RU2265654C2 - Nutrient medium for isolation of hemoculture - Google Patents

Abstract

FIELD: medicine, medicinal microbiology.

SUBSTANCE: invention proposes a medium that comprises para-aminobenzoic acid and agar, citrate or sodium heparinate as an organic salt, tris-(hydroxymethyl)-aminomethane as a buffer, and fish nutritive broth and casein pancreatic hydrolyzate, nutrient yeast as sources of nitrogen nutrition, and glucose. By sensitivity and accumulative effect with respect to strains of microorganisms causing bacteremia and sepsis exceeds the known medium and allows detecting microorganisms in the minimal their parent content in blood. Indicated advantages of the proposed medium enhance the effectiveness in diagnosis in research of blood for hemoculture. Invention can be used in isolation of hemocultures in sepsis and bacteremia.

EFFECT: valuable properties of medium.

2 tbl, 3 ex

Description

The invention relates to medicine, namely to medical microbiology, and can be used to isolate blood cultures in sepsis and bacteremia.

Bacteremia and sepsis continue to be one of the urgent problems of modern medicine due to the steady trend towards an increase in the number of patients and a stable high mortality rate even in the most reputable domestic and foreign clinics.

The causative agents of sepsis and bacteremia can be bacteria, fungi, protozoa and viruses. But bacteria account for more than 95% of cases. The largest share among pathogens of bacteremia and sepsis is made by opportunistic microorganisms, while the proportion of gram-positive and gram-negative bacteria is approximately the same (1).

Registration of bacteremia and sepsis (isolation of blood cultures) is necessary to confirm the diagnosis and determine the etiology of the infectious process, justify the choice of antibiotic therapy regimen and evaluate its effectiveness.

Known nutrient medium for the allocation of blood culture. However, this medium is intended to isolate only salmonella from the blood, which limits its use (2).

A nutrient medium for the isolation of blood cultures is also known (3, 4), containing a source of nitrogen nutrition in the form of a Hottinger or Marten broth and glucose (sugar broth). The disadvantages of this environment include the formation of a fibrin clot during blood culture and a low cumulative effect due to the absence of growth stimulators of highly demanding microorganisms in the medium.

The closest solution to the problem of the same purpose in terms of the set of characteristics and the achieved effect is a nutrient medium for the isolation of blood cultures and cultivation of streptococci containing nitrogenous nutrition sources in the form of an enzymatic blood hydrolyzate and acid casein hydrolyzate, growth stimulator - extract of fodder yeast, organic and inorganic salts (5 )

The reasons that impede the achievement of the technical result indicated below when using a known medium adopted as a prototype are a narrow list of microorganisms (mainly streptococci) that are excluded, the absence of blood anticoagulants and substances that neutralize the antimicrobial effect of drugs that can accumulate in the blood when antibacterial therapy.

The absence of anticoagulants in the medium leads to the formation of a fibrin clot during blood culture, which limits the access of nutrients to the microbial cell, which inhibits the growth of microorganisms. The absence of substances in the environment that neutralize the antimicrobial effect of drugs has a negative effect on the seeding of blood cultures.

The consequence of these drawbacks is poor sensitivity and low cumulative effect of the medium, and a narrow list of allocated microorganisms limits the use of the known medium in bacteriological practice.

The purpose of the invention is to increase the sensitivity, cumulative effect of the environment and the expansion of the spectrum of allocated microorganisms.

The specified technical result during the implementation of the invention is achieved by the fact that the proposed medium additionally contains para-aminobenzoic acid and agar, contains sodium citrate or sodium heparin as an organic salt, Tris (oxymethyl) aminomethane as a buffer, and nitrogen sources as a buffer - nutrient broth fish and pancreatic hydrolyzate of casein in the following ratio of components, in g / l of distilled water:

Nutritional broth fish  10.0-20.0 Pancreatic Hydrolyzate casein  10.0-20.0 Yeast Extract  2.0-4.0 Glucose  8.0-12.0 Sodium citrate or  0.3-0.5 Sodium Heparinate  0.02-0.04 Para-aminobenzoic acid  0.005-0.015 Tris- (hydroxymethyl) -aminomethane  0.5-1.5 Agar  0.5-1.0

The introduction of additional para-aminobenzoic acid into the medium helps to neutralize the antimicrobial effect of drugs that may be contained in the blood during antibiotic therapy.

