Method for simulating prevention of hospital strain formation

FIELD: medicine.

SUBSTANCE: method involves the intramuscular introduction of the preparations Galavite 200 mcg/mouse and Lidocaine 8 mcg/kg of body weight once a day for three days running every 24 hours to experimental animals CBA line mice suffering a burning injury with underlying mycotic and bacterial infections accompanied by anti-infectious protection suppression.

EFFECT: invention enables creating a model of hospital strain formation which is applicable for studying the variations of the properties of opportunistic microorganisms to increasing virulence and antibiotic resistance.

2 tbl

 

The proposed method relates to medicine, namely to Microbiology, immunology and epidemiology, and can be used to simulate prevent the formation of hospital strains.

The problem of nosocomial infection is one of the most serious, as it has a significant impact on the duration of hospitalization, hospital mortality and, ultimately, the cost of treatment. The main reason for the development of nosocomial infections are hospital strains of conditionally pathogenic microorganisms, which are formed in stationary conditions.

Until recently, measures of nosocomial infections was based on the struggle with already incurred by immunocompromising patients and medical staff, inflammatory infections. However, it is possible to prevent the occurrence of nosocomial infections in the formation of hospital strains of pathogens by using modern advances in medicine, in particular, by applying the methods of preventing formation of hospital strains of pathogens.

Literature data about how microbiological monitoring of nosocomial infections have significant shortcomings.

The closest prototype is "a Method for the diagnosis of nosocomial microbe who's associations microbiological monitoring in the framework of the epidemiological surveillance of septic infections (Patent for invention №2298037).

One of the fundamentally important shortcomings proposed in the literature is the study of the properties of pathogens at the level of strains of microorganisms. It is known that populations of all living beings, non-uniform (heterogeneous) on various biological characteristics, which ensures the reliability of the living systems under changing conditions of existence. According to E. Belyakova (1987), the most important driving force of epidemiology (infectious) process and one of the main conditions for the existence of parasitic systems is the heterogeneity of the population of the microorganism and the microorganism (host-parasite).

Set (Vli, Pavolotsky, CIA and other Heterogeneity of microbial populations, M., 2008, 154 C.)that populations of freshly isolated from patients with strains of microorganisms consist of individuals that differ not only by the presence of signs of antibiotic resistance, but also on the level of resistance.

In addition, hospital strains of microorganisms consist of individuals that differ in clinically significant signs of aggression and virulence (antilysocyme, hemolytic, proteolytic activity, and others) [Ephtalou, Vli. Mixed infections of burn injury, Kursk, 1998, 92 S.], which is an important pathogenetic importance for the development of any infection.

Without knowledge and consideration of the mechanisms of creation the hospital strains is very difficult etiological diagnosis of pathogens of nosocomial infections. If not to take into account the heterogeneity of microbial populations in the past, evaluation of different methods of identification, proposed by the authors is incorrect.

Secondly, an essential disadvantage of the proposed methods is that during microbiological monitoring of septic infections, the authors diagnosed strains as hospital solely on the basis of studying the results of the comparative evaluation without studying the past against the background of immunomodulators. And it is known that the selection of the most virulent and multidrug-resistant individuals from the population of any strains of pathogens (i.e. the formation of hospital strains and their associations) occurs only through the body immunocompromised patients. Consequently, the main factor influencing the formation of hospital strains and their associations, is the level of immune protection of an organism. Hence conduct research proving hospital nature of the strains and their associations, without the use of immunomodulators in immunocompromising patients is not significantly probative factor.

In connection with this invention was to develop a simulation method of preventing the establishment of hospital strains that can prevent the change of the properties condition the ESD pathogenic microorganisms in the direction of enhancing their virulence and resistance to antibiotics, what is the basis for the formation of hospital strains in the various types of opportunistic septic processes.

The objective of the invention is achieved by the fact that the experimental animals (mice CBA) with burn trauma on the background of fungal and bacterial infections, accompanied by a sharp inhibition of anti-infective protection, three days in a row with an interval of 24 hours between injections intramuscularly Galavit 1 time per day dose mg/mouse and lidocaine intramuscularly 1 time per day at a dose of 8 mg/kg of body weight. Next studied the population structure of pathogens of nosocomial infections on the signs of drug resistance and the major virulence properties.

The method is as follows. The experiments were performed on mice CBA passed a quarantine regime of the vivarium of the Kursk state medical University and had no external signs of any diseases. In the control and experimental group Requirements of the International Committee, set forth in the Convention for the protection of vertebrate animals used for experimental purposes (, Strasbourg, France, 1986) that fully complied with.

It is known that the formation of hospital strains occurs through the body immunocompromised patients. In this regard, to create immunodefi is that in animals we have chosen the model of a thermal burn, because it is known that this condition is always accompanied by the decline of the immune status of the organism. Thermally burns III-B degree area of 30% of the body surface caused essential Rausch-anesthesia by means of a device for applying a metered burn.

