RU2845276C1 - Method of evaluating resistance of microorganisms to disinfectants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: what is presented is a method for assessing the resistance of microorganisms to disinfectants involving the sequential application on the inner surface of the wells of sterile 12-well flat-bottomed plate of 0.1 ml of a microbial suspension of bacteria containing 1×10⁹ CFU/ml; drying; adding 0.5 ml of a disinfectant solution to the wells; after exposure time of disinfectant solution is introduced by 4.5 ml of melted and cooled down to 47±2 °C neutralizing agar according to Dey-Engley; incubation of crops at 35±2 °C for 24-48 hours; presence of growth of characteristic colonies of a microorganism in the experimental and reference holes testifies to the resistance of the microorganism to the disinfectant, and the absence of growth in the test wells and the presence of growth in the control wells indicates the sensitivity of the microorganism to the disinfectant.

EFFECT: invention provides a wider range of methods for assessing the resistance of microorganisms recovered from surfaces to disinfectants.

2 cl, 8 tbl, 5 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of healthcare, in particular to the field of microbiology and disinfectology, namely to medical microbiology, and concerns a method for assessing the resistance of microorganisms circulating in medical organizations, educational institutions, the production environment of public catering establishments, the food and pharmaceutical industries, to disinfectants.

State of the art

In the modern world, the organization of effective medical care for the population is inextricably linked with the need to address the issues of resistance of microorganisms, primarily infections associated with the provision of medical care, to the effects of antimicrobial drugs. Research and monitoring of the resistance of microorganisms isolated from patients and from objects of the hospital environment to disinfectants (disinfectants, DS), traditionally used in clinical departments, allow us to develop principles for their selection and timely rotation and thereby reduce the possibility of the formation of resistance in microorganisms, the spread of resistant strains and, as a result, reduce the incidence of infections associated with the provision of medical care.

This problem is also relevant for educational institutions, catering enterprises, food and pharmaceutical industries. Massive use of antibiotics in veterinary medicine and animal husbandry changes the ecology and biology of microorganisms, which develop the ability to extrachromosomal transfer of resistance to antimicrobial drugs. A number of studies confirm that the main reason for the emergence of a high level of resistance is the widespread use of antibiotics as an additive to feed. If cleaning, washing and disinfection procedures are not carried out properly and in high quality, opportunistic and pathogenic "industrial" strains of microorganisms resistant to individual active substances of disinfectants may appear at enterprises. Untimely detection of strains resistant to the disinfectants used at the enterprise leads to the spread of resistant microorganisms in the production environment, and, consequently, to the release of low-quality products for the consumer. In this regard, the detection of an increased number of microorganisms in production environment objects or the appearance of microorganisms that are not typical for the organization in any production lines indicates the need to take additional preventive measures, including checking the effectiveness of the disinfectants and antiseptics used.

Methods for determining the sensitivity of microorganisms to disinfectants are known.

A method for determining the effectiveness of disinfectants used in anti-tuberculosis institutions is known [RU 2364629, 2009], which includes the sequential preparation of a suspension of mycobacteria culture, contamination of test objects with mycobacteria, their treatment in the tested disinfectant solution, neutralization of the disinfectant on the test objects after a specified exposure of the disinfectant to the test objects contaminated with mycobacteria, incubation of the neutralized tests on a dense nutrient medium in a thermostat, and recording and analysis of the experimental results. In this case, calico test objects are used as test objects for contamination with mycobacteria, neutralization of contaminated test objects is carried out with Di-Ingley broth, and the Novaya medium is used as a dense nutrient medium. The main disadvantage of the known method is the sequence of operations, including the treatment of contaminated test objects in a disinfectant solution, adding neutralizing broth according to Dee-Ingley to the solution as a universal neutralizer, then transferring the test objects to a dense nutrient medium in test tubes with a slant for incubation. In addition, this method is suitable only for determining the sensitivity of Mycobacterium bacteria to disinfectants and is not suitable for determining the sensitivity of other genera of microorganisms circulating in the production environment at food industry enterprises, including opportunistic and pathogenic microorganisms, to disinfectants.

Methods for determining the sensitivity of microorganisms to a disinfectant are known [RU 2378363, 2010] when it acts in solution or on surfaces. The method for determining in solution includes: exposure of a mixture of a suspension of a microorganism culture with a disinfectant dissolved in distilled water for a period of time known for a given disinfectant as the time required for the manifestation of the disinfectant effect; addition of a neutralizer for the disinfectant; seeding of the suspension on a solid nutrient medium with subsequent assessment of the growth of microorganisms on the nutrient medium. The method of determination on surfaces consists in that the tested suspension of microorganisms is applied to the sterile surface of the test object, to which, after the microbial suspension dries, a disinfectant is applied, the resulting mixture is exposed for a time known for this disinfectant as the time required for the manifestation of the disinfectant effect, then a neutralizer of the disinfectant is applied to the surface of the test object and, after exposure of the neutralizer, the mixture is washed off the surface of the test object with a sterile swab, and the same swab is immediately used to seed the mixture onto a solid nutrient medium. The sensitivity of microorganisms to disinfectants is assessed by the presence of growth of characteristic colonies on the nutrient medium.

The disadvantage of the known method of determination on surfaces is the relatively low degree of reliability of the research results, which is explained by the fact that using a swab it is impossible to ensure sufficient accuracy of quantitative seeding of the mixture on a solid nutrient medium, since some of the microorganisms remain on the tested surface, and some on the swab.

Thus, the known methods are labor-intensive, as they require special preparation of test objects for conducting research (washing, packaging, sterilization).

