Patent Application
20190192714
The present disclosure relates to a device including a bacteria cell for monitoring the efficacy of a medical device reprocessing system and a method for using such monitoring device including a bacteria cell.
Healthcare-associated infections are commonly linked to contaminated medical devices, and more healthcare-associated infections are linked to contaminated endoscopes than to any other medical device. A majority of these complex, reusable surgical tools are incompatible with existing sterilization technologies. Minimally, hospitals are required to use high-level disinfection processes to reprocess endoscopes between patients.
One way to monitor medical device reprocessing processes is to use minimum effective concentration strips to test the concentration of a disinfectant prior to running the decontamination cycle. Though it may be useful to know the concentration of disinfectant, these strips provide no information about the effectiveness of bacterial kill during the decontamination cycle.
In one aspect, provided is a device for monitoring the effectiveness of a decontamination process, the device comprising a bacteria cell. In some embodiments, the device may further comprise a substrate.
In another aspect, provided is a method of detecting the presence of viable microorganisms after a high-level disinfection cycle, the method comprising exposing a bacteria cell to a sterilant in a high-level disinfection cycle, contacting the bacteria cell with a growth medium to provide a culture, and correlating a change in the appearance of the growth medium with presence of viable microorganisms.
Features and advantages of the present disclosure will be further understood upon consideration of the detailed description as well as the appended claims.
Repeated use of reference characters in the specification and drawings is intended to represent the same or analogous features or elements of the disclosure. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art, which fall within the scope and spirit of the principles of the disclosure. The figures may not be drawn to scale.
Devices useful for monitoring the effectiveness of decontamination processes, e.g., sterilization, high-level disinfection, commonly use a highly resistant organism, e.g., a bacterial spore, as the indicator test organism. However, in some decontamination processes, such as, for example, high-level disinfection cycles that employ relatively short exposure times (e.g., five minutes) and low temperatures (e.g., 25-30° C.), bacterial spores may be too resistant to function as an effective indicator organism. Provided herein is a device for monitoring the effectiveness of a decontamination process, the device including a bacteria cell as the indicator test organism.
The terms “decontamination” and “decontamination process” as used herein means a process through which instruments and/or supplies may be cleaned, and refer to both sterilization and high-level disinfection processes.
The terms “sterilant” and “sterilant liquid” as used herein refer to a solution, e.g. peracetic acid (“PAA”) solution, ortho-phthalaldehyde (“OPA”) solution, glutaraldehyde solution, that may be used in a decontamination process such as, for example, a sterilization process and/or a high-level disinfection process.
In some embodiments, the bacteria cell of the disclosed monitoring device may be a Mycobacteria cell. Particularly for high-level disinfection, Mycobacteria are considered highly resistant organisms, due in part to their waxy lipid coat that provide resistance to sterilants employed for decontamination kill during a high-level disinfection process. In some embodiments, the Mycobacteria cell may be a Mycobacteria terrae cell, such as those commercially available as “ATCC 15755” from American Type Culture Collection, Manassas, Md. Mycobacteria terrae may be a desirable indicator organism from a safety perspective, as it is generally viewed as a nonpathogenic species of Mycobacteria, and would thus not introduce harmful bacteria into a decontamination system.
Mycobacteria terrae cells useful in embodiments of the present disclosure may be cultivated by methods known in the art. It was surprisingly discovered that the resistance performance of Mycobacteria terrae cells, i.e., resistance to being killed by a sterilant, increased with increased growth time before harvesting. In some embodiments, Mycobacteria terrae cells useful in embodiments of the present disclosure may be cultivated for example, on an agar medium or in a liquid medium, for at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, or at least 35 days before harvesting.
In some embodiments, a device of the present disclosure may further include a substrate. The substrate may comprise a material having a particular characteristic, for example, the substrate may comprise a porous material, a non-porous material, a hydrophilic material, a hydrophobic material, and combinations thereof. In some embodiments, the substrate may be selected from the group consisting of a paper, a polymeric material, and combinations thereof. In some embodiments, the paper may include, for example, a cellulosic paper, a silica paper, and combinations thereof. In some embodiments, the polymeric material may include, for example, a polyethylene, a polypropylene, polyurethane, a cellulose, a nylon, a rayon, and combinations thereof.
