RU2550132C1 - METHOD FOR PHOTODYNAMIC INACTIVATION OF Enterococcus faecalis BACTERIA (VERSIONS) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method for photodynamic inactivation and inhibition of growth of Enterococcus faecalis bacteria, based on pretreatment of the bacteria with a hypericin-containing photosensitiser based on an alcoholic tincture of hypericum "Diahyperon" or "Hypericum tincture" and subsequent exposure of the obtained suspension to optical radiation of laser or LED sources with transmission maxima in the region corresponding to absorption spectrum maxima or maxima of the excitation spectrum of fluorescent components of the preparations with radiation energy density of 0.9-9.0 J/cm², under certain conditions.

EFFECT: method simplifies inactivation of Enterococcus faecalis bacteria.

22 cl, 7 dwg, 3 tbl

Description

The invention relates to biotechnology and medicine, in particular to a method for the photodynamic inactivation and inhibition of the growth of Enterococcus faecalis bacteria in the presence of a drug with sensitizing properties.

Enterococcus faecalis (fecal enterococcus) is a conditionally pathogenic microbe that is characteristic of the normal microflora of the oral cavity, digestive organs and intestines, as well as the human urogenital system.

The pathogenic form of Enterococcus faecalis causes inflammation of the urinary and genital tract, bladder, pelvic organs and kidneys, most nosocomial infections, cystitis, pyelonephritis, adnexitis, prostatitis, urethritis, etc. The specified microorganism is also found in persistent endodontic infections. Its prevalence in such infections, according to various sources, ranges from 24% to 77%. This is due to the particular resistance and virulence of Enterococcus faecalis, the ability to compete with other microorganisms.

The bacteria Enterococcus faecalis belong to the genus of gram-positive enterococci of a subclass of lactobacilli. Enterococcus faecalis is able to carry out cellular respiration both in an oxygen-free and oxygen-saturated environment; resistant to disinfectants and certain antibiotics, they can withstand high temperatures and difficult environmental conditions: they grow at a temperature of 10-45 ° C, pH 4.5-10.0, as well as at high concentrations of sodium chloride. Enterococcus faecalis has certain virulence factors, including lytic enzymes, cytolysin, aggregation material, and pheromones. Enterococcus faecalis is able to suppress the action of lymphocytes, potentially contributing to endodontic failure.

A known method of inactivation of bacteria Enterococcus faecalis, based on the treatment of microflora with a solution of antibiotics (ampicillin, vancomycin, penicillin, cefolosporins, aminoglycosides, etc.), antiseptics or other drugs with antimicrobial action [1].

The disadvantage of this method of suppressing enterococcal activity of Enterococcus faecalis is the high level of resistance of bacteria to these agents [2]. Some enterococcal strains have internal mechanisms of resistance to beta-lactam antibiotics (penicillins and cephalosporins), as well as to many aminoglycosides. In the last two decades, especially virulent enterococcal strains have appeared that are resistant to vancomycin and can cause nosocomial infections.

There is also known a method for the photodynamic inactivation of Enterococcus faecalis bacteria in the presence of a dye with sensitizing properties due to the photodynamic effect of optical radiation corresponding to the dye absorption band [3-16]. The following compounds are used as sensitizing dyes: methylene green, acridine orange and proflavin [3], methylene blue and toluidine blue [3, 4, 5, 6, 7], indocyanine green [7, 8], Bengal pink [6, 7, 9], eosin [9], curcumin [9], chlorin [10], porphyrins [11], phthalocyanines [12, 13], chalcogen-containing benzophenoxazinium dyes [14], nanoparticle conjugates with methylene blue [15], porphyrin [16] or chlorin e ₆ [17].

In these methods for the photodynamic inactivation of Enterococcus faecalis bacteria, microbial cells are grown in culture medium. After preliminary incubation in the dark of Enterococcus faecalis under aerobic conditions for 10-30 min with one of the above dyes at a dye concentration of 1-250 μM, the contents are irradiated with optical radiation whose spectral range corresponds to the absorption band of the photosensitizer. Enterococcus faecalis uses laser diodes, light emitting diodes, broadband lamp sources of white light, or lamp sources from the total emission spectrum of which with the help of light filters select the region corresponding to the maximum of the sensitizer absorption band as radiation sources in these methods for the photodynamic inactivation of Enterococcus faecalis bacteria.

The disadvantages of the known methods for the photodynamic inactivation of Enterococcus faecalis bacteria are: (a) restrictions on the use of some of the above sensitizers (methylene blue, toluidine blue, methylene green, acridine orange, Bengal pink, indocyanine green, eosin, phthalocyanines, chalcogen-containing benzophenazine) medical purposes (antimicrobial photodynamic therapy of diseases of the oral cavity, face, etc.) for aesthetic reasons due to the strong and stable staining I surface of the fabric, subjected to application of the dye; (b) the complexity of chemical synthesis (conjugates of nanoparticles with methylene blue, porphyrin or chlorine e ₆ ) and / or chemical purification (proflavin, curcumin, chlorins, porphyrins) of drugs used as a photosensitizer.

