RU2457254C1 - Method for estimating effectiveness of antimicrobial action of antibiotics and ultrasound on pathogenic bacteria in form of biofilm - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: clinical effectiveness is estimated on an simulation model and provides creating a model of bacterial biofilm grown on biolumenescent bacteria Vibrio fischeri. The biofilm is exposed to antibiotics and ultrasound to estimate an antimicrobial effect. The bacterial viability variation may be controlled by varying luminous intensity of the biolumenescent bacteria with using devices, e.g. lumen meters. The antimicrobial effect is estimated by a degree of luminous intensity inhibition as compared to the reference.

EFFECT: method may be applicable in pre-clinical experiments to specify the effective method of treating chronic bacterial infections complicated by bacterial biofilm formation, reduced costs and labour inputs of experiments.

25 dwg, 5 ex

Description

The invention relates to experimental medicine and biotechnology and can be used to determine effective measures for the treatment of chronic infectious diseases in humans under modeling conditions in preclinical experiments.

Microbial biofilms are responsible for the etiology and pathogenesis of many acute and especially chronic bacterial infections of humans. These infections include chronic rhinosinusitis, periodontitis, tooth decay, middle ear inflammation, cystic fibrosis, bacterial prostatitis, infectious endocarditis, urinary tract infections, and miningococcal disease. Such infectious diseases can be caused not by one type of bacteria, but by their entire community. Biofilms are three-dimensional ordered structures that exhibit high resistance to physical and chemical influences, including antibiotics, sulfa drugs, nitrofurans, and other modern drugs. Very often, bacterial biofilms develop on medical implants. According to many researchers, over 60% of all nosocomial infections occur as a result of the activity of microorganisms in biofilms. For example, infections associated with vascular catheterization are most often caused by Staphylococcus aureus and other gram-positive microorganisms.

Experiments and clinical studies have shown that sonication can be a fairly effective means of sensitizing biofilms to antimicrobial agents.

A known method of exposure to low-frequency ultrasound, which is used in combination with various antimicrobial agents for the treatment of bacterial infections associated with the formation of biofilms. (Bartley, J. Ultrasound as a treatment for chronic rhinosinusitis. [Text] / J. Bartley, D. Young // Med. Hypotheses. - 2009. - V.73. No. 1. - P.15-17.) [ one]. The bioacoustic effect is manifested in a decrease in the viability of bacteria in biofilms as a result of simultaneous exposure to low-frequency ultrasound and antimicrobial agents.

Serious problems in surgery are infections associated with prosthetics. To combat them, bone cement treated with antibiotics is used.

A known method of exposure to a combination of low-frequency ultrasound and antibiotics to reduce the viability of planktonic forms of bacteria and bacterial biofilms in bone cement, used for permanent fixation of joints. (Ensing, GT The combination of ultrasound with antibiotics released from bone cement decreases the viability of planktonic and biofilm bacteria: an in vitro study with clinical strains [Text] / GT Ensing, D. Neut, JR van Horn, HC van der Met, HJ Busscher // J. Antimicrob. Chemother. - 2006. - V. 58. - No. 6. P.1287-1290.) [2]. As a result of treatment of bacteria Escherichia coli ATCC 10798, Staphylococcus aureus 7323, staphylococci (CoNS 7368 and CoNS 7391), Pseudomonas aeruginosa 5148 (planktonic and biofilm forms) with pulsating ultrasound in combination with antibiotics in bone cement for 40 hours ) it was concluded that low frequency ultrasound alone does not affect bacterial viability. However, in combination with antibiotics, it significantly reduces the viability of both planktonic forms of bacteria and bacterial biofilms.