The closest in essence to the claimed method according to the present invention is a method for determining the sensitivity of microorganisms to disinfectants [RU 2650760, 2018], which consists in the fact that a suspension of test microorganisms is applied to the inner surface of a Petri dish with a dispenser, after drying the suspension, the contaminated surface of the dish is treated with an appropriate disinfectant by irrigating, wiping or immersing it, after the end of the disinfection holding, a solution of a neutralizer corresponding to the chemical composition of the disinfectant used is added to the Petri dish, and after a certain holding time, melted and cooled to 45 ° C agar is added, then the dish is placed in a thermostat at an optimal temperature and time for the growth of the test microorganism, after which the number of colonies grown on the Petri dish is recorded and the results are compared with the data obtained with a similarly contaminated dish, treated in the same way as the experimental one, but not a disinfectant solution and sterile tap water; based on the comparison results, a judgment is made on the sensitivity of the tested microorganisms to the disinfectant when the number of microorganisms decreases from the effect of the disinfectant solution by 99.99% - 100%, and when less than 99.99% - on their resistance. The disadvantage of this known method is the need to select a neutralizer corresponding to the tested disinfectant. Thus, to neutralize the antimicrobial action of disinfectants from the oxidizing group, 0.5-1% sodium thiosulfate solutions are used; for aldehyde- and phenol-containing agents - water, a universal neutralizer containing 3% tween-80, 0.3% saponin, 0.1% histidine and 0.1% cysteine; for cationic surfactants - 0.1-1.0% sulfonol solutions or 0.5-1.0% sulfonol solutions with 10% skim milk. The process of preparing a neutralizer from different components is usually labor-intensive and materially expensive, and the components, which are chemical substances, do not have registration certificates permitting their use in medical organizations, as a result of which there are difficulties in implementing the method.

The task, The aim of the present invention is to develop an effective, less labor-intensive and more economically acceptable method for assessing the resistance to disinfectants of microorganisms circulating in the hospital environment of medical organizations, in educational institutions, in the production environment of public catering establishments, and in the food and pharmaceutical industries.

Thus, the technical problem solved by the claimed invention consists in the need to overcome the shortcomings inherent in analogues and the prototype by creating a simple and effective method for assessing the resistance of microorganisms to disinfectants in order to promptly eliminate the risks of the spread of resistant forms of microorganisms and ensure the biosafety of the population.

Disclosure of the essence of the invention

The stated problem is solved by a method of assessing the resistance of microorganisms to disinfectants, which consists of performing a sequence of operations, including introducing a suspension of the tested microorganisms onto the inner surface of a well of a flat-bottomed plate, drying the cell suspension, introducing a working solution of the disinfectant into a contaminated well, disinfection holding, introducing neutralizing agar according to Dee-Ingley into the well, holding in a thermostat, and assessing the result based on the presence or absence of microorganism growth.

The technical result achieved by implementing this invention is a new technical solution for performing a research method by using neutralizing agar, combining the properties of a neutralizer and a nutrient medium, using a flat-bottomed well plate as a test object for contamination, performing all stages of the research in a well of the plate without transferring the contents to another container, and also by eliminating a separate neutralization stage by combining the neutralization process with the stage of seeding on a dense nutrient medium.

The technical result is achieved by a method for assessing the resistance of microorganisms to disinfectants, which consists of performing a sequence of operations, including introducing 0.1 ml of a suspension containing 1×10 using a sterile pipette.⁹CFU/ml of the tested microorganism strains, onto the inner surface of the wells of a 12-well flat-bottomed plate, drying the cell suspension, adding a working solution of the appropriate disinfectant used in the organization where the microorganism was isolated to the contaminated wells with a sterile pipette, disinfection holding, adding 4.5 ml of neutralizing agar according to Dee-Ingley to the wells, holding the plate in a thermostat at a temperature of (35±2)°C for 24-48 hours, evaluating the result based on the presence or absence of microbial growth. In this case, the growth of microorganisms is accompanied by a change in the color of the medium from purple to yellow. The obtained result is compared with the control, which consists of wells into which sterile tap water was added instead of a disinfectant. A production isolate or hospital strain of a microorganism is considered resistant to the disinfectant being studied if the experiment is repeated three times at a certain exposure time and gives a positive result (the presence of microbial growth) in the presence of characteristic growth in the control.

The essential features of the claimed method are:

- the use of a flat-bottomed 12-well plate as a test object for contamination, which allows for the simultaneous determination of the resistance of three microorganisms to the effects of one disinfection regime in three replicates under the same conditions;

- the use of universal neutralizing agar according to Di-Ingley to neutralize the disinfectant, which allows combining the processes of neutralization and subsequent sowing on an agarized growth medium;

- carrying out all stages of determining the resistance of microorganisms in the same flat-bottomed 12-well plate, which eliminates the need to transfer the contaminated test object after treatment with a disinfectant and neutralizer to another container with a dense growth medium.

Thus, the claimed method for assessing the resistance of microorganisms to disinfectants is new and is not described in available literary sources. The combination of features provides an optimal technical solution to the task at hand.

The determination of resistance to disinfectants is carried out in relation to microorganisms circulating in the hospital environment of medical organizations, educational institutions, the production environment of public catering establishments, the food and pharmaceutical industries in relation to those disinfectants that are used to disinfect objects in these organizations. In the experiments, the disinfection modes specified in the instructions for use of a specific disinfectant are used, observing all recommendations: concentration, duration of disinfection, processing method, consumption rate, temperature of the working solution.

To detect microorganisms on objects of the production environment (surfaces of process and auxiliary equipment, inventory and containers), swabs are collected at various sections of the production line in accordance with the methodological recommendations MR 4.2.0220-20 "Methods of sanitary and bacteriological research of microbial contamination of environmental objects". Each subsequent sampling should be carried out at the same points.

To identify microorganisms in the production environment of medical organizations, samples are taken in accordance with the sanitary norms and rules SanPiN 3.3686-21 "Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases", methodological guidelines MU 3.1.3798-22 "Ensuring the Epidemiological Safety of Non-Sterile Endoscopic Interventions on the Gastrointestinal Tract and Respiratory Tracts", methodological guidelines MUK 4.2.2942-11 "Methods of Sanitary and Bacteriological Studies of the Environment, Air and Sterility Control in Medical Organizations", etc.

Before determining the resistance of a microorganism to a disinfectant, a preliminary identification of the isolated microorganism to the species level is carried out by any available method (study of the biochemical, antigenic, phagolytic and other properties of the culture, including the use of commercial test systems or using microbiological analyzers, nucleic acid amplification methods (NAAT), time-of-flight mass spectrometry (MALDI-TOF) in accordance with their operational documentation).

The proposed method for assessing the resistance of microorganisms to disinfectants, proposed for use in microbiological laboratories, provides for the sequential implementation of preparatory stage operations, the performance of the experiment itself, and the recording and analysis of its results.