Closest to the claimed is a method for the photodynamic inactivation of Enterococcus faecalis bacteria, based on the preliminary incubation of isolated bacteria with the dye-photosensitizer hypericin [18]. In a known method, Enterococcus faecalis bacteria are grown on nutrient agar under aerobic conditions at 37 ° C for 18-24 hours. Then, on a sterile phosphate-buffered saline buffer (pH 7.4), a suspension of bacteria is prepared with a cell concentration of 10 ⁷ CFU / ml (CFU - colony forming units). To a bacterial suspension of Enterococcus faecalis in the dark, a solution of hypericin at a final concentration of 1.0 μg / ml is added. The mixture was incubated in the dark at 37 ° C for 5 minutes. Aliquots of 200 μl Enterococcus faecalis cells containing the dye-photosensitizer hypericin are irradiated with light for 3, 5 and 10 minutes. A light emitting diode is used as an irradiation source, from the emission spectrum of which radiation is emitted with a yellow filter with a maximum in the region of 590 nm (the region corresponding to the maximum of the absorption spectrum of hypericin). The radiation power was 30 mW, the power density of the radiation acting on Enterococcus faecalis, 80 mW / cm ² . At exposure times of 3, 5, and 10 min, the energy dose (dose density) was 14.4, respectively; 24; 48 J / cm ² [18].

According to the known method [18] incubation in the dark of bacteria Enterococcus faecalis with hypericin at a concentration of 0.1; 0.3; 0.6; 1.0 μg / ml for 5 min had no toxic effect on them. The impact on Enterococcus faecalis in the absence of hypericin of optical radiation with a maximum at 590 nm at a power density of 80 mW / cm ² , exposure from 3 to 10 minutes and an energy dose of 14.4 to 48 J / cm ² also did not cause changes in the activity of Enterococcus bacteria faecalis.

However, irradiation of Enterococcus faecalis in the presence of hypericin at its concentration of 1 μg / ml for 3 min at a power density of 80 mW / cm ² (energy dose of 14.4 J / cm ² ) caused a decrease in the ability to grow and colony formation. So, if in the control (cells that did not contain hypericin and were not irradiated), the cell concentration was 10 ⁷ CFU / ml, then after their irradiation, it decreased by four orders of magnitude.

The disadvantages of the known method for the photodynamic inactivation of Enterococcus faecalis bacteria are: (a) the use of a commercial chemical preparation (hypericin) as a photosensitizer, which is not approved for use in medical practice; (b) the high cost of the drug due to the complexity of its isolation and chemical purification (according to the catalog of Sigma-Aldrich, the cost of 10 mg of the drug is 326.5 euros).

The objective of the invention is the development of an effective method for the photodynamic inactivation of Enterococcus faecalis bacteria using an official medicinal product as a photosensitizer, which is approved for use in medical practice, and which, due to the photodynamic effect, provides a complete bactericidal effect on Enterococcus faecalis at a lower cost for the method being implemented.

The problem is solved as follows.

Option 1

In the method for the photodynamic inactivation of bacteria Enterococcus faecalis, based on pretreatment of bacteria with a hypericin-containing photosensitizer and subsequent exposure to optical radiation in the visible region of the spectrum corresponding to the absorption band of the photosensitizer, the drug “Dihyperon” based on its alcohol-based tincture of bacteria is used as a hypericin-containing photosensitizer / 30 ± 10 by volume and act on the suspension by radiation from a laser or LED source nicks with a maximum emission in the region corresponding to the maxima of the absorption spectrum or the maxima of the excitation spectrum of the fluorescent components of the suspension at a radiation energy density of 0.9-9.0 J / cm ² .

Option 2

In the method for the photodynamic inactivation of bacteria Enterococcus faecalis, based on pretreatment of bacteria with a hypericin-containing photosensitizer and subsequent exposure to optical radiation in the visible region of the spectrum corresponding to the absorption band of the photosensitizer, an official medicine “St. John's wort herbivore” is used as a hypericin-containing photosensitizer its suspension of bacteria 1/30 ± 10 in volume and affect the sauce enziyu emission laser or LED sources with a maximum emission in the region corresponding to the maxima of the absorption maxima of the spectrum or the excitation spectrum of fluorescent slurry component at a radiation energy density of 0,9-9,0 J / cm ^2.

The exposure is carried out by laser radiation with a wavelength of λ = 665 ± 10 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength of 405 nm with an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength in the region of 435-445 nm with an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength of 532 nm at an energy density of 0.9-9.0 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 405 ± 10 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by radiation of a LED source with a maximum emission at a wavelength of λ = 450 ± 10 nm with an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by radiation of a LED source with a maximum emission at a wavelength of λ = 545 ± 10 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 590 ± 10 nm at an energy density of 0.9-4.5 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 660 ± 15 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by the radiation of an LED white light source with emission maxima at wavelengths λ ₁ = 450 ± 10 nm and λ ₂ = 575 ± 10 nm with an energy density of 0.9-6.0 J / cm ² .