A known method of arthroplasty of large joints (RU 2218886, IPC ⁷ A61B 17/56, A61N 7/00; publ. 2003.12.20) [3], which consists in the fact that at the stage of dissection of tissues, resection of parts of the joint and processing of the acetabulum carry out contact ultrasound hemostasis. After processing the acetabulum, holes are formed in it with ultrasound. Then the acetabulum is filled with a solution of an antiseptic or antibiotic, the working end of the instrument is immersed in the solution and the surface is cleaned and cavitation hemostasis is performed. After fluid removal, ultrasonic fragmentary drying of the bone bed surface is carried out for cementing and installing the pelvic component of the endoprosthesis. Before installing the femoral part of the endoprosthesis and its cementing, the bone marrow canal is filled with an antiseptic or antibiotic. The working end of the instrument is immersed in the solution and the channel is cleaned and cavitation hemostasis is performed.

The known method has established the promise of using a combination of antibiotics and pulsating ultrasound in clinical practice to prevent or treat infections associated with prosthetics.

A known method of studying the effect of low-frequency ultrasound on increasing the effectiveness of the antibiotic effects of gentamicin on gram-negative bacteria. (Pitt, WG Ultrasonic enhancement of antibiotic action on gram-negative bacteria [Text] / WGPitt, MOMcBride, JK Lunceford, RJRoper, RDSagers // Antimicrob. Agents Chemother. - 1994. - V.38. - No. 11 . - P.2577-2582.) [4]. The research results were recommended for use in clinical practice in the treatment of antibiotic-resistant infections on medical implants.

A known method of treating acute pancreatitis (RU 2119769, IPC ⁶ A61B 17/00, A61N 7/00, A61K 31/505, A61K 38/55; publ. 1998.10.10) [5], which consists in the fact that after opening the abdominal cavity an insulated cavity is formed from the stuffing bag. Fill the cavity with a solution of protease inhibitors. Through the solution, the pancreas and retroperitoneal tissue are treated with low-frequency ultrasound. The treated liquid is replaced with a solution of 5-fluorouracil with antibiotics. Repeatedly exposure to ultrasound. The procedure is repeated 1 time per day for 3-5 days. The method allows to reduce the treatment time for acute pancreatitis due to the intensification of drug and physical effects.

A known method of treating adenoiditis in children (RU 2087166, IPC A61N 7/00; publ. 1997.08.20) [6] by applying to the region of the upper cervical lateral lymph node of the first order for the nasopharyngeal tonsil located behind the external auditory canal, 3-5% ointments containing an antibiotic, and exposure to this area with ultrasound alternately on both sides.

Scientists note that exposure to ultrasound enhances the effectiveness of antibiotic exposure, however, the mechanisms of this process are still not fully understood. It is assumed that exposure to ultrasound increases the rate of antibiotic transport to bacterial cells, improves the permeability of bacterial membranes. Pulsating ultrasound is as effective as continuous. Poor knowledge of the effect of ultrasound on a bacterial cell does not allow us to more accurately indicate the mechanisms of the observed phenomena. Obviously, their study is one of the urgent tasks of modern microbiology.

Closest to the claimed invention is a method for studying on a rabbit the complex effects of aminoglycoside antibiotics and low-frequency ultrasound (Rediske. AM Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: an in vivo model [Text] / AMRediske, BLRoeder, MKBrown, JLNelson , RLRobison, DODraper, GBSchaalje, RARobison, WG Pitt // Antimicrob Agents Chemother. - 1999. - V.43. - No. 5. - P.1211-1214) [7], taken as a prototype. In the known method, a biofilm of pathogenic microorganisms is placed on a polyethylene disk and surgically introduced into the body of a white rabbit. The combined effect on the rabbit of low-frequency ultrasound and an antibiotic (gentamicin) by indicators of his well-being made it possible to determine some patterns. It was shown that exposure to ultrasound causes a decrease in the viability of bacterial biofilms (Escherichia coli ATCC 10798) only in the presence of an antibiotic. The method confirmed the possibility of the combined action of ultrasound and gentamicin in clinical practice. The disadvantages of the method are manipulation of the animal, surgical intervention in his body using surgical instruments and causing him surgical injuries. It is difficult, expensive, time-consuming, and can also cause side effects (for example, the formation of purulent abscesses), weakening of the animal's body and even its death.

