RU2289812C2 - Method for evaluation of antibacterial effect of ozonized physiological solution (ops) - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to study of antibacterial effect of ozonized physiological solution (OPS) and can be used in suppurative and combustion surgery. Method involves isolation of bacterial strain from patient blood with severe thermal trauma complicated by hospital infection and its parent properties are assayed. The strain is cultured on slant agar, poured with OPS in the ozone concentration 1, 3 and 5 mg/l and incubated in thermostat for 24 h. Standard suspensions containing 5 x 10⁵ CFU/ml are prepared from diurnal cultures that are incubated in nutrient medium under condition of microbiological analyzer for 18 h. Then dynamic determination of optical density of medium is carried out in combination with plotting curves reflecting the growth of microorganisms and used them for evaluation of antibacterial effect of OPS on the microorganism strain. Also, elongation of LAG-phase, double time of microorganism counts and amount of microorganisms in 1 ml of nutrient medium are determined. Using the proposed method provides enhancing precision and information content of antibacterial effect of the ozonized physiological solution.

EFFECT: improved evaluation method.

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Description

The present invention relates to medicine, in particular to the study of biological fluid, in particular blood, and can be used in purulent surgery and in combustiology in the treatment of patients with severe thermal injury complicated by a hospital infection.

The problem of purulent infection in modern combustiology is of particular importance. Among infectious complications, the greatest danger is early sepsis, characterized by lightning-fast development, severe course and extremely high mortality. According to the NNIIITO during the last decade of the 20th century, the main cause of death of seriously burned people, in whom thermal trauma was accompanied by the development of severe and extremely severe shock, remains a generalized infection, 60% of cases of which are early sepsis. Many authors indicate that sepsis of early periods of burn disease is usually gram-negative and most often caused by Pseudomonas aeruginosa (Pylaeva S.I., Matusis Z.E., Pakhomov S.P., Korablev S.B., Gordinskaya N.A., Samoilov V.A., Khrapunkova G.G. “On the problem of prevention of purulent-septic complications in seriously burned people.” Materials of the 3rd All-Union Conference on the problem of thermal injury “Modern means of first aid and methods of treatment of burn disease”, Moscow, 1986, p.200-201).

The stability of Pseudomonas aeruginosa in the external environment, unpretentious diet, natural resistance to antibacterial drugs determine the likelihood of its wide spread in the hospital environment and the formation of hospital strains, which turns the burn hospital into an "epidemiological focus" of Pseudomonas aeruginosa.

In this regard, hospital pseudomonas infection is a serious medical and epidemiological problem.

This situation requires an active search for effective means and methods of combating hospital microflora in the clinic of thermal injury. The development and improvement of the latest medical technologies has led to the widespread introduction in medical practice of various methods of ozone therapy, successfully used in the treatment of a wide range of diseases. The widespread use of ozone is due, inter alia, to a non-specific bactericidal and anti-inflammatory effect. However, to date, there is debate about the methodology for the use of ozone to achieve the optimal antibacterial effect, about the spectrum of the antibacterial action of ozone in various carrier media.

There is a method of studying the effect of ozonized physiological (OGF) solution on the growth of microorganisms (see Grechko VN "The effect of ozone on the pathogenic microflora of purulent wounds." // Abstracts of 3 All-Russian scientific-practical conference "Ozone and methods of efferent therapy in medicine "N. Novgorod. - 1998 - S. - 28-29).

The author has studied the effect of OGF on the microflora most often sown from the surface of purulent wounds (S.aureus, S.epidermidis, S.haemolyticus, E.coli, E.cloaceae, P.mirabilis, P.vulgaris, P.aeruginosa). The daily culture of microorganisms was inoculated with a lawn on a Petri dish with hungry agar. Next, the control group cultures were washed with 4 ml of 0.89% NaCl and placed in a thermostat. The cultures of the experimental series were washed with 4 ml of prepared ex tempore 0.89% NaCl (the solution was prepared by sparging 400 ml of physiological solution with an ozone-oxygen mixture with an ozone concentration of 5 mg / L for an hour). The cups were also placed in a thermostat. Evaluation of the results was carried out quantitatively by the number of grown colonies. In the control series, there was a continuous growth of microflora on the cup, which was not countable. In the experimental series, germination of certain groups of colonies took place.

