RU2718910C1 - Indicator for detecting biological films of bacteria on medical instruments (versions) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry and specifically to an indicator for detecting biological films of bacteria on medical instruments. Indicator has a gel-like structure and contains hydrogen peroxide, a hydrogen peroxide stabilizer, N-cocoalkyl-N,N-dimethylamine oxide, N,N-bis(3-aminopropyl)dodecylamine, a corrosion inhibitor, a complexing agent, a thickener and water, with the following ratio of components, wt%: hydrogen peroxide 3.0–10.0; hydrogen peroxide stabilizer 0.5; N-cocoalkyl-N,N-dimethylamine oxide 1.5; N,N-bis(3-aminopropyl)dodecylamine 0.25; corrosion inhibitor 0.05; complexing agent 0.5; thickener 2.0; water – balance. Invention also relates to a version of an indicator.

EFFECT: obtaining new indicators which enable more reliable detection of bacteria on surfaces of medical instruments by detecting them in a biofilm state.

3 cl, 2 tbl

Description

The inventions relate to the fields of microbiology, epidemiology, biochemistry, sanitation, hygiene, are compositions and can be used to indicate biological films of bacteria on various surfaces of medical instruments, for example, on the outer surface and channels of flexible and rigid endoscopes.

More than 95% of all bacteria live on abiotic and biotic surfaces in a biofilm state, and not in the form of planktonic (free-living) forms. Biofilms are microbial associations, which are a continuous multilayer of bacterial cells attached to the interface and to each other and enclosed in an exopolysaccharide matrix.

The formation of a biofilm is accompanied by the formation of an exopolymer matrix, a product of the vital activity of the cells themselves. Upon maturation of the biofilm, a significant amount of exopolymer is produced, combining neighboring cells and forming a matrix. Exopolymers make up 85% of the biofilm mass, and 15% are bacteria. Matrix is the main structural component of a biofilm. Exopolysaccharides form a significant part of the matrix. In prokaryotes, as well as in eukaryotes, polysaccharides form the surface layer of the cell membrane (glycocalyx). In bacteria, exopolysaccharides form a capsule, mucous layers and can be released into the external environment, separating from the cell surface. The exopolysaccharide matrix (EPM) also performs such important functions as frame and protective. EPM protects bacteria in the biofilm from antibacterial drugs, negative environmental factors, such as UV radiation, radiation, pH changes, osmotic shock, drying.

Thus, the bacteria in the biofilm are enclosed in a polymer matrix, whose properties determine the relationship within the cell community and with the environment. The matrix in different types of bacteria varies in physical properties and chemical composition, but, as a rule, it is an anionic polymer.

The identification and identification of bacteria in a biofilm state is difficult, since the EPM interferes with the mechanical transfer of bacteria to culture media for subsequent identification. In the practice of routine sanitary surveillance of critical facilities of medical treatment institutions and food industry enterprises, the method of washing off bacteria onto nutrient media is widely used to monitor the effectiveness of sanitary treatment of surfaces, tools, apparatus and equipment.

Due to the increase in the use of minimally invasive high-tech surgical methods and manipulations, endoscopic techniques - rigid and flexible endoscopes are used in the vast majority of surgical interventions on the gastrointestinal tract and respiratory organs.

At the same time, poor-quality disinfection of endoscopic equipment can lead to infection of patients.

Diagnostic procedures are known for detecting bacteria in a biofilm state on the surfaces of endoscopes (SP 3.1.3263-15. Sanitary and epidemiological rules. Prevention of infectious diseases during endoscopic interventions. Approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation A.Yu. Popova of June 8, 2015 N 20 // TEXHEXPERT and CODE, Electronic Foundation for Legal and Normative and Technical Documentation [Electronic resource], URL: http://docs.cntd.ru/document/420283545, accessed date: 08/10/2019), including in taking rinsing, which is carried out using sterile moistened cotton swabs or rinsing of endoscope channels with sterile water, followed by seeding on nutrient media. Sterile cotton swabs on glass, metal or wooden sticks mounted in test tubes with cotton plugs are prepared in advance in the laboratory. However, this method of flushing from surfaces can only determine planktonic, i.e. separately growing, forms of bacteria or fungi that make up an insignificant part of the probable microbial encumbrance of surfaces.