At the preparatory stage, the isolated strain of microorganism is seeded and grown on the appropriate nutrient medium. Bacteria are cultivated on tryptone soy agar, meat-peptone agar or other nutrient media intended for cultivating certain types of bacteria at a temperature of (35-37)°C for 18-24 hours.

To prepare a suspension of the microorganism, the culture is washed off the agar with sterile saline or sterile tap (drinking) water. The resulting suspension of microorganisms is then diluted with sterile saline or sterile tap water to a concentration of 1×10 ⁹ cells in 1 ml, corresponding to a turbidity of 3.3-3.5 McFarland standard units, determined using a densitometer.

The working solution of disinfectants is prepared immediately before conducting the tests on sterile drinking water at room temperature. A measured amount of the disinfectant concentrate is added to the appropriate container, water is added, mixed and closed with a lid. Work with the solution begins after the agent has completely dissolved.

The study uses sterile 12-well flat-bottomed plates. Using a sterile pipette, 0.1 ml of the microorganism suspension is added to the inner surface of the wells of the plate. Then, they are dried for 45-60 minutes at room temperature (22.0±2.0)°C and room air humidity of 40-60%. After drying, 0.5 ml of the working solution of the disinfectant under study, prepared in accordance with the instructions for use, is added to the wells of the first three rows No. 1-3 (test wells) using a sterile pipette, after which the exposure time is maintained according to the tested disinfection mode. In this case, 3 modes can be studied simultaneously: a separate disinfection mode in each row of wells. Since one row of a 12-well plate contains 3 wells, each mode will be tested three times. After the exposure time has elapsed, 4.5 ml of melted and cooled to 45°C Dee-Ingley agar is added to all wells of the plate, including row #4 (control wells), using a sterile pipette. The 12-well plate with the cultures is then placed in a thermostat and incubated at a temperature of (35±2)°C for 24-48 hours. The results are assessed by the presence or absence of microorganism growth. The comparison is made with the control, which is the cultures of microorganisms in the wells of row #4 without treatment with a disinfectant.

Microorganisms are considered sensitive to the effects of the tested disinfectant regimes if there is no characteristic growth of colony-forming units in the test wells in a three-fold repeated experiment and there is a characteristic growth of microorganism colonies in the control wells (similarly contaminated wells, where 0.5 ml of sterile tap water was added instead of a disinfectant solution).

Microorganisms are considered resistant (stable) to the effects of the tested disinfectant regimens if there is a characteristic growth of colony-forming units in the experimental and control wells in a three-fold repeated experiment.

Thus, the essence of the claimed invention, concerning the method for assessing the resistance of microorganisms to disinfectants, consists in using a flat-bottomed 12-well plate as a test object for contamination, treatment with a disinfectant and a neutralizer without transferring to another container, and also in the fact that in order to simplify the process and increase the reliability of the result obtained, universal neutralizing agar according to Dee-Ingley is used to neutralize the disinfectant, which makes it possible to combine the processes of neutralization and subsequent seeding on an agarized growth medium.

The advantages of the claimed method according to the present invention are:

- simplification of the procedure for assessing the resistance of microorganisms;

- elimination of the process of taking samples of various reagents when preparing a solution of a universal neutralizer;

- elimination of the disinfectant neutralization procedure;

- reduction of the duration of the procedure for determining the resistance of microorganisms by combining the stages of neutralization and sowing;

- reduction of the volume of the nutrient medium by 4.4 times due to the use of a flat-bottomed plate instead of Petri dishes;

- significant reduction in the amount of disinfectant and neutralizing agar according to Dee-Ingley due to the use of a well plate;

- suitability of neutralizing agar according to Dee-Ingley for disinfectants of any chemical groups;

- simplification of the process of interpreting the results, which is due to a change in the color of the neutralizing agar when the growth of microorganism colonies appears (there is a change in the color of the medium from purple to yellow, which is due to the fact that microorganisms fermenting dextrose color the medium yellow using the bromocresol red indicator);

- high productivity and reduced labor costs, which is due to the increase in the number of simultaneously tested modes and repetitions when studying one mode in three repetitions (Table 1);

- the possibility of conducting a simultaneous test in one 12-well plate of three modes in three replicates on one microorganism culture, or studying the resistance of three microorganisms to the effects of one disinfection mode in three replicates under the same conditions;

- significant reduction of material and financial costs.

Table 1 provides for comparison the stages of assessing the resistance of microorganisms to disinfectants using the known prototype method (RU 2650760, 2018) and the claimed method according to the present invention.

Table 1. Stages of assessing the sensitivity of microorganisms to disinfectants using the known method (RU 2650760, 2018) and the claimed method according to the present invention

Name of the stage and its procedure Performing procedures* RU 2650760 The claimed method 1. Preparatory: 1.1. Preparation of microorganism culture, preparation of suspension + + 1.2. Taking 6 samples for preparing a universal neutralizer + - 1.3. Preparation of the neutralizer + - 1.4. Preparation of nutrient medium + + 1.5. Preparation of disinfectant solutions + + 1.6. Control of completeness of neutralization + - 2. Study of microorganism resistance to disinfectants: 2.1. Contamination of surfaces with a microorganism suspension Petri dishes, diameter 9 cm 12 well plate 2.2. Adding a disinfectant solution + + 2.3. Exposure + + 2.4. Neutralization + - ** 2.5. Adding agar + + Volume of nutrient medium for testing one mode in one replicate, ml 20 ml 4.5 ml 3. Incubation at a temperature of (35±2)°C for 24-48 hours + + 4. Accounting and interpretation of results + + 4.1. Growth indication by change in the color of the medium - + 4.2. Number of simultaneously tested modes 1 3 4.3. Number of simultaneously set repetitions 1 3 Note: * - executed ( + ); not executed ( - );
**- the neutralization process is combined with the sowing procedure

The results of determining the completeness of neutralization of residual quantities of active substances of disinfectants using a universal neutralizer (RU 2650760, 2018) containing tween-80 (3%), saponin (0.3-3%), histidine (0.1%) and cysteine (0.1%), and neutralizing agar according to Dee-Ingley (the claimed method according to the present invention) in the processes of assessing the resistance of microorganisms are presented for comparison using the example of test microorganisms Escherichia coli ATCC 10536 (synonym Escherichia coli VKPM B-11659) and Staphylococcus aureus ATCC 6538P (synonym Staphylococcus aureus VKPM B-6646) in Table 2. Alkyl dimethyl benzyl ammonium chloride (ADBAC), glutaraldehyde (GA), sodium dichloroisocyanuric acid (DCC) and hydrogen peroxide (HP).

