Patent Application
20170268031
Provided are methods and devices for the detection of bacteriophages.
Bacteriophage (phage) contamination is one of the major causes of fermentation failure in the dairy (1, 2) and biochemical processing industries (3). The presence of phages, especially lytic phages (4) in the starter culture or raw material such as milk can slow the fermentation process (5), reduce lactic acid production (4) and affect quality of the end-product (4). It has been reported that phage contamination is the primary cause of economic loss in the dairy industry (6, 7), affecting up to 10% of all milk fermentation processes (7). To minimize phage contamination, the fermentation industry has implemented several strategies to control phages, including improved sanitation protocols (2, 5), rotation of starter culture (2, 5), and the development of phage-resistant bacterial starter strains (8, 9).
Despite careful control of phages, phage contamination cannot be completely eliminated (2). Therefore, early detection of phages in the starter culture itself or in the raw material is needed. Currently, phage contamination is detected by sampling the whey using traditional microbiological methods such as indicator tests (2, 5) and plaque assays (5, 9). However, these traditional assays often require an extended period of incubation (a few hours to days) before phage contamination could be detected (2). More recently, molecular based techniques such as polymerase chain reaction (PCR) (1, 10) and flow cytometry (11) have been developed to detect phages in starter culture. The reported detection limits for PCR and flow cytometry were between 104-107 PFU/mL (1, 2, 4), 10 and 105 PFU/mL (11), respectively. One of the major drawbacks of PCR is that the technique cannot distinguish between viable and non-viable phages (2), and only known species of phages can be detected or quantified (2, 11). On the other hand, the flow cytometric method by Michelsen, et. al. quantified the number of membrane-compromised bacteria as an indication of phage infection (11). Using the side scatter plots, Michelsen, et. al. determined that the late stage phage infected cells have 30-50% decrease in scattered light compared to the control (11). However, flow cytometry is an expensive technique that requires a highly trained personnel to operate the instrument, gate the events and analyze the data (4); thus, it may not be a suitable method to detect phages in a typical fermentation industry.
In one aspect, provided is a method of assaying for bacteriophage in a sample suspected of comprising bacteriophage. In varying embodiments, the methods comprise:
a) creating a water-in-oil (W/O) emulsion, comprising:
b) detecting the cell viability dye, wherein detectable cell viability dye provides a signal when bacterial cells within the water-in-oil (W1/O) emulsion are non-viable, thereby indicating the presence of bacteriophage in the sample suspected of comprising bacteriophage. In varying embodiments, the method further comprises before step a) i) the step of mixing a sample suspected of comprising bacteriophage with a population of bacterial cells, thereby yielding a bacterial cell mixture.
In a related aspect, provided is a method of assaying for bacteriophage in a sample suspected of comprising bacteriophage. In varying embodiments, the methods comprise:
a) creating a water-in-oil-in-water (W1/O/W2) emulsion, comprising:
b) detecting the cell viability dye, wherein detectable cell viability dye provides a signal when bacterial cells within the water-in-oil-in-water (W1/O/W2) emulsion are non-viable, thereby indicating the presence of bacteriophage in the sample suspected of comprising bacteriophage. In varying embodiments, the method further comprises before step a) i) the step of mixing a sample suspected of comprising bacteriophage with a population of bacterial cells, thereby yielding a bacterial cell mixture.
In another aspect, provided is a method of assaying for bacteria strains that are resistant to bacteriophage lysis, comprising:
a) creating a water-in-oil (W/O) emulsion, comprising:
b) detecting the cell viability dye, wherein detectable cell viability dye provides a signal when bacterial cells within the water-in-oil (W1/O) emulsion are non-viable, thereby indicating the presence of bacteria susceptible to bacteriophage in the bacterial cell culture or mixture; and wherein nondetectable cell viability dye indicates the presence of bacteria resistant to bacteriophage in the bacterial cell culture or mixture.
In a further aspect, provided is a method of assaying for bacteria strains that are resistant to bacteriophage lysis, comprising:
a) creating a water-in-oil-in-water (W1/O/W2) emulsion, comprising:
b) detecting the cell viability dye, wherein detectable cell viability dye provides a signal when bacterial cells within the water-in-oil (W/O) emulsion are non-viable, thereby indicating the presence of bacteria susceptible to bacteriophage in the bacterial cell culture or mixture; and wherein nondetectable cell viability dye indicates the presence of bacteria resistant to bacteriophage in the bacterial cell culture or mixture.
