Methods for eradicating biofilms from plumbing systems

Abstract

Disclosed are advantageous systems and methods for treating building water systems, especially the interior surfaces of premise plumbing, to remove biofilm and inactivate biofilm-associated pathogens, including protozoa, using disinfectant formulations at concentrations at in excess of those used for drinking water treatment, and further, in co-applying complexing agents to mitigate corrosion of the materials treated; and using these in conjunction with off-gas containment devices that allow flushing of taps without the liberation of toxic fumes.

Claims

1. A method for eradicating biofilm from a premise plumbing system, the method comprising contacting the biofilm on interior surfaces of the premise plumbing system with a treatment solution comprising a mixture of chlorine and chlorine dioxide jn a ratio by weight of from 80:20 to 20:80, each component of the mixture having a concentration of at least 1.5 mg/L and the mixture having a total concentration of up to 200 mg/L.

2. The method of claim 1, wherein the mixture of chlorine and chlorine dioxide is in a ratio by weight of 50:50.

3. The method of claim 1, wherein the concentration of each of chlorine and chlorine dioxide is at least 12.5 mg/L, at least 25 mg/L, or at least 50 mg/L.

4. The method of claim 1, wherein the treatment solution has a temperature of between 20 to 55° C.

5. The method of claim 1, wherein the treatment solution has a pH of about 6.5 to about 9.0.

6. The method of claim 1, wherein the treatment solution has a pH of between 7.2 and 9.0.

7. The method of claim 1, wherein the treatment solution has a pH of >7.5.

8. The method of claim 1, wherein the premise plumbing system has a free residual concentration of 0.8 mg/L or less chlorine dioxide and 0.4 mg/L or less chlorine.

9. The method of claim 1, wherein the interior surfaces of the premise plumbing system comprise a material selected from the list consisting of copper, brass, iron, galvanized steel, stainless steel, PVC, HDPE, and combinations thereof.

10. The method of claim 1, wherein the biofilm comprises a microorganism selected from the group consisting of Acinetobacter, Elizabethkingia (Flavobacterium), Escherichia coli, Klebsiella, Legionella, non-tubercular Mycobacteria (NTM), Pseudomonas, Stenotrophomonas, protozoa, and combinations thereof.

11. The method of claim 1, wherein the treatment solution further comprises a complexing agent.

12. The method of claim 11 wherein the complexing agent comprises sodium silicate.

13. The method of claim 1, wherein the contacting is carried out under turbulent flow conditions.

14. The method of claim 13, wherein the turbulent flow conditions have a Reynolds value of at least 4,000.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A is a schematic illustration of a conduit between the tap and the drain. The upper dashed line indicates the level of the tap and the lower dashed line indicates the level of the drain. The arrow indicates the direction of water flow, which is from the tap to the drain.

(2) FIG. 1B is a schematic illustration of a conduit between the tap and the drain. The conduit contains a check valve. The upper dashed line indicates the level of the tap and the lower dashed line indicates the level of the drain. The arrow indicates the direction of water flow, which is from the tap to the drain.

(3) FIG. 1C is a schematic illustration of a conduit between the tap and the drain. The conduit is attached to the inverted end of a funnel. The wide end of the funnel is at the level of the drain. The upper dashed line indicates the level of the tap and the lower dashed line indicates the level of the drain. The arrow indicates the direction of water flow, which is from the tap to the drain.

(4) FIG. 1D is a schematic illustration of a conduit between the tap and the drain. The conduit is fitted through a sponge and the sponge is at the level of the drain. The upper dashed line indicates the level of the tap and the lower dashed line indicates the level of the drain. The arrow indicates the direction of water flow, which is from the tap to the drain.

(5) FIG. 1E illustrates a conduit attached to the tap of a sink and leading into the drain of the sink.

DETAILED DESCRIPTION

(6) For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” The technical and scientific terms used in herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise.

(7) Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”, etc.

(8) It is to be understood that both the foregoing general summary and the following detailed description, including the drawings and examples, are exemplary and explanatory only and are not restrictive of the inventions of this disclosure.