The disadvantages of this method are:

a) the initial biological properties of microorganisms are unknown;

b) the initial seed dose of microorganisms is unknown;

c) washing the cups with sowing could mechanically remove some part of the microbial cells;

d) assessment of the results is visual, approximate.

For the prototype of the present invention, a well-known method for evaluating the antibacterial effect of ozone therapy was selected by examining the effects of OGF on bacterial growth, including preparing the strain for research, the effect of OGF and evaluating the results (see G.V. Rodoman, V.N. , A.L. Korotaev, V.G. Nikitin, I.R.Sumedy, T.V. Chernenkaya, A.K. Ivanov "Problems of combined antibacterial therapy of surgical infections" // Everest website the automated information system for hospitals and polyclinics of City Clinical Hospital No. 13 in Moscow).

The authors conducted an experimental study of the antibacterial effect of OGF on museum strains of microorganisms S.aureus ATCC 25923, E.coli ATCC 25922, P.aeruginosa ATCC 27853. A decreasing concentration of microorganisms in physiological saline was prepared 10 ⁸ 10 ⁷ 10 ⁶ 10 ⁵ , i.e. the degree of contamination of the experimental samples corresponded to a critical level of bacterial contamination of wounds or exceeded it. To each sample with microorganisms was added OFR in a ratio of 1: 1. The ozone concentration in the solution corresponded to 20 μg / ml (20 mg / l). Samples with the same microorganism content were used as a control, but unzoned physiological saline was added in the same volume. After a 20 minute exposure, all samples were subjected to bacteriological examination by plating on solid nutrient media. In the samples exposed to ozone, microflora growth was not detected in any of the cases, regardless of the generic affiliation of the microorganisms. In the control samples, microflora growth was noted in accordance with the initial level of microbial contamination. Similar results were obtained in series with an ozone content in solution of 10 μg / ml (10 mg / l).

The known method has the following disadvantages:

a) the saturating concentrations of ozone used by the authors for physiological saline - 10-20 mg / l can be dangerous for parenteral use due to the damaging effect on the vascular endothelium, in addition, in experimental studies T.A. Vasina et al. 1995 (T.A. Vasina, E.B. Lazareva, V.E. Shestoperov, B.M. Bogos “Correction of antibacterial therapy in the treatment of purulent-inflammatory processes in emergency surgery by ozone.” // Abstracts of 2 All-Russian scientific -practical conference "Ozone in Biology and Medicine. - N.Novgorod. - 1995. - P.32), inactivation of antibiotics was noted with their combined use with ozone concentrations of more than 6 mg / l;

b) currently OFR, due to its bactericidal effect, is used as an antiseptic mainly for external use. In this situation, high concentrations of ozone in saline (OZ) = 2-4-6 mg / l provide the maximum bactericidal effect on the microflora of wounds. In this regard, revealing research Kozhinova V.F. et al. ("Ozonation of water", Moscow. - 1974.), which indicate a high bactericidal effect of ozone in relation to mixed microflora in concentrations of 0.95-2.2 mg / L. These data confirm the antiseptic properties of ozone for the conditions of its direct contact with microflora in the aquatic environment.

For our specific situation, when patients are diagnosed with a septic state, confirmed by the release of a specific pathogen from the blood - Pseudomonas aeruginosa of hospital origin, antibiotic therapy is not always effective due to the microorganism's high resistance to antibacterial agents. Complex therapy, including the use of ozonized physiological solution with an ozone concentration of 10-20 mg / l, is not acceptable, because the introduction into the vein of highly concentrated solutions of oxidizing agents is fraught with complications in the form of rapidly progressive phlebitis.

The objective of the invention is the possibility of including ozone therapy with intravenous administration of FGF with ozone concentrations of 1 mg / l, 3 mg / l and 5 mg / l in the complex treatment of patients with thermal injury complicated by a hospital infection.

