RU2590713C1 - STRAIN OF Staphylococcus xylosus USED AS TEST CULTURE FOR DETERMINING ANTI-INTERCIDAL ACTIVITY OF MICROORGANISMS - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: strain of bacteria Staphylococcus xylosus No. 5/55 is deposited in National Collection of Normal Microflora of federally funded G. N. Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology under registration number No. 1254. Strain has high sensitivity to cationic protein of intericide leukocytes and can be used to determine anti-intericide activity of microorganisms.

EFFECT: invention makes it possible to inactivate cationic protein leukocytes.

1 cl, 2 ex

Description

The invention relates to microbiology, in particular to a strain of Staphylococcus xylosus No. 5/55, which can be used as a test culture to determine the "anti-bactericidal" activity of microorganisms.

It is known to use strain Corynebacterium xerosis No. 181, which is sensitive to the bactericidal fraction of the preparation of human leukocyte interferon, for determining the anti-interferon activity of bacteria (SU 1564191). However, the present preparation of human leukocyte interferon does not contain an antibacterial component (Bukharin O.V. Persistence of pathogenic bacteria. M., 1999. - P. 94-95), which limits the use of this strain to determine the "anti-intercidal" activity of microorganisms.

The objective of the present invention is to search for a strain of bacteria that is sensitive to the bactericidal action of the cationic leukocyte protein "intercide".

New in the claimed invention is the use of a strain of Staphylococcus xylosus No. 5/55, highly sensitive to the bactericidal effect of the cationic protein of leukocytes "intercide".

The technical result from the implementation of the invention is to obtain a strain of Staphylococcus xylosus No. 5/55, sensitive to the bactericidal action of the cationic protein of leukocytes "intercid".

Staphylococcus xylosus strain No. 5/55 was isolated from the vagina of a woman with uterine myoma with submucous localization of the myomatous node, which was hospitalized in the gynecology department in the Municipal City Clinical Perinatal Center (Orenburg).

The strain Staphylococcus xylosus No. 5/55 was deposited in the State collection of microorganisms of normal microflora (GKNM) FBUN "MIIIIEM them. G.N. Gabrichevsky Rospotrebnadzor "under No. 1254

Cultural, morphological and biochemical properties of the strain

Cells are motionless gram-positive non-spore-forming cocci located singly in the smear, in pairs and groups of clusters of irregular shape, with a diameter of 0.5-1.5 microns.

Optional anaerobic; optimal conditions for cultivation are aerobic. The cultivation temperature is + 30-37 ° C. Maximum growth rate at + 35-37 ° C. In liquid and solid nutrient media grows within 18-24 hours. On yolk-salt agar (+ 37 ° C, 24-48 hours), the strain forms isolated opaque cream-colored colonies with a diameter of 2-4 mm with smooth edges without a rainbow whisk, which indicates the absence of lecithinase activity of microorganisms. In a salt broth with 6.5% sodium chloride, the strain causes diffuse turbidity of the medium with the formation of a loose flocculent precipitate after 48 hours of cultivation.

The biochemical activity of the strain was determined using STAPHYtest 16 (Lachema, Czech Republic):

VPT - positive; Urease (URE) - positive; Arginine (ARG) - negative; Ornithine (ORN) - negative; β-galactosidase (GAL) - negative; β-glucuronidase (GLR) - positive; Nitrates (NIT) - positive; Phosphatase (PHS) - positive; Pyrrolidonylamidase (PYR) - positive; Aesculin (ESL) - positive; Sucrose (SUC) - positive; Trehalose (TRE) - positive; Mannitolum (MAN) - positive; Xylose (XYL) - positive; Maltose (MLT) - positive; Mannose (MNS) - positive; Glucose / Novobiocin (GLN) - positive.

The strain does not have plasmocoagulase, lecitovitellase and hemolytic activities, the oxidase test is negative, growth using (NH ₄ ) ₂ SO ₄ as a nitrogen source is positive.

The strain is resistant to the antagonistic effect of Staphylococcus aureus and coagulase-negative.

Storage conditions: culture grown during the day at 37 ° C in test tubes with 0.3% ΜΠΑ (pH 7.2-7.6) and top-filled with sterile liquid paraffin, in a domestic refrigerator at 4 ± 2 ° C for at least 6 months or freeze-dried culture at a temperature of 4 ± 2 ° C in a place protected from direct sunlight with a relative humidity of not more than 60% for at least 2 years. A sucrose-gelatin medium (0.5% sucrose, 2.0% gelatin, distilled water. Autoclaving at 1 atmosphere for 20 minutes) is used as a protective medium for ensuring long-term preservation during freeze-drying.

A comparative determination of the sensitivity of Staphylococcus xylosus strain No. 5/55 and Corynebacterium xerosis strain No. 181 to the bactericidal effect of the cationic leukocyte protein "intercide" was carried out using the "cup" method.

Experiment progress: 2 series of experimental Petri dishes were prepared with 5 ml of 1.5% meat-peptone agar (Nutrient agar for culturing dry microorganisms; NPO Nutrient Sredy, Makhachkala) containing a cationic white blood cell protein "intercid" at a final concentration of 0.3 ; 0.6; 0.9, 1.2; 1.5 and 1.8 mg / ml.