The invention is illustrated by drawings, where:

figure 1 shows the absorption spectrum of "Diaperone";

figure 2 - absorption spectra of hypericin (curve 1) and chlorophyll (curve 2) fractions "Diaperone" after their preliminary isolation using thin layer chromatography;

figure 3 - fluorescence spectra of diaperone at an excitation wavelength λ _exc = 405 (curve 1), 445 (curve 2), 532 (curve 3) and 545 nm (curve 4);

figure 4 - excitation spectra of fluorescence "Diaperone" at a wavelength of registration λ _reg = 653 (curve 1), 702 nm (curve 2);

figure 5 - absorption spectrum of "St. John's wort tincture" after dilution with alcohol in a ratio of 200 μl of tincture / 4000 μl of ethanol;

figure 6 - fluorescence excitation spectra of "St. John's wort tincture", diluted in a ratio of 50 μl tincture / 4000 μl of ethanol at a recording wavelength λ _reg = 620 (curve 7) nm and diluted in a ratio of 200 μl tincture / 4000 μl of ethanol at a wavelength registration λ _reg = 720 nm (curve 2);

Fig.7 - emission spectra of LED sources with maximum intensity at λ _max = 405 (curve 1); 450 (curve 2); 545 (curve 3); 600 (curve 4); 660 (curve 5); 450 and 575 nm (curve 6).

The medicinal product Diagiperone is a tincture (70% ethyl alcohol) of St. John's wort herb for internal and external use. It has anxiolytic (relieves anxiety, tension) and antidepressant effect (improves mood). Improves the functional state of the central and autonomic nervous system. It has astringent and anti-inflammatory effects, as well as antibacterial activity (in dark conditions) in relation to a number of microorganisms that are resistant to antibiotics.

As follows from the data presented in figure 1, in the absorption spectrum of Diagiperone three maxima are distinctly recorded: at λ _pogl = 551; 590 and 664 nm. Separation of the components by thin-layer chromatography showed that the first two peaks belong to hypericin, which is part of Diaperone, and the long-wavelength maximum at λ _pogl = 664 nm is mainly due to the presence of chlorophyll a in the preparation (Fig. 2). This conclusion is supported by the fact that the maxima of the absorption spectra of the purified preparations of chlorophyll a in ethanol are located at λ _pogl = 431 and 665 nm, chlorophyll b at λ _pogl = 463 and 649 nm, and hypericin at λ _pogl = 477, 510, 548 and 590 nm. After chromatographic separation of the components, one of the fractions (hypericin-containing, curve 1, _figure 2) has maxima in the absorption spectrum at λ _damp = 548 and 591 nm, the other (chlorophyll-containing, curve 2, _figure 2) - at λ _damp = 408, 504, 535, 607, 666 nm. The presence of several types of chromophores, absorbing in the visible region of the spectrum, in the composition of Diaperone "is also confirmed by the data of the fluorescence spectra and excitation of fluorescence of the drug (figure 3 and figure 4). In this case, the fluorescence spectra of chemically pure ethanol solutions of chlorophyll a are located at _λfl = 674 and 725 nm; chlorophyll b - at λ _fl = 650 and 710 nm; hypericin - with λ _fl = 594; 642 and 702 nm. Therefore, a change in the wavelength of Diagiperone fluorescence excitation leads to a change in the contributions of hypericin and chlorophyll to the total fluorescence of diaperone tincture, and, accordingly, to a change in the position of the maxima of the fluorescence spectrum. Moreover, if the excitation in the _WHO λ ≥630 nm (at which the absorption of hypericin almost absent) in the fluorescence spectrum "Diagiperona" is registered (3) only chlorophyll component _(fl λ = 673 nm), then the _cart at λ = 545 nm - the hypericin component prevails ( _λfl = 595 and 643 nm, curve 4). Upon excitation in the region λ _w = 405 (curve 1), 445 (curve 2) and 532 nm (curve 3), both hypericin and chlorophyll components are recorded in the fluorescence spectrum. Note that by changing the excitation wavelength, it is possible to register both the fluorescence of chlorophyll a (maximum in the spectrum in the region _λfl = 672 nm, curve 3), and chlorophyll b ( _λfl = 658 nm, curve 2) present in the Diaperone composition ".

Information on the optimal excitation range of the photosensitizing components in Diagiperone was obtained from the data of fluorescence excitation spectra (Fig. 4): when registering fluorescence in the region λ _reg = 653 nm (curve 1), the corresponding maxima are located at λ _max = 430; 546 and 590 nm; when registering in the region of 702 nm (curve 2) λ _max = 405; 547; 589 and 664 nm.

