Dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol, and the Mini Extruder were purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.). Cholesterol, triethylamine, chloroform, methanol, HEPES, EDTA, dimethyl sulfoxide, Sephadex G-50, Sepharose CL-4B, sulforhodamine B (SRB), Tween 20, and n-octyl-β-d-glucopyranoside were purchased from Sigma Chemical Co. (St. Louis, Mo.). N-Succinimidyl-S-acetylthiopropionate (SATA), N-[κ-maleimidoundecanoyl-oxy]sulfosuccinimide ester (sulfo-KMUS), hydroxylamine hydrochloride, N-ethylmaleimide, and blocker casein (1% casein in Tris-buffered saline) were purchased from Pierce (Rockford, Ill.). A DispoDialyzer membrane (molecular weight cutoff, 15,000) was purchased from Spectrum Lab, Inc. (Rancho Dominguez, Calif.). Durapore membrane filters (0.22- and 0.45-μm pore sizes), potassium phosphate, and 12- by 75-mm polystyrene snap cap tubes were purchased from Fisher Scientific (Pittsburgh, Pa.), while 8- by 40-mm glass vials were purchased from National Scientific Company (Duluth, Ga.). The 0.45-μm-pore-size gridded membrane filters were purchased from Pall Gelman Sciences (Ann Arbor, Mich.), and the 0.2- and 0.4-μm-pore-size Nuclepore Track-Etch membranes were purchased from Whatman (Clifton, N.J.). Sorbitol-containing MacConkey agar (SMAC) was purchased from Becton Dickinson (Sparks, Md.).
Affinity-purified polyclonal goat anti-E. coli O157:H7 antibodies and heat-killed E. coli O157:H7 positive control antigen were purchased from Kirkegaard & Perry Laboratories Inc. (Gaithersburg, Md.). E. coli O157:H7 immunomagnetic beads were purchased from Neogen Corp. (Lansing, Mich.) and Dynal Biotech Inc. (Brown Deer, Wis.). Medi-8 accelerated-growth medium for E. coli O157:H7 was purchased from Eichrom Technologies (Darien, Ill.). Tryptic soy broth (TSB) and tryptic soy agar (TSA) were purchased from Difco Laboratories (Detroit, Mich.). E. coli O157:H7 (ATCC 43895), an E. coli laboratory strain (ATCC 25922), and a Bacillus subtilis strain (ATCC 6633) were purchased from the American Type Culture Collection (Manassas, Va.).
The magnetic separation stand was purchased from Promega (Madison, Wis.). The handheld Aquafluor fluorometer was purchased from Turner Designs (Sunnyvale, Calif.). Pasteurized apple juice and pasteurized apple cider were purchased from Wegman's Food Markets (Rochester, N.Y.). Bottled spring water was purchased from Aqua Valley (Edmeston, N.Y.) or Clearwater Springs (Syracuse, N.Y.), tap water was acquired from the City of Geneva, N.Y., and groundwater used in optimization experiments was collected from a nonchlorinated well in Scottsville, N.Y. Groundwater for experiments conducted at the Erie County Public Health Laboratories were collected from nonchlorinated wells in Hamburg, N.Y.
Preparation of anti-E. coli O157:H7 antibody-tagged immunoliposomes.
