Method of accelerated growth of staphylococcus aureus for diagnostics of infections associated with delivery of health care

FIELD: biotechnology.

SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.

EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.

2 tbl, 7 ex

 

The invention relates to medicine, namely to clinical Microbiology, and relates to a method of fast growing Staphylococcus aureus (Staphilococcus aureus) for use in the diagnosis of infections associated with health care. Known standard method of growing Staphylococcus for the diagnosis of infections caused by these microorganisms, which consists in using as a nutrient medium yolk-salt agar. Duration of cultivation by the standard method [2,6,7] up to 48 hours.

Irrational use of antimicrobial agents has led to the development of resistance in many pathogenic and conditionally pathogenic microorganisms (including Staphylococcus) and wide dissemination of socially relevant staphylococcal infections, receiving threatening and causing the development of nosocomial purulent-inflammatory diseases, especially in surgical wards, maternity hospitals, pediatric hospitals (ulcerative colitis, enteritis, peritonitis, endocarditis, pneumonia, lung abscesses, staphylococcal sepsis, and others) [1,8,9]. The duration of the existing production bacteriology negative impact on the provision of timely medical aid to the patient, complicating quick apply adequate terap is I. Therefore, the development of methods for rapid cultivation of staphylococci for the diagnosis of infections associated with health care.

The aim of the invention is to increase the growth rate of Staphylococcus aureus for the diagnosis of infections associated with health care, and reducing the time of issuance of the analysis for the timely appointment of adequate antibiotic therapy.

This objective is achieved in that when grown Staphylococcus aureus for diagnostic purposes in preparation for analysis in the nutrient medium on the basis of the yolk-salt agar as a growth promoter microorganisms injected chemical compound: Tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfonyl (1) or Tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfonyl (2) or Tris(2-hydroxy-ethyl)ammonium 2-chlorophenoxyacetate (3) or Tris(2-hydroxyethyl)ammonium 2-methyl-4 - chlorophenoxyacetate (4) or Tris(2-hydroxyethyl)ammonium 1-benzyliden-3-yl-sulfonylurea (5) in the form of aqueous solutions at a concentration of 10-4-10-6wt.% (table 1). When using growth stimulants 1-5 growth of Staphylococcus reduced from 48 (without stimulants) up to 9 hours.

Table 1 presents the chemical structure of stimulants 1-5.

Table 2 presents the effect is biostimulators 1-5 on the growth of Staphylococcus aureus.

As growth stimulants 24 strains St. aureus isolated from patients with purulent-septic complications from the Department of surgery, first investigated biologically active compounds are representatives of a new class of biologically active proton (2-hydroxyethyl)ammonium ionic liquids [3-5].

Methods of preparation and properties of stimulants 1-5 are given in examples 1-5. The composition and structure of compounds 1-5 were confirmed by IR, NMR spectroscopy and elemental analysis. IR spectra of (v, cm-1) were recorded on spectrophotometer Varian 3100 FT-IR75 in CVG. NMR spectra (δ, ppm) was shot in the D2O spectrometer DPX 400 (400,13 MHz to1N and 101,62 MHz to13C).

The following examples illustrate the invention:

Example 1. Tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfonyl (1)

To a solution of 2.03 g (0.01 mol) 4-chlorophenylsulfonyl acid in 10 ml of ethanol was bury ethanol solution of 1.49 g (0.01 mol) of Tris-(2-hydroxyethyl)amine (10 ml). The mixture was stirred at 25°C. for 3 hours. The solvent is kept in vacuum. The solid residue was repeatedly washed with ether and dried in a vacuum desiccator over P2About5. Received a colorless powder (yield 3.44 g, 98%) with TPL 91°C, soluble in water and organic solvents (acetone, THF, alcohols). NMR1H: 7.11-6.32 (m, 4H, C6H4S), 3.75 (t,6N, co2), 3.44 (who, 2H, SCH2COO), 3.28 (t, 6N, NCH2). NMR,13From: 177.19 (C=O), 155.81-111.55 (C6H4S), 60.79(SCH2COO), 55.09(och2), 54.83(NCH2). IR: 1560 (C=O), 2550-2700 (H+N), 3300 (OH). Found, %: C, 48.11; H, 6.01; N, 3.93. C14H22ClNO5S. Calculated, %: C, 47.79; H, 6.30; N, 3.98.

Example 2. Tris(2-hydroxyethyl)ammonium 4-chlorophenylsulfonyl (2)

Was obtained from 4-chlorophenylsulfonyl acid and Tris(2-hydroxyethyl)amine same as 1. Colourless powder with TPL 93°C. the Yield is 97%. NMR1H: 7.19-6.22 (m, 4H, C6H4SO2), 4.04 (s, 2H, SO2CH2COO), 3.71 (t,6N, co2), 3.22 (t, 6N, NCH2). IR: 1566 (C=O), 2500-2710 (H+N), 3310(HE). Found, %: C, 44.10; H, 5.50; N, 3.63. C14H22ClNO7S. Calculated, %: C, at 43.80; H, 5.77; N, 3.65.

