INTRODUCTION
The method of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry has numerous advantages, such as accuracy, reproducibility, and speed of research. Meanwhile, this technique also has a sufficient list of shortcomings and difficulties, the overcoming of which is important for optimizing its use in the study of complex and rare microorganisms. First of all, this fact was described in relation to mycobacteria. Developing an additional library of protein spectra for representatives of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), it turned out that the nutrient medium affects the quality of the spectra, their intensity, and the possibility of using them in routine identification. In this regard, the developers decided to include in the library of spectra of microorganisms grown on liquid and solid media – Lowenstein–Jensen and Middlebrook.[1,2] However, data on the possibility of cultivating NTMs for preliminary identification and on universal nutrient media are gradually accumulating.[3]
Another problem with mass spectrometers is the difficulty in identifying some genera of acid-fast bacteria (AFB). Due to the peculiarities of the colony architectonics (the formation of dry colonies with a complex structure) and the structure of the cell wall, the probability of obtaining a sufficient amount of ribosomal proteins is significantly reduced.[4–10] This problem is partially solved using an extended method of direct application and extraction with formic acid, which can significantly improve the quality of the identification.[11]
METHODS
Samples
The study analyzed strains of microorganisms obtained during the examination of patients with suspected tuberculosis. In total, 287 strains of NTM were obtained: Mycobacterium avium 73/287; Mycobacterium gordonae 57/287; Mycobacterium kansasii 41/287; Mycobacterium fortuitum 35/287; Mycobacterium peregrinum 12/287; Mycobacterium abscessus 12/287; Mycobacterium chimaera/intracellulare 8/287; Mycobacterium malmoense 8/287; Mycobacterium senegalense 7/287; Mycobacterium chelonae 7/287; Mycobacterium lentiflavum 3/287; Mycobacterium pseudoshottsii 2/287; Mycobacterium marseillense 2/287; Mycobacterium porcinum 2/287; Mycobacterium frederiksbergense 2/287 and one strain each of Mycobacterium celatum, Mycobacterium gastri, Mycobacterium scrofulaceum, Mycobacterium bohemicum, Mycobacterium interjectum and Mycobacterium seoulense.
In addition, 63 strains of the most common bacteria from the AFB group were analyzed: Streptomyces spp. 39/63, Nocardia spp. 14/63, and Gordonia spp. 10/63. Before carrying out identification procedures, all strains were subcultured on Lowenstein–Jensen media (BioRad, USA) and universal chromogenic medium UriSelect (BioRad, USA). The crops were incubated at a temperature of 37°C on the 1st day and 28°C on the next day of cultivation until growth appeared. Visible growth was obtained for all strains on both media.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method
MALDI was used. The method is based on the repeated action of laser pulses on bacterial cells, precoated with a matrix, followed by protein ionization. The substance that was used as a matrix is α-Cyano-4-hydroxycinnamic acid (HCCA) (Bruker Daltonik GmbH, Germany). For identification, the standard library of spectra from Bruker Daltonik GmbH and additional libraries of Heatable Transmission Module spectra (Mycobacteria Library version 4.0, Bruker Daltonik GmbH, Germany) were used. The latest version of the library for the identification of NTMs contains 880 reference spectra, allowing the identification of 159 species of mycobacteria.
Method of sample preparation of microorganisms for use with the method of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Three main methods of sample preparation of microorganisms according to the manufacturer’s recommendations for use with the MALDI-ToF mass spectrometry method were used in the work: Direct coating method, extended direct coating method, and formic acid extraction method.
The direct application method was performed by directly applying a single colony (or part of it) in a thin layer to a single well of a 96-well target, starting from the middle of the well. After application, the biomaterial was covered with 1 μl of a freshly prepared matrix solution (HCCA). Matrix coating was carried out within an hour after the application of the material.
An extended method of direct application was that after applying a thin layer of the biomaterial directly to the target point, starting from the middle of the point, after drying, the material was covered with 1 μl of 70% formic acid. The material was additionally covered with a matrix solution (HCCA).
The formic acid extraction method has been used to improve the quality of the data allowing the identification of crops with maximum accuracy.[11] The method was implemented by a sequence of actions:
- Biomaterial was introduced into 300 μl of deionized water in an amount of 1–5 colonies
- After vortexing, 900 μl of ethanol was added and repeated vortexing was performed
- After centrifugation for 2 min at maximum speed (13,000 rpm), the supernatant was removed with a pipette. If necessary, the centrifugation procedure was repeated to remove residual ethanol
- The precipitate was dried at room temperature for 2 to 10 min
- Seventy percentage of formic acid was added to the dried precipitate in proportion to the amount of precipitate (from 1 to 80 μl), the material was mixed by pipetting
- Acetonitrile was added to the test tube in an amount equal to the volume of formic acid and gently mixed with a pipette
- After centrifugation for 2 min at maximum speed (13,000 rpm), 1 μl of the supernatant was taken and applied to the target point
- After complete drying of the material on the point, it was covered with 1 μl of the matrix solution (HCCA).
Statistics
To assess the strength of the relationship between the analyzed factors, Pearson’s Chi-square test and the degree of regularity of the event (P) were used. The relationship between the signs was statistically regarded as significant at a significance level of P < 0.001 or P < 0.05, depending on the test statistics.