(9) Mixtures of chlorine and chlorine dioxide in a treatment solution are surprisingly effective for eradicating biofilms on interior surfaces of plumbing systems via killing (or inactivating) of biofilm-associated pathogens and dislodging and/or removing the biofilms, even at high pH (e.g., pH>7.2) which results in minimal plumbing system corrosion. This discovery holds true for treatment solutions of chlorine and chlorine dioxide where the concentrations of these disinfectants are at the relatively-low levels permitted in drinking water (e.g., 0.8 mg/L chlorine dioxide; 4 mg/L chlorine). The method and treatment solutions are also effective to eradicate biofilms at much higher concentrations (e.g., 25 mg/L chlorine dioxide; 25 mg/L chlorine). Additionally, it is surprising that this discovery holds true at pH values much greater than 7.2, (e.g., pH 8.5), and even in domestic cold water (e.g., 0-20° C./32-68° F.).

(10) The co-application of a complexing agent, such as sodium silicate, with treatment solution can provide enhanced protection of the metallic components of premise plumbing materials from corrosion, especially when the treatment solution is applied at higher concentrations and higher temperatures.

(11) The penetration of biofilm on the surfaces of pipes is benefitted by increased flow and most markedly by turbulent flow at the biofilm:treatment solution interface. Without being limited by theory, we believe this is likely is due to enhanced contact between the treatment solution and the surface being treated by eliminating a boundary layer associated with laminar flow and/or stagnant contact. The degree of turbulence that can be achieved is a function of flow rate and pipe diameter; for example, a 2-inch diameter pipe requires a flow rate of approximately 2 feet per seconds to achieve turbulent flow (Reynolds number ˜4,000).

(12) The removal of biofilm on the treated surfaces is also benefitted by increased flow. Without being limited by theory, we believe this is because of increased shear forces that have a scouring effect on the biofilm.

(13) Based on these discoveries, certain embodiments of the invention provide novel means and methods for treatment of premise plumbing systems.

(14) For treatment of premise plumbing systems, it is desirable to remove surface-attached biofilm and to kill biofilm-associated pathogens, such as bacteria, viruses and protozoa, without causing significant physical damage to pipes and other premise plumbing system components and without environmental release of noxious chemical fumes. Removing surface-attached biofilm and killing biofilm-associated pathogens without damaging copper pipes and other system components can be met by flushing the premise plumbing system with a mixture of chlorine and chlorine dioxide in aqueous solution (treatment solution). The treatment solution can comprise a mixture of chlorine and chlorine dioxide at a ratio of 80:20 to 20:80 (weight basis) at a total concentration of up to 200 mg/L for up to 24 hours. The treatment described herein may be advantageously practiced at treatment solution pH values greater than neutral, especially pH>7.2, at typical cold water temperatures (e.g., 0-20° C.; 32-68° F.) up to temperatures at which scalding becomes a risk (43.3° C./110° F.). Co-treating with a complexing agent, such as sodium silicate, can further enhance compatibility of the treatment solution with plumbing system materials, especially metals such as copper and brass.

(15) Environmental release of noxious chemical fumes at the tap can be avoided by utilizing a gas-containment device, such as a hose that contains chlorine and chlorine dioxide vapors, which hose is attached to a tap and terminates near or is attached to a drain or chemical scrubber and provides containment of chemical fumes and directs the flow of treatment chemicals from the tap down a drain or into a chemical scrubber. The gas-containment device can be configured in many ways, and can incorporate advantageous features such as check valves, remotely actuated valves, and sensors for temperature, pH and disinfectant concentration, as well as data acquisition, storage and transmission means. The gas-containment device can be used in conjunction with flushing the treatment solution, or with other volatile treatment chemicals, such as hypochlorite (bleach) or chlorine dioxide, that may be used to flush plumbing systems.

(16) In certain plumbing systems with surfaces coated by a mixture of lime scale, iron sediment and biofilm, such as those that receive water with high mineral content (hard water), treatment can be carried out by first applying a low pH treatment to dissolve the limescale, and iron, preferably in conjunction with a complexing agent, such as sodium silicate. The first step is followed by utilizing the treatment solution described herein at higher pH (e.g., >7.2) to remove the biofilm. This sequence can be repeated, if necessary, until the limescale, iron sediment and biofilm have been removed as determined by sampling or visual inspection.