Example 3. Tris(2-hydroxyethyl)ammonium 2-chlorophenoxyacetate (3)

Was obtained from 2-chlorophenoxyacetic acid and Tris(2-hydroxyethyl)amine same as 1. Colourless powder with TPL 81-83°C. a Yield of 99%. NMR1N: 7.26-6.02 (m, 4H, C6H4), 4.24 (s, 2H, och2Soo), 3.70 (t,6N, co2), 3.22 (t, 6N, NCH2). NMR13From: 178.09 (C=O), 157.01-111.05 (C6H4O), 60.00(och2MEO)57.19(och2), 55.93(NCH2). IR: 1558 (C=O), 2520-2700 (H+N), 3330(HE). Found, %: C, 50.23; H, 6.50; N, 4.18. C14H22ClNO6. Calculated, %: C, 50.07; H, 6.60; N, 4.17.

Example 4. Tris(2-hydroxyethyl)ammonium 2-methyl-4-chloro-PHENOXYACETIC (4)

Was obtained from 2-methyl-4-the EOS-phenyloxirane acid and Tris(2-hydroxyethyl)amine same as 1. Colourless powder with TPL 88°C, the Yield of 98%. NMR1N: 7.72-6.22 (m, 3H, C6H3), 4.44 (s, 2H, och2Soo), 3.78 (t,6N, co2), 3.38 (t, 6N, NCH2), 2.25 (CH3). NMR13From: 175.19 (C=O), 156.21-111.11 (C6H3O), 62.23(och2MEO)58.29(och2), 57.93(NCH2), 14.71 (CH3). IR: 1563 (C=O), 2500-2720 (H+N), 3310(HE). Found, %: C, 51.72; H, 6.62; N, 4.04. C15H24ClNO6. Calculated, %: C, 51.50; H, 6.91; N, 4.00.

Example 5. Tris(2-hydroxyethyl)ammonium 1-benzyliden-3-ylsulphonyl (5) was Obtained from 1-benzyliden-3-resultdisplay acid and Tris(2-hydroxyethyl)-amine analogously to 1. Colourless powder with TPL 109-110°C. a Yield of 96%. NMR1N (D3C)2SO, (δ, ppm, J/Hz), 3.05 (t, 6N, 3CH2N, J=5.3); 3.63 (t, 6N, SN2HE, J=5.3). IR: 1560, 1600 (C=O), 2500-2720 (H+N), 3290(HE). Found, %: C, 58.05; H, 6.23; N, 5.87. C23H30N2O7S. Calculated, %: C, 57.72; H, 6.31; N,5.85.

Example 6. Preparation of dilutions of growth stimulants for the study. During the dissolution tests were carried out for each potential growth stimulator according to the following procedure:

Was dissolved 0.1 g (100 mg) of the substance in 100 ml of distilled water, getting a 0.1% solution, which was further of the view matrix.

Further breeding:

The first breeding: 0.1 ml of the matrix 0.1% solution was added to 100 ml of the yolk-salt agar (ISA). The concentration of the drug in the environment with the Tawil 10 -4%.

Second breeding: 0.1 ml matrix 0.1% solution is added to 1000 ml ISA. The drug concentration in the medium was 10-5%.

The third breeding: 0.01 ml matrix 0.1% solution is added to 1000 ml ISA. The drug concentration in the medium was 10-6%.

In the experiment using each drug dilution in culture media and pure culture of pathogenic bacteria associated with health care.

Example 7. The experiment on the cultivation of St. aureus.

The capture and culture of infected material and the light of the results produced in accordance with regulatory orders [2,6,7].

Culture St. aureus was obtained by culturing in an incubator at 37°C with the use of the yolk-salt agar, which was prepared in accordance with the instructions on the vial label.

For the preparation of nutrient medium required quantity of dry matter stirred in 1 l of distilled water, boiled for 3 min until complete melting of agar, filtered through a cotton-gauze filter, poured into sterile vials and sterilized by autoclaving at 121°C for 15 min, the Medium was cooled to a temperature of 45-50°C, poured into sterile Petri dishes with a layer of 3-4 mm After solidification of the medium, following the rules of asepsis, cups were dried at the temperature of the round (37±1)°C for 40-60 minutes

Sowing on Petri dishes was performed by standard method. Identification of isolated microorganisms was evaluated using a commercial test systems company LACHEMA (Czech Republic): "STAPHYtest"

Of the identified culture St. aureus was preparing a suspension 104 in physiological solution according to the turbidity standard.

Then took three dilution in GSA, poured agar cups (three cups of each dilution in culture medium). The cups were sown 0.1 ml of the prepared suspension of Staphylococcus aureus and incubated at 37°C, viewing the cups were made every 2-3 hours, noting the growth of the colonies. As a control, were sown on JSA without growth promoter.

Counting colonies grown on the surface of the agar, produced with the help of a magnifying glass (X5) or of the device for counting colonies. To do this, put the Cup upside down on a dark sheet of paper (thus providing better visibility of the colonies). For convenience, each counted colony was noted from the bottom of the Cup with a pencil or marker. 1 When using stimulants 1-5 in the environment IS for accelerated growth of crops St. aureus has established the viability of the microorganisms. The results of the growth-promoting activity displayed in table 2.

