WO1999049731A1

WO1999049731A1 - Methods and compositions for disinfection of heat-sensitive objects

Info

Publication number: WO1999049731A1
Application number: PCT/US1999/006652
Authority: WO
Inventors: Barnet R. Adelman
Original assignee: ADELMAN ASSOCIATES
Current assignee: ADELMAN ASSOCIATES
Priority date: 1998-03-27
Filing date: 1999-03-26
Publication date: 1999-10-07
Anticipated expiration: 2000-09-27
Also published as: AU3117099A

Abstract

Compositions and methods useful in the disinfection of objects, particularly heat sensitive objects are provided. The compositions of the inventions comprise about 0.01-6 % silver nitrate by weight and one or more compound selected from the group consisting of: C2-C10 alcohols and phenolic compounds. Also provided are compositions consisting essentially of one or more C2-C10 alcohol, about 0.01-6 % silver nitrate by weight, 0-15 % water and 0-5 % glycerin. The methods of the invention comprise contacting an object to be disinfected with a composition of the invention.

Description

METHODS AND COMPOSITIONS FOR DISINFECTION OF HEAT-SENSITIVE OBJECTS

This application claims the benefit of U.S. Provisional Application 60/079,673, filed 27 March 1998.

FIELD OF THE INVENTION The invention relates generally to the field of sterilization. More particularly, the invention relates to the disinfection of heat-sensitive objects.

BACKGROUND OF THE INVENTION Historically, objects that are required to be sterile have been heat sterilized to destroy bacteria, viruses and other microorganisms. High heat with steam under pressure is required to inactivate particularly resistant organisms, such as Mycobacterium tuberculosis, because of its thick polysaccharide and lipid membrane, the spores of bacteria, fungi and algae, and the cysts of protozoa. However, heating can damage temperature sensitive surgical instruments such as, for example, endoscopes with plastic components.

Chemical disinfection of heat sensitive objects has been used as an alternative to heat sterilization. Chemicals used for disinfection include formaldehyde, glutaraldehyde, phenol and phenol derivatives, 50-70% alcohol in water, such as 70% ethanol, halogens, such as chlorine and iodine, heavy metals, such as mercuric chloride, silver nitrate and copper sulfate, ethylene oxide and detergents. Desirable features of compositions for use in chemical sterilization are high toxicity to microorganisms, low toxicity to humans and animals, noncorrosiveness, stability and economy. Rarely does one chemical or chemical composition combine all of these features.

SUMMARY OF THE INVENTION Compositions and methods useful in the disinfection of objects, particularly heat sensitive objects are provided. The compositions of the invention are disinfecting solutions comprising about 0.01-6% silver nitrate by weight and one

-1- or more compound selected from the group consisting of: C -C₁₀ alcohols and phenolic compounds. Also provided are disinfecting solutions consisting essentially of one or more C -C₁₀ alcohol, about 0.01-6% silver nitrate by weight, 0-15%) water, 0-5% glycerin, and optionally, a perfume. The methods of the invention comprise contacting an object to be disinfected with a disinfecting solution of the invention.

DETAILED DESCRIPTION OF THE INVENTION The invention comprises methods and compositions that can be used to disinfect objects. The compositions of the invention are particularly suited for the disinfection of heat sensitive objects, and in particular, of heat-sensitive medical equipment. Such objects include, but are not limited to, surgical instruments, thermometers, flexible endoscopes, lensed instruments, polyethylene tubing and catheters, and inhalation and anesthesia equipment. The invention also methods of disinfecting objects through the use of these disinfecting solutions. The invention has the additional advantage that the components of the disinfecting solutions are already approved for use with medical equipment and are non-corrosive to optics and/or electronics.

It has long been standard practice to use 50-70%) alcohol solutions as disinfectants. However, alcohol concentrations greater than 70%) are known to decrease the efficacy of disinfection. Similarly, 2-5% aqueous phenol is also useful as a disinfectant. Silver nitrate is also known to kill microorganisms. However, none of these compounds are suitable for efficient killing of highly resistant microorganisms, such as Mycobacterium tuberculosis. Surprisingly, it has been discovered that the combination of about 0.01 -6%> silver nitrate and >60%>, preferably greater than 80%, of a C -C₁₀ alcohol act synergistically to efficiently inactivate microorganisms, including Mycobacteria. A synergistic killing effect has also been found for the combination of about 0.01- 6%> silver nitrate and about 0.2-10%) of a phenolic compound. As noted above, some of the most resistant pathogens are TB related because they are enclosed in polysaccharides. The disinfecting solutions of the invention are particularly effective in killing TB related pathogens because the C - C₁₀ alcohol and/or the phenolic compound can dissolve the polysaccharide membrane, thereby exposing the interior portion of the pathogen to the silver nitrate.

