RU2156807C2 - Method of determination of antilactoferrin activity in microorganisms - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: method involves culturing analyzed culture on liquid nutrient medium and supernatant from cultures grown in the presence of lactoferrin is obtained. In parallel two control specimens are prepared, incubated and test-culture of M. luteus GISK N 211001 is added to experimental and control specimens. Optical density of specimens is measured and antilactoferrin activity of studied culture is calculated by optical density value change. Method ensures to detect the novel property of microorganisms, antilactoferrin activity. Proposed method promotes to select rational antibacterial therapy, study persistence mechanism in microorganisms and formation of germ carrying. Invention can be used in laboratories of science-research institutes and bacteriological laboratories of clinics studying the assay of persistence properties of microorganisms (in experiment) and for study of their etiological significance in pathological processes (in clinics). EFFECT: improved method of assay. 1 tbl, 2 ex

Description

 The invention relates to microbiology and can be used in the research work of the research institute and the practical work of the bacteriological laboratories of clinical institutions that determine the persistent characteristics of microorganisms to establish their etiological significance in pathological processes.

 Currently, the nature of the course of most infections has changed in the direction of increasing the proportion of protracted and chronic forms [1].

 Long-term experience of bacteria in the host’s body (carriage of bacteria), as well as the transition of the infectious process to a chronic form, are ensured by the mechanisms of persistence of microorganisms that inactivate a number of factors of natural anti-infection resistance: lysozyme, complement, interferon [2], platelet cationic protein [3], etc. At present time, more and more attention is paid to the study of the properties of pathogenic microorganisms aimed at inactivating the factors of natural resistance of the host organism.

 Known methods for determining antilysocyme [4], "anti-interferon" [5], anti-immunoglobulin [6], anti-complementary [7] and antiplatelet cationic protein [3] activities of microorganisms.

 Currently, it has been established that natural glycoprotein lactoferrin has a number of protective properties: bacteriostatic and bactericidal action [8], takes part in the processes of local protection of the mucous membranes [9], in the regulation of humoral and cellular immunological reactions, in anti-inflammatory processes, affects the complement system regulates granulocytopoiesis [10], hematopoiesis according to the feedback principle [11].

 The relevance of developing a method for determining the antilactoferrin activity of microorganisms is determined by the opportunity to improve the accuracy of diagnosis and prediction of diseases of microbial etiology, as well as the choice of rational antibacterial therapy to combat bacterial carriage.

 The objective of the proposed technical solution is to create a method for determining the antilactoferrin activity (ALFA) of microorganisms.

где ΔOD - изменение оптической плотности в опыте между 1 и 2 измерениями;
ΔOD₁- изменение оптической плотности в контроле 1 между 1 и 2 измерениями;
ΔOD₂ - изменение оптической плотности в контроле 2 между 1 и 2 измерениями.To solve this problem, in the inventive method, the studied microorganism culture is grown in a liquid nutrient medium, the grown cultures are treated with chloroform, the supernatant is separated and mixed with lactoferrin, two control samples are prepared in parallel with the experimental one, and liquid nutrient is added to control 1 instead of the supernatant of the studied microorganism cultures medium, and control 2 is prepared from buffered saline and liquid nutrient medium, the experimental and control samples are incubated, after incubation and add the test culture M.luteus GISK N 211001 and conduct the first measurement of optical density in the experiment and controls immediately after reaching the temperature control mode (37 ^o C) and the second measurement after 18-24 hours of incubation, then calculate antilactoferrin activity according to the formula

where ΔOD is the change in optical density in the experiment between 1 and 2 measurements;
ΔOD ₁ - change in optical density in control 1 between 1 and 2 measurements;
ΔOD ₂ - change in optical density in control 2 between 1 and 2 measurements.

 Analogues of the invention in the patent and scientific literature are not found.

The authors experimentally established that microorganisms, regardless of their species origin, are able to inactivate the bactericidal effect of lactoferrin. As a result of the studies, it was suggested that bacteria have certain mechanisms of protection against the influence of lactoferrin. This assumption was confirmed by experiments. Cultures of E. coli, S.aureus, S.epidermidis were grown on meat and peptone agar at a temperature of 37 ^o C for 24 hours, they were prepared in suspension at a concentration of 1 billion / ml. Then, in equal volumes, a suspension and a solution containing 2 mg / ml (sub-bacteriostatic concentration) of lactoferrin were mixed, the mixture was incubated for 30 minutes, centrifuged at 3000 rpm for 20 minutes, a cell-free supernatant was obtained, in which using an iEMS microbiological analyzer MF (Labsystems, Finland) determined the lactoferrin residue. It turned out that the culture of E. coli inactivated (75.6 ± 5.3)% of the drug, S. aureus - (68.8 ± 7.5)%, S.epidermidis - (54.6 ± 3.7)% lactoferrin.