The introduction of the proposed environment additional agar with high sorption ability, helps to neutralize metabolic products and protects microbial cells from their toxic effects.

The introduction of sodium citrate or heparin sodium, which has anticoagulant properties, instead of sodium acetate, which does not have it, prevents blood coagulation (the formation of a fibrin clot), which facilitates the access of nutrients to the microbial cell, thereby contributing to the active reproduction of microorganisms in the medium.

Given that in patients with a positive blood culture, the concentration of pathogens in the blood usually does not exceed 10 ² / ml, the provision of conditions for the penetration of nutrients into the microbial cell is necessary for the active accumulation of microorganisms in the medium.

The use of tris- (oxymethyl) -aminomethane, which has a larger buffer capacity in comparison with sodium phosphate, as a buffer contributes to the fact that the acid resulting from the fermentation of glucose by microorganisms is completely neutralized, as a result of which the pH of the medium does not shift to acidic side that impedes the growth of microorganisms.

The high nutritional value of the nitrogenous components of the proposed environment - the nutrient broth from fish and pancreatic casein hydrolyzate fully provides the nutritional needs of microorganisms of various taxonomic groups that cause bacteremia.

On the whole, the totality of the presented characters contributes to the expansion of the spectrum of microorganisms secreted increases the sensitivity of the medium and provides a high cumulative effect, which makes it possible to isolate blood cultures with a minimum initial amount in the blood.

The environment is prepared as follows.

Example 1. In 1 liter of distilled water, 10 g of fish nutrient broth, 10 g of pancreatic casein pancreatic hydrolyzate, 2 g of yeast extract, 8 g of glucose, 0.3 g of sodium citrate or 0.02 g of heparin sodium, 0.005 g of para-aminobenzoic are successively dissolved acid; 0.5 g of tris- (oxymethyl) aminomethane; 0.5 g of agar. The mixture is heated until the agar is completely expanded, boiled for 1-2 minutes, poured into 100.0 ml vials and sterilized at 120 ° C for 30 minutes. Environmental quality control is carried out by seeding in vials of test strains of microorganisms - the most commonly isolated during sepsis and bacteremia: staphylococci, streptococci, salmonella, Escherichia, meningococci, Klebsiella, brucella, Yersinia, Pseudomonas aeruginosa, Listeria, etc. from ^10-6 and 10 ^-7 (10-10 cells, respectively) (6).

гдеAfter incubation of the crops in the thermostat at 37 ° C for 18-24 hours, growth is noted both visually and by seeding their vials with loops on nutrient, blood and serum agars. The cumulative effect is determined by the formula

Where

n ₁ - the number of colonies on plates seeded from the medium after 18-20 hours of incubation;

n ₀ - the number of viable cells in a seeding dose;

K is the degree of dilution.

Example 2. 15 g of fish nutrient broth, 15 g of pancreatic casein pancreatic hydrolyzate, 3 g of feed yeast extract, 10 g of glucose, 0.4 g of sodium citrate or 0.04 sodium hepinate, 0.01 g of steam are successively dissolved in 1 liter of distilled water -aminobenzoic acid, 1.0 g of tris- (hydroxymethyl) -aminomethane, 0.75 agar. Further according to example 1.

Example 3. In 1 l of distilled water, 20 g of fish nutrient broth, 20 g of pancreatic casein pancreatic hydrolyzate, 4 g of feed yeast extract, 12 g of glucose, 0.5 g of sodium citrate or 0.04 g of heparin sodium, 0.015 g of para- aminobenzoic acid, 1.5 g of tris- (oxymethyl) aminomethane, 1.0 g of agar. Further according to example 1.

The results of bacteriological control of the proposed and known environments are presented in tables 1, 2.

From table 1, 2 it is seen that the proposed environment has a high sensitivity and significant cumulative effect in relation to strains of microorganisms most often causing bacteremia and sepsis, and surpasses the known medium in terms of these indicators, which makes it possible to identify microorganisms with a minimum initial content in the blood.

Sources of information

1. Modern principles of antibacterial therapy of sepsis. V.A. Rudnov. Antibiotics and chemotherapy, vol. 45, 7, 2000, p.3-5.

