Method for differentiating probiotic strains of enterococcuslactis using maldi-TOF mass spectrometry

Abstract

FIELD: biotechnology; microbiology; medical diagnostics.SUBSTANCE: method for differentiating probiotic strains of Enterococcus lactis using MALDI-TOF mass spectrometry. The method involves cultivating the test sample in a nutrient medium, applying the resulting biomass to a metal target, treating it with a 70% aqueous solution of formic acid, then applying a matrix solution based on alpha-cyano-4-hydroxycinnamic acid and subsequent mass spectrometric analysis using the time-of-flight laser ionization method. Identification is carried out using a database of reference mass spectra of Enterococcuslactis, and the belonging of a strain to Enterococcuslactis is judged by the presence in its mass spectrum of at least four values of the mass-to-charge ratio m / z, selected from the group: 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553. If the specified m / z values are not present, the strain is classified as Enterococcusfaecium.EFFECT: reducing the analysis time while maintaining high identification reliability and simplifying the research procedure.3 cl, 3 dwg

Description

[0001] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The invention relates to the field of biotechnology, microbiology and medical diagnostics, in particular to methods for identifying microorganisms using mass spectrometry.

[0003] STATE OF THE ART

[0004] Enterococcus faecium has long been considered one of the most prevalent enterococcal species associated with the human body. Because some members of this species possess significant pathogenic potential and are capable of causing infections in humans, enterococci were long considered opportunistic pathogens [doi: 10.1128 / microbiolspec.gpp3-0053-2018, Pathogenicity of Enterococci]. However, among the representatives of this species there are also representatives of the normal human intestinal microbiota, which have probiotic potential [Strain of enterococci enterococcus faecium 1-3 for the production of therapeutic and prophylactic agents and food products for therapeutic and prophylactic purposes, https: / / patents.google.com / patent / RU2220199С1 / ru?peid=64aa0c2391аа8%3Ac4%3Af543a2ba].Recently, based on whole genome sequencing data, it was established that some Enterococcus faecium strains should be classified as a separate species, Enterococcus lactis, whose representatives are the least pathogenic for humans. E. lactis was first isolated from the E. faecium group in a publication by Morandi et al. in 2012 [Morandi, S. Enterococcus lactis sp. nov., from Italian raw milk cheeses / S. Morandi, P. Cremonesi, M. Povolo, M. Brasca / / International Journal of Systematic and Evolutionary Microbiology. - 2012. - Vol. 62, №Pt_8. -P. 1992-1996. - DOI: 10.1099 / ijs.0.030825-0]. The E. lactis genome does not contain the virulence genes typical of clinical isolates of E. faecium, including IS 16, Esp, and Hyl. E. lactis lacks complete gene clusters encoding pili (PGC), which are associated with pathogenicity. E. lactis also exhibits sensitivity to antibiotics: E.lactis is characterized by the absence of mutations in the PBP5 gene, which determines its sensitivity to ampicillin (PBP5-S phenotype), in contrast to resistant hospital strains of E. faecium (PBP5-R phenotype).

[0005] Another advantage of E. lactis is its psychobiotic potential. Strains of this species are known to produce the postbiotic γ-Aminobutyric acid (GABA), which has many beneficial properties for the nervous system and is useful in the treatment of certain neuropsychiatric disorders. GABA is the main inhibitory neurotransmitter and has a calming and regulatory effect on the central nervous system [Genomic and phenotypic analysis of probiotic properties of GABA-producing Enterococcus lactis A1 in Micropterus salmoides aquaculture Xinru Wang 1, Lei Zhu 2, Zhengyan Du 1, Wencai Zheng 1, Xianghui Kong Affiliations Expand PMID: 40472638 DOT 10.1016 / j.micres.2025.128241].

[0006] Traditionally, enterococci have been identified using classical methods using selective media, but this approach is very difficult to distinguish between representatives within a species.

[0007] Another, newer, alternative approach is the real-time PCR method. However, its use for reliable species identification requires specialized primers specific to strictly defined genetic markers of the species, which can be difficult to find. Furthermore, the PCR method is quite laborious and time-consuming.

[0008] A method is known for differentiating enterococci beneficial to humans from pathogenic enterococci by culturing them on a selective nutrient medium [Suvorov A.N., Simanenkov V.I., Sundukova Z.R., Ermoolenko E.I., Tsapieva A.I., Donetsk V.I., Solovieva O.I. METHOD FOR OBTAINING AN AUTOPROBIOTIC BASED ON ENTEROCUCCUS FAECIUM, A REPRESENTATIVE OF THE INDIGENOUS INTESTINAL MICROFLORA OF THE HOST. Application: 2010154822 / 10, 30.12.2010].

[0009] A method is known for differentiating enterococci beneficial to humans from pathogenic enterococci by distinguishing potentially dangerous strains based on their genetic profile using polymerase chain reaction. Upon detection of genes encoding enterococcal virulence factors, a conclusion is made about the pathogenicity of the enterococcal strain [Vershinin A.E., Kolodzhieva V.V., Ermoolenko E.I., Grabovskaya K.B., Klimovich B.V. et al. Genetic identification as a method for detecting pathogenic and symbiotic strains of enterococci. Journal of Microbiology, Epidemiology and Immunobiology. 2008. No. 5. P. 82].

