RU2738858C1 - Method of extracting uncultivated forms of staphylococci - Google Patents

Abstract

FIELD: microbiology; medicine.

SUBSTANCE: invention refers to medical microbiology and can be used in laboratory diagnostics to isolate uncultivated forms of staphylococci. Method of extracting uncultivated forms of staphylococci involves sowing the analyzed biomaterial, wherein tampon with biomaterial is pre-incubated for 18 to 24 hours in a thermostat at a temperature of + 37 °C in 2.0 ml of liquid nutrient medium - GRM-medium containing 1% to 4% of glucose, then performing the produced microorganisms suspension seeding with the tampon or the Gold calibration loop in parallel for 3 Petri dishes - with the meat infusion agar, 5% blood agar and 10% yolk-salt agar. Crops are cultivated for 24 to 48 hours at a temperature of +37 °C with subsequent qualitative and quantitative recording of growth of staphylococci by species.

EFFECT: advantage of the declared invention is creation of a simple method of extracting uncultivated forms of staphylococci, which does not require considerable material expenditures, which provides reliable results.

1 cl, 4 tbl, 3 ex

Description

The invention relates to medicine, more specifically to the field of medical microbiology and can be widely used in laboratory diagnostics to detect staphylococcal infection.

There is a problem in creating a safe environment for patients to stay in hospitals and ensuring the quality of care as priorities for health care. Staphylococcal infections are recognized as one of the most dangerous, complicating the result of medical care in inpatient and especially in obstetric institutions. The widespread use of antibiotic therapy has led to the emergence of uncultivated forms of microorganisms. The greatest danger lies in the ability of staphylococci to re-cultivate (revive) and restore their pathogenicity when they repeatedly enter the body of other people. In this regard, there is a problem of rapid and reliable detection of staphylococcal infection, both during clinical examination of the population and during hospitalization in hospitals, maternity hospitals.

Known, for example, a method of cultivating a culture of Staphilococcus aureus (See: Labinskaya A.S. "Microbiology with the technique of microbiological research" / publishing house "Medicine", 1978. - 394 S.) on mesopatamia agar (MPA) and on yolk-salt agar at a temperature of + 37 ° C. However, the known method does not allow to identify all possible forms of staphylococcus. Bacteria with non-cultivable properties do not grow on nutrient media used for microbiological research, but have pathogenic properties, due to this, false-negative research results are recorded in microbiological laboratories.

The closest analogue (prototype) of the claimed invention is a method for isolating uncultivated forms of staphylococci under the RF patent No. 2470074 dated 11/14/2011 for an invention (IPC C12Q 1/04). This method is based on the cultivation of the test material on mesopatamia agar for 24 hours at a temperature of 37 ° C, and then the grown culture of bacteria is kept at a temperature of + 4 ° C for 48 hours. The disadvantage of the prototype is that the cultivation of uncultivated forms of staphylococci on mesopatamia agar is not possible. When you reproduce the method, you cannot get a positive result.

The objective of the claimed invention was to develop a new method for reliable isolation of uncultivated forms of staphylococci, devoid of the disadvantages of known analogues. The technical result of the claimed invention is to create a simple method that does not require significant material costs for the isolation of uncultivated forms of staphylococci.

The essence of the invention lies in the fact that the method for isolating uncultivated forms of staphylococci includes sowing the studied biomaterial, and the tampon with the biomaterial is pre-incubated for 18 to 24 hours in a thermostat at a temperature of + 37 ° C in 2.0 ml of a liquid nutrient medium - GRM broth, containing from 1% to 4% glucose, then the obtained suspension of microorganisms is inoculated with the swab itself or with a calibration loop according to the Gold method, in parallel on 3 Petri dishes - with mesopatamia agar, 5% blood agar and 10% yolk-salt agar, after which the crops are cultivated from 24 to 48 hours at a temperature of + 37˚С, followed by qualitative and quantitative registration of the growth of staphylococcal cultures by species characteristics.

