RU2802662C1 - Method for pseudomonas aeruginosa biofilm destruction by a combination of ozone with hydrogen peroxide - Google Patents

Abstract

FIELD: destruction of biofilms.

SUBSTANCE: invention relates to a method for the destruction of Pseudomonas aeruginosa biofilms with a combination of ozone and hydrogen peroxide, which consists in acting on biofilms formed by bacteria in polystyrene plates with a combination of ozone simultaneously in combination with a 3% hydrogen peroxide solution in a disinfection chamber with an ozone output of at least 15 g/h and exposure time of 120 minutes.

EFFECT: invention provides destruction of Pseudomonas aeruginosa biofilm obtained in the wells of polystyrene plates by treatment with a combination of ozone and hydrogen peroxide.

1 cl, 2 ex, 1 dwg

Description

The invention relates to methods for disinfection or sterilization of materials or objects, in particular to the field of disinfection of medical devices.

State of the art

Most microorganisms exist naturally in the form of supracellular structures referred to as “biofilms.” They form on various types of surfaces, including metal, plastic, silicone, polyvinyl chloride and other materials that are widespread in hospital environments. This form of coexistence of microorganisms has its own laws of interaction [2]. As a result, their resistance to environmental factors, including disinfectants and antibiotics, increases hundreds and even thousands of times, which makes it difficult to combat them, especially on medical products with a complex configuration [5, 12, 14].

One of the most important and common causative agents of nosocomial infections is Pseudomonas aeruginosa, and this microorganism has the highest percentage of film formation [3, 6].

To date, there are many methods of combating biofilms, among the most common is the use of various enzymes. Nevertheless, the search for the most effective agents and compositions that cause the destruction of biofilms remains relevant and promising.

Ozonation is an environmentally friendly and effective technology in the fight against microbial pollution, including that represented by biofilms [1, 9, 11, 13]. As a result of the action of ozone, not only the death of planktonic cells occurs, but also the destruction of the biofilm itself [4]. It is known that humidification or the combined use of ozone with liquids potentiates the destructive effect on bacteria [1].

As a result of the destruction of the matrix structure of the biofilm by ozone and its spatial disorganization, direct access of disinfectants to the bacterial cells of the biofilm opens, which in turn can demonstrate an increase in the efficiency of disinfection with this combination.

Analogs

There is a known method of treating a culture of Staphylococcus aureus with oxygen-containing gas from a portable ozonator, which includes sowing a culture of Staphylococcus aureus in a sterile zone on a medium in Petri dishes and treating it with oxygen-containing gas for 5 minutes using an ozonizer tube at a distance of 1 cm from the surface of the agar (Patent RU 2709720 dated December 19 .2019, Bulletin No. 35, “Method of treating Staphylococcus aureus culture with oxygen-containing gas from a portable ozonizer” / Belyaev V.A. et al.). The disadvantages of this method are the need to use a small-diameter tube, ozone leakage due to the lack of sealing of the Petri dish and, as a consequence, the rapid decay of ozone, which does not allow the use of this method for longer-term processing. This method is aimed at assessing the sensitivity of a particular strain to ozone and does not involve assessing the impact of ozone on biofilms.

There is a known method for sterilizing plastic medical supplies with an ozone-oxygen or ozone-air mixture with an ozone concentration of 2-3%. at a rate of 2-4 l/h for 1-30 minutes (Patent RU 2075976 dated March 24, 1992 “Method for sterilizing plastic medical supplies” / Pokrovsky A.G. et al.). This method has the following disadvantages: high labor intensity, the need to monitor the ozone concentration in the air mixture, the duration of incubation, and a visual method for assessing sterility results.