[0010] However, the methods described are very labor-intensive, as they require determining a number of biological properties (virulence and persistence markers) in the isolated strains, as well as determining their antibiotic resistance and culturing them on various nutrient media. Application of this method in clinical practice is difficult due to the complexity of the mathematical calculations.

[0011] Whole genome sequencing is considered the most accurate method, but in practice its use is also difficult due to its labor-intensive nature and high cost.

[0012] We propose a new method for identifying enterococci using MALDI-TOF and a new database of Enterococcus lactis mass spectra. This method allows for rapid, reliable, and accurate identification of enterococci and their probiotic potential within seconds. MALDI-TOF is the fastest and most reliable method among the listed methods. However, until recently, all enterococci were identified by this method as opportunistic Enterococcus faecium, because all spectra in the mass spectral databases were registered as E. faecium.

[0013] SUMMARY OF THE INVENTION

[0014] For the purposes of this description, "opportunistic Enterococcus faecium" means strains that show no characteristic peaks inherent to E. lactis in the MALDI-TOF mass spectrum (e.g., 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553 m / z) and / or whose genome has been found to contain virulence genes (including IS16, Esp, Hyl) typical of clinical isolates of E. faecium, mutations in the PBP5 gene that cause resistance to ampicillin, or other markers associated with pathogenicity and antibiotic resistance.

[0015] For the purposes of this invention, the strain is considered opportunistic due to the absence of peaks characteristic of E. lactis in the mass spectrum, even if specific pathogenicity genes were not directly tested. The scope of protection of the proposed invention specifically encompasses the method for distinguishing E. lactis (peaks 2107 and others present) from E. faecium (peaks 2107 and others absent).

[0016] For the purposes of this invention, opportunistic Enterococcus faecium refers to strains of the Enterococcus faecium species that, under certain conditions (e.g., host immunosuppression, compromised mucosal barriers, or the use of broad-spectrum antibiotics), are capable of causing infectious diseases in humans or animals. Such strains, unlike probiotic Enterococcus lactis, are characterized by the presence of one or more of the following characteristics, confirmed by molecular genetic or phenotypic methods.

[0017] The term "opportunistic" is not limited to strains isolated from clinical material ("clinical isolates"), but also includes strains from the environment, food products, or the microbiota of healthy hosts, if they possess the aforementioned virulence and / or resistance factors. In the context of this application, the distinction between "probiotic Enterococcus lactis" and "opportunistic Enterococcus faecium" serves to distinguish safe strains with probiotic and psychobiotic potential from strains of the same genus that are potentially dangerous to humans and animals.

[0018] A method is proposed for differentiating probiotic strains of Enterococcus lactis bacteria from opportunistic Enterococcus faecium using MALDI-TOF mass spectrometry, which includes culturing the test sample on a nutrient medium, applying the resulting biomass to a metal target, treating with a 70% aqueous solution of formic acid, then applying a matrix solution based on alpha-cyano-4-hydroxycinnamic acid and subsequent mass spectrometric analysis using the time-of-flight laser ionization method, characterized in that the identification is carried out using a database of reference mass spectra of Enterococcus lactis, and the belonging of the strain to Enterococcus lactis is judged by the presence in its mass spectrum of at least four m / z values ​​selected from the group: 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553, and in the absence of the specified m / z values, the strain is classified as Enterococcus faecium.

[0019] The proposed method is distinguished in that the matrix solution is prepared from a mixture of acetonitrile, distilled deionized water and a 10% aqueous solution of trifluoroacetic acid with a volume ratio of components (1.5-2.5): (0.5-1.5): (0.5-1.5).

[0020] The proposed method is distinguished by the fact that the treatment of the biomass on the target with a 70% aqueous solution of formic acid is carried out before applying the matrix solution.

[0021] Technical result of the invention:

[0022] - reduction of analysis time to several minutes while maintaining high identification reliability compared to traditional methods (RT-PCR and whole genome sequencing), which require more than 8 hours;

[0023] Simplification of the research procedure by eliminating the need for: the use of selective media, the development of specific primers, and labor-intensive genetic studies.

[0024] The conventional practice of enterococcal identification did not distinguish between E. lactis and E. faecium. Until recently, the MALDI-TOF method (including commercial databases, e.g., Bruker, Shimadzu) identified all isolates belonging to the E. faecium group (including E. lactis) simply as Enterococcus faecium. A specialist using routine MALDI-TOF diagnostics did not need to distinguish between these two species, since they were considered a single taxon. Even after the publication of Morandi et al. (2012) [Morandi, S. Enterococcus lactis sp. nov., from Italian raw milk cheeses / S. Morandi, P. Cremonesi, M. Povolo, M. Brasca / / International Journal of Systematic and Evolutionary Microbiology. - 2012. - Vol. 62, No. Pt_8. - P. 1992-1996. - DOT 10.1099 / ijs.0.030825-0] on the isolation of E. lactis, commercial databases have not been updated in terms of the separation of these species.Therefore, for a person skilled in the art there was no obvious task of creating a separate method for identifying E. lactis and contrasting it with E. faecium with an assessment of probiotic potential.