With respect to embodiments of the methods, in varying embodiments, the detecting step comprises performing visual inspection. In varying embodiments, the method detects bacteriophage with a sensitivity of about 104 PFU/mL or less by visual inspection. In varying embodiments, the detecting step comprises performing optical microscopy. In varying embodiments, the detecting step comprises performing optical flow cytometry. In varying embodiments, the method detects bacteriophage with a sensitivity of about 102 PFU/mL or less by optical microscopy or flow cytometry. In varying embodiments, the detecting step does not comprise performing one or more of flow cytometry, impedance spectroscopy or nucleic acid amplification. In varying embodiments, the method can be performed in 2 or fewer hours, e.g., in less than 120, 90, 60, 45, 30 minutes. In varying embodiments, the water soluble or hydrophilic emulsifier in the inner aqueous phase (W1) has a hydrophilic lipophilic balance (HLB) value of 10 or greater. In varying embodiments, the emulsifier with a hydrophilic lipophilic balance (HLB) value of 10 or greater comprises a protein-based or proteinaceous emulsifier, e.g., whey protein isolate (WPI), soy protein isolate, caseins and/or milk proteins. In varying embodiments, the hydrophilic emulsifier in the inner aqueous phase (W1) comprises a particle-based emulsifier. In varying embodiments, the cell viability dye is a fluorophore. In varying embodiments, the cell viability dye binds to or intercalates into DNA. In varying embodiments, the cell viability dye is selected from the group consisting of propidium iodide (PI), 7-aminoactinomycin D (7-AAD), DRAQ7™, and TO-PRO®-3 Iodide. In varying embodiments, the cell viability dye is selected from propidium iodide (PI), hexidium iodide, a carbocyanine, rhodamine 123, tetra methyl rhodamine, dialkylaminophenylpolyenylpyridinium, aminonaphthylethenylpyridinium, resazurin, formazan, red-fluorescent ethidium homodimer-1, calcein, tetrasodium (6E,6′E)-6,6-[(3,3′-dimethylbiphenyl-4,4′-diyl)di(1E)hydrazin-2-yl-1-ylidene]bis(4-amino-5-oxo-5,6-dihydronaphthalene-1,3-disulfonate) (Evans blue), (3Z,3′Z)-3,3′-[(3,3′-dimethylbiphenyl-4,4′-diyl)di(1Z)hydrazin-2-yl-1-ylidene]bis(5-amino-4-oxo-3,4-dihydronaphthalene-2,7-disulfonic acid) (Trypan blue), 7-aminoactinomycin D (7-AAD), DRAQ7™, eFluor® 455UV, eFluor® 450, eFluor® 506, eFluor® 520, eFluor® 660, eFluor® 780, Zombie Aqua™, Zombie Green™, Zombie NIR™, Zombie Red™, Zombie Violet™, Zombie UV™, and Zombie Yellow™. In varying embodiments, the cell viability dye is a colorimetric dye. In varying embodiments, the one or more bacteriophages are lytic bacteriophages. In varying embodiments, the one or more bacteriophages are lysogenic or temperate bacteriophages. In varying embodiments, the methods further comprise prior to the detecting step, inducing the lytic cycle of the lysogenic or temperate bacteriophages. In varying embodiments, the one or more bacteriophages are a member of a viral family selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae. In varying embodiments, the one or more bacteriophages are lytic to a bacterial cell selected from the group consisting of Campylobacter, Cronobacter, Escherichia, Salmonella, Lactococcus, Vibrio, Erwinia, Xanthomonas, Shigella, Staphylococcus, Streptococcus, Clostridium, Pseudomonas, Mycobacterium, Neisseria, and Bacilli. In varying embodiments, the bacteriophages are selected from the group consisting of lactococcal phage species (936, c2, c6A, 1483, T187, P087, 1358, KSY1, 949, and P335 phage species), T4 phage, T7 phage, phage A1511, phage Felix-Ol1, phage PHL 4, phage P7, ECML-4, ECML-117, ECML-134, phage A511, phage P100, ATCC accession no. PTA-5372, ATCC accession no. PTA-5373, ATCC accession no. PTA-5374, ATCC accession no. PTA-5375, ATCC accession no. PTA-5376, ATCC accession no. PTA-5377, phage FO1-E2, phage CJ6, phage Φ88, phage Φ35, NgoΦ6 and NgoΦ7, lambdoid prophages, phage β, Lambda phages, Mu-1, lactococcal lysogenic phages (φLC3, Tuc2009, bIL285, bIL286 and bIL309, bIL170, biL167), Lysogenic phages of S. aureus (8325-4, Ps6, 655, 248, W-26, U9, 655C, Oh-SO, 608, N-135, C-72), and mixtures thereof. In varying embodiments, the oil in the oil phase is liquid at room temperature, e.g., at 25-30° C. In varying embodiments, the oil in the oil phase is selected from mineral oil, canola oil, olive oil, corn oil, sunflower oil, safflower oil, peanut oil, coconut oil and fluorinated oils (e.g., perfluorodecalin). In varying embodiments, the hydrophobic emulsifier with an HLB value of 4 or less comprises a polyglycerol ester of fatty acid. In varying embodiments, the hydrophobic emulsifier with an HLB value of 4 or less comprises polyglycerol polyricinoleate (PGPR). In varying embodiments, the outer aqueous phase (W2) comprises a mixture comprising a bile salt, a zwitterionic detergent and a nonionic detergent. In varying embodiments, the outer aqueous phase (W2) comprises one or more bile salts, lecithin and Tween 20. In varying embodiments, the detecting step is performed in comparison to a control comprising the inner aqueous phase without bacteriophage.
In another aspect, provided is a portable device. In some embodiments, the portable device comprises a tubing in fluid communication from the upstream to downstream direction, with (i) a fluidic droplet generator, (ii) an incubator and (iii) a detector. In varying embodiments:
a) the upstream end of the tubing is in fluid communication with a sample reservoir;
b) the tubing within the fluidic droplet generator comprises a first upstream syringe comprising a needle comprising a beveled tip, wherein the beveled tip is pierced into the inner space of the tubing, wherein the inner space of the first syringe comprises an oil phase comprising an emulsifier; and a second downstream syringe comprising a needle comprising a beveled tip, wherein the beveled tip is pierced into the inner space of the tubing downstream from the needle of the first syringe, wherein the inner space of the second syringe comprises an aqueous phase comprising at least one detergent;
c) the incubator can hold a preselected or predetermined temperature in the range of about 4° C. to about 50° C.; and
d) the detector can detect a fluorescent or colorimetric signal. In some embodiments, one or more of the sample reservoir, the first syringe and the second syringe automatically deliver fluid. In some embodiments, the device weighs less than 10 kg, e.g., less than about 9 kg, 8 kg, 7 kg, 6 kg, 5 kg, 4 kg, 3 kg, 2 kg, 1 kg, or less. In some embodiments, the device has a desk or table footprint of less than about 200 in2, e.g., less than about 190 in2, 180 in2, 170 in2, 160 in2, 150 in2, 140 in2, 130 in2, 120 in2, 110 in2, 100 in2, or less. In some embodiments, the inner space of the tubing has a diameter in the range of about 1/32 (0.03125) inches to about 1/16 (0.0625) inches. In some embodiments, the needle of the first syringe and/or the second syringe has a gauge from about 18 G to about 34 G, e.g., from about 25 G to about 30 G, e.g., 25 G, 26 G, 27 G, 28 G, 29 G, 30 G, 31 G, 32 G, 33 G or 34 G. In some embodiments, the portable device is as depicted in
In a further aspect, provided is a microfluidic device for creating water-in-oil (W/O) and/or water-in-oil-in-water (W1/O/W2) emulsion droplets. In varying embodiments the microfluidic device comprises one or more units or modules of channels comprising:
In a related aspect, provided is a microfluidic device for creating water-in-oil (W1/O) emulsion droplets, comprising:
1. Introduction
Bacteriophage contamination of starter culture and raw material poses a major problem in the fermentation industry. Provided are methods for the rapid detection of phage contamination, e.g., in a sample suspected of containing bacteriophage contamination (e.g., a food product sample, e.g., a fermented food product, milk, whey, etc.), using water-in-oil-in-water (W/O/W) and/or water-in-oil (W/O) emulsion microdroplets. Model bacteria with varying concentrations of phages were encapsulated in W/O/W and/or W/O emulsion microdroplets using a simple needle-in-tube setup. The detection of phage contamination was accomplished in one hour using the propidium iodide labeling of the phage-infected bacteria inside the W/O/W emulsion microdroplets. Using this approach, a detection limit of 102 PFU/mL of phages was achieved quantitatively, while 104 PFU/mL of phages could be detected qualitatively based on visual comparison of the fluorescence images.