The EPM does not allow a qualitative assessment of the sanitary condition of surfaces using the swab method, hiding bacteria or pathogenic fungi from inoculation with a tampon, preventing the mechanical transfer of bacteria to nutrient media for subsequent identification.

Thus, a negative result of sanitary-bacteriological control by the method of flushing studies is not sufficiently reliable (false-negative) and does not take into account the possibility of the existence of bacteria and fungi in a biofilm state.

The closest analogue of the invention is an indicator for detecting bacteria, including in the state of a biofilm (Biofilm Remove
[Electronic resource]: Detection of biofilms with BioFinder, URL: https://biofilmremove.com/en/, access date: 10/20/2018), which is a water-alcohol solution of hydrogen peroxide. Such an indicator is ineffective for detecting bacteria that are in a state of mature and dried biofilm due to the presence of a powerful exopolysaccharide matrix, since it does not contain special substances for its destruction.

The objective of the proposed group of inventions is the creation of indicator substances that can improve the quality of evaluation of medical instruments for the presence of bacteria in the state of biofilm, in addition, indicators should not spoil or stain the treated surfaces, be soluble in water and easy to wash off, be environmentally friendly and biodegradable .

The technical result of the invention is to increase the reliability of identifying bacteria in a state of biofilm on various surfaces of medical instruments.

The indicated result is achieved by an indicator for identifying biological films of bacteria on medical instruments with a gel-like structure and containing hydrogen peroxide, a stabilizer of hydrogen peroxide, N-cocoalkyl-N, N-dimethylamine oxide, NN-bis (3-aminopropyl) dodecylamine, a corrosion inhibitor, complexing agent , a thickener and water in the following ratio of components, mass. %:

hydrogen peroxide 3.0-10.0;

hydrogen peroxide stabilizer 0.5;

N-cocoalkyl-N, N-dimethylamine oxide 1.5;

NN-bis (3-aminopropyl) dodecylamine 0.25;

corrosion inhibitor 0.05;

complexing agent 0.5;

thickener 2.0;

water the rest.

The indicator may contain chlorhexidine digluconate in a concentration of 1.5% to 3.0%.

The result is also achieved by an indicator for identifying biological films of bacteria on medical instruments, which is an aqueous solution and contains hydrogen peroxide, colloidal silver, a stabilizer of hydrogen peroxide, a corrosion inhibitor, a complexing agent and water in the following ratio of components, masses. %:

hydrogen peroxide 3.0-10.0;

colloidal silver 0.0012-0.0024;

hydrogen peroxide stabilizer 0.5;

corrosion inhibitor 0.05;

complexing agent 0.5;

water the rest.

Indicators with high reliability make it possible to identify gram-positive and gram-negative bacteria in the state of biofilm on various abiotic surfaces, such as stainless steel, tiles, polypropylene, painted surfaces, etc.

The advantage of this type of detection lies in the fact that biofilms, as a rule, have a poly-species structure and one type of catalase-positive bacteria in a biofilm is sufficient for detection (detection, detection). However, indicators also allow mono-species biofilms to be detected.

Catalase is the primary primary antioxidant protection system that catalyzes the decomposition of hydrogen peroxide to water. Hydrogen peroxide is destroyed by two classes of related enzymes that catalyze its two-electron reduction to H ₂ O and use H ₂ O ₂ as an electron donor in the case of catalase or various organic compounds in the case of peroxidase.

Catalase and peroxidase activities were found in all aerobic and facultative anaerobic prokaryotes. Among obligate anaerobes, these enzymes are much less common than superoxide dismutase.