The objective of the invention is the replacement of animal experiments to determine effective treatment of chronic infectious diseases, experiments on a model of bacterial biofilm grown from bioluminescent bacteria, the pathogenic properties of which are identical for microorganisms that cause infectious diseases.

The technical result of the claimed invention is to reduce costs, complexity, increase safety by assessing the effectiveness of treatment of chronic infectious diseases not on animals, but on a model of a bacterial biofilm grown from bioluminescent bacteria, the pathogenic properties of which are identical for microorganisms that cause infectious diseases. In this case, a decrease in the viability of bacteria under the influence of physical and chemical factors, for example, ultrasound and antibiotics, can be controlled by reducing the intensity of their glow with the help of devices, for example, luminometers.

The stated result is achieved by the fact that in the known method for evaluating the effectiveness of the antimicrobial effect of antibiotics and ultrasonic radiation on pathogenic bacteria existing in the form of a biofilm, which consists in creating a model of a bacterial biofilm, selecting doses of the effect of antibiotics and ultrasonic radiation on a bacterial biofilm, recording and evaluating the antimicrobial effect, according to the invention, as a model of a bacterial biofilm, a biofilm of bioluminescent bacteria is used, which is grown they are removed from Vibrio fischeri bioluminescent bacteria that mimic morphological, physiological and biochemical properties similar to pathogenic bacteria, and the effect of antibiotics and ultrasound on the biofilm is recorded by measuring the luminescence intensity of Vibrio fischeri bioluminescent bacteria, and the antimicrobial effect is estimated by the degree of suppression of luminescence control.

The formation of biofilms is characteristic of many microorganisms. In the course of studies, it was found that the main mechanisms of biofilm formation, as well as the regulatory systems of this process that exist in pathogenic microorganisms, are similar to those in the marine luminescent bacteria Vibrio fischeri. Bacteria of the genus Vibrio are able to form biofilms with typical morphological, physiological and biochemical parameters similar to pathogenic microorganisms, which combines Vibrio fischeri with pathogenic vibrios. This allows the use of biofilm grown from the cells of the microorganism Vibrio fischeri as a simulation model to study the effects of physical and chemical factors on pathogenic bacterial species. A significant advantage of Vibrio fisheri is that the formation of a biofilm is accompanied by a sharp increase in luminescence, and the influence of physical and chemical factors, such as ultrasound radiation and antibiotics, is recorded by a change in luminescence, which allows recording the corresponding processes in real time. Thus, on the model of a biofilm grown from the Vibrio fischeri bacterium, it is possible to record and evaluate the antibacterial effect of physical and chemical factors, for example, ultrasonic radiation and antibiotics on pathogenic bacteria, which will greatly simplify and speed up the process of choosing the optimal treatment.

A method for evaluating the effectiveness of the antimicrobial effects of antibiotics and ultrasonic radiation on pathogenic bacteria existing in the form of biofilms is illustrated by the drawings.

Figure 1 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of tetracycline (final concentration in the medium of 1.62 μg / ml).

Figure 2 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of tetracycline (final concentration in the medium of 0.44 μg / ml).

Figure 3 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of tetracycline (final concentration in the medium of 0.032 μg / ml).

Figure 4 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of tetracycline (final concentration in the medium of 0.0012 μg / ml).

Figure 5 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 1.62 μg / ml).

Figure 6 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.44 μg / ml).

Figure 7 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.032 μg / ml).

On Fig shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.0012 μg / ml).

Figure 9 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 1.0 μg / ml) and sonication after 5.5 hours after the start of cultivation.

Figure 10 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.5 μg / ml) and sonication after 5.5 hours after the start of cultivation.

Figure 11 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.25 μg / ml) and sonication after 5.5 hours after the start of cultivation.

12 is a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.1 μg / ml) and sonication after 5.5 hours after the start of cultivation.

Figure 13 shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.05 μg / ml) and sonication after 5.5 hours after the start of cultivation.

On Fig shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) to the addition of chloramphenicol (final concentration in the medium of 0.025 μg / ml) and sonication after 5.5 hours after the start of cultivation.