Thus, in one embodiment, the invention provides a disinfecting solution comprising about 0.01-6% silver nitrate by weight and one or more compound selected from the group consisting of: a C₂-Cιo alcohol and a phenolic compound.

The disinfecting solution may contain about OJ-6% silver nitrate by weight. Preferably the disinfecting solution contains about 0.5-4%> silver nitrate by weight, more preferably about 1-2% silver nitrate by weight, and most preferably about 1.5% silver nitrate by weight. Any C₂-C₁₀ alcohol may be used in the disinfecting solutions of the invention. By alcohol is meant a compound in which a hydroxyl group is attached to a saturated carbon. The saturated carbon may be a carbon of a simple alkyl group, such as ethanol, isopropanol, 4-methyl-l-pentanol, 3-chloro-l-l-propanol, 4-penten-2-ol and the like. Alternatively, the carbon may be a saturated carbon of an alkenyl or alkynyl group, such as 2-propenol, 2-propynol and the like. Or, the carbon may be a saturated carbon that is attached to an aromatic ring, such as benzyl alcohol, phenyl alcohol and the like. In addition, the alcohol may contain two hydroxyl groups, such as ethylene glycol, propylene glycol, trimethylene glycol and the like. Preferably the C₂-Cjo alcohol is ethanol, a propyl alcohol or a butyl alcohol. Most preferably, the C₂-C₁₀ alcohol is isopropanol.

The C₂-C₁₀ alcohol can present in the disinfecting solutions of the invention at a concentration of about 60-99.5%ι by weight. Preferably the concentration of C₂-C₁₀ alcohol is about 80-98% by weight or 85-96% by weight. Even more preferably, the concentration of C₂-C₁₀ alcohol is about 89-95% by weight or even 90-92%) by weight. Most preferably, the concentration of C₂-Cιo alcohol is about 90% by weight.

By "phenolic compound" is meant a compound based on the substitution product of phenol. Phenolic compounds include, but are not limited to, phenol, chlorophenol, nitrophenol, bromophenol, cresols, dihydroxybenzenes, phenylphenol, hexylresorcinol, hexachlorophene, methylphenol, ethylphenol, dimethylphenol, propylphenol, amylphenol, hexylphenol, heptylphenol and the like. Preferably the phenolic compound is phenol.

The phenolic compound may be present in the disinfecting solutions of the invention at a concentration of about OJ-99% by weight. Preferably the

-3- concentration of the phenolic compound is about 0.2 to 10% by weight. Most preferably, the concentration of the phenolic compound is about 1-5% by weight. In a preferred embodiment, the phenolic compound is present at a concentration of about 2-5%. In one embodiment of the disinfection solution, one or more phenolic compound and/or one or more C₂-Cι₀ alcohol are combined with silver nitrate.

Other components that may useful additions in the disinfecting solutions of the invention are glycerin and perfume. Glycerin is helpful in dissolving the silver nitrate. Glycerin may be present in the disinfecting solutions of the invention at a concentration of about 0-5% by weight, preferably at about 0.5-2%> by weight, most preferably at about 1% by weight. Perfume aids in masking the odor of the C₂-Cιo alcohols and phenolic compounds having an offensive smell. A perfume may be included in the disinfecting solutions of the invention at a concentration effective in partially or completely masking the offensive odor due to the C -C₁₀ alcohol and/or phenolic compound.

In another embodiment, the invention provides a disinfecting solution consisting essentially of about 0.01-6%> silver nitrate by weight, a C -C₁₀ alcohol, 0-15%) water, 0-5%> glycerin and optionally, a perfume.

The compositions of the invention are useful in disinfecting objects. Thus, in one aspect, the invention is directed to a method of disinfecting an object comprising contacting said object with a disinfecting solution comprising about 0.01-6%) silver nitrate by weight and one or more compound selected from the group consisting of: C₂-C₁₀ alcohols and phenolic compounds.

To carry out the methods of to the invention, the object to be disinfected is exposed to the disinfecting solution of the invention. The exposure of the object may be by immersion, dipping, submersion or spraying. Preferably the exposure is for at least 10 minutes, more preferably at least 20 minutes, even more preferably at least 30 minutes, and most preferably, at least 40 minutes. Ultrasonic stimulation of the articles and composition can be useful to expedite the sterilization process.

Preferred embodiments of the invention include exposing the object to the disinfecting solutions of the invention at a temperature of about 15-100°C. Even more preferably the temperature of said disinfecting solution is about 20-30°C.