Table 2. Results of determining the completeness of neutralization of residual quantities of active substances of disinfectants using a universal neutralizer (RU 2650760, 2018) and neutralizing agar according to Dee-Ingley (the claimed method according to the present invention) in relation to test strains of Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 6538P

Operation Research Name of the method (implementation procedure) Quantity S. aureus ATCC 6538P (CFU) grown on nutrient medium (M±m) **, after exposure to DS Quantity E. coli ATCC 10536 (CFU) grown on nutrient medium (M±m) **, after exposure to DS ADBAKH, 0.02% GA,
0.005% DHCK,
0.015% PV,
1.0% ADBAKH, 0.02% GA,
0.005% DHCK,
0.015% PV,
1.0% Control 1 destructive action of DS (to 9 ml of suspension (10³CFU/ml) + 1 ml of DS solution, without adding a neutralizer) RU 2650760 0 0 0 0 0 0 0 0 The claimed method Not applicable, since the process of seeding the microorganism on the nutrient medium and the neutralization process are combined Not applicable, since the process of seeding the microorganism on the nutrient medium and the neutralization process are combined Control 2 completeness of neutralization of DS RU 2650760
(to 9 ml of suspension (10 ³ CFU/ml) on a neutralizer + 1 ml of disinfectant solution) 98.0±3.4 39.0±0.95 40.9±0.88 99.6±0.98 92.0±3.4 49.0±0.95 39.9±0.88 29.6±0.98 The claimed method
(to the bottom of a 12-well plate +0.1 ml suspension (10 ³ CFU/ml) + 0.5 ml DS + 4.5 ml Di-Ingly) 99.1±2.3 65.3±0.8 77.5±0.85 99.4±1.2 98.1±2.3 65.3±0.8 50.5±0.85 38.4±1.2 Control 3 lack of antimicrobial effect of the neutralizer RU 2650760
(to 9 ml of suspension (10 ³ CFU/ml) on a neutralizer + 1 ml of neutralizer solution) 97.0±2.39 89.3±0.9 95.4±0.95 91.9±1.2 91.0±2.39 92.4±3.6 90.4±0.95 89.9±1.2 The claimed method
(to the bottom of a 12-well plate + 0.1 ml suspension (10 ³ CFU/ml) + 4.5 ml Di-Ingly) 99.4±1.6 98.4±3.6 99.4±4.3 99.8±3.3 96.4±1.6 100.3±0.9 99.4±4.3 99.4±3.3 Control 4 number of bacteria RU 2650760
(to 9 ml of suspension (10 ³ CFU/ml) per active ingredient* + 1 ml active ingredient) 98.7±6.0 92.7±5.4 93.7±2.3 94.7±3.5 89.7±6.0 92.7±5.4 90.7±2.3 94.7±3.5 The claimed method
(same as in control 3) 99.8±0.9 99.4±2.4 98.4±4.3 99.9±3.3 96.4±1.6 99.4±3.6 98.4±4.3 99.4±3.3 Note: * - a.i. - distilled water;
** p<0.05 - differences are statistically significant when comparing the growth intensity of strains in controls 1 (only for method RU 2650760) and 2 with the growth intensity in controls 3 and 4 in all experiments

As can be seen from the data in Table 2, the neutralizing capacity of agar according to Dee-Ingley in most samples does not differ from the completeness of neutralization of the known method (p>0.05), and in a number of samples it demonstrates greater effectiveness (for example, in the control of neutralization of GA and DCCK both when usingE. coliATCC 10536, and when usedS. aureusATCC 6538P (p<0.05)).

In addition, the possibility of excluding from the method for monitoring the completeness of neutralization such operations as control 1 (the destructive effect of the neutralizer) and control 4 (the number of bacteria), since the procedure for monitoring the number of bacteria is identical to the procedure for control 3 (the absence of an antimicrobial effect of the neutralizer), demonstrates the obvious advantages of the claimed method, concerning the reduction in the number of procedures and, as a consequence, the reduction in the consumption of reagents, other consumables, labor costs and the reduction in the duration of the studies.

Results of a comparative study of the sensitivity of test microorganismsS. aureusATCC 6538Pand E. coliATCC 10536 to ADBAC (0.02%), GA (0.05%), PV (2%) and DHCC 0.015% using neutralizing agar according to Di-Ingley, carried out as part of the validation of the methodology for the claimed method for assessing the resistance of microorganisms to DS, are presented in Table 3.

Table 3.Results of comparative tests to assess the resistance of test microorganismsS. aureusATCC 6538Pand E. coliATCC 10536 to DS using neutralizing agar according to Dee-Ingley

Validation parameters Acceptance Criteria Acceptable values of the criterion Results obtained Precision Coefficient of variation (CV) ≤35% 3.1-15.6% Right Percentage of microorganism recovery >70% 75-95% Limit of quantification Coefficient of variation (CV) ≤35% 4.4-25%

As follows from Table 3, during the validation of the claimed method for assessing the resistance of the test microorganisms S. aureus ATCC 6538P and E. coli ATCC 10536 to disinfectants, no deviations of the test results from the established acceptance criteria were found. This proves the possibility of using the neutralizing agar according to Dee-Ingley as a neutralizer in assessing the resistance of microorganisms to disinfectants. Based on the obtained results, it was established that the neutralization of active substances from various chemical groups when using the neutralizing agar according to Dee-Ingley does not affect the percentage of microorganism recovery for samples inoculated with microorganisms in an amount of 10 ³ CFU/ml, which confirms the suitability of the nutrient agar with neutralizing components in terms of the "resistance" parameter. These data also indicate the effectiveness of neutralization by the nutrient agar with neutralizing components for assessing the effectiveness of combined DS. In addition, the use of neutralizing agar according to Di-Ingley allows to exclude the stage of double washing of microorganisms from DS residues and to use the medium for non-selective enrichment of microorganisms, which allows to reduce the labor intensity of the method. The use of sterile 12-well flat-bottomed plates for the implementation of the procedure for assessing the resistance of microorganisms to disinfectants allows to reduce the consumption of the nutrient medium by 4.4 times, i.e. from 20 ml when using Petri dishes to 4.5 ml, and also to conduct a simultaneous study of three modes in three replicates, or simultaneously study the resistance of three strains to one disinfection mode.