On Fig shows a graph of the bioluminescent response of Vibrio fischeri (the initial number of cells in 1 ml of medium is 10 ⁵ ) without adding chloramphenicol without and with ultrasound treatment 5.5 hours after the start of cultivation.

Figure 16 shows the luminescence frame of the culture samples of luminous bacteria obtained at the initial (15 hours after seeding) stage of biofilm formation.

Negative image.

On Fig shows the luminescence frame of the samples of the culture of luminous bacteria, observed at 19 hours of cultivation. Negative image.

On Fig shows a luminescence frame of samples of a culture of luminous bacteria, observed at 22 hours of cultivation. Negative image.

Figure 19 shows a luminescence frame of a culture sample of luminous bacteria observed at 26 hours of cultivation. Negative image.

Figure 20 shows a graph of the kinetic curve of the luminescence of bacterial cultures that underwent a 60-minute ultrasound treatment directly in the installation of video recording. Sample 1 - suspension culture of bacteria in a liquid medium (in the luminescence decay phase), sample 2 - wet bacterial biofilm on the glass surface (luminescence growth phase).

↑ Ultrasound, ↓ Ultrasound - turning on and off the ultrasound generator.

On Fig shows a graph of the kinetic curve of the luminescence of bacterial cultures subjected to a 60-minute treatment with ultrasound reduced power directly in the installation of video recording. Sample 1 - suspension culture of bacteria in a liquid medium, sample 2 - wet bacterial biofilm on the glass surface.

↑ Ultrasound, ↓ Ultrasound - turning on and off the ultrasound generator.

On Fig is a graph of the dynamics of the luminescence of samples subjected to the combined effects of ultrasound (prolonged exposure) and subthreshold doses of the antibiotic. Full timeline. Sample a - without antibiotic, sample b - 0.25 μg / ml chloramphenicol.

On Fig shows a graph of the dynamics of the luminescence of samples subjected to the combined effects of ultrasound (prolonged exposure) and subthreshold doses of the antibiotic. Sample a - without antibiotic, sample b - 0.25 μg / ml chloramphenicol. Glow induction period. The luminescence kinetics in the full time scale, see Fig.11.

24 is a graph of the effect of a 60-minute sonication on the kinetics of the luminescence of bacteria treated and untreated with subthreshold doses of the antibiotic. 1 - wet bacterial biofilm on glass treated with an antibiotic (0.25 μg / ml chloramphenicol); 2 - sample in a liquid nutrient medium without antibiotic; 3 - sample in a liquid nutrient medium with an antibiotic. The full timeline of the experiment is shown.

On Fig shows a graph of the effect of a 60-minute treatment with ultrasound on the kinetics of the luminescence of bacteria, treated and untreated subthreshold doses of the antibiotic. 1 - wet bacterial biofilm on glass treated with an antibiotic (0.25 μg / ml chloramphenicol); 2 - sample in a liquid nutrient medium without antibiotic; 3 - sample in a liquid nutrient medium with an antibiotic. A limited timeline is shown at the ultrasound exposure site.

The method is implemented as follows.

As a simulation model for studying the effects of antibiotics and ultrasound on pathogenic microorganisms existing in the form of biofilms, one can use biofilms of any bioluminescent bacteria of the genus Vibrio. In this case, a biofilm formed by the cells of the luminescent bacterium Vibrio fisheri isolated from the Black Sea water was used. To study the sensitivity of biofilms to the effects of antibiotics, tetracycline and chloramphenicol were used. The mechanism of action of these drugs on bacteria has been studied in detail. They non-specifically inhibit protein synthesis by interacting with ribosomes. A similar mechanism of action is characteristic of most antibiotics, including more modern ones. The microorganism Vibrio fischeri is sensitive to both drugs, which allows it to be used as an experimental simulation model. Ultrasound was used to enhance the effect of antibiotics. An ultrasonic generator was used as a source of ultrasonic radiation, giving oscillations with a frequency of 44.5 kHz. These frequency characteristics have the majority of modern devices for the destruction of biofilms. To study the luminescence of bacteria, an LM-01T microplate luminometer (Immunotech) was used.