Compartmentalization of biomolecules in discrete W/O/W and/or W/O emulsion microdroplets is an attractive approach for detection and screening, and has been used in applications such as PCR reactions (12-15), phage display amplification (16, 17), and quantification of enzyme activity (18) and protein expression (19) at the single cell level. While many studies have demonstrated that uniformly sized emulsion microdroplets can be produced rapidly using chip-based microfluidics (20-25), the commercialization this technology has been limited due to scale up cost (26), and handling of large sample volumes. Therefore, large-scale methods such as homogenization (27), rigorous stirring (28), and tubing-based setup (needle-in-tube) (29-33) are simpler and more practical approaches to the chip-based technology for industrial use.
Using the needle-in-tube setup, a model bacteria and phage were encapsulated in discrete W/O/W emulsion microdroplets. The presence of phages in each W/O/W emulsion was detected using fluorescence microscopy based on the propidium iodide (PI) labeling of dead bacteria. Since the PI dye can only bind to the DNA inside the bacteria with damaged membrane, a PI-labeled bacterium would be indicative of a phage infection. The high local concentration of bacteria and phages in discrete W/O/W emulsion microdroplets provided a good signal-to-noise ratio for quantification using fluorescence microscopy, and the presence of phages could be detected within one hour.
Given the simplicity and sensitivity of this approach, this method can be readily adapted to any strains of bacteria and phages that are commonly used for fermentation, e.g., using a portable device, and is applicable to rapid detection of phage contamination, e.g., in food products suspected of having bacteriophage contamination.
2. Methods of Bacteriophage Detection
Generally, the methods for bacteriophage detection comprise encapsulating a population of bacterial cells suspected of bacteriophage infection with a cell viability dye in a water-in-oil (W/O) and/or water-in-oil-in-water (W/O/W) emulsion microdroplet. Non-viable bacterial cells, e.g., which have been infected with a bacteriophage, will be stained with the cell viability dye, indicating the presence of bacteriophage contamination. The methods can be performed rapidly, with bacteriophage contamination being detected within 2 or fewer hours, e.g., 120, 90, 60, 45, 30 or fewer minutes, and without requiring performing flow cytometry, impedance spectroscopy or nucleic acid amplification. Detection performed employing visual inspection can achieve a sensitivity of 104 PFU/mL of phages. Detection performed employing optical microscopy, or optionally flow cytometry can achieve a sensitivity of 102 PFU/mL of phages.
a. Suspending the Bacterial Cells in an Inner Aqueous Phase (W1)
The methods provide for the rapid detection of phage contamination, e.g., in a sample suspected of containing bacteriophage contamination (e.g., a food product sample, e.g., a fermented food product, milk, whey, etc.), using water-in-oil-in-water (W/O/W) and/or water-in-oil (W/O) emulsion microdroplets. In varying embodiments, the sample may already be a mixture containing a population of bacterial cells. Such samples can be used directly in the methods, as long as the bacterial cells in the sample are susceptible to lysis in the presence of bacteriophage. In varying embodiments, the sample suspected of containing bacteriophage contamination does not contain bacteria. In embodiments testing samples which do not contain bacteria, a population of bacteria susceptible to lysis in the presence of bacteriophage can be mixed into the sample, thereby creating a bacterial cell mixture.
A population of bacterial cells, e.g., from a bacterial cell culture or mixture suspected of containing or being monitored for the presence of bacteriophage contamination is suspended in an inner aqueous phase (W1) comprising a water soluble emulsifier with a hydrophilic lipophilic balance (HLB) value of 10 or greater and a cell viability dye. Generally, the methods are performed on a scale such that the volume of the inner aqueous phase fits within a microtube, e.g., less than about 2 mls, e.g., less than about 2.0, 1.8, 1.5, 1.2, 1.0, 0.8 mLs. In varying embodiments, the inner aqueous phase comprises about 1-10%, e.g., about 5% (w/v) surfactant (e.g., water soluble emulsifier, e.g., with a hydrophilic lipophilic balance (HLB) value of 10 or greater).