This object is achieved by the fact that in the method for evaluating the antibacterial action of FGM, including the study of the action of FGM on the growth of microorganisms and evaluating the results, a microorganism strain is isolated from the blood of a patient with severe thermal injury complicated by a hospital infection, its initial properties are determined, grown on mowed agar, fill in the RPF with an ozone concentration of 1 mg / l, 3 mg / l, 5 mg / l and incubate in an incubator for 24 hours, from standard cultures, prepare standard suspensions containing 5 × 10 ⁵ CFU / ml, which are incubated they are fed in a nutrient medium under the conditions of a microbiological analyzer for 18 hours, while dynamic measurement of the optical density of the medium is carried out with the construction of graphs of the growth curves of bacteria, which evaluate the antibacterial effect of OGF on the microorganism strain, determining the elongation of the PAH phase, the time of doubling the number of bacteria and the number of microorganisms in 1 ml of culture medium.

The present invention meets the criteria of "novelty" and "inventive step", because in the process of conducting patent information research, no information was found discrediting the novelty of the method, as well as technical solutions with essential features of the proposed method. The well-known methodology of “Growth Models”, incorporated into the WAST 3.5 program (AOZT Analitika, Moscow; certificate of suitability for use in healthcare of the Russian Federation No. 055 of September 5, 2000, valid until September 5, 2005, approved by Order of the Ministry of Health of the Russian Federation No. 359 dated 12/12/1998). The program is designed to automate measurements and perform calculations on the iEMS Reader complex (Labsystems, Finland) - an IBM PC / AT compatible personal computer.

Section of the program “Growth Models” is intended to build kinetic models of the growth of microorganisms in solving various scientific and practical problems (including studying the antimicrobial action of various antimicrobial agents).

To confirm the clinical efficacy of ozone therapy, including its antibacterial properties, we studied the effect of the same concentrations of ozone in saline 1-3-5 mg / l on the growth of hospital pseudomonas bacteria in vitro. The results of the evaluation are preceded by the isolation of a microorganism from the blood of a patient with severe thermal injury complicated by a hospital infection, determination of the initial biological properties of the strain, cultivation on mowed agar, its contact with FG with an ozone concentration of 1 mg / l, 3 mg / l, 5 mg / l in thermostat for 24 hours, re-growing on mowed agar and preparing a standard suspension containing 5 × 10 ⁵ CFU / ml, followed by incubation in a microbiological analyzer.

The proposed method allows to obtain the following positive effect:

1) in the proposed method shows the effect on bacteria of average therapeutic concentrations of ozone (1-3-5 mg / l) in physical. solution which can be applied parenterally. The indicated concentrations of ozonized physiological saline are the most optimal in terms of bactericidal action, and when combined with antibacterial drugs, potentiation of the antibacterial activity of the latter is observed (T.A. Vasina, E. B. Lazareva, V. E. Shestoperov, B. M. Bogos “ Correction of antibacterial therapy in the treatment of purulent-inflammatory processes in emergency surgery by ozone. ”// Abstracts of the 2nd All-Russian scientific-practical conference“ Ozone in biology and medicine. ”- N. Novgorod. - 1995. - P.32);

2) in the proposed method taken hospital strains of Pseudomonas aeruginosa isolated from severe burn patients with sepsis. The biological properties of the isolated strains, including pathogenic ones, were determined. All microorganisms were highly resistant to antibacterial drugs, as evidenced by the presence of resistance plasmids. The experimental strains of pseudomonads had a high IAM adhesion index of 2.5-4.0 and higher (the adhesive ability of microorganisms is one of the markers of pathogenicity and plays an important role in the development of the infectious process, ensuring the attachment of bacteria to the cells and tissues of the macroorganism). Thus, the studies were conducted on especially “problematic” hospital microorganisms, the fight against which is a serious medical problem;