There is a known method of treating crops with ozone (see Effect of low-dose gaseous ozone on pathogenic bacteria / Belchor Pontes, Ana Maria Cattani Heimbecker, Glacus de Souza Brito, Silvia F Costa, Inneke M van der Heijden, Anna S LevinEmail author and Samir Rasslan / / BMC Infectious Diseases. - 2012. - No. 12. - P. 358 [8]). This method is based on two-stage studies on eight bacterial strains (E. coli - ATCC: 25922, Staphylococcus aureus, resistant to oxacillin - ATCC: 29213, Staphylococcus aureus, sensitive to oxacillin - ATCC: 25923, Enterococcus faecalis, resistant to vancomycin - ATCC: 51299 , ESBL producing Klebsiella pneumoniae, sensitive only to carbapenems - clinical isolate from a patient, Acinetobacter baumannii, resistant to carbapenem - clinical isolate from a patient, Acinetobacter baumannii, sensitive only to carbapenem - ATCC: 19606, Pseudomonas aeruginosa, sensitive to imipenem and meropenem - ATCC: 27853) using different concentrations of ozone in the ozone-oxygen mixture (O ₃ /O ₂ ). At the first stage, the minimum effective dose of O ₃ /O ₂ was determined, at the second, its bactericidal activity was assessed when applied to various bacterial strains. Ozone was produced by a medical ozone generator when supplied with pure medical oxygen. The device was equipped with an oxygen flow regulator, in which the oxygen flow passes through a glass cylinder and is subjected to an electrical discharge of a dielectric barrier with controlled energy and voltage. As a result, a gas mixture O ₃ /O ₂ is formed, in which the ozone concentration is regulated by changing the oxygen flow and voltage from the electrodes. Pure cultures were sown on blood agar in Petri dishes at a concentration of microbial cells of 1×10 ⁵ per dish and incubated aerobically at a temperature of 35±2°C for 18-24 hours. A standardized inoculum of these cultures was prepared using direct subculture of colonies from blood agar. Subculture was carried out according to the turbidity standard “0.5 McFarland” (1 to 2×10 ⁸ CFU/ml) using a photometric device (colorimeter). The suspension was diluted 1:1000 in sterile saline to obtain a tube containing approximately 10 ⁵ CFU/ml. Then 10 μl of this suspension was seeded onto prepared Petri dishes with blood agar. Initially, agar plates with the seeded culture were exposed for 60 minutes. It was subsequently found that the minimum dose of ozone mixture and the time required to completely prevent the bacterial growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is 20 μg/ml for 5 minutes. Then a lower concentration of 15 μg/ml was used for 5 min on six bacterial cultures: Escherichia coli, Staphylococcus aureus not resistant to oxacillin, Staphylococcus aureus resistant to oxacillin, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa. The experiments were repeated for each culture 4 times and the results were evaluated. When analyzing this method, the following disadvantages can be noted: a complex technical device, the need to separately supply pure medical oxygen, low concentration of ozone in the gas mixture, low concentration of microbial cells in the inoculation and lack of testing on biofilms.

There is a known method for assessing the effect of ozone on Pseudomonas aeruginosa cultures by exposing bacterial suspensions to ozonated physiological solution of various concentrations (Patent RU 2289812 dated December 20, 2006, Bulletin No. 35, “Method for assessing the antibacterial effect of ozonized physiological solution” / Kuvakina N.A. et al. .). This method is aimed at studying the effect of ozone on actively dividing cells, without taking into account dormant and inactive forms of microorganisms in the structure of the biofilm. In addition, this method is more labor-intensive and time-consuming.

There is also a known method for assessing the antibacterial effect of ozonized saline solution (Patent RU 2425888 dated 08/10/2011, Bulletin No. 22, “Method for assessing the antibacterial effect of ozonized saline solution” / T.I. Karpunina et al.) on formed biofilms of various cultures isolated from patients, through a comparative analysis of the state of biofilms formed by bacteria before and after exposure to ozonated and non-ozonated physiological solution.

The method consists in treating biofilms formed by bacteria in polystyrene tablets with ozonated and non-ozonated physiological solution for 10 minutes. The plates were then washed and the biofilms were stained with a 1% aqueous solution of gentian violet for 45 minutes. The condition of the films was determined by the intensity of staining of the contents of the wells with a dye, which was eluted with 96% alcohol, and the optical density of the eluate was determined on a spectrophotometer. All experiments were carried out at least 3 times and the results obtained were compared with each other and significant differences between them were established (p < 0.05). The disadvantages of this method are: one-time assessment of the structural integrity of the biofilm without taking into account the preservation of cell viability, the lack of a proven effect of ozone under the specified conditions on the biofilm of the Pseudomonas aeruginosa culture.

Task

The objective of the present invention is to create an effective method for the destruction of bipolar cells, based on potentiating the antibacterial properties of ozone through the use of its combination with a solution of hydrogen peroxide.

The novelty lies in the use of a combination of ozone with a 3% hydrogen peroxide solution for the destruction of biofilms of Pseudomonas aeruginosa cell culture.

Technical result

The technical result of the invention is the destruction of Pseudomonas aeruginosa biofilm obtained in the wells of polystyrene plates by treatment with a combination of ozone and hydrogen peroxide. This result is achieved by exposing polystyrene tablets containing a formed Pseudomonas aeruginosa biofilm for 120 minutes to a combination of ozone and a 3% hydrogen peroxide solution.

