. From another set of samples (triplicates), membrane was processed for qPCR as described below. These samples constituted as non-neutralized samples. Samples of filtered water was also collected for determination of free chlorine as described below.

[00147] Neutralized samples: For another set of samples (triplicates), the culture was diluted to obtain approximately 10² cfu/ml and 1 ml of the solution was added to 100 ml of sterile water to obtain approximately 100 cfu. A 47 mm track etched 0.4μ polycarbonate Isopore™

Membrane Filter (cat# HTTP04700; EMD Millipore Corporation) was placed in a

polypropylene filter holder (Cat # 43303020, Advantec 43303020 polypropylene filter holder for 47-mm membranes, available from Cole-Parmer, item # UX-06623-22; Cole-Parmer). The sample containing about 100 cfu of E. coli was filtered through the membrane using a syringe.

[00148] A Thermo Scientific™ Nalgene™ Aspirator Vacuum Pump (Cat # 6140-0010;

Thermo Scientific) was attached to the sink faucet. A venturi device was used to deliver a concentrated neutralizing solution (lOx) directly to achieve lx concentration in flowing water to neutralize residual chlorine typically present in tap water. The 3M™ bleach dispensing system is designed to deliver 1 : 10 bleach solution from a concentrate and it was utilized to deliver lx neutralizing solution to flowing tap water.

[00149] 3M™ bleach dispensing system PI 1 (part # 70-0715-9368-8, 3M Company) was used to deliver neutralizing solution (1% sodium thiosulfate). Four liters of 1% solution of sodium thiosulfate (lOx concentrate) was prepared in sterile deionized water and was dispensed in to a sterile 38 mm neck size 4 liter amber bottle (Boston round narrow-mouth amber jugs with closure, Cat #145-4000, Thermo Scientific). The 3M™ bleach dispenser collar with dip tube was screwed to the neck of the bottle containing sodium thiosulfate solution. The other end of dispenser was connected to the vacuum outlet of the aspirator vacuum pump and the trigger on the dispenser was pressed and held in "on" position. The filter holder was attached to the outlet of aspirator vacuum pump and 10 liters of water was filtered through the membrane by turning on the faucet. The vacuum created by the flowing water allowed venturi device to deliver lx concentration of neutralizing solution. From one set of samples (triplicates), membrane after filtration, was removed and placed on hydrated E. coli/Coliform count plates and incubated at 37°C for 18h for quantitation of bacteria retained on the membrane. The bacterial colonies on the plate were counted manually and per cent recovery was calculated based on input amount of bacteria. From another set of samples (triplicates), membrane was processed for qPCR as described below. These samples constituted as neutralized samples. Samples of filtered water was also collected for determination of free chlorine as described below.

[00150] Free chlorine detection: The amount of free chlorine in tap water and filtered samples was determined using Hach pocket colorimeter TM II, chlorine (free and total) (Cat # 5870000, Hach Company, Loveland, Colorado) following manufacturer's instructions.

[00151] qPCR analysis: The membrane after filtration was removed from the filter holder and folded using a sterile forceps and placed into a sterile 2 ml tube (Cat. No. 522S, Biospec products, Inc.) containing 500 μΐ AE buffer (Qiagen, Inc.) and 300 mg of 0.1 mm

Zirconia/silica beads (Cat # Cat # 11079101z, Biospec Products, Inc.). The sample tubes were bead beaten using a bead beater (Mini-Beadbeater-1, cat # 3110BX, Biospec products, Inc.) for 2 min.

[00152] All the tubes were centrifuged in a microcentrifuge for 2 min at 14000 RPM, and the resulting supernatant was removed from the container and transferred to a sterile 1.5 ml microfuge tube (Plastibrand microtubes). The 1.5 ml microfuge tubes were spun again at 14000 RPM for 2 min to pellet any beads carried over during pipetting and the supernatant fluid was transferred to a Microcon^® centrifugal filter (Microcon^® DNA Fast Flow centrifugal filter unit, cat # MRCFOR100, EMD Millipore Corporation). The Microcon unit was centrifuged at 500 x g to further concentrate the sample to about 50 μΐ. The concentrated sample from the

Microcon^® was recovered by inverting the unit into a new microfuge tube and centrifuging at 1000 x g for 3 min. The filter device was removed and a known aliquot (10 μΐ) was used for PCR.

[00153] PCR was set up as described in Reference Example 2. 10 μΐ of DNA sample was added to 96-well PCR plate containing 40 μΐ of reaction mix (primers, probes, and enzyme mix). Thermal cycling was carried out using ABI 7500 sequence detection system (Life Technologies Corporation) with the following conditions: 2 min at 95^° C for denaturation followed by 40 cycles of 20 sec at 95^° C and 1 min at 60^° C.

[00154] As evidenced by the data in Table 4, the percent recovery of bacteria from control samples and neutralized sample was similar indicating that the neutralization of chlorine in flowing tap water using venturi device was effective. However, the non-neutralized sample had only 16% recovery. As seen in Table 5, the Ct values obtained from neutralized sample again indicated that the neutralization of chlorine using venturi device was effective to enable detection of bacteria from tap water. The free and total chlorine was also measured in tap water and neutralized tap water samples (Table 6). The chlorine was not detected in neutralized samples indicating that the approach of neutralization of flowing tap water using venturi device was effective.