With respect to the emulsifier in the inner aqueous phase (W1), any water soluble emulsifier can be used. In varying embodiments, the emulsifier in the inner aqueous phase (W1) has a hydrophilic lipophilic balance (HLB) value of 10 or higher. In varying embodiments, the emulsifier comprises a protein-based or proteinaceous emulsifier, e.g., whey protein isolate (WPI), soy protein isolate, caseins and/or milk proteins. Other emulsifiers that can find use in the inner aqueous phase include without limitation, e.g., PEG-7 Glyceryl Cocoate (HLB=10); PEG-20 Almond Glycerides (HLB=10); Linoleamide DEA (HLB=10); PEG-25 Hydrogenated Castor Oil (HLB=10.8); Stearamide MEA (HLB=11); Glyceryl Stearate and/or PEG-100 Stearate (HLB=11); Polysorbate 85 (HLB=11); PEG-7 Olivate (HLB=11); Cetearyl Glucoside (HLB=11); PEG-8 Oleate (HLB=11.6); Polyglyceryl-3 Methyglucose Distearate=12 Oleth-10 (HLB=12.4); Oleth-10/Polyoxyl 10 Oleyl Ether NF (HLB=12.4); Ceteth-10 (HLB=12.9); PEG-8 Laurate (HLB=13); Cocamide MEA (HLB=13.5); Polysorbate 60 NF (HLB=14.9); Polysorbate 60 (HLB=14.9); Polysorbate 80 (HLB=15); Isosteareth-20 (HLB=15); PEG-60 Almond Glycerides (HLB=15); Polysorbate 80 NF (HLB=15); PEG-20 Methyl Glucose Sesquistearate (HLB=15); Ceteareth-20 (HLB=15.2); Oleth-20 (HLB=15.3); Steareth-20 (HLB=15.3); Steareth-21 (HLB=15.5); Ceteth-20 (HLB=15.7); Isoceteth-20 (HLB=15.7); Polysorbate 20 (HLB=16.7); Polysorbate 20 NF (HLB=16.7); Laureth-23 (HLB=16.9); PEG-100 Stearate (HLB=18.8); Steareth-100 (HLB=18.8); PEG-80 Sorbitan Laurate (HLB=19.1); starch-based emulsifiers including starch particles; protein-based or proteinaceous emulsifiers including soy protein isolate, or casein; and hydrophilic particles including silica particles.
With respect to the cell viability dyes, both fluorophores and colorimetric dyes find use. In varying embodiments, the cell viability dye is a fluorophore. Illustrative fluorophores to distinguish live versus dead (e.g., non-infected versus infected with bacteriophage, respectively) bacterial cells include without limitation is selected from the group consisting of propidium iodide (PI), 7-aminoactinomycin D (7-AAD), DRAQ7™, and TO-PRO®-3 Iodide. In varying embodiments, the cell viability dye is selected from propidium iodide (PI), hexidium iodide, a carbocyanine, rhodamine 123, tetra methyl rhodamine, dialkylaminophenylpolyenylpyridinium, aminonaphthylethenylpyridinium, resazurin, formazan, red-fluorescent ethidium homodimer-1, calcein, tetrasodium (6E,6′E)-6,6-[(3,3′-dimethylbiphenyl-4,4′-diyl)di(1E)hydrazin-2-yl-1-ylidene]bis(4-amino-5-oxo-5,6-dihydronaphthalene-1,3-disulfonate) (Evans blue), (3Z,3′Z)-3,3′-[(3,3′-dimethylbiphenyl-4,4′-diyl)di(1Z)hydrazin-2-yl-1-ylidene]bis(5-amino-4-oxo-3,4-dihydronaphthalene-2,7-disulfonic acid) (Trypan blue), 7-aminoactinomycin D (7-AAD), DRAQ7™, eFluor® 455UV, eFluor® 450, eFluor® 506, eFluor® 520, eFluor® 660, eFluor® 780, Zombie Aqua™, Zombie Green™, Zombie NIR™, Zombie Red™, Zombie Violet™, Zombie UV™, and Zombie Yellow™. Zombie dyes are available from BioLegend, Inc. (on the internet at biolegend.com).
The methods can be used to detect the presence of any kind of bacteriophage contamination of a bacterial culture or mixture. The bacteriophage can be lytic or lysogenic (e.g., temperate). When detecting lysogenic or temperate bacteriophages, the methods can further entail the step of inducing the lytic cycle of the lysogenic bacteriophages. Induction of the lytic cycle is generally performed prior to the detection step, and can be performed after formation of the water-in-oil (W1/O) emulsion or water-in-oil-in-water (W1/O/W2) emulsion. In varying embodiments, this can be accomplished by exposing the bacterial cell mixture to an external stimulus that induces the lytic cycle. Such external stimuli are known in the art and include without limitation changes in temperature, UV light exposure, chemicals such as antibiotics or combination of these. Because the methods do not employ nucleic acid amplification, the methods do not rely on the knowledge of bacteriophage genomic sequences. In varying embodiments, the one or more bacteriophages subject to detection are a member of a viral family selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae. Illustrative bacteriophages that can be detected using the methods and devices described herein include without limitation, e.g., lactococcal phage species (936, c2, c6A, 1483, T187, P087, 1358, KSY1, 949, and P335 phage species), T4 phage, T7 phage, phage A1511, phage Felix-O1, phage PHL 4, phage P7, ECML-4, ECML-117, ECML-134, phage A511, phage P100, ATCC accession no. PTA-5372, ATCC accession no. PTA-5373, ATCC accession no. PTA-5374, ATCC accession no. PTA-5375, ATCC accession no. PTA-5376, ATCC accession no. PTA-5377, phage FO1-E2, phage CJ6, phage Φ88, phage Φ35, NgoΦ6 and NgoΦ7, lambdoid prophages, phage β, Lambda phages, Mu-1, lactococcal lysogenic phages (φLC3, Tuc2009, bIL285, bIL286 and bIL309, biL170, biL167), Lysogenic phages of S. aureus (8325-4, Ps6, 655, 248, W-26, U9, 655C, Oh-SO, 608, N-135, C-72), and mixtures thereof. See, e.g., Siddiqui et al., Applied Microbiology, January 1974, p. 278-280).
