RU2709625C1 - Method of determining sensitivity of non-fermenting bacteria to disinfectants using mass spectrometry - Google Patents

Abstract

FIELD: microbiology.SUBSTANCE: invention relates to microbiology and can be used to determine non-fermentative bacteria (NFB) sensitivity to several disinfectants simultaneously. For this purpose, the sensitivity of the NFB is determined before and after exposure to disinfectants by a mass spectrometric method. For this purpose, NFB crop profiles are compared by distribution of peaks of different span length in obtained profiles. High-transit peaks have ratio of ion weight to charge >5,000 m/z, medium-span – from 5,000 to 2,500 m/z and low-span – ≤2,500 m/z. If the high- and medium-span peaks are reduced or completely disappear, and the mass-spectrometric profile is smoothed with transition to the low-span interval, the sensitivity of the bacteria to the disinfectant is stated.EFFECT: invention enables to determine sensitivity of NFB simultaneously to several disinfectants.1 cl, 1 tbl, 5 dwg, 5 ex

Description

The invention relates to the field of microbiology and relates to a method for determining the sensitivity of non-fermenting bacteria (NFB) to disinfectants (disinfectants).

Currently, infections associated with the provision of medical care (IASP) are an urgent medical problem, while the NFB dominates in the microbial landscape in the departments of medical institutions of various profiles. The frequency of NFB isolation from clinical material reaches 15% of all aerobic and facultative anaerobic gram-negative bacteria, of which about 70% are P. aeruginosa, Acinetobacter spp. and Stenotrophomonas maltophilia. These bacteria can exist in any of the ecological niches - soil, water, air, tissues and organs of plants and animals, and also have the ability to spread horizontally through objects or hands of patients and medical staff, i.e. to be the causative agent of hospital infections. These capabilities of the NFB are provided by the presence of a high frequency of resistance to various classes of antimicrobial agents, the quorum sensing intercellular signaling system, and the ability to form biofilms, the structure and physiological properties of which provide increased resistance to antibiotics and disinfectants.

The study of the resistance of bacteria to disinfectants is hindered, first of all, by the lack of unified methods of determination and the imperfection of indicators for assessing sensitivity - the resistance of bacteria to this group of drugs.

A known method for determining the sensitivity of microorganisms to a disinfectant (RU 2378363), which is based on assessing the viability of microorganisms by the growth of microorganisms in a nutrient medium after processing the suspension of microbes with a disinfectant for the time necessary for the manifestation of a disinfecting effect and subsequent metered inoculation on a solid nutrient medium. The sensitivity of microorganisms to a disinfectant is judged by the growth of microorganisms on a nutrient medium, while, with growth up to 100 CFU / ml, an incomplete bactericidal effect is judged, with an increase of 100-300 CFU / ml, a subbactericidal effect, with an increase of more than 300 CFU / ml, on the resistance of microorganisms to a disinfectant.

Colony counting of viable bacteria in this method is carried out in manual mode, which is subjective in nature. This method does not allow the simultaneous determination of the sensitivity of crops NFB to various disinfectants, time-consuming and requires expensive consumables.

The aim of the invention was to develop a method that allows you to explore the sensitivity of cultures of non-fermenting bacteria at the same time to different disinfectants, providing objectivity and standardization of the procedure, reducing the time of research and consumables.

This goal is achieved using time-of-flight mass spectrometry with matrix-activated laser desorption / ionization (MALDI-TOF MS), based on the ionization of the atoms and molecules that make up the sample and the registration of the mass spectrum of the ions formed, is widely used in the identification of microorganisms. The resulting mass spectrum profiles of microorganisms (protein profiles) are a unique genus, species-specific characteristic, and, accordingly, allow the microorganism to be identified by genus, species.

At the end of the automatic identification process, based on a comparison of the obtained initial spectrum with the reference spectra of the database, the program displays the identification result, listing the taxonomic database unit most relevant to the original spectrum with the value of the correspondence coefficient (indicator of the level of identification of bacteria) - “Score”. The higher the coefficient, the more likely the classification of the species. After the identification process is completed, the result is displayed in the classification table showing the best identification results of the obtained mass spectra, the reliability of which is confirmed by the correspondence coefficient.