Method for isolating uncultivated forms of staphylococci. First, in accordance with the Methodological Guidelines MUK 4.2.1890-04 "Determination of the sensitivity of microorganisms to antibacterial drugs", biomaterial is taken from the nose, pharynx and palatine tonsils with a disposable sterile swab. Then the method is carried out in two stages. The first stage is preliminary. At this stage, a standard sterile plastic tube with a volume of 10.0 ml is poured in an amount of 2.0 ml of 1-4% glucose broth prepared on the basis of the culture medium of the GRM broth. The GRM broth is intended for the cultivation of various microorganisms, unpretentious in their nutritional needs, when conducting research in sanitary and clinical microbiology. "GRM-broth" is a finely dispersed hygroscopic powder of light yellow color. Produce "GRM-broth" in FBUN "State Research Center for Industrial Medicine", Russia, Moscow region, Serpukhovskaya district, Obolensk, in two versions (based on g / l): 1) pancreatic hydrolyzate of fish meal 18.0 and sodium chloride 2.0; 2) pancreatic hydrolyzate of fish meal 8.0, peptone dry enzymatic 8.0 and sodium chloride 4.0. Then the probe-swab with the biomaterial is immersed and left in a thermostat at a temperature of 37 ° C for 18-24 hours for incubation. Next, a glucose broth is prepared: based on 1 liter of distilled water, add 20.0 g of commercial powder of timing broth, stirring continuously, bring the solution to a boil, add from 1% to 4% glucose powder (10-40 grams, respectively) and in boil for 3 minutes until it is completely dissolved. The thus prepared glucose broth is poured into glass bottles of 500.0 ml, closed with cotton-gauze stoppers and sterilized in an autoclave at 0.5 ATM (121 (C) for 30 minutes. The second stage involves inoculation with a calibration loop (d = 3mm) of a bacterial suspension from a test tube with 1% glucose GRM broth with sectors according to the Gold method on Petri dishes filled with nutrient media: mesopatamia agar (MPA), 5% blood agar (CA) and 10 % yolk-salt agar (YSA).

Table 1

Yolk Salt Agar (YSA) The medium is intended for the isolation of staphylococci, detection of lipochromic pigment and lecitovitellase.
For preparation use mesopatamia agar (pH 7.3 + 0.1) with the addition of 10% sodium chloride. After filling in vials of 100.0-200.0 ml, agar is sterilized in an autoclave for 20 minutes at a temperature of 121 ° C.
Before use, the medium is melted, cooled to 45-50˚C and 20% (by volume) of sterile yolk suspension (1 chicken egg yolk per 150.0-200.0 ml of sterile isotonic sodium chloride solution) is added. The medium is quickly mixed, poured into cups, which can remain in the refrigerator for a long time in plastic bags. The finished medium is translucent, has a whitish color with a light cream shade.
The results are recorded after 36-48 hours of incubation in a thermostat at 35-37 ° C. Blood agar (CA) To 100.0 ml of 2% sterile MPA pH 7.5 + 0.1, melted and cooled to 45 ° C, observing the rules of asepsis, add 5.0 ml of MPA of sterile defibrinated blood of a horse, rabbit, ram or human, which does not contain preservatives or antibacterial drugs. The mixture is thoroughly mixed, being careful not to form foam. The prepared medium is poured into sterile Petri dishes in a uniform layer of not more than 4.0 mm. Meat Peptone Agar (MPA) MPA is used for the cultivation of unpretentious microorganisms and as a basis for the preparation of dense special media for the cultivation of more demanding microbes (serum, blood agar, etc.)
Structure:
Peptone - 10.0 g
Meat water - 1000.0 ml
Agar - 1.0 - 20.0 g
pH 7.3 + 0.2
The mixture is boiled over low heat with constant stirring until the added ingredients are completely dissolved. The broth is filtered, the pH is increased, and crushed agar is added (depending on the quality of the agar and the purpose of the medium). After adding agar, the medium is boiled over low heat with constant stirring until the agar is completely dissolved. When the environment becomes cloudy, it is enlightened by one of the known methods.
The prepared medium is poured into test tubes or vials, sterilized in an autoclave at 121 ° C for 20 minutes.