The methods can be used to detect bacteriophage (e.g., lytic or lysogenic) contamination of any bacterial culture or mixture. In varying embodiments, the one or more bacteriophages are lytic to or infect a bacterial cell selected from the group consisting of Campylobacter, Cronobacter, Escherichia, Salmonella, Lactococcus, Vibrio, Erwinia, Xanthomonas, Shigella, Staphylococcus, Streptococcus, Clostridium, Pseudomonas, Mycobacterium, Neisseria, and Bacilli.
b. Forming Water-in-Oil (W1/O) Emulsion
Droplets of the inner aqueous phase (W1) are suspended into an oil phase (0) comprising an oil and a hydrophobic emulsifier with an HLB value of 4 or less, to yield a water-in-oil (W1/O) emulsion. The water-in-oil (W1/O) emulsion or microdroplets can be formed using any method in the art. In varying embodiments, the droplets of the inner aqueous phase are delivered or suspended into the oil using a needle, e.g., having a needle gauge in the range of about 18 G to about 34 G, e.g., 18 G, 19 G, 20 G, 21 G, 22 G, 23 G, 24 G, 25 G, 26 G, 27 G, 28 G, 29 G, 30 G, 31 G, 32 G, 33 G or 34 G, depending on the desired size of suspended droplet. In varying embodiments, the needle can have a beveled or blunt end. As described herein, the needle-in-tube method for forming water-in-oil emulsions is known in the art. In varying embodiments, water-in-oil (W1/O) emulsion or microdroplets are formed in the inner space of a tubing, wherein a channel containing the fluidic flow of the inner aqueous phase (W1) suspension flows into the confluence or junction of two channels of fluidic flow of the oil phase such that when the inner aqueous phase (W1) meets the confluence of the fluidic flow of the two channels of the oil phase, microdroplets of a water-in-oil (W1/O) emulsion are formed. This is depicted in the portable device of
In varying embodiments, the inner aqueous phase (W1) microdroplets are delivered or suspended into the oil phase via a microfluidic lumen or channel in a microfluidic device, e.g., at a junction in the microfluidic device where the fluidic flow from a channel or lumen containing the inner aqueous phase (W1) flows into the fluidic flow of one or more channels or lumens containing the oil phase. See, e.g., the illustrative microfluidic lumen or channel configurations of a microfluidic device depicted in
Generally, the oil in the oil phase is liquid at 25-30° C. In varying embodiments, the oil in the oil phase is selected from mineral oil, canola oil, olive oil, corn oil, sunflower oil, safflower oil, peanut oil, coconut oil and fluorinated oils (e.g., perfluorodecalin).
With respect to the hydrophobic emulsifier, any hydrophobic emulsifier with a hydrophilic lipophilic balance (HLB) value of 4 or lower can be used. In varying embodiments, the emulsifier is a polyglycerol ester of fatty acid. In varying embodiments, the hydrophobic emulsifier comprises polyglycerol polyricinoleate (PGPR). Other emulsifiers that can find use in the oil phase include without limitation, e.g., Glycol Distearate (HLB=1); Sorbitan Trioleate (HLB=1.8); Propylene Glycol Isostearate (HLB=2.5); Glycol Stearate (HLB=2.9); Sorbitan Sesquioleate (HLB=3.7); Glyceryl Stearate (HLB=3.8); and Lecithin (HLB=4).
c. Forming Water-in-Oil-in-Water (W1/O/W2) Emulsion
The water-in-oil (W1/O) emulsion or microdroplets are then mixed in an outer aqueous phase (W2) comprising at least one water soluble emulsifier, yielding a water-in-oil-in-water (W1/O/W2) emulsion comprising bacterial cells. In varying embodiments, the outer aqueous phase can comprise water alone or an aqueous salt buffer, e.g., having a salt concentration in the range of about 10 μM to about 1.0 M.
With respect to the water soluble emulsifier in the outer aqueous phase (W2), in varying embodiments, the water soluble emulsifier in the outer aqueous phase (W2) comprises an HLB value greater than 7. The water soluble emulsifier in the outer aqueous phase (W2) may or may not be a mixture of emulsifying components. In varying embodiments, the water soluble emulsifier of the outer aqueous phase (W2) comprises a mixture of a bile salt, a zwitterionic detergent and a nonionic detergent. In varying embodiments, the outer aqueous phase (W2) comprises one or more bile salts, lecithin and Tween 20. In varying embodiments, the water soluble emulsifier of the outer aqueous phase (W2) comprises one or more emulsifiers selected from Lecithin; PEG-8 Dioleate (HLB=8); Sodium Stearoyl Lactylate (HLB=8.3±1); Sorbitan Laurate (HLB=8.6±1); PEG-40 Sorbitan Peroleate (HLB=9±1); Lecithin (HLB=9.7±1); Laureth-4 (HLB=9.7±1); PEG-7 Glyceryl Cocoate (HLB=10); PEG-20 Almond Glycerides (HLB=10); Linoleamide DEA (HLB=10); PEG-25 Hydrogenated Castor Oil (HLB=10.8); Stearamide MEA (HLB=11); Glyceryl Stearate and/or PEG-100 Stearate (HLB=11); Polysorbate 85 (HLB=11); PEG-7 Olivate (HLB=11); Cetearyl Glucoside (HLB=11); PEG-8 Oleate (HLB=11.6); Polyglyceryl-3 Methyglucose Distearate=12 Oleth-10 (HLB=12.4); Oleth-10/Polyoxyl 10 Oleyl Ether NF (HLB=12.4); Ceteth-10 (HLB=12.9); PEG-8 Laurate (HLB=13); Cocamide MEA (HLB=13.5); Polysorbate 60 NF (HLB=14.9); Polysorbate 60 (HLB=14.9); Polysorbate 80 (HLB=15); Isosteareth-20 (HLB=15); PEG-60 Almond Glycerides (HLB=15); Polysorbate 80 NF (HLB=15); PEG-20 Methyl Glucose Sesquistearate (HLB=15); Ceteareth-20 (HLB=15.2); Oleth-20 (HLB=15.3); Steareth-20 (HLB=15.3); Steareth-21 (HLB=15.5); Ceteth-20 (HLB=15.7); Isoceteth-20 (HLB=15.7); Polysorbate 20 (HLB=16.7); Polysorbate 20 NF (HLB=16.7); Laureth-23 (HLB=16.9); PEG-100 Stearate (HLB=18.8); Steareth-100 (HLB=18.8); PEG-80 Sorbitan Laurate (HLB=19.1).