Noteworthy are the differences in the fluorescence excitation spectrum (Fig. 4) from the absorption spectrum of the preparations (Fig. 1 and Fig. 2), due to the presence in their composition of non-fluorescent (in the red spectral region) components: essential oils, tannins, phenol aldehydes , cineole, and others. These compounds have a pronounced absorption in the ultraviolet region of the spectrum and are the reason for the high optical density of DiCiperon solutions in the range of 350-400 nm (Fig. 1).

From the analysis of the presented data it follows that the excitation of the hypericin and chlorophyll components of DiHiperone is possible in almost the entire visible region of the spectrum, however, the most effective is the choice of the ranges corresponding to the absorption maxima of its fluorescent components: 405 ± 10 nm, 430 ± 25 nm; 545 ± 15 nm; 590 ± 10 nm; 665 ± 15 nm.

Practically the indicated method for the photodynamic inactivation of Enterococcus faecalis bacteria using DiCiperone as a photosensitizer can be implemented by using the currently most available semiconductor lasers with a radiation wavelength in the region of 405 nm; 435-445 nm; 665 ± 15 nm; solid-state Nd: YAG laser with diode pumping and doubling of the radiation frequency with a wavelength of 532 nm; superbright LEDs, the maximum emission spectrum of which is located in the region of 405 ± 10 nm (curve 1 in Fig. 7); 450 ± 10 nm (curve 2); 545 ± 10 nm (curve 3); 590 ± 10 nm (curve 4); 660 ± 15 nm (curve 5). It is also possible to use a white light LED, one of the maximum emission of which is located at 450 nm, the other at 575 nm (curve 6).

The medicine "St. John's wort tincture" is a tincture of St. John's wort herb perforated with 40% ethyl alcohol for internal and external use. It has an astringent and anti-inflammatory effect, due to the contained flavonoids, azulene and essential oils, as well as a mild antidepressant, sedative and anxiolytic effect.

Data on the absorption spectrum of "St. John's wort tincture" are presented in figure 5. It is seen that in the visible region of the spectrum (400–760 nm), the points of extrema that could be correlated with the presence of photosensitizing components in the solution are weakly expressed. Only a weakly manifest maximum in the region of 591 nm and “shoulders” in the regions of 485 nm and 548 nm, associated with the presence of hypericin in the preparation, are noted. There is no doubt that such masking of the absorption bands in the preparation is due to the multicomponent composition of the drug, including the presence in it of components that are not capable of exerting a photosensitizing effect. For this reason, the most reliable information on the photosensitizing compounds "St. John's wort tincture" was obtained from the analysis of the fluorescence excitation spectra (Fig.6). Moreover, to register the excitation spectrum in the region of 400-600 nm (curve 1) λ _reg = 620 nm, a tincture was used, diluted in the ratio of 50 μl of tincture / 4000 μl of ethanol. The need for such dilution is due to the high optical density of the solution in the region of 400 nm, which (due to the screening effect) does not allow us to determine the true position of the maxima in the excitation spectrum. In the study of dilute solutions in the excitation spectrum (λ _reg = 620 nm), maxima are recorded in the region of 430; 477, 548 and 590 nm. In addition, a maximum is also observed in the region of weak absorption (low intensity in the excitation spectrum at 511 nm). To record the fluorescence excitation spectrum in the red region (λ _reg = 720 nm, curve 2), we used more concentrated solutions of St. John's wort tincture (200 μl of tincture / 4000 μl of ethanol), which made it possible to detect in the fluorescence excitation spectrum along with previously recorded "hypericin" peaks in the region of 548 and 590 nm, a band with a maximum at 659 nm, due to the presence of a chlorophyll component in the solution.

From the presented data it follows that for the activation of the photosensitizing components of “St. John's wort tincture”, radiation sources whose emission spectrum maxima are in the region of 430 ± 25 nm are most suitable; 548 ± 15 nm, 590 ± 10 nm and 659 ± 15 nm.

In practice, the method for the photodynamic inactivation of Enterococcus faecalis bacteria when using St. John's wort tincture as a photosensitizer is implemented by using the most available semiconductor lasers with a radiation wavelength in the region of 435-445 nm; 665 ± 10 nm; solid-state Nd: YAG laser with diode pumping and doubling of the radiation frequency with a wavelength of 532 nm; superbright LEDs, the maximum emission spectrum of which is located in the region of 450 ± 10 nm (curve 2 in Fig.7); 545 ± 10 nm (curve 3); 590 ± 10 nm (curve 4); 660 ± 15 nm (curve 5). It is also possible to use a white light LED, one of the maximum emission of which is located at 450 nm, the other at 575 nm (curve 6).

The essence of the proposed method for the photodynamic inactivation of bacteria Enterococcus faecalis is as follows.

