RU2348037C2 - Method for assessment of clinical significance of clostridia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to the field of medicine, namely to medical toxicology, microbiology, and is related to method for assessment of clinical significance of clostridia in conditions of polymicrobial infection. Method consists in the fact that standard amount of daily clostridia culture is incubated in conditions of anaerostat in SKS medium together with monolayer of human embryo skin fibroblasts, and after triple washing of monolayer cells by Eagle's medium, method of microscopy is used to assess availability and effect of toxin according to extent of monolayer damage in compliance with USA Pharmacopoeia scale.

EFFECT: higher accuracy of detection of clostridial toxins and determination of their effect.

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Description

The invention relates to the field of medicine, medical toxicology, microbiology, and in particular to assessing the clinical significance of clostridia, especially in conditions of polymicrobial infection.

The peculiarity of the biology of pathogenic bacteria indicates that the ability to cause infectious diseases was formed in them in the direction of acquiring functions that allow them to penetrate the host’s body, resist its protective systems, and also cause disturbances in the activity of physiologically important systems. Pathogenicity factors with an invasive function and a function of protection against phagocytosis can be combined into one group of factors that ensure the development of the initial, often not clinically expressed, stage of the infectious process.

To another group of pathogenicity factors can be attributed biologically active substances (toxicants) that cause the syndrome of the disease with a pronounced clinical picture and the possible death of the host (Yu.V. Ezepchuk "Biomolecular basis of the pathogenicity of bacteria" M., 1977, 214 pp.).

Clostridia producing histotoxic exotoxins, except C. perfringens, include: C. novyi, C. septicum, C. histoliticum, C. bifermentans and several others.

The most severe form of clostridial tissue damage is gas gangrene (myonecrosis). Microorganisms produce a number of toxins: alpha, beta, epsilon and iota. Alpha toxin is the main one, it exhibits phospholipase, lecithinase and hemolytic activity, and also has direct lethal and necrotizing effects. Clostridia are typical conditional pathogens because they cannot proliferate and produce toxins in intact tissues.

The main factor contributing to their proliferation is a decrease in tissue oxygenation associated with injuries, surgical interventions, and circulatory disorders. With the formation of these conditions, clostridial damage can develop on almost any part of the human body.

Gas gangrene (clostridial myonecrosis) is a direct toxic destruction of muscle tissue, accompanied by the proliferation of microorganisms and the accumulation of gas (hydrogen and nitrogen), expressed by toxemia, bacteraemia is observed in most cases.

The most typical localization of gas gangrene is the muscle masses of the lower extremities, however, smooth muscle lesions, such as myometrium, developing after a criminal abortion or complicated delivery are also possible.

In addition to gas gangrene, cytotoxic clostridia cause necrotic processes with gas formation in the soft tissues without muscle damage (crepitating anaerobic cellulites). According to the severity of the course, in some cases, these processes may not be inferior to gas gangrene.

It must be borne in mind that soft tissue lesions accompanied by crepitus (gas formation) can also be caused by other microorganisms (anaerobic cocci, Bacteroides spp., Fusobacterium spp., C. bifermentans, C.sordelii, C.tertium and others).

Anaerobic cellulites are characterized by the presence of a polymicrobial flora in the smear and a leukocyte reaction.

Clostridia are typical conditional pathogens due to their inability to proliferate and produce toxins in intact tissues. The leading role of clostridia in the development of necrotic lesions of ischemic tissues is obvious (Sidorenko SV, Yakovlev SV "Infections in intensive care", M., 2000, p.40).

The detection and determination of the degree of action of clostridial toxins in polymicrobial infections (associations of microorganisms) is the basis for assessing the clinical significance of clostridia.

An analogue of the proposed proposal is a method for detecting clostridial toxin by infection of laboratory animals (guinea pigs) (I.E. Kolukanov "Methods for the isolation and identification of anaerobes - causative agents of gas gangrene" (methodological manual for bacteriologists), L., 1970, p. 16). A microbial daily culture of clostridia in the amount of 0.5-1 ml is injected / muscle into the inner thigh of a guinea pig. In dead animals, tissues from the lesion and internal organs (blood from the heart, liver, etc.) are examined. If the animal does not die, then after several days after infection it is killed and the tissue in which the material was injected is examined.

The disadvantage of this method are: the lack of standardization, the duration and complexity of the study, the inability to determine the presence and degree of action of the clostridial toxin in surviving animals.