The water-in-oil-in-water (W1/O/W2) emulsion or microdroplets can be formed using any method in the art. In varying embodiments, water-in-oil-in-water (W1/O/W2) emulsion or microdroplets are formed in the inner space of a tubing, wherein a channel containing the fluidic flow of the oil phase containing water-in-oil (W1/O) emulsion microdroplets flows into the confluence or junction of two channels of fluidic flow of the outer aqueous phase such that when the water-in-oil (W1/O) emulsion microdroplets meet the confluence of the fluidic flow of the two channels of the outer aqueous phase, microdroplets of a water-in-oil (W/O) and/or water-in-oil-in-water (W1/O/W2) emulsion are formed. This is depicted in the portable device of
In varying embodiments, the water-in-oil (W1/O) emulsion microdroplets are delivered or suspended into the outer aqueous phase via a microfluidic lumen or channel in a microfluidic device, e.g., at a junction in the microfluidic device where the fluidic flow from a channel or lumen containing the water-in-oil (W1/O) emulsion microdroplets in the oil phase flows into the fluidic flow of one or more channels or lumens containing the outer aqueous phase. See, e.g., the illustrative microfluidic lumen or channel configurations of a microfluidic device depicted in
d. Detecting Changes in the Cell Viability Dye
Changes in cell viability can be detected using various dye or reporter molecules. Signal from the cell viability dye or reporter encapsulated in the water-in-oil (W/O) water-in-oil-in-water (W1/O/W2) emulsion or microdroplets is detected as an indicator of the presence of bacteriophage in the bacterial cell population. The cell viability dye can be detected using any method known in the art appropriate to the kind of dye employed, e.g., a fluorophore or a colorimetric dye. In varying embodiments, the signal from the cell viability dye is detected by visual inspection. In varying embodiments, the signal from the cell viability dye is detected by optical microscopy. In varying embodiments, the signal from the cell viability dye is detected by flow cytometry. In varying embodiments, the signal from the cell viability dye is detected without employing flow cytometry, nucleic acid amplification or impedance spectroscopy.
In varying embodiments, the detecting step is performed in comparison to a control comprising the inner aqueous phase without bacteriophage.
3. Portable Bacteriophage Detection Device
Further provided is a portable device for detection of the presence of bacteriophage in a bacterial culture or mixture, e.g., using the methods described herein. In varying embodiments, the device comprises a tubing in fluid communication from the upstream to downstream direction, with (i) a fluidic droplet generator, (ii) an incubator and (iii) a detector. Upstream of the fluidic droplet generator, e.g., at the upstream end, the tubing is in fluid communication with a sample reservoir, which can be a syringe. The sample reservoir can contain the inner aqueous phase, with embodiments as described above and herein, e.g., a bacterial cell population suspended in a water soluble emulsifier having an HLB value of 10 or greater.
In varying embodiments, the tubing within the fluidic droplet generator comprises a first upstream syringe comprising a needle pierced into the inner space or inner lumen or inner channel of the tubing, wherein the inner space of the first syringe contains an oil phase comprising an emulsifier; and a second downstream syringe containing a needle pierced into the inner space or inner lumen or inner channel of the tubing downstream from the needle of the first syringe, wherein the inner space of the second syringe comprises an aqueous phase comprising at least one detergent. The first syringe can contain an oil phase, with embodiments as described above and herein, e.g, an oil and an emulsifier having an HLB value of 4 or lower. The second syringe can contain the outer aqueous phase, with embodiments as described above and herein, e.g., an aqueous solution comprising a water soluble emulsifier having an HLB value of about 7 or greater, e.g., that can be a mixture comprising a bile salt, a zwitterionic detergent and a nonionic detergent.
In varying embodiments, the incubator can hold a preselected or predetermined temperature in the range of about 4° C. to about 50° C., e.g., in the range of about 25° C. to about 37° C. In varying embodiments, the detector can detect a fluorescent signal and/or a colorimetric signal. In varying embodiments, one or more of the sample reservoir, the first syringe and the second syringe automatically deliver fluid. For example, the device can further comprise a controller in electrical and/or mechanical communication with one or more of the sample reservoir (which can be a syringe), the first syringe and the second syringe such that the plungers of the syringes can automatically depress to incrementally dispense the inner aqueous phase, the oil phase and/or the outer aqueous phase into the tubing. Fluid flows through the tubing in the upstream to downstream direction from the sample reservoir to the inlet of the first syringe; from the inlet of the first syringe to the inlet of the second syringe; and from the inlet of the second syringe to the incubator, the detector and then the outlet. In varying embodiments, the portable device weighs less than 10 kg, e.g., less than about 9 kg, 8 kg, 7 kg, 6 kg, 5 kg, 4 kg, 3 kg, 2 kg, 1 kg, or less. In varying embodiments, the device has a desk or table footprint of less than about 200 in2, e.g., less than about 190 in2, 180 in2, 170 in2, 160 in2, 150 in2, 140 in2, 130 in2, 120 in2, 110 in2, 100 in2, or less. In varying embodiments, the inner space or inner lumen or inner channel of the tubing has a diameter in the range of about 1/32 (0.03125) inches to about 1/16 (0.0625) inches. In varying embodiments, the needle of the first syringe and/or the second syringe has a gauge from 18 G to 34 G, e.g., from 25 G to 30 G, e.g., e.g., 18 G, 19 G, 20 G, 21 G, 22 G, 23 G, 24 G, 25 G, 26 G, 27 G, 28 G, 29 G, 30 G, 31 G, 32 G, 33 G or 34 G. In varying embodiments, the device is as depicted in
In varying embodiments, the methods described herein are performed using a portable bacteriophage detection device, as described above.
4. Microfluidic Bacteriophage Detection Device
Further provided is a microfluidic device for creating water-in-oil-in-water (W1/O/W2) emulsion droplets. In varying embodiments the microfluidic device comprises one or more units or modules of channels for creating water-in-oil (W/O) and/or water-in-oil-in-water (W1/O/W2) emulsion droplets, one unit comprising:
In varying embodiments, the methods described herein are performed using a microfluidic bacteriophage detection device, as described above.