In experiments on the photodynamic inactivation of Enterococcus faecalis bacteria, five to eight strains of a daily culture of clinically isolated enterococci grown on a beveled nutrient agar are used with mandatory control of culture purity and verification of the main biochemical generic and species characteristics. A suspension of cells at a concentration of 1.5 × 10 ⁸ CFU / ml is prepared from a grown daily culture in sterile physiological saline, which corresponds to 10 IU of the turbidity standard. By successive 10-fold (1:10) dilutions, a suspension was prepared with a dilution of 1: (100000-10000000) for each enterococcal culture.

2 ml of the resulting suspension of each strain are introduced into sterile Petri dishes with a diameter of 3.5 cm (bottom area S = 9.6 cm ² ). Four groups of suspensions are prepared:

- 1st group (control without photosensitizer). In each cup under sterile conditions add 0.5 ml of diluted 1: 5 sterile physiological solution of 70% ethyl alcohol (ethanol),

- 2nd group (experimental without photosensitizer). In each cup, under sterile conditions, 0.5 ml of 1: 5 diluted with sterile physiological solution of 70% ethyl alcohol (ethanol) is added. After 5 minutes, the contents of each cup are exposed to optical radiation (LED source or semiconductor laser),

- 3rd group (control with photosensitizer). In each dish under sterile conditions, add 0.5 ml of diluted 1: 5 sterile physiological solution of Diaperone,

- 4th group (experimental with a photosensitizer). In each dish under sterile conditions, 0.5 ml of 1: 5 diluted with sterile physiological solution of Diaperone is added. After 5 minutes, the contents of each cup are exposed to optical radiation (LED source or semiconductor laser).

When used for the irradiation of Enterococcus faecalis bacteria with the official medicinal product Diagiperone, one of the following sources is used:

- a semiconductor laser with a radiation wavelength in the region of 405 nm (violet region of the spectrum);

- a semiconductor laser with a radiation wavelength in the region of 435-445 im (blue region of the spectrum);

- a semiconductor laser with a radiation wavelength in the region of 665 ± 10 nm (red region of the spectrum);

- solid-state Nd: YAG laser with diode pumping and doubling of the radiation frequency with a wavelength of 532 nm (green region of the spectrum);

- superbright LEDs, the maximums of the emission spectrum of which is located in the region of 405 ± 10 nm (violet region of the spectrum);

- superbright LEDs, the maximums of the emission spectrum of which is located in the region of 450 ± 10 nm (blue region of the spectrum);

- superbright LEDs, the maximums of the emission spectrum of which is located in the region of 545 ± 10 nm (green region of the spectrum);

- superbright LEDs, the maximums of the emission spectrum of which is located in the region of 590 ± 10 nm (yellow region of the spectrum);

- superbright LEDs, the maximums of the emission spectrum of which is located in the region of 660 ± 15 nm (red region of the spectrum).

- superbright LEDs emitting white light, one of the maximum emission of which is located at 450 ± 10 nm, the other at 575 ± 10 nm.

The optical radiation source is placed above the Petri dish so that the optical axis of radiation is perpendicular to the surface of the cup, and the size of the light spot is slightly larger than the bottom area (S = 9.6 cm ² ) of the culture cup, providing irradiation of the entire suspension of microorganisms.

The optical radiation power of each source is measured using an IMO-2H meter and adjusted by changing the pump current of the semiconductor emitter. The laser radiation power is changed in the range of 100-1000 mW, and the radiation power of the LED is 75-225 mW. Moreover, the power density of the acting radiation is 5-100 mW / cm ² ; and the exposure time of optical radiation is from 1 to 5 minutes

Then from each cup of the control and experimental groups, 1 ml of the content is taken and plated in sterile Petri dishes with a diameter of 9 cm with enterococcus agar. Enterococcus agar cultures are incubated in an incubator at 37 ° C for 24 hours. After 24 hours of incubation, colony forming units are counted in control (n _k ) plates and in plates containing irradiated cells (n _o ). The residual activity of bacteria is defined as γ = n _o / n _to · 100%.

The results obtained when exposed to bacteria Enterococcus faecalis radiation of a semiconductor laser with a wavelength of λ = 665 nm are summarized in table 1.

Table 1 Culture, strain The number of colony forming units (CFU) in the group 1st group (control without "Diaperone") (n _k ) 2nd group (irradiation without "Diaperone") (n _o ) 3rd group (control with "Diaperone") (n _k ) 4th group (irradiation with "Diaperone") (n _o ) Enterococcus faecalis No. 1 40 27 26 0 Enterococcus faecalis No. 2 35 twenty eighteen 0 Enterococcus faecalis No. 3 40 thirty 29th 0

Enterococcus faecalis No. 4 twenty 10 10 0 Enterococcus faecalis No. 5 25 fifteen 13 0 Enterococcus faecalis No. 6 thirty twenty 21 one Enterococcus faecalis No. 7 fifty 35 36 one Enterococcus faecalis No. 8 twenty 17 16 0 Average value thirty 21.8 21.1 0.25 Average value, % one hundred 72.7 70.3 0.8

Table 1 shows the photodynamic inactivation of Enterococcus faecalis bacteria when exposed to radiation from a semiconductor laser with a wavelength of λ = 665 nm at a power density of 100 mW / cm ² for 1 min in the absence and presence of Diagiperone added to the bacterial suspension in a ratio of 1/30 by volume. The laser energy density is 6.0 J / cm ² .