EXAMPLES

(17) Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.

Example 1: Eradication of Pseudomonas Biofilms Using Solutions of Chlorine, Chlorine Dioxide, and a Mixture of Both

(18) This example illustrates the use of a mixture of chlorine and chlorine dioxide for removing biofilms of Pseudomonas aeruginosa. These results show the efficacy of this treatment solution even at pH 6.5, and the surprising advantage of such mixtures at pH 7.5 relative to treatment solutions comprising either chlorine or chlorine dioxide alone for the treatment of biofilms.

(19) Overview of Testing Method

(20) Pseudomonas aeruginosa biofilms are challenged with the reagents chlorine dioxide (“CD”), Chlorine (sodium hypochlorite; “Bleach”) or a mix of both reagents in equivalent volume proportions (“Mix”), at approximately pH 6.5 and pH 7.5 to assess their ability for biofilm inactivation, as measured by a Minimal Biofilm Eradication Concentration (MBEC) assay. This MBEC assay measures the ability of a treatment solution to eliminate bacteria from already grown biofilms by killing the microbial cells and/or dislodging the biofilm.

(21) The MBEC assay protocol used is an adaptation of the ASTM-standardized MBEC method. Briefly, biofilms are grown in a Calgary biofilm device, which is a 96 well plate with a lid that when in place has 96 individual pegs that protrude into the wells of the plate (see e.g., Ceri et al., 1999). The bacterial cells in the wells form biofilms on the submerged pegs. After a 20 h growth period, these biofilms formed on the pegs are challenged by placing the pegged lid on a 96-well plate of serial dilutions of the disinfectants to be tested (e.g., the CD, Bleach, or Mix), along with the required controls. All challenges were performed in a Biosafety Level 2 cabinet with a challenge exposure time of 30 minutes at a temperature of 22-25° C. In the case of CD, products are prepared fresh from highly concentrated stock solution minutes before the challenge. Reagent pH and concentration stability are tested for the CD working stock solutions, using either water or a borate-boric acid buffer as solvents. The concentration ranges for disinfectant reagents are from approximately 3 ppm to approximately 50 ppm.

(22) After the challenge, the pegged lid is transferred to a recovery plate containing a sodium bisulfite solution. Sonication of the recovery plate with the pegged lid induces detachment of the biofilm from the pegs into the wells of the recovery. Viable bacterial cell counts from each peg are determined by culturing the recovery plate. The extended MBEC assay protocol and methods of preparing the disinfectant reagent stock solutions used in the challenge are included in the Materials and Methods section provided below.

(23) Results

(24) Results for CD, Bleach and the Mix challenges can be seen in Tables 1, 2 and 3, respectively. Bleach challenge shows a pH-dependent effect for biofilm reduction, consistent with the literature, except for anomalous results at −3 ppm. When Mix (Bleach and CD) was used (Table 3), at both pH 6.5 and pH 7.5 efficacy against biofilms was superior to the efficacy of either of these disinfectant reagents alone. This is most clearly demonstrated by comparing the dose of the Mix of 6.25 ppm of each component (12.5 ppm total) vs. 12.5 ppm of either component individually.

(25) TABLE-US-00001 TABLE 1 Results of challenge of P. aeruginosa biofilms with CD. pH 6.5 pH 7.5 Concentration Log.sub.10 reduction Concentration Log.sub.10 reduction (ppm) (CFU/mm.sup.2) (ppm) (CFU/mm.sup.2) 50.0 Total inhibition 50.0 total inhibition 12.5 2.34 12.5 2.49 6.25 2.56 6.25 2.38 3.12 0.82 3.12 1.63 Log.sub.10 reduction = logarithm of the number of Colony Forming Units (CFU) per mm.sup.2 after culturing the ultrasound-recovered biofilm.