The Vibrio fischeri strain was grown in Luria-Bertani (LB) liquid medium (peptone - 10 g, yeast extract - 5 g, sodium chloride - 10 g per 1 liter of solution; pH 7.0) in the presence of 3% NaCl. In the case of using LB agar medium, microbiological agar in the amount of 20 g / l was added to its composition. Bacteria were grown at a temperature of 25 ° C.

To study the bioluminescent response of Vibrio fischeri when exposed to antibiotics, solutions of chloramphenicol and tetracycline in various concentrations in ethanol were prepared.

Example 1. Measurement of the bioluminescent "response" of the strain Vibrio fischeri at different concentrations of antibiotics

Vibrio fischeri night culture was grown on LB liquid medium with 3% NaCl at 25 ° C.

A suspension of the Vibrio fischeri strain was prepared using a turbidity standard of 10 units (microbial cell concentration of 1 billion) and ten-fold serial dilutions (4 dilutions inclusive) in LB liquid medium with 3% NaCl. In further work, 4 dilutions (10 ⁵ CFU / ml) were used. 195 μl of Vibrio fischeri suspension was added to the wells of the luminometer plate and grown for 13-17 hours at 25 ° C. 5 μl of ethanol was added to the part of the wells that served as a control, and 1.62 μg / ml, 0.44 μg / ml, 0.032 μg / ml, 0.0012 μg / ml solutions were added to the other wells (in triplicate) antibiotics in ethanol. Immediately after antibiotic administration, Vibrio fischeri cells were placed in a luminometer and the bioluminescence intensity of the cell suspension was measured every 20 minutes. Samples were incubated at room temperature. After the completion of antibiotic administration (26 hours), the measurements continued for another 16 hours. The units for measuring the intensity of bioluminescence are arbitrary units.

Figure 1-4 presents the results of the intensity of the bioluminescence of the cells of the strain Vibrio fischeri after processing the strain with various concentrations of the tetracycline antibiotic.

The concentration of tetracycline 1.62 μg / ml, the highest of all studied, caused a complete suppression of the bioluminescence of cells of the strain Vibrio fischeri. A tetracycline concentration of 0.44 μg / ml led to an earlier “outbreak” of cell luminescence for 5 hours compared to the control. At a tetracycline concentration of 0.032 μg / ml, a similar effect is observed, but the difference between the onset of bioluminescence growth in the experiment and control was a shorter time — 1 h 20 min. The concentration of tetracycline 0.0012 μg / ml, the lowest of all studied, did not cause changes in the intensity of bioluminescence of the strain Vibrio fischeri.

Figure 5-8 presents the results of the intensity of the bioluminescence of the cells of the strain Vibrio fischeri after processing the strain with different concentrations of the antibiotic chloramphenicol.

As can be seen from the data obtained, the concentration of chloramphenicol 1.62 μg / ml, the highest of all studied, caused, as in the case of tetracycline, a complete suppression of the bioluminescence of cells of the strain Vibrio fischeri NB 15. The concentration of tetracycline in the medium of 0.44 μg / ml also caused a suppression of the bioluminescence of the cells of the strain Vibrio fischeri, but not 100%. At a concentration of chloramphenicol of 0.032 μg / ml, a later “outbreak” of cell luminescence was recorded — approximately 2 hours and 40 minutes compared with the control. The concentration of chloramphenicol 0.0012 μg / ml, the lowest of all studied, on the contrary, caused an earlier increase in the bioluminescence intensity of the Vibrio fischeri strain in the experiment (for 1 h 20 min) compared with the control.