Liposome nanovesicles were prepared by a method involving hydration and freezing and thawing and extrusion (1
). Briefly, 28.9 mg of dipalmitoylphosphatidylcholine, 3.7 mg of dipalmitoylphosphatidylglycerol, 16.8 mg of cholesterol, and 0.5 ml of a solution of DPPE-ATA (thiolated phospholipid) in chloroform were added to 10 ml of chloroform and 4 ml of methanol (the DPPE-ATA was prepared in advance by reacting 5 mg of DPPE and 3.5 mg of SATA in 1.0 ml of 0.7% [vol/vol] triethylamine in chloroform for 30 min at room temperature, followed by evaporation under vacuum and reconstitution in 1.0 ml of chloroform). After being mixed thoroughly, the solution was evaporated under vacuum on a rotary evaporator, resulting in a thin lipid film. To this was added 4 ml of a 150 mM solution of SRB in 20 mM HEPES buffer (pH 7.5), and the resulting solution was subjected to five freeze-thaw cycles as described previously (1
) to produce large multilammelar liposome nanovesicles. The liposomes were subsequently extruded sequentially through a 0.4- and 0.2-μm Nuclepore Track-Etch membranes, and any unencapsulated SRB was removed by applying the liposomes to a Sephadex G-50 size exclusion column equilibrated with Tris-buffered saline (pH 7.4, same osmolality as SRB encapsulant). Preparation of sulforhodamine-containing liposomes via this procedure generally resulted in the formation of vesicles with a mean (± standard deviation [SD]) diameter of 203 ± 14.8 nm, as shown by particle size analysis (LS130 particle size analyzer; Coulter, Hialeah, Fla.). Assuming the formation of unilammelar vesicles and a width of 2 nm for the phospholipid bilayer, the amount of SRB contained in each liposome is approximately 6 × 10−10
O157:H7 antibody-tagged liposomes were prepared by reacting activated liposomes with sulfo-KMUS-derivatized antibodies. Briefly, anti-E. coli
O157:H7 antibodies were derivatized by reacting 7.2 μl of a 1/50 (wt/vol) solution of sulfo-KMUS in dimethyl sulfoxide with 3 mg (~20 nmol) of anti-E. coli
O157:H7 antibodies in 0.8 ml of 0.05 M potassium phosphate and 1 mM EDTA (pH 7.8) for 3 h in the dark at room temperature. The reaction was quenched by the addition of 12 μl of 0.5 M Tris (pH 7.8), and the derivatized antibody was dialyzed overnight in a DispoDialyzer (molecular weight cutoff, 15,000) against Tris-buffered saline (pH 7.4) at 4°C. Based on the results of a Bartlett assay (2
) conducted on the liposome nanovesicles prepared as described above and a desire to tag 0.4 mol of antibody per 100 mol of total lipid, 250 μl (125 μmol) of 0.5 M hydroxylamine hydrochloride in 0.1 M HEPES and 25 mM EDTA (pH 7.5) were added to 2.5 ml of liposome solution (equal to 100 nmol of surface DPPE-ATA) and allowed to react at room temperature for 2 h in the dark. The pH of the reaction was adjusted to 7.0 with 0.5 M potassium phosphate, and the dialyzed antibodies were added. The solution was allowed to react at room temperature in the dark for 3 h and then quenched by the addition of 20 μl (2 μmol) of N
-ethylmaleimide in phosphate-buffered saline (PBS; pH 7.0). Finally, unconjugated antibodies were separated from antibody-tagged liposomes by means of a Sepharose CL-4B size exclusion column equilibrated with Tris-buffered saline (pH 7.4, same osmolality as SRB encapsulant).
E. coli O157:H7 growth conditions.
E. coli O157:H7 was cultured in TSB for 18 h at 37°C with shaking. Cultures were serially diluted with TSB, and cell concentrations were determined by applying 100 μl of the 10−6 and 10−7 dilutions to 10 TSA plates each and visually counting colonies after incubating the plates for 24 h at 37°C. Typically, a 100-μl aliquot of the 10−6 dilution of the overnight culture resulted in 100 to 200 CFU while a 100-μl aliquot of a 10−7 dilution resulted in 10 to 20 CFU.
Detection of E. coli O157:H7 in water samples and apple juice and cider.
Water samples (100 ml) were spiked with 100 to 1,000 μl of a 10−7 serially diluted overnight culture of E. coli O157:H7 for a final concentration range of 0.1 to 0.2 CFU/ml to 1 to 2 CFU/ml. Spiked and unspiked samples (used as negative controls) were filtered through 0.45-μm-pore-size, 25-mm-diameter Durapore membrane filters with a water aspirator (29 in of Hg, 30 to 40 s/100 ml of water). The membrane filters were placed in 12- by 75-mm polystyrene snap cap tubes to which were added 20 μl of E. coli O157:H7 immunomagnetic beads (1:10 dilution in blocker casein) and 2 ml of Medi-8 accelerated-growth medium for E. coli O157:H7 prepared according to the manufacturer's directions.