5. Kits
Further provided are kits. In varying embodiments, the kits can comprise vials containing an inner aqueous phase, an oil phase and an outer aqueous phase, as described above. In varying embodiments, the kits can comprise vials containing an inner aqueous phase and an oil phase, as described above. In varying embodiments, the kits can further comprise a portable device for the preparation of water-in-oil-in-water emulsions, as described above and herein. In varying embodiments, the kits can further comprise a microfluidic device for the preparation of water-in-oil-in-water emulsions, as described above and herein.
The following examples are offered to illustrate, but not to limit the claimed invention.
Materials.
Clear Tygon PVC tubing ( 1/16″ ID and 3/16″ OD) was from McMaster-Carr. Kendall MonoJect hypodermic needles (27 G×1.5), 5 mL and 1 mL disposable syringes, phosphate buffered saline (PBS) were from Thermo Fisher Scientific (Waltham, Mass.). Mineral oil, bile salt, Tween 20 and propidium iodide (PI) were from Sigma Aldrich (St. Louis, Mo.). Polyglycerol polyricinoleate (PGPR 4175) was from Palsgaard (Morris Plains, N.J.). Whey protein isolate (WPI) was a generous gift from Professor John M. Krochta from the University of California, Davis. Low melting lecithin (ALCOLEC® PC75) was also a gift from American Lecithin Inc. (Oxford, Conn.). The SYBR Green I and BODIPY 665 dyes were from Life Technologies (Carlsbad, Calif.). All chemicals were used as is without further purification.
Bacterial Culture and Growth Conditions.
E. coli BL21 (ATCC, #BAA-1025, Manassas, Va.) was used as a model bacteria in this study and grown according to the manufacturer's protocol. Briefly, the stock bacteria was streaked on LB agar plate and grown overnight at 37° C. to obtain isolated bacterial colonies. Fresh bacterial cultures were prepared weekly by picking one bacterial colony from the LB agar plate and grown overnight in 50 mL LB media at 37° C. and shaken at 250 rpm. Then, an aliquot of this overnight culture was grown in 5 mL LB media until late log-phase (OD600˜0.8, 108 CFU/mL) and used for encapsulation.
Phage Propagation.
The T7 phages (ATCC, #BAA-1025-B2, Manassas Va.) were propagated on LB agar plates. Briefly, an aliquot of stock T7 phages (˜104 PFU/mL final concentration) were mixed with 300 μL of E. coli BL21 added to 3.5 mL molten agar (0.3% w/v). The solution was inverted once to mix and poured over a LB agar plate and incubated overnight at room temperature to form plaques. The soft agar layer was gently scraped off, resuspended with 10 mL of 1×TBS-Mg (50 mM Tris, 150 mM NaCl and 10 mM MgCl2), and placed on a shaking incubator for 30 min at 250 rpm to allow the phages to diffuse out of the agar. The agar/phage mixture was then centrifuged at 10,000 rpm for 10 min to remove bacterial and agar debris and filter using a 0.22 μm syringe filter. The phages were stored at 4° C. until use.
Compositions of the (W1/O/W2) Emulsion Microdroplets.
The inner aqueous phase (W1) was composed of: 5% (w/w) WPI, E. coli BL21 bacteria (108 CFU/mL), and PI dye (1 μg/mL final concentration). Different titers of T7 phages (102, 104 and 106 PFU/mL) were added to the W1 phase no more than 5 min prior to W1/O generation. The oil phase (0) was composed of 6% (w/w) PGPR and 94% mineral oil. A lipophilic BODIPY 665 dye (0.025 μg/mL) was added to visualize the O phase. The outer aqueous phase (W2) was made by first mixing 1.5% (w/w) lecithin with 0.5% (w/w) bile salts in water, and the solution was stirred rigorously at 40° C. for 1 hr to dissolve the lecithin. Next, the solution was probe sonicated using a Qsonica sonicator (Model Q55, 50 W power, 20 kHz frequency; Newton, Conn.) to generate smaller lecithin/bile vesicles. The probe sonicator was set at 50% power, and manually pulse 5 times on a 3 sec on/3 sec off cycle. Then, Tween 20 (0.4 wt. % final concentration) was added to the sonicated lecithin/bile salt solution to make up the final W2 phase (
Generation of the W1/O Emulsion Using a Needle-in-Tube Setup.
The first W1/O emulsion was synthesized by injecting W1 solution into a continuous flow of O phase (
Preparation of the W1/O/W2 Emulsion Microdroplets.
A 1 mL solution of W2 was prepared in a second 1.5 mL microcentrifuge tube. Once the W1/O emulsion had settled to the bottom of the tube, 400 μL of the W1/O emulsion was pipetted into the W2 solution using a cut pipet tip (
Visualization of the W1/O/W2 Emulsion Microdroplets Using Optical Microscopy.
An aliquot of the SYBR Green I dye (5 μg/mL) was added to the W1 to stain the bacteria and BODIPY 665 was added to the O phase to visualize the W1 and O phase, respectively. The fluorescence excitation and emission of the SYBR Green dye in the W1 phase were 480/30 nm and 535/40 nm, respectively (Olympus). The fluorescence excitation and emission of the BODIPY 665 dye in the O phase were 640/20 nm and 680/30 nm, respectively (Olympus). The brightfield and fluorescence microscopy images of the W1/O/W2 emulsion microdroplets were taken using an Olympus IX-71 inverted fluorescence microscope with either a 4× or a 10× objective (Olympus UPlanFLN).
Detection of PI-Labeled Bacteria in the W1/O/W2 Emulsion Microdroplets.
To detect phage contamination, different concentrations of phages (102-106 PFU/mL) were incubated with 108 CFU/mL of bacteria and 5 μg/mL of the PI dye in the W1 phase. The W1/O/W2 emulsion microdroplets were made using the methods described above, and the encapsulated bacteria and phages were incubated at 37° C. for 1 hr. For comparison, a non-encapsulated sample of bacteria (108 CFU/mL) and phages (106 PFU/mL) was also prepared as an aqueous suspension. Then, 40 μL of the W1/O/W2 emulsion microdroplets was added to 100 μL PBS in a 96-well plate. The fluorescence images of the W1/O/W2 emulsion microdroplets were taken using an Olympus IX-71 inverted fluorescence microscope with a 4× objective (Olympus UPlanFLN, NA=0.13). The PI dye-labeled bacteria in the W1 phase was excited using a 540/25 nm filter and the fluorescence emission was measured using a 605/55 nm filter (Olympus).