In some embodiments, the substrate may include a film, a membrane, a woven web (e.g., a cloth), a non-woven web, a metal, a laminate, a glass, and combinations thereof. In some embodiments, the membrane may include a nylon membrane, a cellulose membrane, a polytetrafluoroethylene, membrane, a polyethersulfone membrane, a cellulose acetate membrane, and combinations thereof. In some embodiments, the non-woven web comprises a glass fiber, a polypropylene fiber, a polyester fiber, a rayon fiber, a nylon fiber, a cellulose fiber (e.g., cotton, linen, wood), and combinations thereof. In some embodiments, the nonwoven fabric includes meltblown fibers (e.g., meltblown fibers of a hydrophobic thermoplastic olefin). In some embodiments, the laminate comprises polyethylene terephthalate and paper.
A substrate useful in embodiments of the present disclosure can have any desirable geometry, such as, for example, rectangular, circular, oval, elliptical, trapezoidal, triangular, star, crescent, and combinations thereof. In some embodiments, the substrate may have a surface area of 0.01 cm2-100 cm2, e.g., 0.1 cm2-10 cm2, 0.3 cm2-5 cm2. Desirably, the substrate surface area is sufficient to accommodate a bacteria population of 1×106 CFU /substrate to 1×1010 CFU/substrate, e.g., 1×108 CFU /substrate, where “CFU” refers to a “colony-forming unit.” In some embodiments, the substrate coated with bacteria may be dried, for example, by allowing any solvents in which the bacteria were suspended to evaporate at ambient conditions (about 23° C.) and/or by heating the bacteria-coated substrate in an oven at 40° C. for 30-60 minutes.
A monitoring device of the present disclosure may be readily incorporated into decontamination monitoring systems known in the art such as those described, for example, in PCT/US2017/056250 (Bommarito et al.) and U.S. Patent App. No. 62/592,547 (Bennaars-Eiden et al.), the contents of which are hereby incorporated by reference in their entireties.
In another aspect, provided is a method of detecting the presence of viable microorganisms after the microorganisms have been exposed to a decontamination process. The method includes exposing a bacteria cell to a sterilant in a decontamination cycle, e.g., a high-level disinfection cycle. Sterilants that may be used in embodiments of the present disclosure include, for example, ortho-phthalaldehyde, glutaraldehyde, hydrogen peroxide, peracetic acid, and combinations thereof. In some embodiments, the bacteria cell may be a Mycobacteria cell. In some embodiments, the bacteria cell may be a Mycobacteria terrae cell. In some embodiments, the bacteria cell may be coated on a substrate, as described above, prior to exposure to the decontamination process.
After completion of the decontamination process, the exposed bacteria cell may be contacted with a growth medium, such as, for example, a liquid, solid, or gel growth medium, to provide a culture, such that a viable cell will be capable of growth. The growth culture including the exposed bacteria may be heated, for example, to 20° C. to 56° C. for up to about 21 days, to facilitate growth of any viable bacteria cells. In the event that a cell is capable of growth after exposure to a decontamination cycle, such growth may be evidenced by, for example, a change in appearance of the growth medium, such as, for example, a change in turbidity, opacity, color, luminescence (e.g., chemiluminescence, fluorescence, bioluminescence), and combinations thereof, i.e., a change in the appearance of the growth medium may be correlated with presence of viable microorganisms and potentially the need for additional decontamination activities. Alternatively, a lack of change in the appearance of the growth medium may be correlated with the absence of viable microorganisms following the decontamination process, providing evidence of a successful decontamination process.
Unless otherwise noted, all parts, percentages, ratios, etc. in the Examples and the rest of the specification are by weight.
Mycobacterium terrae ATCC 15755
Dissolve 19 g Middlebrook 7H10 powder in 900 ml deionized water and autoclave 20 minutes at 121° C. Cool for 30 min with stirring and add 100 ml Middlebrook OADC Enrichment solution aseptically to cooled media. Add 10 ml of 50% (w/v) glycerol aseptically to cooled media. The cooled media was added to sterile plates and allowed to solidify.
For 7H10 powder, the approximate amounts of the components per 900 ml are: 0.5 g ammonium sulfate, 1.5 g monopotassium phosphate, 1.5 g disodium phosphate, 0.4 g sodium citrate, 25.0 mg magnesium sulfate, 0.5 g calcium chloride, 1.0 mg zinc sulfate, 1.0 mg copper sulfate, 0.5 g L-glutamic acid (sodium salt), 0.04 μg malachite green, and 15 g Agar.
Tween 80, 20% (v/v)
Add 20 ml Tween 80 (MP Biomedicals, LLC, catalogue #103170) to 80 ml deionized water. If needed, heat Tween solution to 56° C. to speed solubilization of Tween 80. Sterilize by filtration through 0.22-μm membrane. Store up to 2 months at room temperature. The final concentration of Tween 80 used in the media in this unit is 0.05% (v/v).