In the proposed method, to determine the sensitivity of NFB to disinfectants, time-of-flight mass spectrometry with matrix-activated laser desorption / ionization (MALDI-TOF MS) is used, in which the sensitivity of the NFB to disinfectants is determined by assessing the degree of denaturation of bacterial proteins, recorded by comparison of mass spectrometric profiles of NFB cultures obtained before and after exposure to disinfectants.

The method is as follows.

Mass spectrometric method (identification of molecules by measuring the ratio of their mass to charge (m / z) in the ionized state) using a bench-top Microflex mass spectrometer with the MALDI Biotyper database (Bruker Daltonics Germany) studies daily diurnal pure culture of NPS before and after exposure to a disinfectant.

An isolated colony of the test culture from a Petri dish is introduced into a test tube with nutrient microbiological broth and incubated at 37 ° C for 24 hours. A drop of the tested daily pure NPF culture is applied to the target in duplicate, dried at room temperature for 5-15 minutes. On each sample, 1 μl of a matrix of СНСА (α-cyano-4-hydroxycinnamic acid) is applied, dried. Samples are placed in a mass spectrometer and subjected to laser pulses (ionization).

The initial (before exposure to disinfectants) mass spectrometric profile is considered as an indicator of the level of identification of NSWs and evaluated in accordance with the obtained compliance coefficient:

- with a compliance coefficient of 2,300-3,000 - a high probability of identification of the species;

- 2,000-2,299 - reliable identification of the genus, probable identification of the species;

- 1,700-1,999 - probable identification of the genus;

- 0.00-1.699 - unreliable identification.

Determine the mass spectrometric profile of the daily broth net culture of the NFB after exposure to disinfectants.

Daily broth pure cultures of the NFB are exposed to various disinfectants: 6% hydrogen peroxide, 70 ° ethanol; drug "Ultradon" (Russia); the drug Sulfochlorantin D (Russia), using the concentration of working solutions of disinfectants, and, as a sublethal dose, 10-fold dilution of their working solutions. Test cultures are incubated in drug solutions at an exposure recommended by appropriate instructions. The action of disinfectants is stopped with a 0.5% sodium thiosulfate solution (10 μl of a 0.5% sodium thiosulfate solution + 90 μl of culture suspension in a disinfectant) for 15 minutes at 20 ° C. Then they grow for 3 hours at 37 ° C in a nutrient broth (NPO Microgen) in a ratio of 1 ml of a suspension of a microorganism with a disinfectant + 1 ml of nutrient broth, and centrifuged at 3000 rpm for 15 minutes. The sediment of microorganisms is applied to the target (10 μl) and analyzed by mass spectrometric method (Bruker Daltonik MALDI Biotyper) according to the scheme described above.

At the same time, metered cultures on microbiological agar (NPO Microgen) are carried out from culture solutions with disinfectants to count viable bacterial cells.

The variation in the distribution of peaks of different span lengths in the obtained profiles is evaluated. Peaks of different span lengths are conventionally divided into groups, depending on the ratio of ion mass to charge (m / z):

- high-span - (m / z≥5000), major;

- mid-span - (m / z from 5000 to 2500), intermediate;

- low-span (m / z≤2500), minor.

The mass spectrometric profile of various P. aeruginosa strains before exposure to disinfectants is shown in Figure 1.

Method description

1. Used strains:

1) P. aeruginosa - 35,

2) Acinetobacter iwoffii -1,

3) Acinetobacter baumannii - 2,

4) Stenotrophomonas maltofilia - 2.

2. Used disinfectants

1) 6% hydrogen peroxide

2) 70 ° ethanol;

3) Ultradon (Russia);

4) the drug "Sulfochlorantin D" (Russia).

Stage 1. Preparation of crops.

Preparation of working suspension. Daily pure broth cultures of non-fermenting bacteria are used to prepare the main working suspension using an optical density standard corresponding to 1 billion mt / ml in an isotonic sodium chloride solution.

Stage 2 Preparation of disinfectants.

Preparation of working standard solutions of disinfectants. Working disinfectant solutions are prepared from concentrated solutions in accordance with the instructions.

Preparation of sublethal concentrations of disinfectant solutions. From working solutions of disinfectants, a 1:10 solution is prepared by successive dilution in an isotonic sodium chloride solution. In this case, 1 ml of a working solution of a disinfectant is combined and mixed with 9 ml of an isotonic sodium chloride solution.

Thus, to study drug resistance, 2 concentrations of the tested disinfectant are used: a working standard solution and 1:10.