The claimed invention is illustrated by the examples below of a specific implementation of a method for assessing the resistance of microorganisms to a disinfectant.

The results of determining the resistance of microorganisms to disinfectants by the claimed method according to the present invention are given to illustrate the effectiveness of the proposed method. However, the present invention is not limited to these specific embodiments of its implementation, and a specialist in this field of technology can make various changes and modifications to it without going beyond the scope or essence of the invention, as defined in the attached claims.

Implementation of the invention

The strains Escherichia coli ATCC 10536 (sin. Escherichia coli VKPM B-11659), Staphylococcus aureus ATCC 6538P (sin. Staphylococcus aureus VKPM B-6646), as well as Pseudomonas aeruginosa ATCC 15442, used to evaluate the bactericidal activity of disinfectants in accordance with Guide No. P 4.2.3676-20 "Methods of laboratory research and testing of disinfectants to evaluate their effectiveness and safety", and Enterococcus faecalis ATCC 29212 were used as control test microorganisms.

Neutralizing agar according to Dee-Ingley, as well as flat-bottomed 12-well culture plates with lids, are commercially available and can be purchased, for example, from HiMedia ( India).

A solution is used as a disinfectant (DS) "Ultradiz-Forte", "Divosan Forte" solution, "NIKA-2" solution, "Ph disinfectant" solution and "Chlorexel" disinfectant tablets. DS "Ultradiz-Forte" with a cleaning effect manufactured by OOO "Biosfera" (Russia) contains as active substances (AS) quaternary ammonium compounds (QAC) alkyl dimethyl benzyl ammonium chloride (ADBAC), 4.5%, polyhexamethylene biguanide hydrochloride (PHMB), 3.5%, as well as surfactants. DS "Divosan Forte" is a transparent colorless liquid with a sharp smell of acetic acid. DS "Divosan Forte" manufactured by OOO "Daiverse" (Russia) contains 10-15% peracetic acid (PAA) and 17-27% hydrogen peroxide (HP) as active ingredients. DS "NIKA-2" with a cleaning effect manufactured by OOO NPF "Genix" (Russia) contains 12% alkaline components (in terms of sodium hydroxide) and 1% alkyl dimethyl benzyl ammonium chloride as active ingredients. "Ph disinfectant" manufactured in Russia contains a complex of QAC andN,N-bis(3-aminopropyl)dodecylamine. Soluble disinfectant tablets "Chlorexel" produced by Felicata Holding LLC (Russia) contain sodium salt of dichloroisocyanuric acid (99.5%) as an active ingredient.

Drinking water means tap water supplied to subscribers using water supply systems, the quality of which meets the requirements of SanPiN 2.1.3684-21.

All procedures, unless otherwise stated, are carried out at room temperature or ambient temperature, i.e. in the range from 20 to 24°C.

Example 1. Evaluation of resistance of hospital microorganisms to the disinfectant "Ultradez-Forte"

Bacteria were isolated and identified from the surface of the handwashing tap in ward No. 2 in the therapeutic department of a general medical institutionStaphylococcus aureus3 andPseudomonas aeruginosa7. A study of the resistance of isolates was conducted in the microbiological laboratoryS. aureus3 andP. aeruginosa7 to the disinfectant "Ultradiz-Forte" used in this department of the medical organization. According to "Instructions No. 8 for the use of the disinfectant with a cleaning effect "Ultradiz-Forte" (hereinafter referred to as the Instructions), the agent is recommended for use in bacterial infections in a working solution concentration of 0.1% with an exposure time of 60 minutes.

Conducting an experiment

1. Preparation of a working suspension of microorganisms S. aureus 3 and P. aeruginosa 7

The P. aeruginosa strain ATCC 15442 used for assessing the bactericidal activity of disinfectants in accordance with Guideline R 4.2.3676-20 “Methods of laboratory research and testing of disinfectants to assess their effectiveness and safety” was used as a control test microorganism.

Working suspensions of microorganism culturesS. aureus3,P. aeruginosa7 andP. aeruginosaATCC 15442 were prepared from cultures of these microorganisms grown on a dense medium at a temperature of 37°C for 24 hours. To prepare a microbial suspension of each type, several identical colonies of microorganisms were selected with a loop, then a small amount of material was transferred to a test tube with sterile saline, bringing the density of the suspension to 3.3-3.5 according to the McFarland standard, which corresponds to 1×10⁹CFU/ml. The density of the suspension was measured with a densitometer.

2. Preparation of a working solution of the disinfectant "Ultradiz-Forte"

The working solution of DS "Ultradez-Forte" was prepared immediately before the study using sterile tap water at room temperature in a bactericidal concentration of 0.1% in accordance with the Instructions.

3. Addition of the suspension of the studied clinical isolates into the wells of a 12-well flat-bottomed plate

0.1 ml of microbial suspension of each type of microorganism was added to the wells of a sterile 12-well flat-bottomed plate using a sterile pipette. Isolate was added to wells A1-A4.S. aureus3, isolate was added to wells B1-B4P. aeruginosa7, in wells C1-C4 were added control test microorganismP. aeruginosaATC 15442.Next The microbial suspension of each microorganism was dried for 60 min at room temperature.

4. Adding a working solution of disinfectant to the wells

After drying the microbial suspension, 0.5 ml of 0.1% working solution of the disinfectant was added to wells A1-A3, B1-B3, C1-C3. The disinfection holding time of the disinfectant was 60 minutes according to the Instructions. 0.5 ml of sterile tap water was added to control wells A4, B4, C4 using a sterile pipette.

5. Adding neutralizing agar according to Dee-Ingley into the wells

After completion of the exposure, 4.5 ml of melted and cooled to 45°C neutralizing agar according to Dee-Ingley were added to all wells. Then, after the agar cooled, the plate with the cultures was placed in a thermostat at a temperature of 37°C for incubation.

6. Accounting for results

After 24 hours of incubation of the crops, the results were recorded for the presence or absence of microorganism growth. The results are presented in Table 4.