The essence of the invention

The essence of the proposed invention is the effect of a combination of ozone with a solution of hydrogen peroxide on biofilms of Pseudomonas aeruginosa cell culture for the purpose of destruction. Biofilms of pure culture were formed in polystyrene plates, simultaneously filled with a 3% hydrogen peroxide solution and placed in a disinfection chamber with an ozone capacity of at least 15 g/h for 120 minutes. Control samples were filled with hydrogen peroxide and exposed to normal atmospheric air. After exposure, all samples were incubated for a day, and then the planktonic cells and the density of the biofilm itself were assessed.

We used a formed biofilm of a Pseudomonas aeruginosa cell culture, a 3% hydrogen peroxide solution (Iodine technologies and marketing, Russia), a sterile polystyrene tablet (Medpolymer, Russia), a Densi-La-Meter densitometer (Erba, Czech Republic), LB broth, and a Multiscan tablet photometer FC (Thermo Scientific, USA), 0.9% sodium chloride solution, phosphate-buffered saline, 0.1% gentian violet solution, 95% ethanol, 40 L disinfection chamber connected by two air ducts to the Ozonbox Air-15 ozonizer (Russia ) with a productivity of 15 g/h.

Carrying out the invention

The test material suspected of containing Pseudomonas aeruginosa is cultured on Colombian sheep blood agar for 24 hours at 37°C. The subsequently obtained pure culture is identified in an accessible and generally accepted way (biochemical, mass spectrometric, genotypic, etc.). To obtain a biofilm from a daily culture of Pseudomonas aeruginosa, a suspension is prepared in LB broth and incubated for 24 hours in polystyrene plates at 37°C. The planktonic cells are then washed three times with a sterile sodium chloride solution. 200 μl of a 3% hydrogen peroxide solution is added to the wells with the finished biofilm and placed in a 40-liter disinfection chamber connected by two air ducts to an Ozonbox Air-15 ozonizer (Russia) with a capacity of 15 g/h for 120 minutes. Control samples containing saline instead of 3% hydrogen peroxide solution are prepared in the same way, as well as samples containing saline or 3% hydrogen peroxide solution placed in an air atmosphere. The contents of the wells are then aspirated, washed three times with sterile 0.9% sodium chloride solution and 200 μl of LB broth is added and the plate is incubated for 24 hours at 37°C. The absorbance is then measured at 620 nm using a plate photometer to assess the yield of planktonic cells. Then the liquid phase is removed and the wells are washed three times with phosphate-buffered saline and filled with 0.1% gentian violet solution. After a 15-minute exposure, the dye is removed, the plate is washed in a bath of water, dried, and 200 μl of 95% ethanol is added to the wells to extract the bound dye. After 10 minutes, the liquid fraction is transferred to a new plate and the optical density is measured at 540 nm on a plate photometer. (O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp.2011 Jan 30;(47): 2437. doi: 10.3791/2437. PMID: 21307833; PMCID: PMC3182663 [10]).

The results are processed using the Excel 2010 package (Microsoft, USA) with the calculation of the arithmetic mean, the error of the arithmetic mean, as well as determining the level of significance of differences using the Student's test.

To assess the degree of destruction of the Pseudomonas aeruginosa biofilm, we compared the actual density values of the biofilm itself and planktonic cells with control samples depending on different types of active factors: 3% hydrogen peroxide solution, ozone and a combination of ozone with a 3% hydrogen peroxide solution. As can be seen from Figure 1, with the same exposure of 120 minutes, the combination of ozone with a 3% hydrogen peroxide solution had the maximum destructive effect on the biofilm; the biofilm density decreased by 83% compared to control samples, while no release of planktonic cells was recorded. When exposed to a 3% solution of hydrogen peroxide, this density decreased by only 38.9%, and when treated with ozone alone by 64.8%, which indicates a joint potentiation of the effect of the combination of ozone with a 3% solution of hydrogen peroxide for the purpose of destruction of the Pseudomonas aeruginosa biofilm and high effectiveness of the proposed method.