After incubation, inoculations performed at 37 ° С for 18-24

hours, carry out qualitative and quantitative accounting of staphylococci (in

microbial units - IU). Qualitative accounting of the growth of microorganisms

carried out visually, assessing the morphological features of golden and

coagulase-negative staphylococci, traditionally confirming

the reliability of the assessment by the plasma coagulase test. Thus, in

test tube with biomaterial in 1% glucose GRM broth is carried out

the transition of staphylococcus from an uncultivated form to a cultivated one

by increasing the nutritional properties of the environment.

The claimed method is illustrated by specific examples of execution,

not limiting the scope of legal protection.

Example 1 . The growth of uncultivated staphylococci on

different options for nutrient media. The experiment compares

quantitative growth of staphylococci on the media of MPA, CA, IVA after

direct seeding of biomaterial from sick people and after preliminary

incubation of the material (24 hours at 37 ° C) in 1% glucose GRM broth.

Growth of NF staphylococci as a result of cultivation of biomaterial from

people on BCH, 1% glucose BCH and 1%, 2%, 3%, 4% glucose GRM-

broths are presented in table 2 and indicates a positive

as a result, in the case of incubation with both 1% glucose BCH and 1-4%

glucose timing broths. However, timing broth is cheaper than mesopatamia

(BCH), in this connection, and was chosen by us for use.

table 2

Evaluation of the growth of uncultivated forms of staphylococci on different variants of liquid culture media

Type of microbe isolated from a person The presence of growth on nutrient media BCH 1% glucose BCH 1% GRM glucose broth 2% GRM glucose broth 3% GRM glucose broth 4% glucose GRM broth SF S.aureus - + + + + + NF staphylococci coagulase-negative - + + + + + CFU / ml
S. aureus NF on JSA - 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ number
CFU / ml coagulase-negative staphylococci on VCA - 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ CFU / ml
SF S.aureus
take that - 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ The number of CFU / ml coagulase-negative staphylococci per CA - 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸

Example 2. Reflects the quantitative growth (in IU) of uncultivated

forms of staphylococci with different cultivation methods. Biomaterial,

taken from the throats of people in the amount of 200 samples, was seeded on

universal medium - 5% blood agar (CA) and 10% yolk-salt agar.

After cultivation of crops at 37 ° C after 24-48 h on the YSA and CA growth

staphylococci were absent. At the same time, an increase in another

microflora, among which there were: non-pathogenic Neisseria,

coryneform bacteria, streptococci of all groups (α-, β-, γ-hemolytic),

hemophilic sticks, sometimes enterobacteria. In accordance with

methodological recommendations, the biomaterial on the tampons was introduced into

a test tube with BCH and cultured for a day at 37 ° C. As

experiment, the second swab with the same biomaterial was introduced into a test tube with

1% glucose GRM broth (37 ° C, 24 hours). After the daily

incubation, in parallel, from both tubes were repeated

inoculation on media (JSA, CA, Sabouraud-agar) and cultured again at 37 ° C 24

hours. On plates of culture media with inoculation from the first tube with MPB growth

staphylococci were absent, and on nutrient media with inoculation from the second

tubes with 1% glucose GRM broth - was present. High quality

diagnostics using rabbit plasma made it possible to establish

species of staphylococci and state that the detection

Staphylococcus aureus occurred as often as

coagulase-negative species. Staphylococcal cultures grew on

both liquid media - MPB and 1% glucose GRM broth, only in that

if earlier they grew on the spacecraft, which means they were

cultivated. When inoculated from two test tubes

a calibration loop (d = 3mm) with sectors according to the Gold method on the media of ZhSA and

CA, a quantitative advantage of the growth of staphylococci was established,

grown in the second test tube. The same pattern was traced

regarding the growth of yeast-like fungi of the genus Candida on MPA media and

Sabouraud with antibiotic chloramphenicol after seeding of two

compared environments (see table 3).