[00155] Table 4. Recovery of bacteria following filtration.

[00156] Table 5. Detection of E. coli by qPCR using direct tap water filtration

¹ ND = Not detected. No DNA was amplified.

[00157] Table 6. Detection of chlorine in tap water and filtered samples

[00158] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. [00159] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[00160] Various modifications may be made without departing from the spirit and scope of the invention. These and other embodiments are within the scope of the following claims.

Claims

CLAIMS:

1. A method for detecting a microorganism present in water discharged from a distribution pipe, the method comprising:

connecting components of an assembly to an outlet of a water distribution pipe, the assembly having a first opening, a second opening, and components that include:

a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway;

wherein the filter is configured to retain a target microorganism from a water sample; and

a static mixer disposed between the first opening and the filter housing;

wherein, after the components are connected, the assembly forms a liquid flow path that extends from a first end that receives water from the distribution pipe to a second end from which water received from the distribution pipe exits the assembly;

wherein, after the assembly is connected to the outlet, the static mixer is disposed between the outlet and the filter housing;

after connecting the components, flowing a water sample from the distribution pipe through the filter;

collecting a viable target microorganism or a component thereof, if present, from the filter housing; and

detecting the viable target microorganism or the component thereof.

2. The method of claim 1, wherein connecting components of the assembly to the outlet comprises connecting to the outlet an assembly that further comprises a pressure governor disposed between the first opening and the filter housing, wherein, after the assembly is connected to the outlet, the pressure governor and the static mixer are disposed between the outlet and the filter housing.

3. The method of claim 1 or claim 2, wherein flowing a water sample from the distribution pipe through the filter comprises flowing a water sample having a volume in the range 100 mL to 25 L, inclusive, through the filter.

4. The method of any one of the preceding claims; wherein collecting a viable target microorganism or a component thereof from the filter housing comprises removing the filter from the filter housing.

5. The method of any one of claims 1 through 4, wherein collecting a microorganism, if present in the sample, from the filter housing comprises dislodging a microorganism from the filter into a predetermined volume of an elution liquid.

6. The method of any one of the preceding claims, wherein collecting a

microorganism, if present in the sample, from the filter housing further comprises contacting the filter with a cell-lysis reagent.

7. The method of any one of claims 1 through 5, further comprising treating the filtrand with a process that renders a nucleic acid from nonliving microorganisms incapable of being replicated in a nucleic acid amplification process.

8. The method of any one of the preceding claims, wherein detecting the viable microorganism or a component thereof comprises detecting a polynucleotide that indicates a presence of a target microorganism, detecting a polypeptide that indicates a presence of a target microorganism, detecting a cell wall component that indicates a presence of a target microorganism, or detecting an antigen that indicates a presence of a target microorganism.

9. The method of any one of the preceding claims, wherein detecting a presence of a target microorganism further includes enumerating how many target microorganisms, if present, are in the sample.

10. An assembly for collecting a viable microorganism present in water discharged from a distribution pipe, the assembly comprising:

a conduit having a first opening and a second opening, the conduit comprising:

a filter housing comprising a passageway extending therethrough and a filter disposed in the passageway;

wherein the filter is configured to retain microorganism from a water sample; and

a static mixer disposed between the first opening and the filter housing;

wherein the filter housing and the static mixer are operatively connected to form a liquid flow path that extends from the first opening to the second opening;

wherein the first opening is configured connect with a distribution pipe or an adaptor configured to operatively connect the distribution pipe to the first opening;

wherein the first opening or the adaptor is configured to receive a water sample from the distribution pipe as water is flowing through the distribution pipe.

11. The assembly of claim 10, further comprising a pressure governor disposed between the first opening and the filter housing, wherein the filter housing, the pressure governor, and the static mixer are coupled together to form a liquid flow path that extends from the first opening to the second opening.

12. The assembly of claim 10 or claim 11, further comprising an adaptor configured to operatively connect the distribution pipe to the first opening.

13. The assembly of any one of claims 10 through 12, further comprising a container holding antimicrobial neutralizer, wherein the container is operatively connected to the static mixer.

14. A kit, comprising:

a filter housing comprising a passageway extending therethrough;

a static mixer; and

a filter dimensioned to be held in the passageway of the filter housing so that water moving through the passageway passes through the filter.

15. The kit of claim 14, wherein the kit is provided with the filter disposed in the passageway of the filter housing such that water moving through the passageway passes through the filter.

16. The kit of claim 14 or claim 15, further comprising a pressure governor.

17. The kit of any one of claims 14 through 16, further comprising a first connector, wherein the first connector is adapted to operatively connect the pressure governor to the filter housing or to operatively connect the static mixer to the filter housing.

18. The kit of any one of claims 14 through 17, further comprising a second connector that is adapted to operatively connect the pressure governor to the static mixer.

19. The kit of any one of claims 14 through 18, further comprising a reagent for detecting a viable microorganism; wherein the reagent is selected from the group consisting of a polynucleotide primer, a polynucleotide probe, a peptide nucleic acid, a locked nucleic acid, an aptamer, an antibody, a viability dye, a DNA polymerase, luciferin, luciferase, and a nutrient for culturing a microorganism.

20. The kit of any one of claims 14 through 19, further comprising a container holding an antimicrobial neutralizer.