From the data presented in table 2, you can see significant differences in germination strains St. aureus on the media with growth factors at different concentrations, and n is the control environment, ISA. So, when you use any stimulants growth at concentrations of 10-4-10-5weight. %of stimulator No. 5 at concentrations of 10-4-10-6weight. % after 3 hours was pulverized growth of microorganisms after 6 hours there were single colony, 9 hours after seeding, growth was observed St. aureus expressed lecithinase activity. When added to the growth medium growth stimulants 1-4 at a concentration of 10-6weight. % after 3 and 6 hours after seeding was observed powdery growth of microorganisms, 9 hours - growth of single colonies.

When sowing the studied cultures in the yolk-salt agar no growth stimulants after 3 and 6 hours of growth was not observed, and only 9 hours appeared powdery growth of microorganisms. Over 9 hours of cultivation St. aureus in the control were observed no growth of single colonies of microorganisms or the growth of the studied cultures with a strong lecithinase activity.

The method of growing microorganisms tested on the 24 strains St. aureus isolated from patients with purulent-septic complications from the Department of surgery with positive results in the implementation of microbiological monitoring

Studies made it possible to establish that the investigated compounds 1-5 stimulate the growth of culture St. aureus, substantially reducing Vremya growth compared to the control on the standard nutrient medium (GSA). These results can be used to develop an improved methodology for rapid diagnosis of nosocomial infections caused by S. aureus, which will greatly reduce the time of issuance of the analysis and timely prescribe an adequate treatment of the patient.

Literature

1. Briskin BS, N. Nachtman. Nosocomial infections and their prevention: a view of the surgeon /Surgery: - 2005. No. 2. - 26-30.

2. Aserinsky, Lpina, Asesino, Private medical Microbiology techniques microbiological studies // M, JSC "Publishing house "Medicine", 2005. - 600 C.

3. Mirkova A.N., Mirkov RG, Adamovich, S., Voronkov M.G., Synthesis and pharmacological activity of 2-hydroxyethylammonium salts organosulfur(sulfonyl) acetic acid - new pharmacologically active compounds. / / Chemistry for sustainable development. - 2011. - So 19. No. 5. - S-478.

4. Mirkova A.N., Levkovskaya GG, Stupina A.G., Chkhenkeli, VA, Voronkov M.G. Influence of Tris(2-hydroxyethyl)ammonium, araxi, aaltio and arylsulfonate on the activity of bifidobacteria // Dokl. An. - 2003. - 390 so. No. 2. - S-282.

5. Mirkova A.N., Levkovskaya GG, Mirkov RG, Voronkov M.G. Alkanolammonium salt organosulfur (sulfonyl) acetic acid - new stimulants biological processes // Zhur.org.chem.. - 2008. - So 44. - VIP. - S-1508.

6. About invites the medical care of patients with purulent surgical diseases and strengthening of measures to combat hospital-acquired infections: order No. 720 the USSR Ministry of health. M., 1978.

7. About the unification of microbiologic methods used in clinical diagnostic laboratories medical and diagnostic institutions: order No. 535 the USSR Ministry of health. M., 1985.

8. Onishchenko GG national concept of prevention of infections associated with health care. - 2011. p.21.

9. Rock L., Sidorenko SV, Nekhoroshev A.G. Practical aspects of modern clinical Microbiology. - M.: Labelform, 2004. P.64-69.

Table 1
ConnectionFormula
1. Tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfonylated
2. Tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfonylated
3. Tris(2-hydroxyethyl)ammonium 2-chlorophenyl-oxyacetic
4. Tris(2-hydroxyethyl)ammonium 2-methyl-4-chloro-PHENOXYACETIC
5. Tris(2-hydroxyethyl)ammonium-1-benzyliden-3-ylsulphonyl

Table 2
no growth stimulatorTime, hConcentration %
10-410-510-6
13andandand
6bband
9b
23andandand
6bband
9b
33andand and
6bband
9b
43andandand
6bband
9b
53andandand
6bbb
9
Control (no growth factor)3d"-""-"
6d"-""-"
9and"-""-"
Notes: - powdered microbial growth, b - isolated colonies of microorganisms, microbial growth with a strong lecithinase activity, Dr. no growth of microorganisms, "-" - studies have not been conducted.

Way fast growing Staphylococcus aureus Staphylococcus aureus for the diagnosis of infections, which consists in the cultivation of Staphylococcus aureus in nutrient medium containing yolk-salt agar with the addition of the biostimulator, characterized in that as biostimulant use the following compound: Tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfonylated or Tris(2-hydroxyethyl)ammonium 2-chlorophenoxyacetate or Tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenoxyacetate or Tris(2-hydroxyethyl)ammonium 1-benzyliden-3-yl-sulfonylurea that bring nutrients the environment in concentrations of 10-4-10-6wt.% in aqueous solution.



 

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