Apple juice samples (100 ml) were prepared identically to the water samples (above) except that samples were spiked with 100 to 1,000 μl of a serially 10−5-diluted stock solution of E. coli O157:H7 in apple juice (the stock solution was prepared in advance by adding 20 ml of an overnight culture to 200 ml of apple juice and placing the solution in a refrigerator [4°C] for 5 to 6 days). Apple cider samples (30 ml) were likewise spiked with 100 to 1,000 μl of a serially 10−5-diluted stock solution of E. coli. However, spiked and unspiked samples were centrifuged at 3,800 × g for 15 min at 4°C rather than filtered. Following centrifugation, the supernatant was removed, and 2 ml of Medi-8 accelerated-growth medium for E. coli O157:H7 was added to each pellet. After brief vortexing (5 s), the resulting suspensions were transferred to 12- by 75-mm polystyrene snap cap tubes to which was added 20 μl of E. coli 157:H7 immunomagnetic beads (1:10 dilution in blocker casein).
The snap cap tubes containing the filter membrane or pellet (apple cider), immunomagnetic beads, and Medi-8 accelerated-growth medium were placed on a Labquake rotating mixer (8 rpm) in an incubator at 42°C and rotated for 4 h to both enrich and capture the E. coli O157:H7 with the immunomagnetic beads. At the end of the simultaneous enrichment and immunomagnetic capture steps, the tubes were removed from the incubator and positioned in the magnetic separation stand. After 3 min (10 min for apple cider), the supernatant was aspirated from the tubes with a vacuum aspirator and 2 ml of blocker casein was added to the magnetic bead-E. coli O157:H7 pellet. The tubes were returned to the rotating mixer and rotated at room temperature for 5 min (10 min for apple cider). After 5 min, the tubes were once again positioned in the magnetic separation stand, and the magnetic separation was repeated and the supernatant was removed as described above.
Following the second magnetic separation (above), the filter membranes were removed (for water and apple juice samples) and 200 μl of the anti-E. coli O157:H7 immunoglobulin G-tagged liposomes (1:40 dilution in blocker casein) was added to each tube. The tubes were replaced on the rotating mixer and rotated at room temperature for 1 h, followed by magnetic separation and supernatant removal (as described above). The resulting pellet was washed again with 2 ml of blocker casein for 5 min as described above and subjected to magnetic separation as described above, and finally 200 μl of 30 mM n-octyl-β-d-glucopyranoside in Tris-buffered saline was added to each of the tubes, followed by vigorous vortexing for 5 s. After a final magnetic separation, the resulting supernatant containing released SRB was transferred to 8- by 40-mm glass vials, and the fluorescence of each sample was measured with a handheld Aquafluor fluorometer (Turner Design) at an excitation wavelength of 540 nm and an emission wavelength of 570 nm.
To enumerate the actual number of E. coli O157:H7 cells added to the various water and apple juice and cider samples, 10 replicate 100-μl samples of the dilution used to spike each sample matrix were plated onto TSA plates. The plates were incubated overnight at 37°C (~20 h), and colonies were counted the next day with the aid of a touch counter (Fisher Scientific).
Validation of the assay at the Erie County Regional Public Health Laboratories and comparison to a standard microbiological assay.
To both validate the IMB/IL fluorescence assay and determine the preliminary specificity of the assay, 100-ml samples of commercial spring water were spiked with various concentrations of B. subtilis, an E. coli laboratory strain, or E. coli O157:H7 at Cornell University and transferred along with unspiked samples to the Erie County (N.Y.) Public Health Laboratories for a blinded analysis. Laboratory staff at the public health laboratory were given detailed protocols with minimal training beforehand. Samples were processed by the IMB/IL fluorescence assay by staff at the public health laboratory as described above for water matrices with the exception that the enrichment-capture step was carried out for 3.5 h and the incubation with anti-E. coli O157:H7 liposomes was carried out for only 1/2 h. In addition, negative and positive controls were included in the analysis. The positive control consisted of a 1-ml sample of a 1:10,000 dilution of heat-killed E. coli O157:H7 positive control antigen in Tris-buffered saline (~7 × 105 cells/ml) incubated directly with the immunomagnetic beads and processed as described above; the negative control consisted of a 50-ml sample of Tris-buffered saline, filtered through a 0.45-μm Durapore membrane filter, which was further incubated with immunomagnetic beads and processed as described before.