The developed formula of indicators implies greater efficiency compared to analogues due to the presence of special substances that destroy the protective EPM of the biofilm and thereby increase the number of bacterial cells that react.

An indicator formulation based on hydrogen peroxide was developed, pilot samples of the indicator were created, and working concentrations of the active ingredients of the aqueous solution of the sample were selected by serial dilutions with identification of the maximum activity on mature biofilms of gram-positive and gram-negative bacteria.

The first indicator for detecting biological bacteria films on medical instruments has a gel-like structure and contains hydrogen peroxide, a stabilizer of hydrogen peroxide, N-cocoalkyl-N, N-dimethylamine oxide, NN-bis (3-aminopropyl) dodecylamine, a corrosion inhibitor, complexing agent, thickener, additionally includes the dye E133, and water in the following ratio of components, mass. %:

hydrogen peroxide 3.0-10.0;

hydrogen peroxide stabilizer 0.5;

N-cocoalkyl-N, N-dimethylamine oxide 1.5;

NN-bis (3-aminopropyl) dodecylamine 0.25;

corrosion inhibitor 0.05;

complexing agent 0.5;

thickener 2.0;

dye E133 0.05;

water the rest.

Additionally, the first indicator may contain chlorhexidine digluconate in a concentration of from 1.5% to 3.0%.

The second indicator for identifying biological films of bacteria on medical instruments is an aqueous solution and contains hydrogen peroxide, colloidal silver, a stabilizer of hydrogen peroxide, a corrosion inhibitor, complexing agent and water in the following ratio of components, mass. %:

hydrogen peroxide 3.0-10.0;

colloidal silver 0.0012-0.0024;

hydrogen peroxide stabilizer 0.5;

corrosion inhibitor 0.05;

complexing agent 0.5;

water the rest.

The first indicator for detecting bacterial biofilms is a thick gel-like solution, which contributes to the prolongation of the visualization of the bubbles (bubbling). It is practical for use on the external surfaces of various medical instruments, for example, on endoscopic technology. The second indicator, due to its liquid structure, is suitable for application to existing internal surfaces of medical instruments, for example, endoscope channels. The described use of indicators is recommended, but not required.

As a stabilizer of hydrogen peroxide for two indicators, salicylic acid or sodium pyrophosphate 0.5%, corrosion inhibitors - alkyl dimethylbenziammonium chloride 0.05% or other anionic, cationic and mixed corrosion inhibitors, complexing agents - silicates / sodium metasilicates, for example sodium carbonate, can be used , thickener - polyacrylic acid.

We give examples of indicators with various possible concentrations of constituent substances.

Table 1. Examples of concentrations of the constituent (wt.%) Substances of the first indicator

Example 1 Example 2 Example 3 Example 4 N-Cocoalkyl-N, N-Dimethylamine Oxide 1.5% 1.5% 1.5% 1.5% Hydrogen peroxide 3.0% 6.0% 8.0% 10.0% Chlorhexidine digluconate - 1.5% 3.0% 3.0% NN-bis (3-aminopropyl) dodecylamine 0.25% 0.25% 0.25% 0.25% Hydrogen peroxide stabilizer 0.5% 0.5% 0.5% 0.5% Corrosion inhibitor 0.05% 0.05% 0.05% 0.05% Complexing agent 0.5% 0.5% 0.5% 0.5% Thickener 2.0% 2.0% 2.0% 2.0% Dye E133 0.05% 0.05% 0.05% 0.05% Water rest rest rest rest

Table 2. Examples of concentrations of the constituent (wt.%) Substances of the second indicator