Table 4. Results of the assessment of the resistance of microorganisms isolated from the surface of the handwashing tap in ward No. 2 in the therapeutic department of a general medical institution to a 0.1% solution of the disinfectant "Ultradiz-Forte" with an exposure of 60 minutes

Microorganism Presence/absence of growth of microorganism colonies in wells 1 A-B-C 2 A-B-C 3 A-B-C 4 A-B-C S. aureus 3 No growth No growth No growth Height P. aeruginosa 7 Height Height Height Height P. aeruginosa ATCC 15442 (control test microorganism) No growth No growth No growth Height

As can be seen from the data presented in Table 4, the isolateP. aeruginosa7 is resistant to the effects of 0.1% working solution of the disinfectant "Ultradiz-Forte" with an exposure of 60 minutes, isolateS. aureus3 and test microorganismP. aeruginosaATC 15442 are sensitive to the effects 0.1% working solution of the disinfectant "Ultradiz-Forte" with an exposure of 60 minutes.

Example No. 2. Evaluation of the resistance of microorganisms isolated from the surfaces of the internal environment in a school institution to the disinfectant "NIKA-2"

Bacteria isolated and identified from surface of desk at schoolStaphylococcus haemolyticus And Bacteria were isolated and identified from the surface of the door handleStaphylococcus epidermidis. The microbiological laboratory conducted a determination of the resistance of isolatesS. haemolyticus And S. epidermidisto the disinfectant "NIKA-2", which is used for preventive disinfection and washing of surfaces in the premises of a school institution.

Working solutions of the disinfectant were prepared according to the manufacturer's "Instructions No. 112 for the use of the disinfectant with a cleaning effect "NIKA-2" by adding a calculated amount of the agent taken with a pipette to tap water and then mixing the solution. The resistance of S. haemolyticus and S. epidermidis isolates was determined by exposure to working solutions of "NIKA-2" at a concentration of 2% of the preparation with a disinfection exposure time of 60 minutes. For comparison, the control test strain Staphylococcus aureus АТСС 6538P was used.

Conducting an experiment

1. Preparation of a working suspension of microorganisms

The working suspension of S. haemolyticus , S. epidermidis and S. aureus ATCC 6538P bacterial cultures was prepared from a daily culture of these microorganisms grown on a dense nutrient medium at a temperature of 37°C for 24 hours. To prepare a microbial suspension of each type, several identical colonies of microorganisms were selected with a loop, then a small amount of material was transferred to a test tube with sterile physiological solution, bringing the suspension density to 3.3-3.5 according to the McFarland standard, which corresponds to 1×10 ⁹ CFU/ml. The suspension density was measured with a densitometer.

2. Preparation of a working solution of the disinfectant "NIKA-2"

The working solution of the disinfectant was prepared using sterile tap water at a bactericidal concentration of 2.0% of the preparation in accordance with “Instructions No. 112 for the use of the product with a cleaning effect “NIKA-2”.

3. Adding a bacterial culture suspension to the wells of a 12-well flat-bottomed plate

0.1 ml of microbial suspension of each type of microorganism was added to the bottom of the wells of a sterile 12-well flat-bottomed plate using a sterile pipette. Isolate was added to wells A1-A4.S. haemolyticus, in wells B1-B4 were added isolateS. epidermidisthe control reference strain was added to wells C1-C4S. aureusATCC 6538P, then the microbial suspension of each microorganism was dried for 60 min at room temperature.

4. Adding a working solution of disinfectant to the wells

After drying the microbial suspension, 0.5 ml of a 2% working solution of the disinfectant was added to wells A1-A3, B1-B3, C1-C3. The disinfection exposure time of the disinfectant was 60 minutes (the exposure time is specified in "Instructions No. 112 for the use of the disinfectant with a cleaning effect "NIKA-2"). 0.5 ml of sterile tap water was added to control wells A4, B4, C4 using a sterile pipette.

5. Adding neutralizing agar according to Dee-Ingley into the wells

After completion of the exposure, 4.5 ml of melted and cooled to a temperature of (47±2)°C neutralizing agar according to Dee-Ingley were added to the wells, then after the agar cooled, the 12-well plate was placed in a thermostat at a temperature of 37°C for incubation.

6. Accounting for results

After 24 hours of incubation of the crops, the results were recorded for the presence or absence of microorganism growth. The results are presented in Table 5.

Table 5. Results of the assessment of the resistance of microorganisms isolated from surfaces in the premises of a school institution to a 2% solution of the disinfectant "NIKA-2" with an exposure of 60 minutes

Microorganism Presence/absence of growth of microorganism colonies in wells 1 A-B-C 2 A-B-C 3 A-B-C 4 A-B-C S. haemolyticus No growth No growth No growth Height S. epidermidis No growth No growth No growth Height S. aureus ATCC 6538P No growth No growth No growth Height

As can be seen from the data presented in Table 5, isolates of S. haemolyticus and S. epidermidis are sensitive to the effects of a 2% working solution of the disinfectant "NIKA-2" with an exposure of 60 minutes.

Example No. 3. Evaluation of the resistance of microorganisms isolated at a public catering establishment to the disinfectant "Ph"

Escherichia coli bacteria were isolated and identified from the surfaces of distribution tables at a public catering establishment. The microbiological laboratory determined the resistance of the E. coli isolate to the disinfectant "Ph" used at the establishment, which is used to disinfect the outer surfaces of kitchen equipment, cutting and distribution tables, etc.

Working solutions of the disinfectant were prepared according to "Instructions No. 1/22 for the use of the disinfectant "Ph" in children's organizations, public catering establishments and in organizations in the field of public services" (hereinafter - Instructions 1/22) by adding the appropriate amount of the agent to tap water at room temperature. The resistance of the E. coli isolate was determined by exposure to working solutions of the "Ph" agent at a concentration of 0.5% of the preparation and with a disinfection exposure time of 15 minutes. For comparison, the control test strain Escherichia coli ATCC 10536 was used.