The IMB/IL fluorescence assay was also compared to a standard microbiological method utilized in the Erie County Public Health Laboratories. Briefly, 1-liter samples of groundwater were collected by the staff at the public health laboratory. From each liter, three 100-ml samples were spiked with 100 μl of a 10−7
overnight dilution of E. coli
O157:H7 containing approximately 15 CFU, and three 100-ml samples were prepared without spiking. One set of spiked and unspiked samples from each 1-liter sample was assayed by the IMB/IL fluorescence assay, one set was assayed by a standard microbiological assay employed by the public health laboratory, and the third set was assayed by an additional assay (modeled after the procedure of Bopp et al. [5
]) intended to confirm the absence of E. coli
O157:H7 in the groundwater samples utilized in this validation experiment. Samples assayed by the IMB/IL fluorescence assay were processed as described above for water matrixes with the exception that the enrichment-capture step was carried out for 5 h and the incubation with anti-E. coli
O157:H7 immunoliposomes was carried out for 30 min. Positive and negative controls were included as described above. The standard microbiological method employed at the Erie County Public Health Laboratories consisted of filtration of the spiked and unspiked samples with 0.45-μm-pore-size gridded membrane filters onto SMAC plates. The SMAC plates were incubated for 18 to 24 h at 35°C, and the resulting sorbitol-nonfermenting colonies were counted under 10× magnification with the aid of a Quebec colony counter. Any sorbitol-nonfermenting colonies were considered suspect because most O157 Shiga toxin-producing E. coli
isolates do not ferment the carbohydrate d
-sorbitol overnight. The third set of spiked and unspiked groundwater samples from each 1-liter sample was processed as follows. Samples were filtered, and any E. coli
O157:H7 organisms present were captured as described for the IMB/IL fluorescence assay except that samples were filtered with 0.22-μm-pore-size 25-mm-diameter Durapore membrane filters prewetted with PBS-0.02% Tween 20, and the filter membranes were incubated with anti-E. coli
O157:H7 immunomagnetic beads in the presence of 1 ml of PBS-0.02% Tween 20 for 2 h at room temperature. The resulting immunomagnetic bead-E. coli
O157:H7 complexes were then isolated by magnetic separation and applied to SMAC plates after suspension in 100 μl of PBS-0.02% Tween 20. Finally, any sorbitol-nonfermenting colonies appearing after 18 to 24 h at 35°C were tested by the API 20E biochemical test method (6
) (bioMérieux, Inc., Durham, N.C.) according to the manufacturer's instructions. The API 20E biochemical test method is a standardized identification system for Enterobacteriaceae
and other nonfastidious, gram-negative rods that utilizes 21 miniaturized biochemical tests. It can confirm the presence of E. coli
but not the exact serotype.
Descriptive statistics (means ± SD) were reported where appropriate. t tests were used to compare data presented in Figures 5 to 8. Linear regression analyses were conducted using individual data points rather than means (Prism, version 3.02; GraphPad Prism Software, Inc.). Standard deviations for signal to noise and percent control in the experiment testing different filters were calculated using the propagation of error equation Δz/z = [(Δx/x)2 + (Δy/y)2]1/2, where Δz, Δx, and Δy are the SDs of z, x, and y, respectively. Finally, the limits of detection for E. coli O157:H7 in various aqueous matrices were determined by interpolation from the linear regression equations fitted to the respective dose-response curves with the mean (plus 3 SDs) fluorescence signal of the blank sample.