From table 1 it follows that the effect on the suspension of bacteria Enterococcus faecalis without the addition of photosensitizers of radiation of a semiconductor laser with a wavelength of λ = 665 nm at a power density of 100 mW / cm ² for 1 min (laser energy density of 6.0 J / cm ² ) leads (2nd group of the table) to reduce the number of colony forming units. The residual activity in this case is γ = n _o / n _k · 100% = 72.7%. The presence of the photodynamic inactivation effect without the addition of Diaperone may be due to the sensitizing effect of endogenous porphyrin sensitizers, which are present in Enterococcus faecalis bacteria.

Introduction to the suspension of Enterococcus faecalis “Diaperone” in a ratio of 1/30 by volume and subsequent incubation with it lead (see group 3 of the table) to partial inactivation of the cells. The residual activity in this case is γ = n _o / n _k · 100% = 70.3%. The indicated inactivation may be due to the antiseptic properties of Diagiperone, which is confirmed by the available data on the ability of one of the components of the drug, hypericin, to inactivate Enterococcus faecalis in dark conditions (without exposure to light). However, when exposed to a suspension of bacteria Enterococcus faecalis in the presence of Diagiperone, previously added in a ratio of 1/30 by volume, radiation from a semiconductor laser with a wavelength of λ = 665 nm at a power density of 100 mW / cm ² for 1 min (laser energy density radiation 6.0 J / cm ² ) is observed (4th group of the table) almost complete photodynamic inactivation of cells. The residual activity in this case is γ = n _o / n _k · 100% = 0.8%. Complete photodynamic inactivation of bacteria (γ = 0) was observed in 6 out of 8 experiments, and only 2 experiments revealed single colony forming units.

Thus, the presented data indicate that the photodynamic effect sensitized by chlorophyll included in Diaperone, absorbing radiation at a wavelength of λ = 665 nm, leads to the photodynamic inactivation of Enterococcus faecalis bacteria.

Inactivation of Enterococcus faecalis bacteria is also observed when exposed to violet spectral radiation, which is caused by the excitation of both the chlorophyll and hypericin components of Diaperone.

The results obtained when the Enterococcus faecalis bacteria is exposed to radiation from an LED source with a maximum emission at a wavelength of λ = 405 nm at a power density of 15 mW / cm ^{2 are} summarized in Table 2. It can be seen from the data presented that the effect of radiation at an energy density of 4.5 J / cm ² leads (see group 4) to a complete loss of the ability to colony cells (residual activity γ = n _o / n _k · 100% = 0.

table 2 Culture, strain The number of colony forming units (CFU) in the group 1st group (control without "Diaperone") (n _k ) 2nd group (irradiation without "Diaperone") (n _o ) 3rd group (control with "Diaperone") (n _k ) 4th group (irradiation with "Diaperone") (n _o ) Enterococcus faecalis No. 1 220 140 165 0 Enterococcus faecalis No. 2 200 130 150 0 Enterococcus faecalis No. 3 190 130 140 0 Enterococcus faecalis No. 4 250 one hundred 175 0 Enterococcus faecalis No. 5 180 90 125 0 Average value 208 118 151 0 Average value, % one hundred 56.7 72.5 0

Table 2 shows the photodynamic inactivation of Enterococcus faecalis bacteria when exposed to LED source radiation with a maximum emission at a wavelength of λ = 405 nm at a power density of 15 mW / cm ² for 5 min in the absence and presence of Diagiperone added to the bacterial suspension in the ratio 1/30 by volume. The laser energy density is 4.5 J / cm ² .

It should be noted that the photodynamic action sensitized by the dye does not depend on the degree of monochromaticity of the optical radiation and the degree of its polarization, but is determined only by the energy density (energy dose density) absorbed by the sensitizer. For this reason, the photodynamic inactivation of the Enterococcus faecalis bacterium is observed both under the action of monochromatic radiation of laser sources (wavelength 405 nm, 435-445 nm, 532 nm, 665 ± 15 nm) and quasimonochromatic radiation of LED sources (with a maximum emission spectrum at 405 ± 10 nm, 450 ± 10 nm, 545 ± 10 nm, 590 ± 10 nm, 660 ± 15 nm), the emission range of which corresponds to the maxima in the absorption spectrum or fluorescence excitation spectra of the chlorophyll and hypericin components of Diaperone. The data shown in table 2 confirm what was said when using radiation from an LED source with a maximum of 405 nm.

