RU2292398C2 - Method for presentation of hospital staphylococcus strain staphylococcus aureus - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: claimed method includes culturing of tested Staphylococcus aureus for 12 h, concentration of Staphylococcus aureus microbial slurry is adjusted to 1 bln CFU/ml. Prepared microbial slurry in amount of 0.1 ml is introduced into test tube with 0.3 ml of plasma diluted with isotonic sodium chloride solution in ratio of 1:5. Staphylococcus aureus strain is recognized as hospital one when coagulation time is not more 50 min.

EFFECT: accelerated investigation method with improved accuracy.

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Description

The invention relates to medicine, namely to microbiological laboratory diagnostics of pathogens of hospital staph infections.

In the Tyumen region in 2000, 1098 cases of nosocomial infections were registered: in obstetric institutions (51%), surgical departments (22%) and outpatient clinics (22%). (Marchenko A.N. et al. Scientific Bulletin of the Tyumen Medical Academy. Medicine and Health. Tyumen, 2001. - No. 4, - p. 85). In the surgical departments of the Tyumen Regional Clinical Hospital, hospital infections with purulent-inflammatory processes caused by pathogenic staphylococci predominate (Timokhina T.Kh et al. Etiology of purulent infections in surgical hospitals of the Tyumen Regional Hospital / Scientific Bulletin of the Tyumen Medical Academy. Medicine and health. Tyumen, 2002. - P.105).

A known method for the identification of pathogenic staphylococci, which includes the sowing of pathological material on nutrient media, followed by isolation of pure culture and the study of biological properties. Identification of the selected culture is carried out for 6-7 days according to morphological, cultural, biochemical, hemolytic, plasma-coagulating properties, lecithinase activity and the presence of enterotoxin. Intraspecific identification of the isolated bacterial culture is carried out by determining the phagotype, sensitivity to antibiotics and other signs (Labinskaya A.S. Microbiology with the technique of microbiological research. M .: Medicine. - 1978. - S.164-174).

The above-described method of laboratory diagnosis of strains of the species Staphylococcus aureus is laborious, requires large material costs for nutrient media and reagents. To determine whether the selected strain belongs to the hospital one, it is necessary to conduct additional studies on the isolation of pathogens from patients, bacterial carriers, and from the environment with their subsequent identification. Laboratory studies are carried out for a long time (6 days or more). The above disadvantages of the method of laboratory diagnosis of hospital strains of S.aureus do not allow public health authorities to widely use this method for laboratory diagnosis of hospital strains.

Yafaev R.Kh and Zueva L.P. (Epidemiology of nosocomial infection. L .: Medicine. - 1989. - P.26-31) hospital strains include bacterial cultures isolated from patients in a hospital with a pronounced resistance "to a certain number of antibiotics."

Shevchenko Yu.L. with co-authors (Shevchenko Yu.L., Grishanin V.A., Matveev S.A., Mazayshvilli K.V. // Hospital infection and some aspects of its immunoprophylaxis / Herald of surgery. - 1996, No. 5. P.104-106 ) microorganisms with the same taxonomic position according to 3 or more traits and resistance markers to at least 5 antibacterial drugs are assigned to hospital strains in case of isolation of bacteria from different patients for a limited period of time.

There is a method of identifying hospital strains by determining the sensitivity of strains to antibiotics, compiling antibioticograms and comparing antibioticograms of bacteria cultures isolated from patients and from the environment (RU 2003101899 A. Kozlov LB S 12 Q 1/04. Publ. 10.02.2005. Bull. No. 4), selected as the closest analogue of the claimed invention. The disadvantage of the proposed method is the need for preliminary identification of pathogens isolated from patients and environmental objects with the subsequent determination of the sensitivity of the selected cultures to antibiotics.

A known method for determining the plasma-coagulating properties of pathogenic staphylococci, which in combination with other properties to determine the type of pathogenic pathogen: Staphylococcus aureus. The method consists in the following: 0.5 ml of plasma diluted 1: 5 with isotonic sodium chloride solution is poured into two tubes, then an 18-20-hour staphylococcus culture is introduced into each tube by a bacteriological loop. One test tube (experimental) is inoculated with the studied culture, the second (first control) is a known pathogenic, plasma-coagulating staphylococcus strain. Inoculated tubes are kept in a thermostat (37 ° C) for 3 hours. If plasma coagulation does not occur during the indicated time, the tubes are left at room temperature for another 18 hours. If plasma coagulation does not occur even after this time, the test culture is coagulase-negative. At the same time, several tubes with unplated plasma are placed in the thermostat (second control). If plasma coagulation due to microbial contamination occurs in one of the tubes of the second control, the result of the experiment is not taken into account. In total, the result of the experiment is taken into account after 21 hours (Labinskaya A.S. Microbiology with the technique of microbiological research. M .: Medicine. - 1978. - P.167).