Infection of mice were produced by intraperitoneal injection of 0.2 ml of 2 billion mist daily culture of microorganisms (fungi of the genus Candida and Pseudomonas aeruginosa), isolated from patients with burn injury and inflammatory processes. Two days after infection, animals were taken out of their experience using ether anesthesia and sterile conditions removed spleen. Produced blood cultures and suspensions of the spleen on the elective environment (CPH - agar for Pseudomonas bacteria and the environment Saburo - for fungi). A suspension of the obtained cultures were infected the next batch of animals, i.e. produced passages. Just spent 6 passages. After each second passage took the blood on the study and produced the seeding suspension of the spleen to obtain isolated colonies. To study the structure of the population on grounds of drug resistance obtained colonies (at least 100) velvet stamp transferred to plates with meat-peptone agar containing 25 μg/ml of antibiotics: carbenicillin, streptomycin, polymyxin b and kanamycin (for Pseudomonas bacteria); amphotericin b,levorin and nystatin (for fungi of the genus Candida). To study the population structure of the studied strains on the basis of pathogenicity same 100 colonies were seeded on 5% blood agar (hemolytic activity) and on Petri dishes with a collapsed horse serum (proteolytic activity). Cups with crops withstand a day in thermostat at 37°C and took into account the results of drug susceptibility in the presence of growth on media with antibiotics and the presence of hemolysis on cups with blood agar and proteolysis - on cups with rolled serum.

To study the prevention of the formation of hospital strains of the structure of microbial populations was studied on the background of the application of Galavit with lidocaine.

Galavit - domestic immunomodulator, has immunomodulatory, anti-inflammatory and regenerative effects (Khaitov R.M P.M., Pinegin B.V. Immunomodulators: mechanism of action and clinical use // Immunology. - 2003. No. 4. - S-202). Furthermore, of great importance in the early days of the disease with burn injury has a painful shock, which in turn also causes suppression of the immune response. Considering the pathogenesis of burn disease, we recommend the use of immunomodulatory drugs in combination with lidocaine, which not only suppresses the expression of a painful syndrome in burn injuries, but also prevents the post of the managing in the bloodstream of toxic and IMMUNOSUPRESSIVE substances during the development of infection on the background of the burn injury. Significant positive lidocaine is also the fact that lidocaine is not difficult ether, it is slowly metabolized in the body and thus has a prolonged effect.

Due to the fact that during its metabolism in the body does not generate para-aminobenzoic acid, it does not antisulfanilamidnoe actions and can be used in patients receiving antibacterial sulfa drugs (Medmaravis. Drugs // Manual for doctors. - 2001, Vol. 1. - S).

Indicators to examine the status of the level of immune protection were: phagocytic activity of neutrophils in peripheral blood (% of active phagocytes), phagocytic number (the average number fagocitaria microorganisms per one fagozyt), which was determined according to standard techniques. She has also studied phagocytic reserve neutrophils (NEF) and the state of bactericidal systems of neutrophils, which is, as you know, significant indicators in pyo-inflammatory processes of any etiology. NGF was determined by reduction reaction of nitrocine of tetrazole (Waxman M.E. Ter-Minassian, mayansky A.N. The way to assess the functional activity of human neutrophils by the reaction of recovery narasinga of tetrazole // Methodical recommendations. - Kazan. - 1979. - 17 S.). The bactericidal activity of si is the neutrophil - the presence of lysosomal cationic proteins in the cytoplasm of leukocytes (Subic MG Identification of cationic proteins in the cytoplasm of leukocytes using bromthymol blue // Questions of medical chemistry. - 1090. No. 10, T.16.. - S).

Statistical processing of results was performed using the software package "Microsoft Office XP" using student's criterion.

Specific example

Experimental animals were divided into groups of 25 animals each):

I. Intact.

II. Animals with burn trauma, infected by Pseudomonas aeruginosa (P. aeruginosa) is a bacterial infection.

III. Animals with burn trauma. Infected by yeast-like fungi (Candida albicans) is a fungal infection.

IV. Animals with burn trauma, infectious bacterial infection and treated with Galavit in combination with lidocaine;

V. Animals with burn trauma infected with a fungal infection and treated with Galavit in combination with lidocaine.

Infected animals passages cultures, the study of population structure of the studied strains and level of immune protection of an organism, as described above, page 4-6.

Galavit introduced vnutrimyshecnam at a dose of 200 μg/mouse immediately after playback of burn injury 1 times a day for 3 days.

Lidocaine intramuscular injection at a dose of 8 mg/kg of body weight immediately after playing ogogo the second injury 1 times a day for 3 days.