(26) TABLE-US-00002 TABLE 2 Results of challenge of P. aeruginosa biofilms with Bleach. pH 6.5 pH 7.5 Concentration Log.sub.10 reduction Concentration Log.sub.10 reduction (ppm) (CFU/mm.sup.2) (ppm) (CFU/mm.sup.2) 50.5 Total inhibition 50.5 Total inhibition 12.6 Total inhibition 12.6 3.03 6.31 Total inhibition 6.31 1.91 3.15 0.16 3.15 0.96 Log.sub.10 reduction = logarithm of the number of Colony Forming Units (CFU) per mm.sup.2 after culturing the ultrasound-recovered biofilm.

(27) TABLE-US-00003 TABLE 3 Results of challenge of P. aeruginosa biofilms with a Mix (CD and Bleach). pH 6.5 pH 7.5 Concentration Log.sub.10 reduction Concentration Log.sub.10 reduction (ppm) (CFU/mm.sup.2) (ppm) (CFU/mm.sup.2) 50.0 Total inhibition 50.0 Total inhibition 12.5 Total inhibition 12.5 Total inhibition 6.25 3.08 6.25 Total inhibition 3.13 2.18 3.13 1.99 Log.sub.10 reduction = logarithm of the number of Colony Forming Units (CFU) per mm.sup.2 after culturing the ultrasound-recovered biofilm.

(28) Detailed Materials & Methods

(29) Preparation of concentrated CD solution: If available, pour approximately 50 mL of a CD solution in two 1 L glass containers that can be airtight sealed. Otherwise, use commercial bleach to treat this glassware. Make the liquid moisten all of the inner surface of the flasks, and let them stand in the dark at room temperature overnight. Add 2 L of sterile, distilled water to a pouch containing the CD-producing reagents. Cap the pouch, and mix by inverting it several times. Let stand for 2 hours at room temperature. Rinse with sterile, distilled water the glassware from point 2, and cover them with tin foil. Transfer the Concentrated CD solution to the glass bottles, so that the air space on top of the solution is as minimum as possible. Store the solution at 4° C. until needed. Prepare 30 mL of 1:500 to 1:1000 dilutions. Transfer 10 mL aliquots to 20 mL bottles of the quantitation system (CD colorimeter). Use aluminum foil to cover the bottles. Add 3 drops of glycine to each bottle, cap it, and mix. Add the content of one bag of DPD to each bottle with a CD solution. Mix by gently inverting the tubes. Use a bottle with water to blank the colorimeter (make sure you are using the CD, and not the chlorine (Bleach) colorimeter), and then measure the concentration. If reading is >2.5, prepare a higher secondary dilution (1:1500) and repeat the process. Average the readings and multiply by the dilution factor to determine the power (in ppm) of the concentrated solution. Label the bottles from with date and concentration.

(30) Preparation of working CD stock solution: Fill a 50 mL polypropylene tube with the concentrated solution, and cover it with aluminum foil. Let the solution reach room temperature while protected from light. In a fresh 50 mL polypropylene tube, mix the concentrated solution with sterile, distilled water, so that it is diluted to the working concentration (e.g., 100 ppm), in a final volume of 50 mL. Close tightly the tube, and mix gently by inversion. Add 0.1M NaOH in 10 μL increments, mix and measure pH. Repeat this step until the desired pH is reached. Also, estimate pH using strips of pH indicator paper. Prepare 1:200 dilutions (30 mL), and verify the working concentration using the colorimeter (as described in making concentrated CD solutions). Use concentration estimation strips for double checking. Submerge the quantitation end of the strip in the solution for 2 seconds. Holding the colorimetric coupon upright, wait for 10 seconds, and then compare its color with the reference pallet. Label the tube, and store it at 4° C.

(31) Preparation of 1× borate-boric acid buffer working CD stock solutions: In a fresh 50 mL polypropylene tube, place concentrated CD solution so that the final concentration after dilution to a volume of 50 mL will be the working concentration (e.g. 100 ppm). Add 1× borate buffer to reach 40-45 mL. Close tightly the tube, and mix gently by inversion. Measure pH, and add 0.5M boric acid in 1 mL increments (or less if needed) until the desired pH is reached. Complete to 50 mL volume with sterile, distilled water. Determine final concentration as described above for CD stock solution.