2. Z.Z. Sultanov, E. D. Stepanova, E. A. Kakulina. Culture medium for the isolation of blood cultures in the diagnosis of typhoid fever and paratyphoid fever. Patent No. 2175671. Zaregist. November 10, 2001

3. Handbook of microbiological and virological research methods. Under. ed. M.O. Birger. M., 1982, p. 73, 255.

4. R.K. Cherkes, L.B. Epiphany, N.A. Belskaya. Microbiology. M., 1986, p. 240-241, 249, 261.

5. Handbook of microbiological culture media and microtest systems. Under. ed. M.M. Mejidova. Makhachkala, 1999, p.41 (prototype).

6. V.D. Badikov, L.E. Zhuravleva, V.P. Bolekhan. Improving the methods of microbiological blood tests in patients with purulent-septic infections. Wedge. lab. Diagn., 1998, No. 7, pp. 17-19.

Table 1.
Comparative characteristics of the growth of test strains of microorganisms on the proposed and known environments The name of the test strains On the proposed environment On a famous medium Sowing dose Sowing dose 100 cells 10 cells 100 cells 10 cells S.pyogenes Dick 1 ++ + + - S.pyogenes 1512 ++ + + - S.epidermidis ATCC 14990 +++ ++ ++ - S.aureus 46 +++ ++ ++ + S.aureus 209 "P". +++ ++ ++ + P.aerogenes 27/99 +++ ++ +++ ++ S.marcescens 1 +++ ++ +++ ++ E.coli 055 +++ ++ +++ ++ P.mirabilis 71 (2) +++ ++ +++ ++ P.vulgaris H19 +++ ++ +++ ++ K.pneumoniae 51 +++ ++ +++ ++ S.typhi H 901 +++ ++ +++ ++ S.pneumoniae LD ++ + + - B.abortus 19BA ++ + + - L.monocytogenes 56 +++ ++ ++ + J.enterocolitica 335 +++ ++ +++ ++ J.pseudotuberculosis 111 +++ ++ +++ ++ N.meningitidis 637 ++ - - - Designations: +++ - intensive growth ++ - average height + - weak growth - lack of growth

Table 2.
Comparative characteristics of the cumulative effect of the proposed and known environments The name of the strains Cumulative effect On the proposed environment On a famous medium S.pyogenes Dick 1 740 ± 15 360 ± 12 S.pyogenes 1512 750 ± 20 250 ± 9 S.epidermidis ATCC 14990 845 ± 18 470 ± 15 S.aureus 46 780 ± 16 540 ± 12 S.aureus 209 "P" 874 ± 20 560 ± 18 P.aerogenes 27/99 1284 ± 24 900 ± 22 S.marcescens 1 933 ± 22 850 ± 16 E.coli 055 3346 ± 50 1900 ± 22 P.mirabilis 71 (2) 3076 ± 45 1500 ± 21 P.vulgaris H19 2448 ± 35 1400 ± 26 K.pneumoniae 51 1000 ± 25 860 ± 12 S.typhi H 901 3140 ± 42 1800 ± 26 S.pneumoniae LD 766 ± 10 420 ± 16 B.abortus 19BA 950 ± 21 130 ± 14 L.monocytogenes 56 1330 ± 32 900 ± 24 J.enterocolitica 335 866 ± 26 420 ± 11 J.pseudotuberculosis 111 960 ± 30 560 ± 13 N.meningitidis 637 540 ± 12 -

Claims (1)

A culture medium for the isolation of hemocultures containing nitrogen sources, yeast extract, glucose, buffer, organic salt, distilled water, characterized in that it additionally contains para-aminobenzoic acid and agar, contains sodium citrate or sodium heparin as an organic salt, as buffer - tris- (oxymethyl) -aminomethane, as sources of nitrogen nutrition - nutrient broth fish and pancreatic hydrolyzate of casein in the following ratio of components, g / l distilled Noah water: Nutritional broth fish 10.0-20.0 Casein Pancreatic Hydrolyzate 10.0-20.0 Yeast Extract 2.0-4.0 Glucose 8.0-12.0 Sodium Citrate 0.3-0.5 or Sodium Heparinate 0.03-0.05 Para-aminobenzoic acid 0.005-0.015 Tris- (hydroxymethyl) -aminomethane 0.5-1.5 Agar 0.5-1.0