Table 3

Assessment of quantitative growth on solid nutrient media

cultivated and non-cultivated forms of microorganisms after

their preliminary incubation on BCH and 1% glucose GRM broth__

Type of microorganism Name of culture media BCH 1% GRM glucose broth MPA 5% CA 10% YSA Saburo-agar S.aureus6538рАТСС = 209р + + 10 ⁸ 10 ⁸ 10 ⁸ SF S.aureus neg neg neg neg + 10 ⁸ 10 ⁸ 10 ⁸ NF Staphylococcus coagulase-negative neg neg neg neg + 10 ⁸ 10 ⁸ 10 ⁸ Yeast-like fungi of the genus Candida + 10 ⁵ + 10 ⁸

Note: NF is an uncultivated form

Table 3 shows that the cultivation on 1% glucose GRM-

broth allows you not only to isolate uncultivated forms

golden, coagulase-negative staphylococci, but also increase

the number of populations of cultivated forms of both staphylococci and

yeast-like fungi of the genus Candida (not using as an additive

antibiotics, as is done with Sabouraud's liquid medium).

Growing two types of microorganisms on one medium without participation

additional components, increases economic value

presented option.

Example 3. Shows the presence of different sensitivity to

antibiotics in uncultivated strains of staphylococci (golden and

coagulase-negative) isolated from humans and strain (S. aureus No. 6538

pATCC = 209p) taken from the collection of museum cultures obtained from

GISK. Due to the fact that 199 strains isolated from humans

staphylococci did not grow during the primary inoculation on CA, MPA and YSA, they

were classified as uncultivated forms and studied for

resistance which is presented in table 4.

Table 4

Antibiotic sensitivity of the museum strain of aureus

staphylococcus and strains of uncultivated coagulase-negative and

Staphylococcus aureus, isolated from humans to antibiotics,

Microbe type Number of strains Antibiotic name oxacillin methicillin amoxiclav meropenem imipenem azithromycin levofloxacin gentamicin amikacin chloramphenicol ciprofloxacin cefepime Ciprofloxacin-sulbactam clarithromycin S.aureus 6538рАТСС = 209р one in in in in in in in in in in in in in in SF S.aureus 99 R R at at at at at at at at at at in at NF Staphylococcus coagulase-negative one hundred R R at at in at at in at in at in at at

Note: NF is an uncultivated form; b-high sensitivity, y-moderate sensitivity, p-resistance

The results presented in table 4 indicate the presence

resistance of isolated staphylococci to many antibiotics, including

of 75.8% of the strains showed resistance to oxacillin

(semi-synthetic penicillin) and methicillin (beta-lactam

antibiotic of the penicillin series), which indicates that these

cultures to MRSA strains, indicating their infectious

aggressiveness, which may be mediated by the possibility of plasmid

transfer with genes of pathogenicity and antibiotic resistance.

The use of the proposed invention makes it possible to increase

reliability of isolating pathogens of staphylococcal infections,

reduce the number of negative research results and

ensure in a fuller extent the implementation of anti-epidemic

activities in the foci of staphylococcal infections.

Claims (1)

A method for isolating uncultivated forms of staphylococci, including sowing a test biomaterial, characterized in that a tampon with a biomaterial is preincubated for 18 to 24 hours in a thermostat at a temperature of + 37 ° C in 2.0 ml of a liquid nutrient medium - GRM broth containing from 1% up to 4% glucose, then the resulting suspension of microorganisms is inoculated with the swab itself or with a calibration loop according to the Gold method in parallel on 3 Petri dishes - with mesopatamia agar, 5% blood agar and 10% yolk-salt agar, after which the inoculations are cultured for 24 to 48 hours at a temperature of + 37 ° C, followed by qualitative and quantitative accounting of the growth of staphylococcal cultures by species.