RU2393229C1 - Method for estimating adhesion staphylococcus spp to haemoproteins - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is intended for a quantitative estimation of adhesive properties of staphylococci recovered from a human and environment objects, with its bacteriological identification and for characterisation of its virulent properties in vitro by the adhesion to biopolymeric molecules. The staphylococcus culture being investigated is incubated in wells of a polystyrene plate with immobilised haemoproteins (muscle haemoglobin, haemoglobin) in a nutrient medium 199 for 1 hour; thereafter, nonadherent bacteria are removed by triple washing in the medium 199 with Twin-20, and a number of adhesive staphylococci is estimated by harvest cells and shown by optical density of the medium and by counting the cells sown on the dense nutrient medium from a series of tenfold cultivations, or by analysing the adhesive strains for the activity of bacterial enzymes.

EFFECT: good results reproducibility, high sensitivity of the method, ease of the reaction directly in wells of the polystyrene plate and possible express estimation of adhesive capacity of strains.

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Description

The invention relates to microbiology and can be used in medical bacteriology for the quantitative assessment of the adhesive properties of staphylococci isolated from humans and environmental objects during their bacteriological identification, to characterize its virulent properties in vitro by the level of adhesion to biopolymer molecules.

Staphylococcus aureus, in particular the pathogenic species S.aureus, differentiates sources of exogenous iron. Hemic iron is the preferred source for pathogenic staphylococcus species, it is they who prefer non-heme iron when growing and multiplying in the tissues of the human or animal body (Skaar EP Humayun M., Wae T., DeBord K.L., Schneewind O. Iron-source preference of Staphylococcus aureus infections. Science. 2004, 305: 1626-8). The molecular basis for the preferred capture of heme iron by pathogenic staphylococcus species is due to the presence of an iron-regulated Isd system that encodes a heme utilization mechanism that consists of three surface membrane proteins of staphylococcal cells (IsdA, IsdB, IsdH) (Skaar EP, Schneewind O. Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme. Microbes Infect. 2004, 6 (4): 390-7). The key role in binding to hemoproteins in the species S.aureus belongs to the IsdB protein, which is actively expressed on the cell surface as a receptor for hemoproteins such as hemoglobin and myoglobin in the most virulent strains (Torres VJ, Pishchany G., Humayun M., Schneewind O ., Skaar EP Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization. J. Bacteriol. 2006, 188 (24): 8421-8429).

A known technique for studying the adhesive process of microorganisms (Brilis V.I., Brilene T.A., Lenzner H.P. and others. Method for studying the adhesive process of microorganisms. Lab. Business 1986; 4: 210-212.). Closest to the claimed invention relates to a method for determining adhesion of Streptococcus pneumoniae by contacting sections of the lungs of mice with pneumococcus (RU (21) 96100972, IPC C12Q 1/14, 1/20 (22) 1996.01.16 (54). Method for determining adhesion of streptococcus pneumoniae and blocking it).

Its disadvantages are low sensitivity and reproducibility, as well as higher economic costs necessary for working with tissue culture.

The aim of the claimed invention is to develop a standardized method that allows you to quantify the rate of adhesion of staphylococci to hemoproteins (hemoglobin, myoglobin) to characterize their virulent potential.

The essence of the invention lies in the fact that the studied culture of staphylococcus is incubated in the wells of a polystyrene tablet with immobilized hemoproteins (myoglobin, hemoglobin) in a nutrient medium 199 for 60 ± 15 minutes, then non-adherent bacteria are removed by washing with 199 medium tween-20 three times and adhesives are determined :

1) by taking into account the grown cells in a nutrient medium according to the optical density of the medium, or

2) by counting cells seeded on a solid nutrient medium from a series of ten-fold dilutions, or

3) by determining the activity of bacterial enzymes in adherent strains.

The technical result of the claimed invention is the development of a method for the quantitative assessment of adhesion, characterized by good reproducibility of results, high sensitivity, ease of reaction directly in the wells of the tablet and the ability to conduct an express evaluation of the adhesive ability of the strains.

An advantage of the patented method for determining the adhesion of Staphylococcus spp. to hematoproteins is the detection of adhesion level in virulent staphylococci to hematoproteins, the high sensitivity of the method due to registration of the enzymatic activity of adherent bacteria and the possibility of an express assessment of the adhesion value.

The method is as follows. A suspension of staphylococcus culture at a concentration of 1 million mt / ml is incubated in the wells of a polystyrene tablet with immobilized hemoproteins (myoglobin, hemoglobin) in nutrient medium 199 for 1 hour, then non-adherent bacteria are removed by washing three times with medium 199 with tween-20 and subsequent determination the number of adhered staphylococci by counting the grown cells in a nutrient medium or by counting cells seeded on a solid nutrient medium from a series of ten-fold dilutions, or by determining the activity of bacte rial enzymes in adherent strains. Adhesive activity is expressed in arbitrary units.

On the basis of the laboratory of immunology and microbiology of the Federal State Budget Institution “KNIIEM" of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare, the adhesiveness of staphylococcus clinical isolates and museum strain S.aureus ATCC No. 25923 was analyzed using the patented method, which showed that clinical staphylococcus aureus isolated from the nasal mucosa of patients with seasonal allergic rhinitis and from the skin of patients with atopic dermatitis (AT) in terms of adhesion, are characterized by greater adhesion to hemoprotein, which correlates with the clinical activity of these strains, museum strain S .aureus ATCC No. 25923, long passaged on solid agar media (MPA), was less adhesive than clinical strains of this species. Using the patented method, various adhesive activity between staphylococcus species was detected, so that coagulase-negative staphylococcus S.epidermidis species, normally colonizing the skin and mucous membranes of healthy individuals, were characterized by significantly lower adhesion to hemoglobin than pathogenic strains of S.aureus species.