 When developing the present method, the authors conducted studies on the choice of indicator culture, which allows testing the presence or absence of lactoferrin in the medium, since a microorganism strain highly sensitive to lactoferrin is required to carry out the method.

The authors, using the analysis of the growth curves of various types of microorganisms in a medium with lactoferrin, experimentally selected a test culture. For this, a suspension of daily agar cultures of the studied microorganisms (10 ⁶ CFU / ml; 50 μl) and a solution of lactoferrin in 0.01 M phosphate buffer (2 mg / ml, 50 μl) were inoculated into nutrient broth (300 μl). Samples without lactoferrin (50 μl of 0.01 M phosphate buffer) served as a control. Growth parameters (OD) were determined using automated photometry using an iEMS-MF microbiological analyzer (Labsystems, Finland). At the end of incubation in a thermostatic reader unit (after 18 hours), the change in optical density in the experimental sample was evaluated compared to the control (table).

 Thus, as a result of the studies, the authors selected the M.luteus strain as the test culture, which is most sensitive to the bactericidal effect of lactoferrin. The strain is deposited in the GISK them. L.A. Tarasevich under registration number 211001 [12].

 The proposed strain of M. luteus grows on simple nutrient media, the cells are spherical, 0.5-3.5 μm in diameter, motionless, gram-positive, aerobic, forms catalase, does not ferment glucose.

To implement the method, lactoferrin from cow's milk, crystalline, manufactured by Fluka (Switzerland) is used, the purity of the preparation (according to gel electrophoresis) is more than 75%, solubility 10 mg / ml H ₂ O, catalog number 61323 [13] .

 The method is as follows.

1) The studied cultures of microorganisms are grown in 3 ml of liquid nutrient medium (for each type of microorganism the appropriate type of nutrient medium is used) at 37 ^o C for 24-36 hours.

 2) Add 0.1 ml of chloroform to all samples, mix and incubate at room temperature for 40-60 minutes, shaking occasionally.

 3) The supernatant is separated from the bacterial cells by centrifugation for 20 minutes at 3000 rpm.

4) In a buffered saline solution (Sigma, P4417. Composition: 0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH 7.4 at 25 ^o C), a solution of lactoferrin with a concentration of 2.5 mg / ml is prepared.

 5) The supernatant of the studied cultures of microorganisms with a volume of 0.8 ml is mixed with 0.1 ml of the prepared solution of lactoferrin.

6) In parallel with the experimental 2 control samples are prepared:
Control 1. To 0.1 ml of lactoferrin add 0.8 ml of a liquid nutrient medium. Control 2. To 0.1 ml of buffered saline is added 0.8 ml of a liquid nutrient medium.

7) Incubate samples for 2 hours at 37 ^o C.

 8) After incubation, 0.1 ml of suspension of the test culture M.luteus GISK N 211001 is added to the experimental and control samples. For this, a 16-18-hour agar culture of M.luteus is washed off with buffered saline and a suspension with an optical density of 0.160 is prepared at 450 nm wavelength. The resulting suspension is diluted 10,000 times.

где ΔOD - изменение оптической плотности в опыте между 1 и 2 измерениями;
ΔOD₁ - изменение оптической плотности в контроле 1 между 1 и 2 измерениями;
ΔOD₂ - изменение оптической плотности в контроле 2 между 1 и 2 измерениями.9) Next, 100 μl of the experimental and control samples are inoculated into the wells of a polystyrene tablet with 200 μl of nutrient broth. The tablet is placed in a temperature-controlled unit of the iEMS-MF microbiological analyzer (Labsystems, Finland) and the first measurement of optical density in the experiment and controls is carried out immediately after reaching the temperature control mode (37 ^o C) and the second measurement after 18-24 hours of incubation (when the maximum optical density is reached indicator culture in accordance with generally accepted microbiological standards [14]), then antilactoferrin activity is calculated by the formula

where ΔOD is the change in optical density in the experiment between 1 and 2 measurements;
ΔOD ₁ - change in optical density in control 1 between 1 and 2 measurements;
ΔOD ₂ - change in optical density in control 2 between 1 and 2 measurements.