3) we have shown the possibility of the influence of average therapeutic concentrations of ozone on the growth phases of hospital microorganisms, which is expressed graphically in the form of growth curves and objectively shows a decrease in the number and rate of bacterial reproduction over time. The method increases the information content of evaluating the results of the inhibitory effect of ozonized saline solution (1-3-5 mg / l) on hospital strains of microorganisms in order to further develop optimal modes (multiplicity and dose) of ozonized solution administration to patients with severe thermal injury complicated by hospital infection;

4) the proposed method improves the accuracy of the evaluation of the antibacterial effect of OGF on microorganisms, because The inactivating effect of OGF of therapeutic concentrations on the growth of Pseudomonas bacteria isolated from a particular patient has been proven, which will be of great practical importance for the further development of optimal regimens (multiplicity and dose) for the administration of ozonated solutions to patients with severe thermal injury complicated by hospital infection.

The method is as follows: a patient with severe thermal injury with a suspicion of sepsis is taken blood and a microorganism is isolated (according to the standard method regulated by Order No. 535 of the Ministry of Health of the Russian Federation of 04.22.1985 "On the unification of microbiological (bacteriological) research methods used in the clinic -diagnostic laboratories of medical institutions ").

The initial properties of the microorganism are determined: cultural, biochemical, adhesive, determine the sensitivity of the microorganism to antibacterial drugs. Microorganisms grown on mowed agar are poured with OGF with an ozone concentration of 1-3-5 mg / l at the outlet of the device, i.e. 3 derivatives of the initial sample strain are prepared, treated with RPF with physiological saline, and incubated in a thermostat for 24 hours. Then prepare a standard suspension containing 5.0 × 10 ⁵ CFU / ml, inoculate 100 μl in a plastic tablet with the same amount of culture medium - Mueller-Hinton broth. Incubated in an iEMS Reader analyzer (Labsystems, Finland) for 18 hours. At the same time, every 10 minutes, a dynamic measurement of the increase in optical density in the wells of the tablet occurs. After incubation, the antibacterial effect of the ozonized physiological solution is evaluated by the growth curves of microorganisms on the display screen in the graphic image mode: along the x-axis - time (hh: mm); along the ordinate axis is the increment of optical density in units of optical density. The phases of the development of microorganisms that are most indicative of their growth and reproduction are analyzed: the adaptive or PAH phase (characterizes the adaptability of microorganisms to the conditions of the nutrient medium, normally 4-5 hours), and the exponential or phase of logarithmic growth (characterizes the maximum growth and reproduction rate bacteria and is normal 5-6 hours), in accordance with the data obtained, a parenterally ozonized physiological solution with an ozone concentration of 1-3-5 mg / l is prescribed.

The method was tested on 10 strains of hospital pseudomonas aeruginosa isolated from patients.

The averaged data is shown in the drawing.

When evaluating the growth curves, an extension of the PAH phase is noted, which characterizes the adaptation of bacteria to environmental conditions. Normally, its duration is 4-5 hours. In microorganisms taken in the experiment, before ozonation, it was, on average, 5 hours. After the contact of the same bacteria with OGF, the concentration of 1 mg / L the PAH phase increased to 6 hours 50 minutes, 3 mg / L - 7 hours 20 minutes, 5 mg / L - 7 hours 30 minutes. In addition, the time for doubling the number of bacteria (phase of logarithmic growth) after exposure to FGF concentrations of 3 mg / L and 5 mg / L increased by an average of 1 hour, FGF concentrations of 1 mg / L did not affect this growth phase.

The data obtained show the dose-dependent effect of the action of OGF on the growth of microorganisms (with an increase in the concentration of ozone in physiological saline from 1 mg / l to 5 mg / l, the growth phases of the microorganisms lengthened over time). In addition, the number of colony forming units (CFU) of bacteria in the nutrient broth of the experimental samples after 18 hours of incubation was 1-2 orders of magnitude lower than in the control. In accordance with the data obtained, patients with thermal trauma complicated by generalized Pseudomonas infectus are prescribed parenterally FG with an ozone concentration of 1-3-5 mg / L.

An example of a specific implementation.

