RU2842396C1 - Method for determining haemolytic activity of staphylococcus epidermidis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a method of determining haemolytic activity of strains Staphylococcus epidermidis. Method involves recording spectra by Raman scattering spectroscopy using an optical probe, which is placed above the analysed Staphylococcus epidermidis strain at distance of 7 mm, wherein laser power at exposure of 60 s is 350 mW, normalization of spectra by Extended multiplicative signal correction method, smoothing using Maximum Like Lihood Estimation Savitzky-Golayfilter with parameter σ=4, use of modified method of subtracting fluorescent component by polynomial approximation Improved Modified Multi-Polynomial Fitting with degree of polynomial 11 to eliminate contribution of autofluorescence in Raman spectrum, analysis of Raman spectra in range from 450 to 2000 cm^-1, examining the presence of haemolytic activity in the analysed strain according to the most pronounced spectral differences observed on line 1647 cm^-1 according to the results of the following LD-1 discriminant analysis.

EFFECT: invention provides improved determination of haemolytic activity of Staphylococcus epidermidis strains by analysing Raman spectra.

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Description

Field of technology to which the invention relates

The invention relates to microbiology and can be used to determine the hemolytic activity of Staphylococcus epidermidis strains using Raman spectroscopy.

State of the art

A method for determining the hemolytic activity of bacterial strains using the culture method is known: 5% defibrinated blood is added to meat-peptone agar. The Petri dish is inoculated with the strain of the test microorganism. The dishes are incubated at 37 ° C for 20-24 hours. If there is no change in the color of the medium around the colony, this indicates the absence of hemolysis. If zones of discolored medium form around the colonies, this indicates the presence of beta-hemolysis. Alpha-hemolysis is characterized by partial hemolytic activity and the formation of a greenish or greenish-brown halo around the colonies. [Handbook of Medical Microbiology. General and Sanitary Microbiology. Book 1 / Coll. Authors // Edited by Labinskaya A.S., Volina E.G. - M .: BINOM Publishing House, 2020. - 1080 p: ill.]

The disadvantages of this method are the subjective evaluation of the results obtained, the time required to cultivate the strains, and the lack of standardized evaluation protocols.

A method for determining the hemolytic activity of Staphylococcus aureus by identifying the genes responsible for the production of toxins with the ability to hemolyze is known using the polymerase chain reaction method. The genome of the studied strains is analyzed for the presence of the hla and hlb genes, responsible for the production of α-toxin and β-toxin, respectively. If amplicons with the expected size of 704 bp are detected in PCR with primers for the hla gene, the studied strain is considered capable of producing α-toxin. If amplicons with the expected size of 496 bp are detected in PCR with primers for the hlb gene, the studied strain is considered capable of producing β-toxin. [Abaev I.V., Skryabin Yu.P., Korobova O.V., Polosenko O.V., Shepelin A.P. COMPARISON OF HEMOLYTIC ACTIVITY AND HEMOLYTIC TOXIN GENES OF CLINICAL STRAINS OF STAPHYLOCOCCUS AUREUS ISOLATED IN THE TERRITORY OF THE RUSSIAN FEDERATION // Clinical laboratory diagnostics. 2019. No. 5.]

The disadvantages of the method are the impossibility of determining the presence of hemolysin genes in Staphylococcus epidermidis strains.

Disclosure of invention

The objective of the invention is to improve the determination of the hemolytic activity of Staphylococcus epidermidis strains using Raman spectra analysis.

This is achieved by the fact that, unlike the known methods, the hemolytic activity is studied using Raman spectroscopy: Raman spectra are recorded using an optical probe located 7 mm above the object. The laser power during 60-second exposure is 350 MW. The spectra are normalized using the Extended multiplicative signal correction method. Smoothing is performed using the Maximum Like Lihood Estimation Savitzky-Golayfilter with the parameter σ=4. To eliminate the contribution of autofluorescence in the Raman spectrum, a modified method of subtracting the fluorescence component by polynomial approximation Improved Modified Multi-Polynomial Fitting with a polynomial degree of 11 was used. The Raman spectra were analyzed in the range from 450 to 2000 cm ^-1 . The presence of hemolytic activity in the studied strain can be judged by the most pronounced spectral differences, which appear on the line 1647 cm ^-1 (Amide I vibration (collagen-like proteins)). With subsequent discriminant analysis by LD-1. In this case, positive LD-1 values (from 0 to 10) characterize the spectra of Staphylococcus epidermidis with hemolytic activity, and negative values (from -10 to 0) characterize Staphylococcus epidermidis without hemolytic activity. The discriminant function LD-1 describes the dispersion by 100%.

Implementation of the invention

The essence of the proposed method is as follows: the hemolytic activity of Staphylococcus epidermidis strains is studied by Raman spectroscopy using a set of hardware: diode laser LML-785.0RB-04 (PDLD Inc., USA), optical module PBL-785 (InPhotonics, USA), spectrometer SharmrockS R-303i (Andor Technology Ltd., UK), digital cooled camera ANDOR DV-420A-OE (Andor Technology Ltd., UK) and a personal computer. The diode laser focuses the radiation on the object at a distance of 7 mm from the output window and collects the backscattered radiation, which is then directed through the optical fiber to the spectrometer. Registration of spectra is carried out in the range of wave numbers from 450 to 2000 cm ^-1 . The spectra are processed in the WolframMathematica 10 software package (Wolfram Research, USA). The presence of hemolytic activity in the strain under study can be judged by the most pronounced spectral differences that appear on the 1647 cm ^-1 line (Amide I vibration (collagen-like proteins)). Subsequently, discriminant analysis is performed by LD-1. In this case, positive LD-1 values (from 0 to 10) characterize the spectra of epidermal staphylococci with hemolytic activity, and negative values (from -10 to 0) characterize the spectra of epidermal staphylococci without hemolytic activity. The discriminant function LD-1 describes the dispersion by 100%.

The proposed method can be used in microbiology to determine the hemolytic activity of Staphylococcus epidermidis strains.

Claims (1)

A method for determining the hemolytic activity of Staphylococcus epidermidis strains, characterized in that the Raman spectroscopy method records the spectra using an optical probe, which is positioned above the Staphylococcus epidermidis strain under study at a distance of 7 mm, while the laser power during an exposure of 60 s is 350 mW, the spectra are normalized using the Extended multiplicative signal correction method, smoothing is performed using the Maximum Like Lihood Estimation Savitzky-Golayfilter with the parameter σ = 4, a modified method of subtracting the fluorescence component by polynomial approximation Improved Modified Multi-Polynomial Fitting with a polynomial degree of 11 is used to eliminate the contribution of autofluorescence in the Raman spectrum, the Raman spectra are analyzed in the range from 450 to 2000 cm ^-1 , the presence of hemolytic activity in the strain under study is determined by the most pronounced spectral differences that appear on the 1647 cm ^-1 line based on the results of subsequent discriminant analysis by LD-1; in this case, LD-1 values from 0 to 10 characterize the spectra of Staphylococcus epidermidis with hemolytic activity, and negative values less than 0 and up to -10 characterize the spectra of Staphylococcus epidermidis without hemolytic activity.