 Antilactoferrin activity is expressed as a percentage (%) of inactivation of lactoferrin in the experiment compared with the control.

 Examples of specific performance of the method.

 Example 1. Patient K. with a diagnosis of acute mastitis was isolated in a monoculture of Staphylococcus aureus. However, the lack of hemolytic activity, as well as antilysocyme, "anti-interferon" and anti-complementary activity, cast doubt on the etiological role of this microorganism in the development of the inflammatory process. To address the etiological significance of the isolated microorganism in Staphylococcus aureus, the ability to inactivate lactoferrin was determined. The presence of such a property, even in the absence of other persistence factors, determines the ability of the etiological agent to remain in the body for a long time and initiate the inflammatory process.

где 0,038 - изменение оптической плотности в опыте между 1 и 2 измерениями;
0,02 - изменение оптической плотности в контроле 1 между 1 и 2 измерениями;
0,075 - изменение оптической плотности в контроле 2 между 1 и 2 измерениями.To achieve this goal, the studied culture was grown in meat and peptone broth at 37 ^o C for 24 hours. 0.1 ml of chloroform was added to the tubes with the grown culture, mixed and kept for 40 minutes. The supernatant was separated from the bacterial cells by centrifugation at 3000 rpm for 20 minutes. A 2.5 mg / ml lactoferrin solution was prepared on buffered saline. The supernatant of the test culture of 0.8 ml was mixed with 0.1 ml of the prepared lactoferrin solution. In parallel, 2 control samples were prepared: a) control 1. 0.8 ml of a liquid nutrient medium was added to 0.1 ml of lactoferrin in the above concentration. b) control 2. To 0.1 ml of buffered saline was added 0.8 ml of a liquid nutrient medium. All samples were incubated for 2 hours at 37 ^° C. After incubation, 0.1 ml of suspension of the M.luteus GISK test culture No. 211001 was added to the experimental and control samples. For this, the 16-18-hour agar culture of M.luteus was washed with buffered physiological saline and a suspension was prepared with an optical density of 0.160 at a wavelength of 450 nm, the resulting suspension was diluted 10,000 times. Next, 100 μl of the mixture was inoculated into the wells of a polystyrene plate with 200 μl of nutrient broth. The tablet was placed in a thermostatically controlled unit of the iEMS-MF microbiological analyzer (Labsystems, Finland) and the first measurement of optical density in the experiment and controls was carried out immediately after reaching the temperature control mode (37 ^o C) and the second measurement after 18 hours of incubation, then antilactoferrin activity was calculated by the formula:

where 0,038 is the change in optical density in the experiment between 1 and 2 measurements;
0.02 - change in optical density in control 1 between 1 and 2 measurements;
0,075 - change in optical density in control 2 between 1 and 2 measurements.

 It was revealed that Staphylococcus aureus inactivated 32.7% of lactoferrin, i.e., its antilactoferrin activity was 32.7%.

 In the Staphylococcus aureus strain, sensitivity to gentamicin, benzylpenicillin, erythromycin, monomycin, rifampicin was established by the method of indicator discs. The course of antibiotic therapy using monomycin at 1 million units 2 times a day did not lead to the elimination of the pathogen and was not accompanied by positive dynamics of clinical symptoms. In this regard, the effect of sub-inhibitory concentrations of these antibiotics on the antilactoferrin activity of S. aureus was studied. It was established that monomycin and benzylpenicillin do not have a pronounced effect on it (which, obviously, explains the ineffectiveness of using monomycin in this case); erythromycin and rifampicin slightly reduce antilactoferrin activity, and gentamicin has a pronounced inhibitory effect: a staphylococcus strain grown in contact with a sub-bacteriostatic concentration of gentamicin lost its ability to inactivate lactoferrin. A course of antibiotic therapy using gentamicin as a drug that reduces the pathogen's antilactoferrin activity has led to clinical recovery.

 Thus, the identification of S. aureus ability to inactivate lactoferrin made it possible to establish its etiological significance, and the study of the spectrum of sensitivity to antibiotics and targeted antibiotic therapy led to the elimination of the pathogen.