Example 1 Example 2 Example 3 Example 4 Example 5 Hydrogen peroxide 3.0% 3.0% 6.0% 6.0% 8.0% Colloidal silver 0.0012% 0.0024% 0.0012% 0.0024% 0.0012% Hydrogen peroxide stabilizer 0.5% 0.5% 0.5% 0.5% 0.5% Corrosion inhibitor 0.05% 0.05% 0.05% 0.05% 0.05% Complexing agent 0.5% 0.5% 0.5% 0.5% 0.5% Water rest rest rest rest rest

Table 2. Continuation

Example 6 Example 7 Example 8 Hydrogen peroxide 8.0% 10.0% 10.0% Colloidal silver 0.0024% 0.0012% 0.0024% Hydrogen peroxide stabilizer 0.5% 0.5% 0.5% Corrosion inhibitor 0.05% 0.05% 0.05% Complexing agent 0.5% 0.5% 0.5% Water rest rest rest

The possibility of carrying out the invention is confirmed by the following examples.

The first example is a solution of the first indicator. To obtain 100 ml of the first indicator in the form of a gel-like solution ready for use at room temperature, 1.5 ml of N-cocoalkyl-N, N-dimethylamine oxide (ar Barlox-12 ʺ (Barlox-12), catalog of ʺLonzaʺ (Lonza) is dissolved in 100 ml water, add 6.0 ml of 50% hydrogen peroxide, 1.5 g of NN-bis (3-aminopropyl) dodecylamine, sodium pyrophosphate 0.2 ml, sodium carbonate 0.5 g, polyacrylic acid 3 ml, 0.01 g of dye E133.

The second example of obtaining a solution of the first indicator. Carry out similarly to the first example of obtaining a solution of the first indicator, but the tool includes 1.5 ml of chlorhexidine digluconate, 1.0 ml of N-cocoalkyl-N, N-dimethylamine oxide, 12 ml of 50% hydrogen peroxide.

The third example of obtaining a solution of the first indicator. Carried out analogously to the first example, but the tool includes 2.0 ml of chlorhexidine digluconate, 2.0 ml of N-cocoalkyl-N, N-dimethylamine oxide, 16 ml of 50% hydrogen peroxide.

The fourth example of obtaining a solution of the first indicator. Carried out analogously to the first example, but the tool includes 2.5 ml of chlorhexidine digluconate, 2.0 ml of N-cocoalkyl-N, N-dimethylamine oxide, 20 ml of 50% hydrogen peroxide.

The first example is a solution of the second indicator. To obtain 100 ml of the second indicator in the form of a ready-to-use liquid solution at room temperature, 6.0 ml of 50% hydrogen peroxide is dissolved in 100 ml of water, 6.0 ml of 50% hydrogen peroxide are added, 1.2 ml of 10 are added. % colloidal silver, sodium pyrophosphate 0.2 ml, sodium carbonate 0.5 g.

The second example of obtaining a solution of the second indicator. Carried out similarly to the first example of obtaining a solution of the second indicator, but the tool includes 2.4 ml of 10% colloidal silver.

The third example of obtaining a solution of the second indicator. Carried out similarly to the first example, but the product includes 12 ml of 50% hydrogen peroxide and 2.4 ml of 10% colloidal silver.

The fourth example of obtaining a solution of the second indicator. Carried out analogously to the first example, but the tool includes 12 ml of 50% hydrogen peroxide and 2.4 ml of 10% colloidal silver.

The fifth example of obtaining a solution of the second indicator. Carried out analogously to the first example, but the tool includes 16 ml of 50% hydrogen peroxide and 1.2 ml of 10% colloidal silver.

The sixth example of obtaining a solution of the second indicator. Carried out analogously to the first example, but the tool includes 16 ml of 50% hydrogen peroxide and 2.4 ml of 10% colloidal silver.

Seventh example of obtaining a solution of the second indicator. Carried out analogously to the first example, but the tool includes 20 ml of 50% hydrogen peroxide and 1.2 ml of 10% colloidal silver.

The eighth example of obtaining a solution of the second indicator. Carried out analogously to the first example, but the tool includes 20 ml of 50% hydrogen peroxide and 2.4 ml of 10% colloidal silver.