Moreover, taking into account the multicomponent composition of photosensitizing compounds, a change in the wavelength of optical radiation leads to a change in the total light absorption by the photosensitizing components of DiHiperone, as well as the contribution of the chlorophyll and hypericin components of the drug. However, since the photodynamic inactivation of bacteria is caused by both the excitation of chlorophyll components and the hypericin component, a change in the wavelength of a laser (405 nm, 435-445 nm, 532 nm, 665 ± 15 nm) or LED source (405 ± 10 nm, 450 ± 10 nm, 545 ± 10 nm, 590 ± 10 nm, 660 ± 15 nm) within the absorption bands of these components provides photodynamic inactivation of bacteria at comparable radiation energy densities. For the same reason, the photodynamic inactivation of Enterococcus faecalis is also observed when bacteria are exposed to the presence of DiCiperone by an LED white light source, one of the maxima of the emission spectrum of which is located at 450 nm, and the other at 575 nm.

It should be noted that the choice of concentration of the photosensitizer during its final dilution in relation to the bacterial suspension 1 / (30 ± 10) in volume is due to the following considerations. A 1/30 dilution is optimal, which ensures effective photodynamic inactivation of Enterococcus faecalis bacteria. A decrease in the concentration of the photosensitizer due to an increase in its dilution of 1/40 volume bacterial suspension leads to a sharp decrease in the photodynamic effect. An increase in the concentration of the photosensitizer due to a decrease in its dilution of 1/20 bacterial suspension is undesirable due to the pronounced dark (in the absence of light) inactivation of the bacteria with alcohol.

Along with Diagiperone, the official medicinal product St. John's Wort tincture can also be used as a photosensitizer for the photodynamic inactivation of Enterococcus faecalis bacteria. Photodynamic inactivation of bacteria during the final dilution of the St. John's Wort tincture preparation in relation to a bacterial suspension of 1 / (30 ± 10) in volume is realized when one of the following sources is exposed: lasers (with a radiation wavelength of 405 nm, 435-445 nm, 532 nm, 665 ± 10 nm) or LEDs (with a maximum emission spectrum at 405 ± 10 nm; 450 ± 10 nm; 545 ± 10 nm, 590 ± 10 nm and 660 ± 10 nm), the emission range of which corresponds to the maxima (430 ± 25 nm; 548 ± 15 nm, 590 ± 10 nm and 659 ± 15 nm) in the absorption spectrum or excitation spectra of the chlorophyll fluorescence and hypericin component nt "St. John's wort tinctures."

When using Diagiperone as an official medicinal product based on the alcohol tincture of St. John's wort or an official medicine "Hypericum tincture" based on the alcohol tincture of St. John's wort perforated in relation to a suspension of bacteria 1 / (30 ± 10) in volume:

The exposure is carried out by laser radiation with a wavelength of λ = 665 ± 10 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength of 405 nm with an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength in the region of 435-445 nm with an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by laser radiation with a wavelength of 532 nm at an energy density of 0.9-9.0 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 405 ± 10 nm at an energy density of 0.9-6.0 J / cm ² .

The exposure is carried out by radiation of a LED source with a maximum emission at a wavelength of λ = 450 ± 5 nm at a dose density of 0.9-6.0 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 545 ± 10 nm at a dose density of 0.9-6.0 J / cm ² .

The exposure is carried out by the radiation of an LED source with a maximum emission at a wavelength of λ = 590 ± 10 nm at a dose density of 0.9-4.5 J / cm ² .

The exposure is carried out by radiation of a LED source with a maximum emission at a wavelength of λ = 660 ± 10 nm at a dose density of 0.9-6.0 J / cm ² .

The exposure is carried out by the radiation of an LED white light source with emission maxima at wavelengths λ ₁ = 450 ± 10 nm and λ ₂ = 575 ± 10 nm at a dose density of 0.9-6.0 J / cm ² .

The results of the effect of radiation from each of the above laser or LED sources on a suspension of Enterococcus faecalis bacteria in the presence of the official medicine "St. John's wort tincture" based on the alcohol tincture of St. John's wort perforated when diluted 1/30 by volume with respect to the bacterial suspension are presented in Table 3.

Table 3 Type of optical radiation source Radiation wavelength, nm The energy density of the incident radiation, J / cm ² The number of colony forming units (CFU) after exposure to radiation Laser 665 ± 10 6.0 0 Laser 405 6.0 0 Laser 435-445 6.0 0 Laser 532 9.0 0 Light-emitting diode 405 ± 10 6.0 0 Light-emitting diode 450 ± 5 6.0 0 Light-emitting diode 545 ± 10 6.0 0 Light-emitting diode λ = 590 ± 10 4,5 0 Light-emitting diode 660 ± 10 6.0 0 Light-emitting diode 450 ± 10 and 575 ± 10 6.0 0

When using the official medicine Diagiperone as the photosensitizer or the official medicine Hypericum tincture based on the alcoholic tincture of St. John's wort and exposing one of these sources to a suspension of Enterococcus faecalis bacteria at an energy density of 0.9-9 , 0 J / cm ² there is a photodynamic inactivation of bacteria. When the energy density of optical radiation of any of the sources is 9.0 J / cm ² , the complete photodynamic inactivation of Enterococcus faecalis bacteria is ensured, which excludes the possibility of their growth.