Image Processing and Analysis.
The fluorescence intensity of the PI dye was analyzed using ImageJ (http://rsbweb.nih.gov/ij/). Each image was filtered using a rolling ball filter (ball radius of 500 pixels) to eliminate background noise and correct for image tilt. All image were thresholded and set to 0-255 (min-max) pixel intensity. A region of interest (ROI) was drawn around each W1/O/W2 emulsion microdroplets from each fluorescence image, and the mean pixel intensity (MPI) of the ROI was determined using Image J.
Enumeration Ofphage Amplification.
The W1/O/W2 emulsion microdroplets were centrifuged at 13,200 rpm for 10 min to disrupt the emulsion. A clear phase separation between the oil and aqueous phase was obtained after centrifugation and the released phages were located in the aqueous phase. Titers of the released phages were enumerated using the standard plate plaque assay. Briefly, released phages were serially diluted with sterile PBS and added to 3 mL molten soft agar (3% w/v) with 300 μL of an overnight E. coli BL21 culture. The agar solution was poured over pre-warmed LB agar plate and incubated at 37° C. for 2 hr. Visible phage plaques on the LB agar plates were subsequently counted.
Statistical Analysis.
Three independent experiments were conducted and the mean pixel intensities (MPI) from at least 170 W1/O/W2 emulsion microdroplets were quantified for each sample. All data was reported as mean±SEM. A p-value of <0.05 using a Student's t-test was considered as statistically significant. The size distribution, boxplot of MPI and statistical analyses were determined using the Minitab 16 statistical software (Minitab, Inc., State College, Pa.).
Encapsulation of Bacteria and Phages in W1/O/W2 Emulsion Microdroplets.
Bacteria and phages were encapsulated in W1/O/W2 emulsion microdroplets using a tubing-based setup (i.e., needle-in-tube) as demonstrated (
W1/O/W2 Emulsion Microdroplets Improve Signal Contrast for Detection.
To demonstrate the effect of signal enhancement using the W1/O/W2 emulsion microdroplets, a direct comparison between the encapsulated (W1/O/W2) and non-encapsulated (aqueous suspension) sample was performed (
Qualitative Analysis of PI-Labeled Bacteria in the W1/O/W2 Emulsion Microdroplets.
Since the T7 phages have diameters (60-65 nm) that are below the resolution for optical microscopy (34), an indirect approach was used to determine the levels of phages in the W1/O/W2 emulsion microdroplets (
Quantification of MPI Inside W1/O/W2 Emulsion Microdroplets.
The increase in MPI inside the W1/O/W2 emulsion microdroplet for each sample was further quantified and compared (
Enumeration of Amplified Phages Inside the W1/O/W2 Emulsion Microdroplets.
The number of amplified phages in the W1/O/W2 emulsion microdroplets was enumerated using a standard plate plaque assay (Table 1). A 3 log increase in phage count was determined for the 104 and 106 PFU/mL initial phage concentration samples (Table 1). On the other hand, incubation with 102 PFU/mL of initial phage concentration resulted in a 4 log increase in phage count (Table 1). The level of phage amplification at each starting phage titer was comparable to a standard phage propagation system, where the bacteria and phages were incubated together in a bulk solution (Table 1).
In this study, a rapid method to detect the presence of phages in a model bacterial culture was described. The encapsulation of bacteria and phages in W1/O/W2 emulsion microdroplets used in this study were produced by a simple and inexpensive setup using a syringe needle and a piece of tubing (
The microdroplets generated using the simple needle-in-tube setup can produce O/W emulsion (31) and polymeric microbeads (30, 33) with sizes ranging from 200-600 μm. Consistent with previous studies, the W1/O/W2 emulsion microdroplets generated in this study using the needle-in-tube method had an average diameter of 150 μm (
Given the high encapsulation efficiency6, high local concentration (38) and success of phage amplification in microdroplets demonstrated in this study and in prior studies (16, 17), it is envisioned that the W/O/W emulsion microdroplets could be used over a large concentration range of the encapsulant. This encapsulation system would also be especially useful when the concentration of the target analyte (e.g., phage) is low. To detect the presence of phages in a bacterial starter culture, three different titers of phages (102, 104 and 106 PFU/mL) were encapsulated with 108 CFU/mL of bacteria in a W1/O/W2 emulsion microdroplet (
Using this W1/O/W2 emulsion microdroplet and imaging approach, the phage detection could be achieved within one hour after phage infection (
In current fermentation practice, a starter culture would be rotated when 105 to 106 PFU/mL of phages was detected in the whey (8). Using this approach, a detection limit of 102 PFU/mL phages could be achieved quantitatively (
In summary, this method is an attractive approach for the fermentation industry to rapidly detect phage contamination. It is a relatively simple and inexpensive method, and can be used with any strain of bacteria and phage, or in a mixed culture without a priori knowledge of the phage DNA sequence. Together, the results of this study demonstrated that phage infection in a bacterial culture can be detected using a simple W1/O/W2 emulsion microdroplet and imaging approach. Given the simplicity, high sensitivity and relatively low cost of this imaging approach compared to flow cytometry and PCR methods, it is useful for rapid detection or routine screening of phage contamination in starter culture in the fermentation industry.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application is the U.S. national phase under 35 U.S.C. §371 of Intl. Appl. No. PCT/US2015/041400, filed Jul. 21, 2015, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/031,091, filed on Jul. 30, 2014, which are hereby incorporated herein by reference in their entireties for all purposes.
This invention was made with government support under Grant No. 2011-67021-20034 awarded by the United States Department of Agriculture—Agriculture and Food Research Initiative (USDA-AFRI). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/41400 | 7/21/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62031091 | Jul 2014 | US |