Patient X was treated in the Russian Burn Center for a burn with a flame of III degree AB on an area of 40% of the body surface, a burn disease in the stage of septicotoxemia. The course of burn disease was complicated by the development of sepsis. Pseudomonas aeruginosa (No. 480) was isolated from the blood. The initial properties of the microorganism were determined:

a) culture - on 5% blood agar, the colonies are mucoid, flat, irregular in shape, prone to fusion with the presence of the pigment of pyocyanin;

b) biochemical - saccharolytic activity is low, pronounced proteolytic and hemolytic activity;

c) the antibioticogram showed the presence of resistance to almost all antibacterial drugs with anti-Pseudomonas orientation in this strain, with the exception of carbenicillin and polymyxin. Antibiotic resistance is confirmed by the presence of an R plasmid with a molecular weight of 40 ppm;

g) the strain has a high adhesive ability (IAM = 4.0). The microorganism grown on mowed agar was in contact with OGF with an ozone concentration of 1, 3, 5 mg / l for 24 hours. Then, standard suspensions containing 5.0 × 10 ⁵ CFU / ml were prepared from diurnal cultures, 100 μl inoculated into a plastic plate with a nutrient medium — Mueller-Hinton broth. A microorganism sample plate was incubated under analyzer conditions for 18 hours. At the same time, every 10 minutes a dynamic measurement of the increment of optical density in the wells of the tablet took place. It is believed that the increment of the optical density of the nutrient medium with microorganisms directly proportionally correlates with their number (see graphs 3,4,5). After incubation, growth curves were evaluated. An extension of the LAG phase, which characterizes the adaptation of bacteria to environmental conditions, was noted. Normally, its duration is 4-5 hours. In strain No. 480, it was 5 hours and 45 minutes before ozonation. After contact with FGF, the concentration of 1 mg / L the PAH phase increased to 7.20 min, 3 mg / L - 8 h 35 min, 5 mg / L - 8 h 45 min. In addition, the time for doubling the number of bacteria (phase of logarithmic growth) from 8 hours to 15 minutes (control) increased after contact with FGF concentrations of 1 mg / l and 3 mg / l to 9 hours, 5 mg / l to 10 hours. in 1 ml of the nutrient medium grown after contact of bacteria with OGF of the same concentration, it decreased equally from 5.0 × 10 ⁵ to 5.0 × 10 ³ .

Our studies showed the effectiveness of OFR with ozone concentrations of 1 mg / L, 3 mg / L and 5 mg / L in relation to a specific strain of Pseudomonas aeruginosa No. 480 isolated from a particular patient.

The patient underwent a two-week course of ozone therapy, which included on / in the introduction of OFR with an ozone concentration of 3 mg / l at the exit of the device. The amount of solution is 200 ml / day 3 times a week. As a result, there was a clinical improvement in the patient's condition, which was expressed in a decrease in body temperature from hectic to subfebrile, improved sleep, and appetite. From the local course of the wound healing process - the wounds were cleansed of purulent discharge, signs of active regional epithelization of the wounds appeared, the bottom of the wounds was covered with bright fine-grained granulations, so the wounds were prepared for autodermoplasty. After several stages of autodermoplasty and conservative complex therapy, the skin was restored, the patient was discharged in satisfactory condition for outpatient treatment. The duration of inpatient treatment was 39 days.

Claims (1)

A method for evaluating the antibacterial effect of ozonized physiological saline (OGF), including a study of the action of OGF on the growth of microorganisms and evaluating the results, characterized in that a strain of microorganisms is isolated from the blood of a patient with severe thermal injury complicated by a hospital infection, its initial properties are determined, grown on beveled agar pour ODF ozone concentration of 1 mg / l 3 mg / l, 5 mg / liter and incubated in an incubator for 24 hours, cultures are prepared from diurnal standard suspension containing 5 x 10 ⁵ CFU / m which are incubated in a nutrient medium under the conditions of a microbiological analyzer for 18 h, and dynamic measurement of the optical density of the medium is carried out with the construction of graphs of the growth curves of bacteria, which evaluate the antibacterial effect of OGF on a strain of microorganisms, determining the elongation of the PAH phase, the doubling time bacteria and the number of microorganisms in 1 ml of culture medium.