Thus, the data presented show that the photodynamic inactivation of Enterococcus faecalis bacteria is achieved through the use of an official medicinal product based on the alcohol tincture of St. John's wort as a photosensitizer, which is approved for use in medical practice, the minimum volume of which is hundreds of times lower than the cost of an analogue (according to the catalog the company Sigma-Aldrich the cost of a minimum portion of hypericin (10 mg) is 326.5 euros). Therefore, the inventive method provides photodynamic inactivation of Enterococcus faecalis bacteria using, as a photosensitizer, affordable drugs that are approved for use in medical practice.

Literary sources

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Claims (22)

1. A method for the photodynamic inactivation of Enterococcus faecalis bacteria, based on pretreatment of bacteria with a hypericin-containing photosensitizer and subsequent exposure to optical radiation in the visible spectrum corresponding to the absorption band of the photosensitizer, characterized in that the drug “Dihiperone” based on an alcohol-based grass is used as a hypericin-containing photosensitizer dilute it with a suspension of bacteria 1/30 ± 10 by volume and act on the suspension with laser radiation or LED sources with a maximum emission in the region corresponding to the maxima of the absorption spectrum or the maxima of the excitation spectrum of the fluorescent components of the suspension at a radiation energy density of 0.9-9.0 J / cm ² . 2. The method according to claim 1, characterized in that the effect is carried out by laser radiation with a wavelength of λ = 665 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 3. The method according to claim 1, characterized in that the effect is carried out by laser radiation with a wavelength of 405 nm with an energy density of 0.9-6.0 J / cm ² . 4. The method according to claim 1, characterized in that the effect is carried out by laser radiation with a wavelength of 435-445 nm with an energy density of 0.9-6.0 J / cm ² . 5. The method according to claim 1, characterized in that the effect is carried out by laser radiation with a wavelength of 532 nm at an energy density of 0.9-9.0 J / cm ² . 6. The method according to claim 1, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 405 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 7. The method according to claim 1, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 450 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 8. The method according to claim 1, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 545 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 9. The method according to claim 1, characterized in that the effect is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 590 ± 10 nm with an energy density of 0.9-4.5 J / cm ² . 10. The method according to claim 1, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 660 ± 15 nm with an energy density of 0.9-6.0 J / cm ² . 11. The method according to claim 1, characterized in that the exposure is effected by the emission of an LED white light source with emission maxima at wavelengths λ ₁ = 445 ± 10 nm or λ ₂ = 590 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 12. A method for the photodynamic inactivation of Enterococcus faecalis bacteria, based on pretreatment of bacteria with a hypericin-containing photosensitizer and subsequent exposure to optical radiation in the visible region of the spectrum corresponding to the absorption band of the photosensitizer, characterized in that they use the official medicinal solution “Zverokojoba” as the hypericin-containing photosensitizer St. John's wort grass perforated, dilute it with a suspension of bacteria 1/30 ± 10 by volume affect slurry laser or LED light source emitting radiation with a maximum at the area corresponding to the maxima of the absorption maxima of the spectrum or the excitation spectrum of fluorescent slurry component at a radiation energy density of 0,9-9,0 J / cm ^2. 13. The method according to claim 1 or 2, characterized in that the effect is carried out by laser radiation with a wavelength of λ = 665 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 14. The method according to p. 12, characterized in that the effect is carried out by laser radiation with a wavelength of 405 nm with an energy density of 0.9-6.0 J / cm ² . 15. The method according to p. 12, characterized in that the effect is carried out by laser radiation with a wavelength of 435-445 nm with an energy density of 0.9-6.0 J / cm ² . 16. The method according to p. 12, characterized in that the effect is carried out by laser radiation with a wavelength of 532 nm at an energy density of 0.9-9.0 J / cm ² . 17. The method according to p. 12, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 405 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 18. The method according to p. 12, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 450 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 19. The method according to p. 12, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 545 ± 10 nm with an energy density of 0.9-6.0 J / cm ² . 20. The method according to p. 12, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 590 ± 10 nm with an energy density of 0.9-4.5 J / cm ² . 21. The method according to p. 12, characterized in that the exposure is carried out by radiation of an LED source with a maximum emission at a wavelength of λ = 660 ± 15 nm with an energy density of 0.9-6.0 J / cm ² . 22. The method according to p. 12, characterized in that the exposure is effected by the emission of an LED white light source with emission maxima at wavelengths λ ₁ = 445 ± 10 nm or λ ₂ = 590 ± 10 nm with an energy density of 0.9-6.0 J / cm ² .

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