A significant drawback of the proposed method is that the method is intended to detect in pathogenic staphylococci one of the most common enzymes of aggression, and not to indicate hospital strains.

The objective of the present invention is to develop a new, economical express method for indicating hospital strains of Staphylococcus aureus.

The technical result is a simple, low-cost express method for indicating hospital staphylococcus strains, based on the properties of hospital strains to have an earlier plasma-coagulating ability compared to community-acquired strains of pathogenic staphylococci. As a criterion for the indication of hospital strains, the determination of plasmocoagulase in pathogenic staphylococci by the time of plasmocoagulation is proposed. The well-known property of pathogenic staphylococci was used for a new purpose, namely, to indicate hospital strains.

The technical result is achieved by the fact that the method of indicating hospital strains of S.aureus according to the invention is carried out by coagulation of plasma in up to 50 minutes with a 12-hour culture of staphylococcus, standardized by any conventional method to a working concentration of microbial suspension equal to 1 billion CFU / ml.

The essence of the method is achieved by the fact that according to the invention, hospital strains in a shorter time cause plasma coagulation compared to community-acquired S.aureus strains, and the use of a standardized 12-hour bacterial culture compared to an 18-20-hour culture allows to obtain genetically more uniform bacterial populations .

The proposed method is as follows.

On standard culture media for culturing S.aureus, a 12-hour staphylococcus culture is grown at a temperature of 37 ° C. The microbial suspension by any conventional method is adjusted to a working concentration of microbial suspension equal to 1 billion CFU / ml. The resulting suspension of bacteria in an amount of 0.1 ml is introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with an isotonic sodium chloride solution. Coagulation of plasma occurs in a thermostat at a temperature of 37 ° C. Analysis of the results of the reaction is observed for 85 minutes. Plasma coagulation time is taken into account using a stopwatch.

The proposed method for indicating hospital strains of staphylococci in comparison with analogues can save material resources and reduces the time of research.

Examples of specific performance of the proposed method

Example 1

From patient B., 26 years old, being treated at the Department of Purulent Surgery of the Regional Clinical Hospital (OKB) of Tyumen, S.aureus culture was isolated. The pathogen was identified in a bacteriological laboratory in the Tyumen Design Bureau according to generally accepted tests for staphylococci: the presence of hemolysins, pigments, lecithinase on yolk-salt agar, plasmocoagulase, catalase, mannitol fermentation under aerobic and anaerobic conditions, and aerobic and aerobic glucose fermentation under aerobic conditions.

After establishing the belonging of the selected culture to the species S.aureus, the strain was examined for sensitivity to antibiotics to establish the belonging of the strain to the hospital variant. The isolated S.aureus strain turned out to be resistant to gentamicin, oxacillin, lincomycin, amikacin, norfloxacin, ciprofloxacin and is therefore assigned to the hospital and assigned serial number 2888.

The isolated S.aureus culture was grown on a standard nutrient medium (nutrient agar for the cultivation of microorganisms, dry. Ministry of Health of the Russian Federation NGOs "Nutrient media", Makhachkala). The composition of the nutrient medium, g / l: pancreatic sprat hydrolyzate - 17.9; agar - 11.2 ± 1.2; sodium chloride - 7.7 ± 0.3. pH 7.3 ± 0.2.

Sowing of the selected culture was done with a stroke on mowed agar. Bacterial growth occurred within 12 hours at a temperature of 37 ° C, which corresponds to the stationary phase of the maximum bacterial growth (Pyatkin K.Yu., Krivoshein Yu.S. Microbiology. M .: Medicine, 1980).

Then, a working concentration of microbial suspension in physiological saline was prepared using a bacterial suspension turbidity meter — a Biomerie densitometer. The working concentration of microbial suspension was 3.3 units by MF (MacFarland), which corresponded to 1 billion CFU / ml.

A working concentration of microbial suspension in the amount of 0.1 ml was introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with isotonic sodium chloride solution. Plasma coagulation took place at a temperature of 37 ° C. The coagulation rate of plasma was taken into account using a stopwatch.

Plasma coagulation was observed after 44 minutes. The selected strain by the time of plasma coagulation is assigned to the hospital.