The activity of the indicators was tested on mature formed biofilms of the following strains:

Gram-positive:

- Staphilococcusaureus 15 and

Gram-negative:

- E.coli 717,

- Pseudomonas aeruginosa 32,

- Acinetobacter baumanii 503,

- Klebsiella pneumoniae 1553,

- Salmonella typhimurium C53,

SalmonellatyphimuriumC53rpoS with a mutation in the rpoS gene, leading to impaired synthesis of enzyme catalase.

As you know, the enzyme catalase is a catalyst in the decomposition of hydrogen peroxide, in which water and molecular oxygen are formed: Н2О2 + Н2О2 = О2 + 2Н2О.

The biological significance of catalase lies in the decomposition of hydrogen peroxide, which is formed in cells upon exposure to a number of flavoprotein oxidases, which provides effective protection of cellular structures from the destruction that hydrogen peroxide provides. Catalase is found in the tissues of plants, animals and humans, as well as in bacteria, although this enzyme is absent in a number of anaerobic bacteria.

Catalase is one of the fastest enzymes. Just one of its molecules is capable of converting millions of hydrogen peroxide molecules into water and oxygen in a second. From the point of view of enzymology, this means that the catalase enzyme is characterized by a large number of revolutions. As for bacteria, this is visually easy to observe - when bacterial cells interact with the enzyme, active release of oxygen bubbles begins.

In order for this to be clearly visible in our case, an isogenic pair of S. typhimurium strains, the initial wild strain and a strain carrying the rpoS mutation, a global regulator of gene expression, which leads to disruption of catalase synthesis, was included in the tested strain collection.

Indicator sensitivity testing

Option I

1. The cells of diurnal cultures of S. aureus 15 and P. Aeruginosa 32 were besieged by centrifugation (10 min at 7.000 rpm), resuspended in 200 μl of physical. solution and in transparent test tubes in 5 ml of physical. a solution of the prepared initial suspension of bacteria at a concentration of n x 10 ⁸ cells / ml

2. In test tubes, Eppendorf made ten-fold dilutions of the original bacterial suspensions in physical. solution and / or water (from 10 ⁸ to 10 ³ cells / ml).

3. In the large wells of the 24-well plates, 100 μl of ten-fold serial dilutions of bacterial suspensions of each culture were carefully introduced into the center of the flat bottom.

Option II

After preparation of the initial bacterial suspensions (10 ⁸ cells / ml), equal volumes of both cultures were mixed, as in the first embodiment, serial dilutions of ten times were made and 100 μl of each dilution of the mixed culture was added to the bottom of large wells.

Both tablets with two test options set to dry in a thermostat at 37 ° C for 18 hours. The next day, a solution of the second indicator was added in one row of wells with dilutions of each culture or mixed culture, and in another solution of the first indicator.

Visually observed the formation of bubbles in samples from 10 ⁷ to 10 ⁴ cells / 100 μl. Moreover, in wells with a high concentration of cells (10 ⁷ -10 ⁶ cells / sample), bubbles form rather quickly - within 1 min. Lower concentrations of cells form visible vesicles somewhat later - after 5 minutes. Such small bubbles can only be identified with a magnifying glass, because they are very small and rare.

Therefore, we can only talk about the sensitivity of this reaction as the sensitivity of microscopic testing: n × ^{10 4} - n × ^{10 6} cells / sample.

Samples of mixed cultures give the same sensitivity of visual observation.

It should be noted that the first gel indicator has a stabilizing effect on microbubbles for a longer time, which may slightly increase the sensitivity of detecting cells in a dried bacterial biofilm.

A study was conducted of the effectiveness of indicators in relation to biofilms grown at the distal end of the endoscope, a plastic body element and the head of the distal end of the endoscope.

For the experiment, a mixture of cultures of S.aureus 15 and P.aeruginosa 32 in LB (1: 100) was initially prepared.