RESULTS
Evaluation of the cultivation environment study on the result of the determination of nontuberculous mycobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
The identification made on the day of the detection of culture growth is sufficient to determine the direct hit method. Strains inoculated on Lowenstein–Jensen medium were identified with sample preparation by direct application; in the absence of a result by species or group identification, sample preparation was performed by extraction with formic acid. Microorganisms, the growth of which was obtained on a universal chromogenic medium, were studied with sample preparation by the method of direct application. If an acceptable result of species identification was not obtained by this method, sample preparation by the extended direct application method was additionally used. The identification results are presented in Table 1.
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Table 1: Comparison of the results of identification of mycobacteria depending on the method of application of the material and the medium for cultivation
When calculating the reliability of differences in the results of NTM identification on the Lowenstein–Jensen medium, the value of the criterion was χ2 = 92.698 and the significance level was P < 0.001. When assessing the significance of differences in the results of NTM identification on a universal chromogenic medium with different sample preparation methods, the value of criterion was χ2 = 16.451 and the significance level was P < 0.001. When calculating the significance of differences in the results of NTM identification on the Lowenstein–Jensen medium and the universal chromogenic medium with sample preparation by direct application, the value of the criterion was χ2 = 51.060 and the significance level was P < 0.001.
Evaluation of the influence of the cultivation medium on the result of identification of other microorganisms from the group of AFB (acid-fast bacteria) of the Actinomycetales order by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
The cultivation conditions and identification methods for other AFBs (Streptomyces spp., Nocardia spp., and Gordonia spp.) were comparable to those described for NTMs. When analyzing the influence of the cultivation medium on the result of identification, the results presented in Table 2 were obtained.
Table 2: Comparison of the results of the determination of mycobacteria depending on the method of applying the material and the medium for cultivation
When calculating the assessment of the significance of differences in the results of identification of streptomycetes, Nocardia, and gordonia on the Lowenstein–Jensen medium by various methods, the value of criterion was χ2 = 23.166 and the significance level was P < 0.001. When calculating the assessment of the significance of differences in the results of the identification of streptomycetes, Nocardia, and gordonia on a universal chromogenic medium by various methods, the value of the criterion was χ2 = 19.651 and the significance level was P < 0.001. When calculating the assessment of the significance of differences in the results of identification of the analyzed microorganisms on the Lowenstein–Jensen medium and the universal chromogenic medium by direct application, the value of the criterion was χ2 = 23.166 and the significance level was P < 0.001.
DISCUSSION
When evaluating the influence of the cultivation medium on the result of NTM identification by MALDI-ToF mass spectrometry, statistically significant results of the influence of the medium on the result of NTM identification were revealed for all compared parameters. The data comparing sample preparation by the direct application method with the extraction method and the advanced direct application method turned out to be less acceptable in terms of the final identification result. However, the results of identification, with sample preparation by the method of direct application, depending on the medium, showed a significantly higher level when cultivating NTMs on a universal chromogenic medium.
Negative results obtained during identification by direct application and by extraction with formic acid when working with AFB are due to the specific cultural properties of these microorganisms. If the culture was obtained in the form of ingrown colonies or with dysgonic growth, the use of standard tools for applying the material to the target caused certain difficulties. To solve this problem, new tools have been developed and tested for collecting colonies of microorganisms from the surface of dense nutrient media (Patent RF No. 187404, Patent RF No. 187421).
This greatly simplifies the process of working with this group of microorganisms in the case of obtaining growth on agar media and expands the possibility of working with NTM in laboratories not associated with anti-tuberculosis services.
When evaluating the influence of the cultivation medium on the result of identification of other microorganisms from the AFB group of the Actinomycetales order by MALDI-ToF mass spectrometry, for all compared parameters, statistically significant results of the influence of the medium on the result of identification of other microorganisms from the AFB group were also revealed. It can be concluded that the Lowenstein–Jensen medium is not optimal for the identification of representatives of the genus Streptomyces, and the method of direct application using a universal chromogenic medium is acceptable only for species identification of gordonii.
CONCLUSIONS
Thus, mass spectrometry is an important element in the modern system of species identification of microorganisms. Optimization of sample preparation protocols and assessment of the impact on the identification of new methods of cultivating microorganisms can significantly improve the quality of the identification of both clinically significant microorganisms from the AFB group and saprophytic microflora, the clinical significance of which has not been proven at the moment. In this case, accurate species identification and the development of algorithms for its application will improve the diagnosis of diseases caused by AFB.
Limitations of study
We analyzed the possibility of identifying only 22 NTM species and 3 genera of other AFBs. For some microorganisms, the study was limited to one strain. The study used a single manufacturer’s mass spectrometer and used only one universal chromogenic medium out of many commercially available. Care should be taken when extrapolating the results obtained without additional studies using equipment and environments from other manufacturers. The work did not analyze the results of the identification of strains isolated directly from clinical material, which must be taken into account in further research in this direction.
Ethical clearance
This research was approved by the Bioethics Committee at Samara State Medical University with Approval Number 196; October 31, 2018.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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