Example 2

As a control, the plasma-coagulating activity of the community-acquired museum strain S.aureus of the American collection of typical cultures of microorganisms (ATCC No. 209-M) was studied. For the growth of bacteria used a standard nutrient medium (nutrient agar for the cultivation of microorganisms, dry. Ministry of Health of the Russian Federation NPO "Nutrient media", Makhachkala). The composition of the nutrient medium, g / l: pancreatic sprat hydrolyzate - 17.9; agar - 11.2 ± 1.2; sodium chloride -7.7 ± 0.3. pH 7.3 ± 0.2.

Inoculation of the museum strain ATS No. 209-M was carried out on mowed agar with a stroke. Bacterial growth occurred within 12 hours at a temperature of 37 ° C. Then, the working concentration of microbial suspension in physiological saline was prepared. The concentration of microbial suspension was determined by the turbidity of the bacterial suspension using a Biomerie densitometer. The working concentration of the microbial suspension was 3.3 units by MF (MacFarland), which corresponded to 1 billion CFU / ml.

A working concentration of microbial suspension in the amount of 0.1 ml was introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with isotonic sodium chloride solution. Plasma coagulation took place in a thermostat at 37 ° C. Accounting for plasma coagulation time was carried out using a stopwatch.

Plasma coagulation was observed after 60 minutes. The isolated strain in terms of plasma coagulation is attributed to community-acquired.

Example 3

Patient K., 45 years old, was treated in the intensive care unit of the Tyumen Design Bureau. The S.aureus culture at No. 2891 was isolated from the patient. The pathogen was identified in the bacteriological laboratory of the Tyumen Design Bureau according to generally accepted tests for staphylococci: the presence of hemolysins, pigments, lecithinase on yolk-salt agar, plasmocoagulase, catalase, fermentation of mannitol in aerobic and anaerobic conditions; glucose fermentation under aerobic and anaerobic conditions.

After establishing the affiliation of the isolated cultures to the species S.aureus, the strain was tested for antibiotic sensitivity to establish the affiliation of the strain to the hospital variant. Strain S.aureus No. 2891 was resistant to gentamicin, oxacillin, lincomycin, amikacin, norfloxacin, ciprofloxacin and is therefore classified as hospital.

For the growth of bacteria used a standard nutrient medium (nutrient agar for the cultivation of microorganisms, dry. Ministry of Health of the Russian Federation NPO "Nutrient media", Makhachkala). The composition of the nutrient medium, g / l: pancreatic sprat hydrolyzate - 17.9; agar - 11.2 ± 1.2; sodium chloride - 7.7 ± 0.3. pH 7.3 ± 0.2.

Sowing culture No. 2891 was carried out on mowed agar with a stroke. Bacterial growth occurred within 12 hours at a temperature of 37 ° C. Then, a working concentration of microbial suspension in physiological saline was prepared using a device for determining the turbidity of a bacterial suspension — a Biomerie densitometer. The working concentration of microbial suspension was 3.3 units by MF (MacFarland), which corresponded to 1 billion CFU / ml.

A working concentration of microbial suspension in the amount of 0.1 ml was introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with isotonic sodium chloride solution. Plasma coagulation was carried out at 37 ° C. The reaction results were taken into account using a stopwatch.

Plasma coagulation was observed after 44 minutes. The selected strain by the time of plasma coagulation is assigned to the hospital.

Example 4

Patient T., 48 years old, was treated in the burn department of the Design Bureau of Tyumen. The culture of S.aureus No. 2305 was isolated from the patient. The pathogen was identified in a bacteriological laboratory in the Tyumen Design Bureau according to generally accepted tests for staphylococci: the presence of hemolysins, pigments, lecithinase on yolk-salt agar, plasmocoagulase, catalase, mannitol fermentation under aerobic and anaerobic conditions, and aerobic and aerobic glucose fermentation under aerobic conditions.

It was established that the isolated culture possessed properties characteristic of the species S.aureus. The strain was tested for sensitivity to antibiotics. Strain S.aureus No. 2305 was resistant to gentamicin, oxacillin, lincomycin, amikacin, norfloxacin, ciprofloxacin and is therefore classified as hospital.

For the growth of bacteria used a standard nutrient medium (nutrient agar for the cultivation of microorganisms, dry. Ministry of Health of the Russian Federation NPO "Nutrient media", Makhachkala).

Sowing of the selected culture was done with a stroke on mowed agar. Bacterial growth occurred within 12 hours at a temperature of 37 ° C. Then, a working concentration of microbial suspension in physiological saline was prepared. Using a device for determining the turbidity of a bacterial suspension - densitometer company "Biomerie". The working concentration of microbial suspension was 3.3 units by MF (MacFarland), which corresponded to 1 billion CFU / ml.

A working concentration of microbial suspension in the amount of 0.1 ml was introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with isotonic sodium chloride solution. Plasma coagulation was carried out at 37 ° C. Using a stopwatch, the time during which plasma coagulation occurred was determined.