The distal end of the endoscope was placed and fixed in a glass cup with a prepared mixed culture. The structure was placed in an incubator for incubation at 37 ° C for 20 hours. The next day, the distal end was removed from the bacterial suspension and washed three times with water.

In the first version of the experiment, the washed end of the endoscope was lowered into a glass beaker with a transparent solution of the second indicator.

In the first seconds, it was noted that a violent reaction of the cells that make up the mixed biofilm with the components of the solution began, oxygen bubbles began to actively form.

A sparging reaction after a few minutes showed a decrease in effect, because the catalase reaction was depleted. The most noticeable was the reaction of cells in the biofilm formed at the interface between the air and water phases, because At the boundary of the two phases, aerophilic microorganisms form a more intense biofilm.

In the second version of the experiment, the washed distal end of the endoscope was lowered into a glass beaker with the first gel indicator. As in the first version of the experiment, it was noted that a violent reaction of the cells that make up the mixed biofilm with the components of the solution began, oxygen bubbles also began to form actively.

Individual parts of the endoscope, as well as the distal end of the endoscope itself, were placed in a bacterial suspension of a mixed culture of S. aureus 15 and P. aeruginosa 32 in LB (1: 100). The formation of the bacterial biofilm was also carried out by incubation in a thermostat at 37 ° C for 20 hours. The details of the endoscope were also washed three times in water and dried. Then, 300 μl of the first gel indicator was applied to a separate black plastic part. The reaction of bacterial biofilm cells with an indicator was observed instantly.

The head of the distal end of the endoscope with washed and dried bacterial biofilms was placed in a small Petri dish and the first gel indicator was also introduced. The effect of the interaction of bacterial biofilm cells with a gel peroxide indicator was also observed instantly.

Thus, the following conclusions can be made.

1. The developed indicators are based on the reaction of hydrogen peroxide with the enzyme of the antioxidant defense of a bacterial cell - catalase.

2. Indicators can be used on various abiotic surfaces and have safety characteristics acceptable for use in various fields of the national economy.

3. The effectiveness of indicators depends on the number of bacterial cells, and the quantitative indicator of bacteria visualized with the naked eye is in the range of 10 ⁴ - 10 ⁶ cells per ml.

4. Studies of the effectiveness of indicators conducted on various materials, including endoscopic techniques, confirmed the possibility of using them to determine the presence of bacterial contamination, including the state of biofilm on abiotic surfaces, including medical instruments, apparatus and equipment, including flexible ones and rigid endoscopes.

Claims (5)

1. An indicator for detecting biological bacteria films on medical instruments, having a gel-like structure and containing hydrogen peroxide, a stabilizer of hydrogen peroxide, N-cocoalkyl-N, N-dimethylamine oxide, N, N-bis (3-aminopropyl) dodecylamine, a corrosion inhibitor, complexing agent, thickener and water in the following ratio of components, mass. %: hydrogen peroxide 3.0-10.0 hydrogen peroxide stabilizer 0.5 N-Cocoalkyl-N, N-Dimethylamine Oxide 1,5 N, N-bis (3-aminopropyl) dodecylamine 0.25 corrosion inhibitor 0.05 complexing agent 0.5 thickener 2.0 water  - the rest 2. The indicator according to claim 1, additionally containing chlorhexidine digluconate in a concentration of from 1.5% to 3.0%. 3. An indicator for identifying biological films of bacteria on medical instruments, rigid and flexible endoscopes, which is an aqueous solution containing hydrogen peroxide, colloidal silver, a stabilizer of hydrogen peroxide, a corrosion inhibitor, a complexing agent and water in the following ratio of components, mass. %: hydrogen peroxide 3.0-10.0 colloidal silver 0.0012-0.0024 hydrogen peroxide stabilizer 0.5 corrosion inhibitor 0.05 complexing agent 0.5 water - the rest