(32) Stability testing of working CD stock solutions: The method for pH and concentration stability testing consisted of diluting CD from a concentrated stock solution (approximately 590 ppm) to the working stock solution concentration (100 ppm) using water and adding 0.1M NaOH in small amounts until the desired pH was reached. Alternatively, the reagent was diluted to its working concentration in a combination of 1× borate buffer (measured pH 8.8) and 0.5M boric acid (measured pH 4.14). The desired pH levels were reached by adjusting the proportion of boric acid in the solution.

(33) Initial concentration estimation of starting bleach solution: Bleach can come at concentration spanning 1 to 8% (i.e., 10000 to 80000 ppm). The following initial concentration estimation method is used to determine this starting concentration. In a new polypropylene tube, prepare 15 mL of a 1:500 dilution in Ultra-Pure Water (UPW). Place 10 mL of UPW in the 20 mL bottles of the quantitation system (Cl.sub.2 colorimeter). Put aside one of the bottles to use it as blank. Replace either 100 or 50 μL of UPW with the 1:500 dilution from step (1) (i.e., make 1:100 or 1:200 further dilutions, this will make final dilutions of 1:50000 and 1:100000 respectively). Add the content of one bag of DPD reagent to each bottle with a CD solution. Mix by gently inverting the tubes. Blank the colorimeter (make sure you are using the chlorine, and not the CD colorimeter), and then measure the concentration. If reading is >2.5, prepare a higher secondary dilution (for example 1:1500) and repeat the process. Average the readings and multiply by the dilution factor to determine the power (in ppm) of the concentrated solution. Label the original bottle with date and estimated concentration.

(34) Preparation of bleach working stock solutions: Fill a 50 mL polypropylene tube with the concentrated Bleach solution. Let the solution reach room temperature. In a fresh 50 mL polypropylene tube, mix the concentrated solution with sterile, distilled water, so that it is diluted to the working concentration (e.g. 100 ppm), in a final volume of 50 mL. Close tightly the tube, and mix gently by inversion. Add 1N HCl in 10 μL increments, mix as in (9.2) and measure pH. Repeat this step until the desired pH is reached. Also, estimate pH using strips of pH indicator paper. Prepare 1:200 or 1:100 dilutions, and verify the working concentration as before (steps 2-6). Label the tube, and store it at 4° C. until use.

(35) MBEC Assay Protocol

(36) The following equipment, reagents, and methods are used to carry out the MBEC assay protocol for testing disinfectant efficacy of chlorine dioxide (CD), chlorine (Bleach) and a mixture of chlorine dioxide and bleach (Mix) against Pseudomonas aeruginosa biofilm.

(37) A. Assay method: All steps should be performed using aseptic techniques and in an aseptic environment,

(38) 1. Bacterial culture (2 days before the MBEC assay). 1.1. Thaw an aliquot of a working stock of Pseudomonas aeruginosa (ATCC 27835) and use it to streak plate on tryptic soy agar (“TSA”) prepared according to manufacturer's directions. 1.2. Incubate at 35° C. for 16-18 h. 1.3. Pick an isolated colony and with it inoculate 200 mL of sterile tryptic soy broth (“TSB”) prepared according to manufacturer's directions. 1.4. Incubate at 35° C. 150 rpm for 16 to 18 h. Viable bacterial density should be 10.sup.8 CFU/mL or higher and may be checked by serial dilution and plating. 1.5. Prepare a 25 mL 1:1000 dilution in TSB, to adjust cell density to approximately 10.sup.5 CFU/mL. Vortex the diluted sample for approximately 10 s. 1.6. Perform seven 10-fold serial dilutions from step (1.5) in triplicate. 1.7. Spot plate 20 μL of the serial dilutions from 10° to 10.sup.−7 on a series of TSA plates. Label plates and incubate them at 35° C. for 16-18 h.