1×PBST, Tween 80, 0.01% (w/v)
Weigh out 0.1g Tween 80. Add 100 ml of 10×PBS. Add 900 ml deionized water. If needed, heat to 56° C. to speed solubilization of Tween 80. Sterilize by filtration through 0.22-μm membrane. Store up to 2 month at room temperature. The final concentration of Tween 80 used in the media in this unit is 0.05% (v/v).
OPA Solution Preparation:
RAPICIDE OPA: Dilute titrated 0.55 wt % RAPICIDE OPA to a concentration of 0.35 wt % with deionized water. To make 1 L of a 0.35 wt % RAPICIDE OPA solution, weigh out 636.36 g of 0.55 wt % OPA and weigh out 363.64 g of deionized water. Mix thoroughly before use. Place solution in 25° C. water bath to equilibrate for at least 10 minutes before use.
Glycine Neutralizing Solution Preparation:
Mix Horse serum with 0.07% Lecithin, 1% glycine and 0.5% Tween 80. Filter sterilize the solution with 0.2 micron filter unit. Place solution in 25° C. water bath to equilibrate for at least 10 minutes before use.
PAA Solution Preparation:
Into a 50 ml polypropylene tube, pipet 0.85 ml of RAPICIDE Part A, 0.85 ml of RAPICIDE part B, and mix with 48.3 ml of distilled water. Vortex to mix thoroughly. Place tube in 30° C. water bath to equilibrate for at least 10 minutes before use.
Thiosulfate Neutralizing Solution Preparation:
Neutralizer (1% sodium thiosulfate with 0.05% Tween 80): Weigh out 0.5 g of sodium thiosulfate and dissolve in 50 ml of 0.05% Tween 80. Vortex to mix thoroughly. Place solution in 30° C. water bath to equilibrate for at least 10 minutes before use.
Coating of Carriers with Mycobacteria terrae
M terrae cells were inoculated and incubated for 14-days and 21-days at 37° C. on Middlebrook 7H9 agar plates containing 10% oleic acid albumin dextrose catalase enrichment (“OADC”) and 0.5% of glycerol prepared as described above. The bacterial cells were harvested by scraping the agar surface. The harvested cells were resuspended in either 0.05% Tween 80 or 2.4% Trehalose/1×PBS. The cell population was determined by serial dilution and plating. The cells were adjusted to a concentration of 1×108 cfu/mL prior to coating. For comparison purposes, all the carriers were cut to the same diameter size of 0.25 inch using a Mayhew Pro hollow punch (Mayhew Steel Products, Inc., Turners Falls, Mass.). Ten microliters of cell suspension was pipetted and deposited on individual carrier discs of different materials (see Table 4) with a target population of 1×108 CFU/carrier. The coated carriers were dried at room temperature for 16-24 hours in a Forma 1400 series ventilated biosafety cabinet (Thermo Fisher Scientific Inc., Coon Rapids, Minn.).
Ten carrier discs for each type of material were submerged in a 50 ml Falcon tube containing 10 mL of 1×PBS buffer, pH 7.4 and vortexed for 10 seconds at a maximum speed. 100 μl of supernatant was pipetted and plated on Middlebrook 7H9 agar plates for population recovery in order to assess adhesion of the cells to the carrier material.
Assessment of M terrae Cells Resistance to High-Level Disinfection (“HLD”)
Cell Suspension Testing
OPA Time course: Exposure to OPA for various contact time points at 25° C. at the minimum effective concentration of RAPICIDE OPA (0.35 wt %). 10 μl of M terrae cell samples at a target concentration of at least 1×108 cfu/mL were added to a 1.5 mL Eppendorf tube containing 400 μL of 0.35 wt % OPA for the appropriate contact time (30 seconds to 3 minutes) in a 25° C. water bath. At the end of each contact time, samples were neutralized by adding with 600 μL of glycine neutralizing solution (described above) for 15 minutes at 25° C. Samples were centrifuged for 10 minutes at 14000 rpm at 4° C. The supernatant was decanted off and the bacterial cells were resuspended in 1 mL of PBST buffer. Serial dilutions 1:10 were made of each sample and 1 mL of each dilution was plated on Middlebrook 7H10 agar with OADC enrichment. Plates were incubated at 37° C. for 14- 21 days.