 Example 2. When examining 52 employees of the maternity ward of the medical unit of Orenburggazprom JSC, 52 strains of staphylococci were isolated for staphylococcal bacteriocarriers. Since the main danger is represented by resident-type bacterial carriers, the need arose for their differentiation. For this, the ability to inactivate lactoferrin was determined in all strains. The determination of the antilactoferrin activity of microorganisms was carried out according to Example 1. As a result, antilactoferrin activity was detected in 12 strains in the range from 1.8 to 25.4%, while other strains did not have this feature. A double repeated examination of these patients with an interval of 10 days and subsequent phagotyping confirmed the identity of the isolated strains and allowed them to be classified as resident, while in the remaining patients, one type of staphylococcus was replaced by another.

 Remediation of resident bacterial carriers with an oil solution of vitamin A, which reduces the antilactoferrin activity of staphylococci, turned out to be positive in 95% of cases, which was evidence of the importance of this trait in the development and maintenance of staphylococcal bacterial carriage.

 Thus, the use of the proposed method allows to identify a new property of microorganisms - antilactoferrin activity, and as a result, to improve the accuracy of diagnosis and prediction of diseases of microbial etiology. Also, the inventive method contributes to the selection of rational antibacterial therapy, the study of the mechanisms of persistence of microorganisms and the formation of bacteria.

LIST OF REFERENCES
1. Bukharin OV, Usvyatsov B.Ya. Carrier carrier (medical-ecological aspect) .- Ekaterinburg; UB RAS, 1996, - 208 s.

 2. Bukharin OV Biomedical aspects of the persistence of bacteria. // Journal. microbiol., 1994. - Appendix. - S. 4-13.

 3. RU 2120999 C1, IPC 6 C 12 Q 1/02, 1/04, 10.27.98.

 4. RU 2126051 C1, IPC 6 C 12 Q 1/02, 02/10/99.

 5. Sokolov V.Yu. Anti-interferon activity of microorganisms. // The persistence of bacteria. / Ed. O.V. Bukharin. - Kuibyshev. 1990. - S. 83-93.

 6. Mikhailova E.A., Luda A.P., Bigeev M.I. Anti-immunoglobulin activity of bacteria. // Ed. O.V. Bukharina. - Kuibyshev, 1990, - 107 - 111 s.

 7. RU 2010860 C1, IPC 6 C 12 Q 1/02, 1/04, 04/15/94.

 8. Arnold R.R., Russell J.E., Champion W.I. et al. // Immunity. - 1982. - Vol. 35. - P. 792-799.

 9. Butler L. I., Didkovsky N. A., Charlyev G. Study of the content of lactoferrin in bronchial contents in patients with chronic bronchitis. // Health of Turkmenistan. - 1985. - N 10. - S. 15-18.

 10. Broxmeyer H.E., De Sousa M., Smithyman A. // Blood. - 1980. - Vol. 55. - P. 324.

 11. Bagby G., Bennett R. // Blood. - 1982. - Vol. 60. - P. 108-112.

 12. Catalog of strains of the All-Union Museum of Pathogenic Bacteria. Vol. 1. - M., 1982.

 13. Catalog of the company "Sigma" (chemical company): Biochemicals organic compounds and diagnostic reagents. - 1996 .-- S. 222.

 14. Handbook of microbiological and virological research methods. / M.O. Birger .-M., 1992.- P.114.

Claims (1)

A method for determining the antilactoferrin activity of microorganisms, namely, that the studied microorganism culture is grown in a liquid nutrient medium, the grown cultures are treated with chloroform, the supernatant is separated and mixed with lactoferrin, two control samples are prepared in parallel with the experimental one, while in control 1, instead of the supernatant of the studied cultures microorganisms add liquid nutrient medium, and control 2 is prepared from buffered saline and liquid nutrient medium, experimental and ntrolnye sample is incubated after incubation, culture test M. luteus GISCO N211001 and performed the first measurement of the optical density in the experiment and the control mode immediately after reaching thermostatting (37 ^o C) and a second measurement after 18 - 24 h incubation, then calculating activity antilactoferrin the formula

where ΔOD is the change in optical density in the experiment between 1 and 2 measurements;
ΔOD ₁ - change in optical density in control 1 between 1 and 2 measurements;
ΔOD ₂ - change in optical density in control 2 between 1 and 2 measurements.

Images