Burns III - degree area of about 30% of the body surface were obtained under light ether anesthesia on devoid of hair and skin back using a device that supports the temperature of the burning surface at 100°C With exposure 8 seconds (WMO, 1985).

We found: animals with burn trauma, complicated both bacterial and fungal infection are greatly reduced all parameters of phagocytosis compared with the control (intact) animals, and there is a high microbial colonization of the spleen. In the same conditions, but after applying calavita with lidocaine phagocytosis indices is significantly higher and even higher than those of the control animals; the splenic index (the number of microorganisms on the body) in treated animals was significantly decreased already in the study after 4 passages, and after 6 passages as bacteria and fungi from the spleen were not inoculated (table 1).

Table 1
The effect of immunomodulators on the performance of anti-infective organism defense
The studied parametersGroups of animals
The intact Burn+infectionTreatment with Galavit with lidocaine
The indicators in different study periods (passages) M±m (n=25)
246246246
The activity of phagocytosis60,8±0,1560,2±0,1760,5±0,1232,7±2,530,1±2,735,8±2,9X58,5±1,762,3±1,368,2±0,8XX
Phagocytic number2,0±0,042,1±0,052,2±0,070,8±0,030,6±0,050,9±0,02X1,5±0,072,1±0,043,2±0,02XX
NEF (%)28,0±2,229,0±2,37,0±0,95,0±1,28,0±1,3X12,0±1,417,0±2,330,0±2,8XX
The colonization of the spleen (micrel/body)//////2.4 x 105Solid growth6×1073×102Individual coloniesXXThe lack of growth
Note: X = p<0,05 - reliability of differences with the data of the control group (intact);
XX = p<0,05 - reliability of differences with the data of the control group (burn+infection);
// - studies have not been conducted.

The results of the study structure of the population of microorganisms in the dynamics of infectious process in vivo has shown that in parallel with the improvement of the immune status of the animal organism has been significant changes in the composition of populations septic about what escow. If in the composition of populations obtained from infected animals with burn trauma, experienced a continuous increase in individuals with signs of resistance to antibiotics and signs virulence (signs virulence already to 6 passage populations as Pseudomonas bacteria, and fungi of the genus Candida were statistically significantly (p<0,01) homogeneous, i.e. there is a formation of strains with signs of "hospital" through immunocompromising the body, then after applying calavita with lidocaine (increase immune status), on the contrary, the number of individuals with these characteristics are statistically significantly (p<0,01) was decreased, i.e. the use of immunomodulators promotes reliable elimination of the population of virulent and antibiotic resistant individuals, which leads to superficial pathogens in the body, which is observed from passage 4 of the study (table 2).

Table 2
The effect of immunomodulators on the composition of populations of pathogens
Learn the signs of% of individuals in populations (out of 100 colonies)P
the t untreated animals (burn+infection) from treated animals (Galavit+lidocaine)
The indicators in different study periods (passages)
246246
Resistance to:The population of Pseudomonas aeruginosa
carbenicillin72,095,0100,070,058,047,0<0,01
streptomycin75,098,0100,065,050,030,0<0,01
polymyxin50,065,095,050,042,034,0<0,01
kanamycin 85,098,0100,078,055,040,0<0,01
Hemolysis85,0100,0100,080,050,038,0<0,01
Proteolysis72,0100,0100,065,045,025,0<0,01
Resistance to:The population of Candida albicans
Amphotericin b58,070,095,049,045,030,0<0,01
Levario65,078,0100,062,050,034,0 <0,01
The nystatin72,090,0100,068,055,042,0<0,01
Hemolysis55,062,085,039,030,025,0<0,01
Note: the significance of differences with the data of the control group (untreated animals with burn and infection).

Thus, the obtained results show conclusively that despite the pronounced immunodeficiency in the dynamics of infectious process conditions are created when the original heterogeneous populations of agents of nosocomial infections are generated strains with signs of a hospital. The use of drugs, reducing anti-infective resistance in experimental animals, contributes to the elimination of the composition of microbial populations virulent and antibiotic resistant individuals, which leads to superficial pathogens in the body and 100% survival of animals. There are credible reasons to believe that in incesti conditions improve immune status immunocompromising patients with immunomodulators will prevent the formation of hospital strains of pathogens, will increase the treatment efficiency and reduce the time of their stay in hospital.

The simulation method of preventing the establishment of hospital strains, characterized in that the experimental animals the mice CBA with burn trauma on the background of fungal and bacterial infections, accompanied by a sharp inhibition of anti-infective protection, three days in a row with an interval of 24 h between injections intramuscularly "Galavit" 1 time daily at a dose of 200 μg/mouse and "Lidocaine" intramuscularly 1 time per day at a dose of 8 mg/kg of body weight.



 

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