-4- Preferred embodiments of the invention include exposing the articles to the disinfecting solution at 1 atmosphere. However, the articles can be exposed to the disinfecting solution at lower or higher pressures (e.g., from approximately 0.5 to approximately 5.0 atmospheres). In order to reduce the overall cost of implementing the invention, the silver content of the composition can be at least in part recovered. One way to recover the silver content of spent composition is distillation to obtain silver salts. The resulting silver salts can then be subsequently processed.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention. The following examples are intended to illustrate, rather than to limit, the invention.

EXPERIMENTAL

The following protocol was developed as a test to determine the antimicrobial efficacy of the disinfecting solutions of the invention. Test organisms (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Mycobacterium terrae) were exposed to four concentrations of the disinfecting solution. After the designated exposure intervals, a 0J mL aliquot of each tested sample was transferred to a growth medium. After incubation, the samples were observed for growth. The absence of growth indicates antimicrobial activity of the disinfecting solution.

Inoculum Preparation:

Cultures of Staphylococcus aureus ATCC #6538, Pseudomonas aeruginosa ATCC #115442 and Candida albicans ATCC #10231 were incubated at 37 ± 2°C for 24 hours in nutrient broth. The broth was centrifuged for 20 minutes at 1200 rpm and the pellet of bacteria was removed and resuspended in phosphate buffered solution. The suspension was diluted further to yield a bacterial suspension of approximately 6 x 10⁵ CFU/mL. The titer of each test inoculum suspension was confirmed using plate count techniques.

-5- The Mycobacterium terrae ATCC #15755 culture was prepared by swabbing 7H11 agar plates with the organism. The plates were incubated at 37 ± 2°C for 10 days. The agar plates were flooded with peptone tween (PEPT). A sterile bent glass rod was used to suspend the microbial growth. The suspension was added to a sterile container. A 1-2 mL aliquot of the culture suspension was added to a sterile tissue grinder with an equal volume of buffered gelatin. The suspension was macerated for at least 1 minute and then diluted in PEPT. A 10%o volume of glycerol was added to the diluted culture. The suspension was pipetted into sterile 2 mL cryogenic vials. The culture was then frozen at -10 to -30°C for 2 hours then placed into a -70°C freezer until needed. At the time of the test, a vial was removed from a -70°C freezer and thawed. The thawed culture was diluted in a phosphate buffer to achieve an approximate concentration of 6 x 10⁶ CFU/mL. The titer of the test culture was confirmed using plate count techniques.

Preparation of the Disinfecting Solution:

1.5 gram silver nitrate was dissolved in 1 ml of glycerin and then added to 90 ml isopropanol. This solution was brought to a final volume of 100 ml with water.

Test Performance:

The Disinfecting solution was tested at four concentration levels. The concentration levels were: undiluted, 1/2, 1/4, and 1/8. The dilutions of the disinfecting solution were made with sterile purified water. A control with purified water was also part of the test procedure. One milliliter of each of the above dilutions was pipetted into a 13 x 100 mm sterile test tube and placed in a 20 ± 1°C waterbath and equilibrated for ten minutes. One set of dilution tubes was prepared for each of the four test organisms. A 100 μL volume of a bacterial solution was carefully pipetted into the test solutions and the purified water control. The tubes were then incubated in a waterbath at 20 ± 1°C.

For the S. aureus and C. albicans cultures, a 100 μL volume was removed from each of the dilutions of disinfecting solution after 15 and 30 minutes

-6- incubation, and pipetted into a bottle containing sterile letheen broth. The bottles were incubated for 24 hours at 37 ± 2°C and then checked for bacterial growth. An uninoculated letheen bottle was also incubated as a control.

For the M. terrae culture, a 100 μL volume was removed from each of the dilutions of disinfecting solution at 30 and 60 minutes, and pipetted into a bottle containing sterile letheen broth. The contents of the bottles were filtered through a 0.45 μm membrane filter. A 100 mL aliquot of PEPT was then filtered to rinse the membrane. The membrane filter was then plated on 7H111 agar plates, and the 7H11 plates were incubated at 37 ± 2°C for 21 days. After incubation, the plates were observed for growth.