Example 1

In a city clinical hospital in the intensive care unit, in order to prevent nosocomial infections, swabs were taken from inhalation tubes with a sterile swab for the presence of non-fermenting bacteria. The material was delivered to the microbiological laboratory, where it was inoculated on GRM agar No. 9. After daily incubation at 37°C, a bacterial culture was obtained that formed large green-blue pigmented colonies with a specific odor. Using a biochemical identification test kit, the microorganism was identified as Pseudomonas aeruginosa. A suspension was prepared from the culture in nutrient broth, which was incubated at 37°C for 24 hours in the wells of a polystyrene plate. After this, the wells were washed three times with a sterile 0.9% sodium chloride solution, a 3% hydrogen peroxide solution was added to the test wells and the tablet was placed for 2 hours in a disinfection chamber connected to an Ozonbox Air-15 ozonizer; the control ones were under normal conditions. After this, the wells were washed with a sterile 0.9% sodium chloride solution, 200 μl of nutrient broth was poured into the wells and incubated for 24 hours at 37°C, followed by assessment of the biofilm density. It was found that the biofilm was destroyed by 95% after treatment with a combination of ozone and hydrogen peroxide, which made it possible to recommend this treatment method as one of the possible ways to disinfect inhalation tubes to destroy biofilms and prevent nosocomial infections.

Example 2

Patient M. had an open wound infection of the forearm with the presence of necrotic tissue. A sterile swab was used to take a sample from the surgical instruments used to resect the necrotic masses. And the swab was placed in a sterile swab with Stewart's transport medium. The material was delivered to the microbiological laboratory within two hours, after which it was inoculated on Columbia sheep blood agar. After a 24-hour incubation at 37°C, a bacterial culture was obtained that formed pigmented large colonies with a clear zone of hemolysis and a specific odor. Using the MALDI-ToF method, the microorganism was identified as Pseudomonas aeruginosa with 99.9% confidence. A suspension was prepared from the culture in nutrient broth, which was incubated at 37°C for 24 hours in the wells of a polystyrene plate. After this, the wells were washed three times with a sterile 0.9% sodium chloride solution, a 3% hydrogen peroxide solution was added to the test wells and the tablet was placed for 2 hours in a disinfection chamber connected to an Ozonbox Air-15 ozonizer; the control ones were under normal conditions. After this, the wells were washed with a sterile 0.9% sodium chloride solution, 200 μl of nutrient broth was poured into the wells and incubated for 24 hours at 37°C, followed by assessment of the biofilm density. It was found that the biofilm was destroyed by 89% after treatment with a combination of ozone and hydrogen peroxide, which made it possible to recommend this treatment method as one of the possible ways to disinfect instruments after contact with this microorganism.

Bibliography

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Patents:

1. Patent No. 2709720 C1, Russian Federation, IPC C12N 1/20, A61L 2/20, A61L 101/10. Method for treating Staphylococcus aureus culture with oxygen-containing gas from a portable ozonizer: No. 2018128262: appl. 08/01/2018: publ. 12/19/2019 / V.A. Belyaev, I.I. Naumenko, E.V. Svetlakova [and others]; applicant Federal State Budgetary Educational Institution of Higher Education "Stavropol State Agrarian University". - EDN YVLGZB.

2. Patent No. 2075976 C1, Russian Federation, IPC A61L 2/20. Method for sterilizing plastic medical supplies: No. 5033949/13: application. 03/24/1992: publ. 03.27.1997 / A.G. Pokrovsky, O.A. Plyasunova, N.N. Kabalnova [and others]; applicant Institute of Organic Chemistry, Ufa Scientific Center of the Russian Academy of Sciences. - EDN OOAIRL.

3. Patent No. 2289812 C2, Russian Federation, IPC G01N 33/48, C12Q 1/18. Method for assessing the antibacterial effect of ozonized saline solution (OSS): No. 2004126457/15: application. 08/31/2004: publ. 12/20/2006 / N.A. Kuvakina, S.I. Pylaeva, S.P. Peretyagan, A.A. Struchkov; applicant Association of Russian Ozone Therapists ARO. - EDN KSMGCF.

4. Patent No. 2425888 C1, Russian Federation, IPC C12Q 1/18. Method for assessing the antibacterial effect of ozonized saline solution: No. 2010110825/10: application. 03/22/2010: publ. 08/10/2011 / T.I. Karpunina, M.V. Kuznetsova, N.V. Nikolaeva [and others]; applicant State educational institution of higher professional education "Perm State Medical Academy named after Academician E.A. Wagner of the Federal Agency for Health and Social Development." - EDN DBSVDP.

Claims (1)

A method for the destruction of biofilms of Pseudomonas aeruginosa by a combination of ozone with hydrogen peroxide, which consists in affecting biofilms formed by bacteria in polystyrene tablets with a combination of ozone simultaneously in combination with a 3% solution of hydrogen peroxide in a disinfection chamber with an ozone productivity of at least 15 g/h and exposure time 120 minutes.