In the work used an experimental sample of the drug "intercida" (series C2), obtained on the basis of the Ufa NIIVS them. I.I. Mechnikov from prefabricated liquid human leukocyte interferon by the method of selective ultrafiltration using membrane technology and which is a dry yellow-pink powder, soluble in water. The preparation contains a histone-like thermostable protein with a molecular weight of 11.0-11.5 kDa, an isoelectric point of 10.5-11.0, and a low extinction coefficient at 280 nm (Sokolov V.Yu. Anti-interferon activity of microorganisms. Bacteria persistence. Kuibyshev, 1990. S. 92-93).

The control was 2 cups with 5 ml of culture medium, into which an equivalent volume of sterile distilled water without "intercide" was added.

On the surface of the dried agar, a second layer was poured with 3 ml of 0.7% meat-peptone agar containing 0.2 ml of a suspension of the daily agar culture of the tested Staphylococcus xylosus strain No. 5/55 or Corynebacterium xerosis No. 181 prepared in sterile isotonic (0.85%) solution NaCl at a concentration of 5 · 0 ⁸ CFU / ml. The agar was allowed to harden, the plates were placed in a thermostat, where they were incubated at 37 ° C for 18-24 hours, and then the results of the presence of growth of this strain of bacteria on the surface and in the thickness of the nutrient medium were taken into account. The concentration of "intercide" in the medium on which the culture did not grow was taken as the minimum inhibitory concentration (MIC).

For the Staphylococcus xylosus strain No. 5/55, the minimum inhibitory concentration (BMD) of the "intercid" was 0.6 mg / ml, for the Corynebacterium xerosis strain No. 181 it was 1.2 mg / ml, which allows us to conclude that Staphylococcus xylosus No. 5 is more sensitive / 55 to the "intercid" in comparison with the sensitivity of Corynebacterium xerosis No. 181, as well as the possibility of using the Staphylococcus xylosus No. 5/55 strain as a test culture for determining the "anti-intercidal" activity of microorganisms.

Example No. 1

To 1.5% nutrient agar was added the drug "intercid" in a final concentration of 0.6; 0.9; 1,2; 1.5 and 1.8 mg / ml and poured into Petri dishes. After the medium solidified and dried, daily agar cultures of 12 clinical strains of Escherichia coli isolated from the vagina in women with uterine fibroids and from a purulent wound in patients with diabetic foot syndrome were applied to the surface as a separate “patch”. The cups were incubated for 24 hours at 37 ° C, after which the grown bacterial colonies were killed in pairs of chloroform for 20-25 minutes, and then 3 ml of 0.7% nutrient agar containing 0.2 ml of suspension of daily agar culture of Staphylococcus xylosus No. 5/55 with a concentration of 5 · 10 ⁸ CFU / ml Then re-incubated plates for 24 hours at 37 ° C. Analysis of experience is carried out by the presence of growth of Staphylococcus xylosus No. 5/55. The test culture grew around the colonies of those strains that inactivated the “intercid” cationic leukocyte protein introduced into the nutrient medium, that is, showed “anti-intercidal” activity. 3 strains of E. coli inactivated a cationic leukocyte protein "intercid" at a concentration of 0.6 mg / ml; 1 strain - at a concentration of 0.9 mg / ml; 1 strain - at a concentration of 1.2 mg / ml; 1 strain - at a concentration of 1.5 mg / ml; the remaining 6 strains of E. coli did not inactivate the bactericidal effect of the "intercid". Thus, 6 clinical E. coli strains had different levels of “anti-intercidal” activity, and this property was not detected in 6 E. coli strains.

Example No. 2

To 1.5% nutrient agar was added the drug "intercid" in a final concentration of 0.6; 0.9; 1,2; 1.5 and 1.8 mg / ml and poured into Petri dishes. After the medium solidified and dried, daily agar cultures of 16 clinical strains of Staphylococcus aureus isolated from patients with infectious and inflammatory diseases (a purulent wound in the syndrome of diabetic foot, abscess and phlegmon of soft tissues, purulent sinusitis) were applied to the surface with a loop in separate “spots” . The cups were incubated for 24 hours at 37 ° C, after which the grown bacterial colonies were killed in pairs of chloroform for 20-25 minutes, and then 3 ml of 0.7% nutrient agar containing 0.2 ml of suspension of daily agar culture of Staphylococcus xylosus No. 5/55 with a concentration of 5 · 10 ⁸ CFU / ml Then re-incubated plates for 24 hours at 37 ° C. Analysis of experience is carried out by the presence of growth of Staphylococcus xylosus No. 5/55. The test culture grew around the colonies of those strains that inactivated the “intercid” cationic leukocyte protein introduced into the nutrient medium, that is, showed “anti-intercidal” activity. 3 strains of S.aureus neutralized the cationic leukocyte protein "intercid" at a concentration of 0.6 mg / ml; 2 strains - at a concentration of 0.9 mg / ml; 2 strains - at a concentration of 1.2 mg / ml; 1 strain - at a concentration of 1.5 mg / ml; 1 strain - at a concentration of 1.8 mg / ml; the remaining 7 strains of S.aureus did not inactivate the bactericidal effect of the "intercid". Thus, 9 clinical strains of S.aureus had different levels of “anti-intercidal” activity, and this property was not detected in 7 strains of S.aureus.

Therefore, the proposed strain of Staphylococcus xylosus No. 5/55 can be used as a test culture to determine the "anti-intercidal" activity of microorganisms.

Claims (1)

The strain Staphylococcus xylosus, used as a test culture to determine the anti-bactericidal activity of microorganisms, was deposited in the State collection of microorganisms of normal microflora (GKNM) FBUN "MIIIIEM im. G.N. Gabrichevsky Rospotrebnadzor "under No. 1254