Neutralization Control:

In order to detect false positive scores of antimicrobial activity, a neutralization control was performed with each of the test organisms. If no growth was detected for an organism transferred to a growth medium after incubation with the disinfecting solution, the media was reinocculated with fresh, untreated cultures of the test microoganism. Growth of the test organism in the reinoculated medium ensured that the absence of original growth was due to the antimicrobial activity of the disinfecting solution, rather than to a defect in the growth medium. For the S. aureus and C. albicans cultures, a bottle containing the 100 mL of inoculated letheen growth medium was reinoculated with approximately 10-100 CFU of the appropriate test organism. A sample of the inoculum was plated to confirm the titer. The bottles and plates were incubated at 37 ± 2°C for 24 hours. After incubation the plates were counted and the bottles scored for growth. The M. terrae neutralization control was performed by adding 0J mL of the undiluted test sample to 100 mL letheen broth. An additional bottle containing 100 mL letheen broth was included as a control. Each bottle was inoculated with approximately 10-100 CFU of M. terrae, filtered and plated on 7H11 agar as before. The plates were incubated at 37 ± 2°C for 21 days. After the incubation period, the plates were counted. The percent recovery was then calculated.

-7- Results:

The antimicrobial activity of the disinfecting solution is shown in Table 1. All tested organisms, including M. terrae, were inactivated after 30 minutes exposure to the undiluted disinfecting solution. The inoculating titer of each test organism is reported in Table 2. Neutralization control results are reported in Table 3.

TABLE 1. Mini-Kill Time Screen Results

DILUTION Staphylococcus Pseudomonas Candida Mycobacterium aureus aeruginosa Albicans terrae

15 min 30 min 15 min 30 min 15 min 30 min 15 min 30 min

Undiluted 0 0 0 0 0 0 0 0

Vl 0 0 0 0 0 0 + 0

% 0 0 0 0 0 0 + +

1/8 0 0 0 0 0 0 + +

Positive + + + + + + + + Control

Negative 0 0 0 0 0 0 0 0

Control

Growth - + No Growth - 0

TABLE 2. Organism Titers

TEST ORGANISM TITER

Staphylococcus aureus 8.5 x 10° CFU/mL

Pseudomonas aeruginosa 3.4 x 10' CFU/mL

Candida albicans 3.1 x 10° CFU/mL

Mycobacterium terrae 5.3 x 10° CFU/mL

-8- TABLE 3. Neutralization Results

ORGANISM RESULTS INOCULATING TITER (CFU)

Staphylococcus aureus +,+ 92

Pseudomonas aeruginosa +,+ 73

Candida albicans +,+ 22

Mycobacterium terrae 191 CFU 128 CFU

-9-

Claims

THAT WHICH IS CLAIMED:

1. A composition comprising about 0.01 -6% silver nitrate by weight and one or more compound selected from the group consisting of: C -C₁₀ alcohols and phenolic compounds.

2. The composition of claim 1 , wherein said composition contains 60- 99.5%) by weight of one or more C -C₁₀ alcohols.

3. The composition of claim 2, wherein said composition contains about 80-98%) by weight of one or more C₂-Cιo alcohols.

4. The composition of claim 3, wherein said composition contains about 89-95%) by weight of one or more C₂-C₁₀ alcohols.

5. The composition of claim 4, wherein said composition contains about 90%) by weight of one or more C₂-C₁₀ alcohols.

6. The composition of claim 1 , wherein said composition contains isopropanol.

7. The composition of claim 4, wherein said C₂-C₁₀ alcohol isopropanol.

8. The composition of claim 5, wherein said C₂-C₁₀ alcohol isopropanol.

9. The composition of claim 1 , wherein said composition contains phenol.

10. The composition of claim 9, wherein the concentration of phenol is about 2-5%o by weight.

-10-

11. The composition of claim 1 , wherein said composition contains about 0.5-5%o silver nitrate by weight.

12. The composition of claim 11, wherein said composition contains about 1-2%) silver nitrate by weight.

13. The composition of claim 12, wherein said composition contains about 1.5% silver nitrate by weight.

14. A method of disinfecting an object comprising contacting said object with a composition comprising about 0.01-6%) silver nitrate by weight and one or more compound selected from the group consisting of: C₂-Cιo alcohols and phenolic compounds.

15. The method of claim 14, wherein said silver nitrate comprises about

0.5-2%) of said composition.

16. The method of claim 14, wherein said C -C₁₀ alcohol is isopropanol and comprises 80-99%) by weight of said composition.

17. The method of claim 14, wherein the temperature of said composition is about 15-100°C.

18. The method of claim 17, wherein the temperature of said composition is about 20-30°C.

19. The method of claim 14, wherein said object is heat sensitive.

20. A disinfecting solution consisting essentially of about 0.01 -6%> silver nitrate by weight, a C₂-Cιo alcohol, 0-15%) water, 0-5%> glycerin and optionally, a perfume.

21. A method of disinfecting an obj ect comprising contacting said object with the composition of claim 20.

-11-