Conducting an experiment

1. Preparation of a working suspension of microorganisms

The working suspension of E. coli bacterial cultures and the reference strain E. coli ATCC 10536 was prepared from a daily culture of these microorganisms grown on a dense nutrient medium at a temperature of 37°C for 24 hours. To prepare a microbial suspension of each type, several identical colonies of microorganisms were selected with a loop, then a small amount of material was transferred to a test tube with sterile physiological solution, bringing the suspension density to 3.3-3.5 according to the McFarland standard, which corresponds to 1×10 ⁹ CFU/ml. The suspension density was measured with a densitometer.

2. Preparation of a working solution of the disinfectant "Ph"

The working solution of the disinfectant was prepared using tap water at a concentration of 0.5% of the preparation in accordance with the manufacturer's Instructions 1/22 for the use of the disinfectant "Ph".

3. Adding a bacterial culture suspension to the wells of a 12-well flat-bottomed plate

0.1 ml of microbial suspension of each type of microorganism was added to the bottom of the wells of a sterile 12-well flat-bottomed plate using a sterile pipette. Isolate was added to wells A1-A4.E. coli, the control reference strain was added to wells B1-B4E. coliATCC 10536, hereinafter The microbial suspension of each microorganism was dried for 60 min at room temperature.

4. Adding a working solution of disinfectant to the wells

After drying the microbial suspension, 0.5 ml of 0.5% of the preparation of the working solution of the disinfectant was added to wells A1-A3, B1-B3. The disinfection exposure time of the disinfectant was 15 minutes (disinfection modes (concentration of the working solution and exposure) are specified in the instructions for use of the disinfectant "Ph"). 0.5 ml of sterile tap water was added to control wells A4, B4 using a sterile pipette.

5. Adding neutralizing agar according to Dee-Ingley into the wells

After completion of the exposure, 4.5 ml of melted and cooled to a temperature of (47±2)°C neutralizing agar according to Dee-Ingley were added to the wells, then after the agar cooled, the 12-well plate was placed in a thermostat at a temperature of 37°C for incubation.

6. Accounting for results

After 24 hours of incubation of the crops, the results were recorded for the presence or absence of microorganism growth. The results are presented in Table 6.

Table 6. Results of the assessment of the resistance of the Escherichia coli microorganism isolated at a public catering establishment to a 0.5% solution of the disinfectant “Ph” with an exposure of 15 minutes

Microorganism Presence/absence of growth of microorganism colonies in wells 1 A-B-C 2 A-B-C 3 A-B-C 4 A-B-C Escherichia coli Height Height Height Height Escherichia coli ATCC 10536 No growth No growth No growth Height

As can be seen from the data presented in Table 6, the Escherichia coli isolate, isolated from the surface of the distribution table at a public catering establishment, is resistant to the effects of 0.5% of the working solution of the disinfectant “Ph” with an exposure of 15 minutes, the reference strain Escherichia coli ATCC 10536 is sensitive to the effects of 0.5% of the working solution of the disinfectant “Ph” with an exposure of 15 minutes.

Example No. 4. Evaluation of the resistance of microorganisms isolated from the surfaces of technological equipment at a meat processing plant to the disinfectant "Divosan Forte"

Pseudomonas fragi and Enterococcus faecalis bacteria were isolated and identified from the surfaces of process equipment at a meat processing plant. The microbiological laboratory determined the resistance of P. fragi and E. faecalis isolates to the disinfectant "Divosan Forte", which is used to disinfect process equipment at the plant.

Working solutions of the disinfectant were prepared according to the manufacturer's instructions No. 3/11 for the use of Divosan Forte in meat industry enterprises by adding a calculated amount of the agent taken with a pipette to tap water and then mixing the solution. The resistance of the isolates of microorganisms isolated from the surfaces of process equipment was determined by exposure to working solutions of Divosan Forte at a concentration of 0.03% NUK with a disinfection exposure time of 20 minutes. For comparison, the control test strain Enterococcus faecalis АТСС 29212 was used.

Conducting an experiment

1. Preparation of a working suspension of microorganisms

The working suspension of bacterial cultures P. fragi , E. faecalis and E. faecalis ATCC 29212 was prepared from a daily culture of these microorganisms grown on a dense nutrient medium at a temperature of 37°C for 24 hours. To prepare a microbial suspension of each type, several identical colonies of microorganisms were selected with a loop, then a small amount of material was transferred to a test tube with sterile physiological solution, bringing the suspension density to 3.3-3.5 according to the McFarland standard, which corresponds to 1×10 ⁹ CFU/ml. The suspension density was measured with a densitometer.

2. Preparation of a working solution of the disinfectant "Divosan Forte"

The working solution of the disinfectant was prepared using sterile tap water at a bactericidal concentration of 0.03% NUK in accordance with the manufacturer's Instructions 3/11 for use of the product.

3. Adding a bacterial culture suspension to the wells of a 12-well flat-bottomed plate

0.1 ml of microbial suspension of each type of microorganism was added to the bottom of the wells of a sterile 12-well flat-bottomed plate using a sterile pipette. Isolate was added to wells A1-A4.P. fragi, isolate was added to wells B1-B4E. faecalis, in wells C1-C4 were added control reference strainE. faecalisATCC 29212,further The microbial suspension of each microorganism was dried for 60 min at room temperature.

4. Adding a working solution of disinfectant to the wells

After drying the microbial suspension, 0.5 ml of 0.03% working solution of the disinfectant was added to wells A1-A3, B1-B3, C1-C3. The disinfection exposure time of the disinfectant was 20 minutes (the exposure time is specified in the manufacturer's instructions 3/11 for the use of DS "Divosan Forte"). 0.5 ml of sterile tap water was added to control wells A4, B4, C4 using a sterile pipette.

5. Adding neutralizing agar according to Dee-Ingley into the wells

After completion of the exposure, 4.5 ml of melted and cooled to a temperature of (47±2)°C neutralizing agar according to Dee-Ingley were added to the wells, then after the agar cooled, the 12-well plate was placed in a thermostat at a temperature of 37°C for incubation.

6. Accounting for results

After 24 hours of incubation of the crops, the results were recorded for the presence or absence of microorganism growth. The results are presented in Table 7.