(39) 2. Growth of biofilm 2.1. Open a package containing a new MBEC device. 2.2. Transfer 25 mL of the inoculum prepared in (1.5) into a sterile reagent reservoir. 2.3. Add 150 μL to each well of the 96-well plate packaged with the MBEC device, excluding columns 9 to 11 and A12, B12 and C12. 2.4. Place the peg lid onto the microplate, making sure that the orientation of the wells matches that of the peg lid (i.e., peg A1 must be inserted into A1 well). 2.5. Using the orbital shaker and humidified incubator, keep the device at 33-37° C. 2.6. For best biofilm quantitation, the replicate MBEC devices should be prepared according to section 7.

(40) 3. Biofilm growth check 3.1. Using sterile (flamed) pliers, grab peg D12 close to the lid to avoid contact with the biofilm. Break off the peg and place it in a sterile microfuge tube containing 1.0 mL of buffered water (“buffered water”=0.0425 g KH.sub.2PO.sub.4/L distilled water, filter-sterilized and 0.405 g MgCl.6H.sub.2O/L distilled water; filter-sterilized as according to ASTM Method 9050 C.1.a). 3.2. Repeat step (3.1) with wells E12 to H12 into respective microfuge tubes. 3.3. Place the stainless steel insert tray into the sonicator. Place the tubes from 3.1 and 3.2 into the tray and sonicate on high for 25-35 min. 3.4. Make 1.0 mL 10-fold serial dilutions in buffered water and spot plate on TSA. Incubate at 35° C. for 16-18 h.

(41) 4. Preparation of challenge plate 4.1. Use a sterile 96-well, two corners plate, to prepare the challenge plate according the challenge plate set-up map shown below.

(42) TABLE-US-00004 1 2 3 4 5 6 7 8 9 10 11 12 A 100 100 100 100 100 50:N N UC SC B 50 50 50 50 50 50:N N UC SC C 25 25 25 25 25 50:N N UC SC D 12.5 12.5 12.5 12.5 12.5 50:N N UC BGC E 6.25 6.25 6.25 6.25 6.25 50:N N UC BGC F 3.13 3.13 3.13 3.13 3.13 50:N N UC BGC G 1.56 1.56 1.56 1.56 1.56 50:N N UC BGC H 0.78 0.78 0.78 0.78 0.78 50:N N UC BGC 4.2. Prepare 20 mL of the desired disinfectant stock solution. 4.3. Add 200 μL of sterile TSB to well A12 of the challenge plate. This will be the sterility control (SC). 4.4. Add 200 μL of sterile neutralizer to column 7 and well B12. These will be the neutralizer toxicity control (N) and sterility control. 4.5. Add 100 μL of sterile neutralizer to column 6, followed by 100 μL of disinfectant. This will be the neutralizer effectiveness control. 4.6. Add 200 μL of buffered water to column 8 and well C12. These will be untreated control (UC) and buffered water sterility control. 4.7. Add 100 μL of buffered water to columns 1 through 5 (rows B through H). 4.8. Add 200 μL of stock disinfectant to columns 1 through 5 (row A). 4.9. Add 100 μL of the disinfectant stock solution to columns 1 through 5 (rows B and C). 4.10. Use a multichannel micropipette to mix the contents of columns 1 through 5 (row C) by pipetting up and down. Keep the tips in the micropipette for the next step. 4.11. Transfer 100 μL from the wells in row C to the corresponding wells in row D. discard the tips. 4.12. Using fresh tips, mix by pipetting the contents in row D, columns 1 through 5. 4.13. Transfer 100 μL from row D to row E. Discard the tips after each transfer and mix with fresh ones. 4.14. Repeat the process down the length of the plate until row H. 4.15. Discard 100 μL from row H, columns 1 to 5. 4.16. Add 100 μL of buffered water to rows C through H of columns 1 through 5.

(43) 5. Disinfectant challenge of biofilm 5.1. Prepare a rinse plate by adding 200 μL, of buffered water to each well of a new 96-well, 2-corners plate. 5.2. Prepare recovery plate by adding 200 μL, of neutralizer to each well of a new 96-well, 2-corners plate. 5.3. Rinse the planktonic bacteria from the biofilm that formed on the lid of the MBEC device by setting the lid into the rinse plate for 10 s. 5.4. Transfer the MBEC lid to the challenge plate and incubate at room temperature during the contact time recommended by the manufacturer. 5.5. After the contact time, transfer the MBEC lid to the recovery plate containing the neutralizer.