The closest analogue (prototype) of the proposed proposal is the determination of C. perfringens toxin in tissue culture (I.E. Kolukanov "Methods for the isolation and identification of anaerobes - causative agents of gas gangrene" (methodological manual for bacteriologists), L., 1970, p. 24). The method consists in the following: tissue culture is prepared by trypsinization of 11-day-old chicken embryos, followed by growing cell suspension at 37 ° C in a nutrient medium consisting of 7.5% bovine serum and 92.5% Hanks solution. The material is inoculated on Kitt-Tarozzi medium and grown for 4-6 hours at a temperature of 37 ° C. Then the culture fluid is diluted twice with physiological saline and antibiotics are added (50 units / ml of penicillin and streptomycin). A dilution of the toxin thus prepared is introduced in an amount of 0.2 ml into a tissue culture tube, which is then placed in a thermostat at a temperature of 37 ° C. It is taken into account that the addition of 0.2 ml of toxin to 1.8 ml of the nutrient mixture in test tubes leads to a further 10-fold dilution of the toxin. For each sample, 4 test tubes with tissue culture are taken. As a control, a tissue culture tube is used, to which physiological saline solution used for dilution is added. The presence of a cytotoxic effect is determined at a small magnification of the microscope (7 × 10). This action is manifested in the destruction of a significant number of cells and sometimes the delamination of the cell layer from the walls of the tube. It is better to take the daily tissue culture obtained by plating a suspension of cells containing 1 million cells per ml of culture medium.

The presence of a cytotoxic effect is determined 4 hours after the start of the study.

The disadvantage of this method is that it is designed to evaluate only C. perfringens toxin. The test is carried out without the use of anaerostats (without reducing the level of tissue oxygenation), the assessment of the toxin is not standardized either in relation to the number of microbial cells, or in relation to cells of a human macroorganism. In addition, the assessment is carried out visually on the "destruction of a significant number of cells and sometimes the detachment of the cell layer from the walls of the tube."

In this way, it is possible to assess the presence of only the potent toxin C. perfringens, and not other, often found in microbial associations, species of clostridia, for example C. bifermentans, C.sordelii, C. tertium and others.

The technical result of the invention is the identification under standard conditions and with a short exposure of the presence and degree of action of the clostridial toxin under conditions close to the conditions of cells of the human macroorganism, especially in clostridial strains, the role of which was not previously taken into account if they were present in the infected wound.

The result of the invention is achieved due to the fact that under anaerostat conditions for 2 hours with a standard amount (0.2 ml) of SCS storage medium, with a standard number of microorganisms, they are incubated together with a monolayer of fibroblasts of human skin embryos (PCEC). Cells of the monolayer are washed three times with Eagle’s medium and microscopied under magnification (40 × 10) or (90 × 10). The presence and potency of the toxin is assessed by the state of the monolayer on the US Pharmacopoeia scale.

The tables show: in table 1 - the degree of cytotoxic effect of the toxin and cell response in accordance with the US Pharmacopoeia; table 2 - the results of applying the method for assessing the clinical significance of clostridia isolated from wounds of patients and environmental objects. (Methodical instructions МУ 1.2.1105-02 Assessment of toxicity and danger of disinfectants. Journal “Disinfection business”, 2002, p. 58.)

The method is as follows.

Under anaerostat conditions, 0.2 ml of a daily culture of clostridia (1 × 10 ⁶ CFU / ml) in SKS medium (sterility control medium) was placed in 1.8 ml of medium. A needle in a Layton test tube on a cover glass with a monofolder of PCEC (human skin embryo fibroblasts ) The tube was incubated for 2 hours at 37 ° C in an anaerostat, the cells of the monolayer were washed with a 3-fold change in Eagle’s medium and microscopied at a magnification of (40 × 10) or (90 × 10), evaluating the state of the monolayer in accordance with Table 1.

Thus, the proposed method allows to detect the presence and degree of action of the toxin not only known producing histotoxic exotoxins, C. perfringens and C. histoliticum, but also quite common in both monoculture and microbial associations of C.tertium, C.inocuum, C. baratii, C. butyricum. In the case of the presence of the latter in microbial wound associations, clinicians should take into account the possible participation of clostridial strains with a degree of cytotoxic effect of 1-4 in the dynamics of the infectious process, not only as a wound contamination.

Table 1 METHOD FOR EVALUATING CLINICAL SIGNIFICANCE OF CLOSTRIDIA The degree of cytotoxic action (CTD) Cell response The state of cells in culture four heavy 100% cell monolayer destruction 3 moderate 70-75% of the cell monolayer contains rounded and / or lysed cells 2 soft 50% cell death one weak 20-25% of lysed cells 0 no Lack of DHT

table 2 No. Type of clostridia Degree of central heating plant one C.perfringens four 2 C. histoliticum 3 3 C.tertium 2 four C.inocuum 3 5 C. baratii 2 6 C. butyricum one

Claims (1)

A method for assessing the clinical significance of clostridia, characterized in that under anaerostat conditions for 2 hours, a standard amount of daily culture of microorganisms (1 × 10 ⁶ CFU / ml) in a standard amount of SCS storage medium 0.2 ml is incubated together with a monolayer of fibroblasts of the skin of a human embryo and after washing the cells of the monolayer three times with Eagle’s medium, the presence and strength of the toxin are evaluated by the microscopy method according to the degree of destruction of the monolayer in accordance with the US Pharmacopoeia scale.