[0025] Brief description of figures:

[0026] Fig. 1 shows the individual characteristic signals inherent to E. lactis, signals characteristic of all representatives of enterococci are highlighted in green, signals characteristic only of E. lactis are highlighted in pink;

[0027] Fig. 2 shows the individual characteristic signals inherent to E. Faecium, the signals characteristic of all representatives of enterococci are highlighted in blue, the signals characteristic only of E. Faecium are highlighted in pink;

[0028] Fig. 3 shows an example of the transmitted analysis results.

[0029] The illustrations show that E. lactis is characterized by the following individual m / z values: 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553.

[0030] Research procedure protocol:

[0031] This method is based on the isolation of enterococci, growing individual colonies on any suitable medium, followed by a colony preparation procedure:

[0032] 1. Sample preparation procedure:

[0033] 1.1 Preparation of the matrix,

[0034] 1.1.1 A 50 mg sample of alpha-cyano-cinnamic acid is placed in a 2.5 ml Eppendorf flask,

[0035] 1.1.2. In the same vessel are placed 1 ml of ultra-pure acetonitrile, 0.5 ml of distilled and deionized water and 0.5 ml of a 10% aqueous solution of ultra-pure trifluoroacetic acid. The sample is vigorously mixed using an ultrasonic bath. The ratio of the components of the matrix solution can vary within certain limits, maintaining the ability to effectively crystallize and ionize. The optimal, but not the only, volume ratio is acetonitrile : water : 10% trifluoroacetic acid solution = 2:1:1. It is obvious to a person skilled in the art that the ratio of the components of the matrix solution can vary within certain limits without loss of efficiency, in particular, deviations are possible, for example, in the range of (1.5-2.5): (0.5-1.5): (0.5-1.5).

[0036] 2. Sample preparation

[0037] 2.1. A colony fragment is placed on a special well of a steel plate using a sterile platinum loop.

[0038] 2.2. The sample is filled with 1 µl of 70% formic acid solution and left on the laboratory bench until dry.

[0039] 2.3. Apply 1 µl of matrix solution to the sample and leave it on the lab bench until dry. Once dry, the sample plate is placed in a plate holder, from where it enters the ionization unit of the mass spectrometer, and is vacuumed.

[0040] 3. Samples are sequentially irradiated with a 355 nm laser beam. This causes the evaporation and ionization of matrix molecules, along with the enterococcal proteins, which are then transported to a time-of-flight mass analyzer and separated by their mass-to-charge ratio.

[0041] 4. The obtained data are presented as graphical and mathematical relationships between the protein fragment mass and its intensity. These relationships are called mass spectra and signal intensity tables, respectively. Using software algorithms, the resulting mass spectrum is compared with the mass spectra of our own database of enterococci, determining the species of the enterococcus, as well as its biotic and psychobiotic potential.

[0042] E. lactis is characterized by the following individual m / z values: 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553, the presence of which allows one to confidently confirm their identification.

[0043] Examples of application of this method: medical diagnostics of the gastrointestinal system, etc., veterinary industry, food industry - assessment of the probiotic potential of bacterial products.

[0044] The authors express their gratitude to the shared use center “Cellular and Molecular Technologies” at the Federal State Budgetary Scientific Institution “IEM” for the research conducted.

Claims

1. A method for differentiating probiotic strains of Enterococcus lactis bacteria from opportunistic Enterococcus faecium using MALDI-TOF mass spectrometry, comprising culturing a test sample on a nutrient medium, applying the resulting biomass to a metal target, treating with a 70% aqueous solution of formic acid, then applying a matrix solution based on alpha-cyano-4-hydroxycinnamic acid and subsequent mass spectrometric analysis by laser ionization-desorption time-of-flight mass spectrometry, characterized in that the identification is carried out using a database of reference mass spectra of Enterococcus lactis, and the belonging of the strain to Enterococcus lactis is judged by the presence in its mass spectrum of at least four values ​​of the mass-to-charge ratio m / z, selected from the group: 2107, 2311, 4429, 5355, 6053, 6890, 7345, 9062, 9553, and in the absence of the specified m / z values, the strain is classified as Enterococcus faecium.

2. The method according to paragraph 1, characterized in that the matrix solution is prepared from a mixture of acetonitrile, distilled deionized water and a 10% aqueous solution of trifluoroacetic acid in a volume ratio of components (1.5–2.5): (0.5–1.5): (0.5–1.5).

3. The method according to paragraph 1, characterized in that the treatment of the biomass on the target with a 70% aqueous solution of formic acid is carried out before applying the matrix solution.

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