Table 7. Results of the assessment of the resistance of microorganisms isolated from the surfaces of technological equipment at a meat processing plant to a 0.03% solution of the disinfectant "Divosan Forte" with an exposure of 20 minutes

Microorganism Presence/absence of growth of microorganism colonies in wells 1 A-B-C 2 A-B-C 3 A-B-C 4 A-B-C E. faecalis No growth No growth No growth Height P. fragi Height Height Height Height E. faecalis ATCC 29212 No growth No growth No growth Height

As can be seen from the data presented in Table 7, the isolateP. fragiis resistant to the effects of 0.03% working solution of the disinfectant "Divosan Forte" with an exposure of 20 minutes, isolateE. faecalisand reference strainE. faecalisATCC 29212 are sensitive to the effects of 0.03% working solution of the disinfectant "Divosan Forte" with an exposure of 20 minutes.

Example No. 5. Evaluation of the resistance of microorganisms isolated from the surfaces of production facilities at a pharmaceutical plant to the disinfectant "Chlorexel"

Staphylococcus aureus and Micrococcus spp bacteria were isolated and identified from the surfaces of production facilities at a pharmaceutical plant. The microbiological laboratory determined the resistance of Staphylococcus aureus and Micrococcus spp isolates to the disinfectant "Chlorexel" used at the plant, which is used to disinfect surfaces of production facilities.

Working solutions of the disinfectant were prepared according to the manufacturer's instructions No. 01/15 for the use of the disinfectant "Chlorexel" by dissolving the appropriate number of tablets in tap water at room temperature until they were completely dissolved. The resistance of S. aureus and Micrococcus spp . isolates was determined by exposure to working solutions of "Chlorexel" at a concentration of 0.03% active chlorine and with a disinfection exposure time of 30 minutes. The control test strain Staphylococcus aureus ATCC 6538P was used for comparison.

Conducting an experiment

1. Preparation of a working suspension of microorganisms

Working suspensions of bacterial cultures S. aureus , Micrococcus spp . and S. aureus АТСС 6538P were prepared from daily cultures of these microorganisms grown on a dense nutrient medium at a temperature of 37°C for 24 hours. To prepare a microbial suspension of each type, several identical colonies of microorganisms were selected with a loop, then a small amount of material was transferred to a test tube with sterile physiological solution, bringing the suspension density to 3.3-3.5 according to the McFarland standard, which corresponds to 1×10 ⁹ CFU/ml. The suspension density was measured with a densitometer.

2. Preparation of a working solution of the disinfectant "Chlorexel"

The working solution of the disinfectant was prepared using tap water at a concentration of 0.03% active chlorine in accordance with the manufacturer's Instructions No. 01/15 for the use of the disinfectant "Chlorexel".

3. Adding bacterial culture suspensions to the wells of a 12-well flat-bottomed plate

0.1 ml of microbial suspension of each type of microorganism was added to the bottom of the wells of a sterile 12-well flat-bottomed plate using a sterile pipette. Isolate was added to wells A1-A4.S. aureus, in wells B1-B4 were added isolateMicrococcus spp., the control reference strain was added to wells C1-C4S. aureusATCC 6538P,further The microbial suspension of each microorganism was dried for 60 min at room temperature.

4. Adding a working solution of disinfectant to the wells

After drying the microbial suspension, 0.5 ml of 0.03% active chlorine working solution of the disinfectant was added to wells A1-A3, B1-B3, C1-C3. The disinfection exposure time of the disinfectant was 30 minutes (disinfection modes (concentration of the working solution and exposure) are specified in Instructions No. 01/15 for the use of the disinfectant "Chlorexel"). 0.5 ml of sterile tap water was added to control wells A4, B4, C4 using a sterile pipette.

5. Adding neutralizing agar according to Dee-Ingley into the wells

After completion of the exposure, 4.5 ml of melted and cooled to a temperature of (47±2)°C neutralizing agar according to Dee-Ingley were added to the wells, then after the agar cooled, the 12-well plate was placed in a thermostat at a temperature of 37°C for incubation.

6. Accounting for results

After 24 hours of incubation of the crops, the results were recorded for the presence or absence of microorganism growth. The results are presented in Table 8.

Table 8. Results of the assessment of the resistance of microorganisms isolated from surfaces in the premises of a school institution to a 2% solution of the disinfectant "Chlorexel" with an exposure of 60 minutes

Microorganism Presence/absence of growth of microorganism colonies in wells 1 A-B-C 2 A-B-C 3 A-B-C 4 A-B-C S. aureus Height Height Height Height Micrococcus spp. No growth No growth No growth Height S. aureus ATCC 6538P No growth No growth No growth Height

As can be seen from the data presented in Table 8, the isolateS. aureus,isolated from the surfaces of production facilities at a pharmaceutical plant is resistant to the effects of the working solution of the disinfectant "Chlorexel" (0.03% active chlorine) with an exposure of 30 minutes, the isolateMicrococcus spp. and reference strainS. aureusATCC 6538P are sensitive to the effects working solution of the disinfectant "Chlorexel" (0.03% active chlorine) with an exposure of 20 minutes.

Thus, the use of the method proposed by the present invention allows for the most reliable and accurate assessment with minimal time and cost of the effectiveness of disinfectants used to disinfect objects in the production environment using known and available preparations, nutrient media, technical equipment and devices, as well as more effective monitoring of the resistance of microorganisms to disinfectants in medical organizations, educational institutions, public catering establishments, and the food and pharmaceutical industries.

Claims (2)

1. A method for assessing the resistance of microorganisms to disinfectants, including the sequential application of 0.1 ml of a microbial suspension of bacteria containing 1×10 ⁹ CFU/ml to the inner surface of the wells of a sterile 12-well flat-bottomed plate; drying; adding 0.5 ml of a disinfectant solution to the wells; after the action time of the disinfectant solution has elapsed, adding 4.5 ml of melted and cooled to 47±2°C neutralizing agar according to Dee-Ingley; incubating the crops at a temperature of 35±2°C for 24-48 hours; recording and interpreting the results by comparing the number of grown colonies of microorganisms in the experimental and control wells, into which sterile tap water was added instead of a disinfectant; Moreover, the presence of growth of characteristic colonies of the microorganism in the experimental and control wells indicates the resistance of the microorganism to the disinfectant, and the absence of growth of colonies of the microorganism in the experimental wells and the presence of growth in the control wells indicates the sensitivity of the microorganism to the disinfectant. 2. The method according to paragraph 1, characterized in that microorganisms are isolated from the surfaces of objects.