(44) 6. Biofilm growth quantitation (from replicate biofilm plate) 6.1. Prepare a staining plate by adding 200 μL, of 0.1% crystal violet solution into columns 1 through 8 and column 12 of a fresh 96-well, 2 corners plate. 6.2. Transfer the pegged lid of the replicate recovery plate to the staining plate. 6.3. Incubate at room temperature for 30 minutes. 6.4. Prepare two rinse plates by adding 200 μL, of ultrapure water into columns 1 through 8 and column 12 of two 96-well, 2 corners plates. 6.5. Transfer the lid to the first rinse plate and let it settle for 10 s, to remove excess of stain and planktonic bacteria. Transfer to the second rinse plate and repeat. 6.6. Let the pegged lid air-dry, upside down, for 30 minutes. 6.7. Add 150 μL of 95% ethanol to a new 96-wells, 2 corners plate, in columns 1 through 8, and column 12. 6.8. Once dry, place the pegged lid into the ethanol plate and incubate for 10 minutes. 6.9. Remove the pegged lid and discard it, along with the staining and rinse plates used in this section. 6.10. Transfer 100 μL of each well from the plate in (6.8) into the corresponding well of a fresh 96-wells (ONE CORNER) plate. 6.11. Use a plate reader to determine absorbance at 600 nm.

(45) 7. Quantitative determination of the MBEC 7.1. Place the recovery plate with the pegged lid (from step 6.5) in the stainless steel tray, and the tray, in the sonicator. Sonicate on high for 25 to 35 min to remove and disaggregate the biofilm. 7.2. Eight sterile 96-well, ONE CORNER plates are used for this step (columns 1 through 8 only). 7.2.1. Add 180 μL of buffered water to rows B through H in all 8 plates. 7.2.2. Following sonication and using multichannel micropipette, transfer 100 μL from each well of row A of the recovery plate to row A of a sterile plate prepared in 7.2.1. 7.2.3. Transfer 100 μL from each well of row B of the recovery plate to row A of a second sterile plate prepared in 7.2.1. 7.2.4. Repeat for rows C through H of the recovery plate. 7.2.5. Serially dilute with a multichannel pipette (10° to 10.sup.−7) by transferring 20 μL down each of the 8 rows for each plate. 7.3. Spot plate the dilution series from each of the eight microtiter plates on TSA for viable cell counts. Use one square TSA plate per microtiter plate. Using a multichannel pipette, remove 5 μL from each well and dispense on TSA plate. 7.4. Incubate the TSA plates at 33-37° C. during 18 to 20 h and enumerate colonies. 7.5. Discard the pegged MBEC lid and 96 plates used to create the serial dilutions.

(46) 8. Qualitative determination of the MBEC 8.1. Add 100 μL of sterile TSB to each well of the recovery plate. 8.2. Cover recovery plate with a new sterile, non-pegged lid and place in a humidified incubator at 33-37° C. for 24 h.

(47) 9. Data Analysis 9.1. Quantitative MBEC results using Log.sub.10 reduction: 9.1.1. Count the 5 μL spots on each of the 8 spot plates where individual colonies are visibly distinct from each other within the plated spot. Record the column (1-8) and dilution row (10.sup.0 to 10.sup.7) in which each spot is located. 9.1.2. Calculate the log.sub.10 density for each peg as follows:
Log.sub.10(CFU/mm.sup.2)=Log.sub.10[(X/B)(V/A)(D)] where: X=CFU counted in the spot, B=volume plated (0.01 mL), V=well volume (0.20 mL), A=peg surface area (46.63 mm2), and D=dilution 9.1.3. Average the counts from columns 1 through 5 spot plated for Row A to determine the mean log.sub.10 density for the undiluted disinfectant. 9.1.4. Average the counts from columns 1 through 5 spot plated for Row B to determine the mean log.sub.10 density for the 50% disinfectant. Repeat calculation for the remaining rows (C-H). 9.1.5. Average the counts from column 6, Rows A through H to determine the mean log.sub.10 density for the neutralizer effectiveness control according to the procedure described in TSA Test Method E1054. 9.1.6. Average the counts from column 7, Rows A through H to determine the mean log.sub.10 density for the neutralizer toxicity control. 9.1.7. Average the counts from column 8, Rows A through H determine the mean log.sub.10 density for the untreated control. 9.1.8. Calculate the log.sub.10 reduction for each disinfectant concentration as follows:
Reduction=Mean Log.sub.10 Untreated Control Pegs−Mean Log.sub.10 Treated Pegs 9.2. Qualitative MBEC Results are determined following the 24 h incubation of the recovery plates by visual scoring (+/−growth). To determine the minimum biofilm eradication concentration (MBEC) values, check for turbidity (visually) in the wells of the recovery plate. Alternatively, use a microtiter plate reader to obtain optical density measurements at 650 nm (OD.sub.650). Clear wells (OD.sub.650=0.1) are evidence of biofilm eradication. The MBEC is defined as the minimum concentration of disinfectant that eradicates the biofilm. This is the lowest concentration in which there is no growth observed in the majority of the five wells.