PAA Time course: Exposure to PAA for various contact time points at 30° C. at the minimum effective concentration of RAPICIDE PAA (850 ppm). 10 μl of M. terrae cell samples at a target concentration of at least 1×108 cfu/mL were added to a 1.5 mL Eppendorf tube containing 400 μL of 850 ppm PAA for the appropriate contact time ranging from 30 seconds to 5 minutes using duplicate samples for each sample in a 30° C. water bath. At the end of each contact time, samples were neutralized by adding 600 μL of 3% sodium thiosulfate with 0.05% Tween 80 for 15 mins at 30° C. Samples were centrifuged for 10 mins at 14000 rpm at 4C. The supernatant was decanted off and samples were resuspended in lml of BBL buffer. Serial dilutions (1:10) were made of each sample and 1 mL of each dilution was plated on Middlebrook 7H10 agar with Middlebrook OADC enrichment. Plates were incubated at 37° C. for 14- 21 days.
M terrae cells were inoculated and incubated for 14 and 21 days at 37° C. on Middlebrook 7H9 agar plates containing 10% oleic acid albumin dextrose catalase enrichment (“OADC”) and 0.5% of glycerol. The bacterial cells were harvested and OPA (high-level disinfectant) was applied at different time points (0 minutes; 30 seconds; and 3 minutes). The resistance was tested as describerd above and the performance data was collected and plotted.
The M. terrae cells harvested at 14 and 21 days incubation for growth were exposed to the OPA for 30 seconds (survival cycle) and 3 minutes (kill cycle). A control with cells not exposed to the OPA was also tested. The population of the cells harvested at day 7 was too low to allow for testing of these cells using a target population of 1×108 cfu/mL. Results are shown in Tables 2 and 3.
As the data in Table 2 show, the results from 14 days growth may be classified into two groups: 1) three to four log reduction for cells exposed to OPA for 30 seconds, and 2) six log reduction when cells are exposed to OPA for 3 minutes.
The recommendation of the Food and Drug Administration (FDA) is 6 log reduction for a kill cycle and 0 log reduction for a survival cycle. The data show that M. terrae cells responded to the OPA treatment. The OPA treatment for 30 seconds could be considered as a fractional cycle since it showed a log reduction between 0 and 6. The 3-minute exposure was a kill cycle (i.e., 6 log reduction).
M. terrae Cells performance against OPA after 14 Days Growth Culture
Data presented in Table 3 show that 21-days growth exhibited two groups of data: 1) about one log reduction for cells exposed to OPA for 30 seconds, and 2) six log reduction for cells exposed to OPA for 3 min with 6 log reduction. The OPA treatment for 30 seconds could be considered as a survival cycle since it showed no log reduction. M. terrae cells exposure to OPA for 3 minutes showed a kill cycle (i.e., 6 log reduction).
M. terrae Cells Performance against OPA after 21 days Growth Culture
Resistance performance of cells after 14-days growth compared against 21-days growth surprisingly showed that the resistance to the OPA increases with the age of the culture. This is illustrated by higher log reduction for 14-days growth compared to 21-days growth when cells were exposed for 30 seconds to the OPA solution.
Twenty-one different types of carriers including paper, nonwovens, and films were screened and tested according to the protocol described above. The experiments were replicated twice. Carrier performance for cells suspended in both 0.05% Tween 80 and 2.4% trehalose/1×PBS are presented respectively in Tables 4 and 5. The resistance performance to cell removal was tested by subtracting the number of cells removed, during vortexing, from the population initially coated.
A plot of M. terrae cell resistance performance to removal from a coated carrier as a function of type of carrier in two different buffers is shown in
The procedure of Example 1 was repeated using 21-days growth cells, with the M. terrae cells exposed to PAA instead of OPA. Data presented in Table 6 and
M. terrae cells performance against PAA after 21 days growth culture
M. terrae Population
Examples 1-3 demonstrate that Mycobacterium terrae can be used as a predicate to monitor endoscope reprocessing with high-level disinfectant, such as, for example, OPA and PAA, as well as sterilization of surgical instruments. These resistant microbial cells can be tested in suspension form or immobilized by coating on different carriers, such as films and nonwoven materials and used as independent biological indicators.
All cited references, patents, and patent applications in the above application for letters patent are herein incorporated by reference in their entirety in a consistent manner. In the event of inconsistencies or contradictions between portions of the incorporated references and this application, the information in the preceding description shall control. The preceding description, given in order to enable one of ordinary skill in the art to practice the claimed disclosure, is not to be construed as limiting the scope of the disclosure, which is defined by the claims and all equivalents thereto.
This application claims priority from U.S. Provisional Application Ser. No. 62/609,624, filed Dec. 22, 2017, the disclosure of which is incorporated by reference in its entirety herein.
| Number | Date | Country | |
|---|---|---|---|
| 62609624 | Dec 2017 | US |