Examples of the method for determining the adhesion of staphylococcus spp. to hemoproteins

Example 1. Determination of the adhesive ability of staphylococci isolated from the mucous membranes and skin of various groups of individuals. We studied isolates of staphylococci isolated from the nasal mucosa of patients with seasonal allergic rhinitis (CAP), from the skin of patients with atopic dermatitis, and from the nasal mucosa of carriers of Staphylococcus aureus. The studied live culture of staphylococcus was bred to a concentration of 1 million MT per 1 ml of 199 medium and introduced in a volume of 200 μl into the wells of a polystyrene 96-well plate with immobilized hemoglobin, incubated in a thermostat at 37 ° C for 1 hour, then the contents of the wells were removed and washed three times with 199 medium containing 0.05% tween -20, and with physiological saline, the wells were drained and 150 μl of substrate solution (0.03% H ₂ O ₂ solution) was added to each well, after 10 minutes, 50 μl of 4% ammonium molybdate solution was added and examined photometrically at 414 nm on a small photometer for enzyme immunoassay AIFR-01 "Unipla "Measured indicator OP _issled sample. In parallel, the _background OD was determined; for this, 150 μl of a substrate solution (0.03% H ₂ O ₂ solution) and 50 μl of a 4% solution of ammonium molybdate were added to control wells of a 96-well plate (wells into which live staphylococcus cultures were not introduced), photometric at 414 nm. Zero photometer was installed in wells containing 150 μl of distilled water and 50 μl of a 4% solution of ammonium molybdate. The calculation of the adhesion rate of the strains was carried out according to the formula:

PA = OP _background -OP _issled

The results of the adhesion test of clinical strains of staphylococci and the museum strain S.aureus ATCC No. 25923 (from the collection of the microbiology laboratory of the Federal State Institution of Health Sciences Kazan NIIEM) by this method are presented in table 1.

Example 2. Determination of the adhesive ability of staphylococci was carried out analogously to example 1, but to determine the number of adherent cells after the washing step, the same medium with glucose was added to each well of the plate, incubated for 5 hours in an incubator at 37 ° C. After incubation, the wells were photometered at 595 nm on a small photometer for enzyme immunoassay AIFR-01 "Uniplan". The optical density index in the studied wells (OD _assay ) and the optical density of the control (OD _control ) in the zero well (i.e., in the well into which the cell culture was not introduced) were determined. The adhesion index (PA) was calculated by the formula:

PA = OP _research- OP _control

The results are presented in table 2.

Example 3. Determination of the adhesive ability of staphylococci was carried out analogously to example 1, but to determine the number of adherent cells after the washing step with medium 199 with tween-20, the same medium with glucose was added to each well of the plate, incubated for 5 hours in an incubator at 37 ° C. After incubation in a thermostat, 50 μl of culture medium was taken from the well, ten-fold dilutions were prepared in sterile physiological saline, and blood agar was sown from each dilution by the lawn method. After 12 hours, the number of colonies on plates was counted and the number of CFU / ml was calculated according to the dilution. The results of the study of adhesion of staphylococcus isolates are presented in table 3.

Table 1 Staphylococcus strain adhesion indicators for example 1 Type of microorganism PA, M ± m S.aureus (with nasal mucosa of carriers) 0.71 ± 0.07 S.aureus (skin of patients with AD) 0.6 ± 0.06 S.aureus (from the mucosa of patients with CAP) 0.63 ± 0.05 S.aureus (healthy skin) 0.54 ± 0.09 S.aureus ATCC N 25923 0.48 ± 0.07 S.epidermidis (from the nasal mucosa of healthy individuals) 0.38 ± 0.07 S.epidermidis (healthy skin) 0.32 ± 0.05

table 2 Staphylococcus strain adhesion indicators for example 2 Type of microorganism PA, M ± m S.aureus (with nasal mucosa of carriers) 0.37 + 0.07 S.aureus (skin of patients with AD) 0.35 ± 0.06 S.aureus (from the mucosa of patients with CAP) 0.32 ± 0.05 S.aureus (healthy skin) 0.28 ± 0.09 S.aureus ATCC N 25923 0.25 ± 0.04 S.epidermidis (from the nasal mucosa of healthy individuals) 0.15 ± 0.07 S.epidermidis (healthy skin) 0.12 ± 0.05

Table 3 The adhesion indicators of staphylococcus strains for example 3 Type of microorganism PA (CFU / ml) S.aureus (with nasal mucosa of carriers) 1.3 × 10 ⁶ S.aureus (skin of patients with AD) 2.3 × 10 ⁵ S.aureus (from the mucosa of patients with CAP) 2 × 10 ⁵ S.aureus (healthy skin) 6 × 10 ⁴ S.aureus ATCCN 25923 4 × 10 ⁴ S.epidermidis (from the nasal mucosa of healthy individuals) 2 × 10 ² S.epidermidis (healthy skin) 1.1 × 10 ²

Claims (1)

Method for determination of adhesion of Staphylococcus spp. to hemoproteins, which consists in the fact that the studied staphylococcus culture is incubated in the wells of a polystyrene tablet with immobilized hemoproteins in the nutrient medium 199 for (60 ± 15) min, then unadhesive bacteria are removed by triple washing with medium 199 from tween-20 and adhesive adhesives are determined by taking into account staphylococci grown cells in a nutrient medium by the optical density of the medium or by counting cells seeded on a solid nutrient medium from a series of ten-fold dilutions, or by determining the activity of the bacterium ialnyh enzymes in adherent strains.