Example 2: Biofilm Eradication in a Recirculating Domestic Hot Water System Using a Treatment Solution Mixture of Chlorine and Chlorine Dioxide

(48) This example illustrates method for applying a mixture of chlorine and chlorine dioxide to a recirculating domestic hot water system for treatment of biofilms. The physical and chemical parameters in the example, such as the chemical composition of the treatment solution, temperature of the treatment solution, pH of the treatment solution, flow rates, treatment time, and sequence, are for illustration purposes and are not intended to limit the scope of the invention.

(49) A dosing tap is installed at the output side of the building's centralized water heater. A chemical feed pump compatible with the treatment solution is connected to the dosing tap. A sample tap is installed at the hot water return. Fixtures (taps, showerheads) throughout the building are prepared by removing aerators and point-of-use filters. Off-gas prevention devices—e.g., flexible hoses that serve as a conduit from the point where the treatment solution exits the fixture to the drain—are attached to each outlet. Unheated domestic water is circulated through the hot water distribution system at 2-8 feet per second (fps).

(50) Chlorine and chlorine dioxide are applied to the circulating water at the dosing tap such that the resultant composition is a treatment solution with a concentration of 50 mg/L (˜25 mg/L each of chlorine and chlorine dioxide) at pH 7.5. Sodium silicate, a complexing agent is applied to the circulating water to achieve a concentration of 25 mg/L. The treatment solution is circulated through the domestic hot water system for one hour. The concentration of the treatment solution is measured at the hot water return every 5 minutes; if the concentration is >5% less than the 50 mg/L set point, additional chemicals are applied at the dosing tap until the target concentration of the treatment solution, measured at the hot water return, is achieved.

(51) Progressing through the facility, starting at the dosing point, taps are opened to full flow until the treatment solution concentration reaches the 50 mg/L set point; the flow is then reduced to 0.25 gallons per minute (gpm), and the water is allowed to flow for an additional 5 minutes, then turned off. Owing to the design of the off-gas containment device, the treatment solution remains in contact with all wettable surfaces of the tap.

(52) After all the taps have been flushed with the treatment solution and closed, the treatment solution is circulated through the system for additional 1 hour. The chemical feed pump is turned off, and the hot water system is flushed with clear potable water for 30 minutes.

(53) Starting at the dosing point and progressing through the facility, all taps are opened to full flow and flushed with clean, unheated domestic water until the concentrations of chemicals in the water are below the EPA Maximum Contaminant Level (MCL) and Maximum Disinfectant Residual Level (MRDL) limits, which are the levels to which disinfectants or disinfection by-products are regulated. Clean water is then allowed to flow through the tap for an additional 5 minutes. The concentration of the treatment solution is re-measured and documented to be below the MRDL/MCL for each regulated disinfectant/disinfection by-product. The tap is turned off and the gas-containment device is removed.

(54) After the acute treatment, the domestic hot water system may be treated to provide ongoing microbial control.

(55) All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. Full citations for these references may be found at the end of the specification immediately preceding the claims.

(56) While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

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