Stage 3 the implementation of the method

1. The daily pure broth culture of bacteria is prepared in accordance with the instructions for conducting biotyping of bacteria using a mass spectrometer. The initial (control) mass spectrometric profile of the strain is obtained.

2. Bacterial cultures (microbial suspension - 1 billion mt / ml) are exposed to various disinfectants (6% hydrogen peroxide, 70 ° ethanol; Ultradon (Russia); Sulfahlorantin D (Russia), in working and sublethal concentrations preparations.

3. The test cultures are incubated in drug solutions at an exposure recommended by appropriate instructions at 20 ° C.

4. The action of disinfectants is stopped with a 0.5% sodium thiosulfate solution. Then they grow for 3 hours at 37 ° C in a nutrient broth (NPO Microgen), adding 3.0 ml of a suspension of 3.0 ml of a nutrient medium to 1.0 ml of suspension. Then centrifuged at 13000 rpm-10 min.

5. The sediment of microorganisms is re-analyzed by mass spectrometry (Bruker Daltonik MALDI Biotyper).

Stage 4 Accounting for mass spectrometric profiles.

Comparison the obtained mass spectrometric profiles using the MALDI-TOF Biotyper program and then conduct a visual analysis of the changes.

Example 1. The study of the mass spectrometric profile of non-fermenting bacteria after exposure to ethanol.

Changes that occurred under the influence of ethanol for P. aeruginosa are presented in Figure 2.

For a working ethanol solution, the almost complete disappearance of high-span peaks, the absence of mid-span and the prevalence of low-span peaks were revealed. For sublethal concentration, all peaks were low-span. At the same time, the transition of the values of the initial peaks to lower values (mark 1) or their complete disappearance (mark 2) was noted.

In general, smoothing of the mass spectrometric profile is observed with a transition to the low-span interval, which indicates the denaturing effect of the drug.

Example 2. The study of mass spectrometric profiles of non-fermenting bacteria after exposure to hydrogen peroxide.

Mass spectrometric profile of P. aeruginosa after the action of hydrogen peroxide takes the form shown in Figure 3.

The mass spectrometric profile of non-fermenting bacteria after exposure to a working solution of hydrogen peroxide is not presented, this is determined by the fact that the disinfectant in most cases (97.3%) was lethal to crops. Exposure to sublethal concentration in P. aeruginosa led to a decrease in the number of high- (mark 1) and mid-span peaks (mark 2); they transformed into peaks with lower values of span, which indicates the effectiveness of the drug.

Example 3. The study of mass spectrometric profiles of non-fermenting bacteria after the action of "Sulfochlorantin D".

Changes in the mass spectrometric profile for P. aeruginosa after exposure to Sulfochlorantin D are shown in Figure 4.

As can be seen from Figure 4, the P. aeruginosa mass spectrometric profile for the working solution and sublethal concentration of Sulfochlorantin D leads to a reduction in the number of high (peak 1), mid-span peaks (mark 2) and their transition to low-span values.

Example 4. The study of the mass spectral profiles of non-fermenting bacteria after the action of "Ultradon".

The observed changes in the mass spectrometric profile of P. aeruginosa are shown in Figure 5.

When using Ultradon, as can be seen in Figure 5, there was a decrease in the number of high- and mid-span peaks (mark 1), as well as the disappearance of some low-span (mark 2).

Example 5. The study of mass spectrometric profiles in different non-fermenting bacteria under the influence of different concentrations of disinfectants

Table 1 shows the data on the study of mass spectrometric profiles of different non-fermenting bacteria by the action of different concentrations of ethanol, hydrogen peroxide, Sulfochlorantin D, Ultradon compared to the serial dilution method with control sowing

In General, the results showed that the methods are significantly similar.

Claims (1)

A method for determining the sensitivity of non-fermenting bacteria (NFB) to several disinfectants at the same time, in which their sensitivity is determined before and after exposure to disinfectants using the mass spectrometric method by comparing the profiles of NFB cultures by the distribution of peaks of different span lengths in the obtained profiles, where there are high-span peaks have a ratio of ion mass to charge> 5000 m / z, mid-span - from 5000 to 2500 m / z and low-span - ≤ 2500 m / z, and with a reduction or complete disappearance of high- and mid-span peaks and when smoothing the mass spectrometric profile with a transition to the low-span interval, they conclude that bacteria are sensitive to this disinfectant.

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