Plasma coagulation was observed after 45 minutes. The strain is assigned to the hospital.

Example 5

Patient C., 29 years old, was treated in the burn department of the Design Bureau of Tyumen. S.aureus culture No. 1719 was isolated from the patient. The pathogen was identified in the bacteriological laboratory of the Tyumen Design Bureau according to generally accepted tests for staphylococci: the presence of hemolysins, pigments, lecithinase on yolk-salt agar, plasmocoagulase, catalase, mannitol fermentation in aerobic and aerobic fermentation of glucose under aerobic and anaerobic conditions.

The bacterial culture is classified as S.aureus. The strain was tested for sensitivity to antibiotics and was sensitive to gentamicin, oxacillin, lincomycin, amikacin, norfloxacin, ciprofloxacin, and therefore it was assigned to community-acquired.

For the growth of bacteria used a standard nutrient medium (nutrient agar for the cultivation of microorganisms, dry. Ministry of Health of the Russian Federation NPO "Nutrient media", Makhachkala).

Sowing of the selected culture was done with a stroke on mowed agar. Bacterial growth occurred within 12 hours at a temperature of 37 ° C. Then, a working concentration of microbial suspension in physiological saline was prepared. Using a device for determining the turbidity of a bacterial suspension - densitometer company "Biomerie". The working concentration of microbial suspension was 3.3 units by MF (MacFarland), which corresponded to 1 billion CFU / ml.

A working concentration of microbial suspension in the amount of 0.1 ml was introduced into a test tube with 0.3 ml of plasma diluted 1: 5 with isotonic sodium chloride solution. Plasma coagulation was carried out at 37 ° C. Using a stopwatch, the time during which plasma coagulation occurred was determined.

Plasma coagulation was observed after 81 minutes. The strain is classified as community-acquired.

With strains of ATCC 209-M, 2888, 2891, 2305, 1719, 10 experiments were conducted to determine the plasma-coagulating activity. The results of the experiments are presented in the table.

Studies on the identification of S.aureus strains were carried out in the bacteriological laboratory of the Design Bureau of the city of Tyumen according to generally accepted methods.

In 2004, 1233 bacterial strains isolated from patients being treated in the Design Bureau of the city of Tyumen were studied. The incidence of S.aureus was 18%. Most often, S.aureus was isolated from patients in the burn department (32.4%), purulent surgery (26.5%), resuscitation and intensive care (10.3%). Isolated staphylococci in 32.4% had multiple antibiotic resistance. Based on resistance to oxacillin, gentamicin, amikacin, ciprofloxacin, lincomycin, these strains were assigned to hospital. So, the most common hospital strains were allocated in the departments of surgery, burn and resuscitation and intensive care. Therefore, 3 strains of S.aureus species isolated from these departments were taken for the study. Studies on the plasma-coagulating activity of the strains were carried out on the basis of the bacteriological laboratory of the Department of Microbiology, GOU VPO of the Tyumen State Medical Academy of Roszdrav.

The above examples illustrate the possibility of using the proposed method for indicating hospital strains.

The proposed method for indicating hospitalized staphylococcal strains does not require large material costs, it is easy to reproduce, it shortens the detection time for hospitalized staphylococcal strains and allows the indication of hospitalized staphylococcal strains with plasma-coagulating activity with an accuracy of 100%.

Table
Plasma coagulation time (in min) of hospital and community-acquired S.aureus strains p / p
Experience number Strains of the species S.aureus ATCC 209-M 2888 2891 2305 1719 Plasma coagulation time (in min) one 60 44 44 45 81 2 63 41 43 47 80 3 61 45 41 46 86 four 60 43 45 48 83 5 61 45 44 41 87 6 64 43 45 45 81 7 60 44 44 45 81 8 64 45 46 44 87 9 64 44 46 46 86 10 63 43 47 46 81 The average time coagulation of strains 62 ± 1.6 43.7 ± 1.06 44.5 ± 1.3 45.3 ± 1.3 83.3 ± 2.5

Claims (1)

A method for indicating hospital strains of Staphylococcus aureus, characterized in that the studied Staphylococcus aureus is cultured for 12 hours, bring the concentration of microbial suspension of Staphylococcus aureus to a working concentration of 1 billion CFU / ml, the prepared microbial suspension in the amount of 0.1 ml is introduced into a test tube with 0.3 ml ml of plasma diluted 1: 5 with isotonic sodium chloride solution, according to plasma coagulation within a period of up to 50 min, the Staphylococcus aureus strain is classified as a hospital strain.