Example 2. Measurement of the bioluminescent "response" of the strain Vibrio fischeri at different concentrations of antibiotics after treatment with ultrasound

Vibrio fischeri night culture was grown on LB liquid medium with 3% NaCl at 25 ° C. A suspension of the Vibrio fischeri strain was prepared using a turbidity standard of 10 units (microbial cell concentration of 1 billion) and ten-fold serial dilutions (4 dilutions inclusive) in LB liquid medium with 3% NaCl. In further work, 4 dilutions (10 ⁵ CFU / ml) were used. 195 μl of Vibrio fischeri suspension was added to the wells of the luminometer plate and grown for 5-6 hours at 25 ° C. 5 μl of ethanol was added to the part of the wells that served as a control, and antibiotic solutions in ethanol at a concentration of 1.0 μg / ml were added to the other wells (in triplicate); 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml; 0.025 mcg / ml. Immediately after antibiotic administration, samples with Vibrio fischeri cells were sonicated. For this, the tablet containing the wells combined with the strips with the cultures was immersed in a bath (9 × 5.5 × 15.6 cm) with water (772 ml). An ultrasonic (ultrasound) generator was placed at the bottom of the bath. The frequency of the generated sound was 44.5 kHz, the average acoustic power was 30 watts. The processing time in all experiments with the registration of luminescence using a photoelectron multiplier was 60 min. Then, samples with Vibrio fischeri cells were placed in a luminometer and the bioluminescence intensity of the cell suspension was measured every 20 minutes. Samples were incubated at room temperature. After antibiotic administration and sonication, measurements continued for another 20-30 hours. The units for measuring the intensity of bioluminescence are arbitrary units. The luminescence was measured on a LM-01T microplate luminometer (Immunotech). Figures 9-15 show the results of studies of the intensity of fluorescence of Vibrio fischeri cells at various concentrations of antibiotics after sonication.

As can be seen from FIG. 15, ultrasound treatment without additional antibiotic exposure only to some extent — accelerates the onset of bioluminescence by about 1.5 hours. At the same time, the level of luminescence both in the control and in the experiment practically does not differ. The results of introducing chloramphenicol in different concentrations, followed by ultrasound processing can be seen in Fig.9-14.

Figure 9 shows that the combined action of ultrasound and an antibiotic at a maximum concentration of 1.0 μg / ml led to a complete suppression of the luminescence of the Vibrio fischeri strain, while an outbreak of cell bioluminescence was observed without sonication. Those. the simultaneous action of ultrasound and antibiotic prevents the formation of biofilms. With the simultaneous action of an antibiotic at a concentration of 0.5 μg / ml and ultrasound, a bioluminescence “flash” delay of about 6 hours is observed compared to the action of a single antibiotic (Figure 10). At concentrations of chloramphenicol 0.25-0.1 μg / ml, a decrease in the intensity of the bioluminescence of the strain is observed by about 20-30% in comparison with the control (11, 12). At concentrations of chloramphenicol 0.05-0.025 μg / ml, in addition to reducing the level of cell luminescence by 30-50% of the Vibrio fischeri strain (Figs. 13, 14), the delay of the “flash” of bioluminescence by 1.5-3 hours is also recorded.

Thus, the most significant antimicrobial effect of ultrasound is recorded when combined with the antibiotic chloramphenicol at its maximum investigated concentration of 1.0 μg / ml. Moreover, the degree of decrease in the intensity of bioluminescence of the Vibrio fischeri strain when exposed to the antibiotic chloramphenicol depends on its concentration and time of application during the growth of the culture. The maximum decrease in bioluminescence is recorded when the antibiotic is introduced into the nutrient medium after 21-26 hours of its growth.

The design of the luminometers does not make it possible to observe the morphology of the formation of biofilms and does not allow to observe the bacteria directly when exposed to ultrasound. Modern CCD-cameras allow the observation of bio-chemiluminescence with a sensitivity of up to 0.0004 lux, comparable with the sensitivity of photomultipliers. Methods have also been developed for the quantitative analysis of this kind of video data, providing for automatic frame-by-frame measurement of the brightness of the highlighted areas and data output in the form of text files and graphs.

Example 3. Registration bioluminescent "response" using a video camera

A 2 ml bacterial suspension was placed in 20 ml tubes mounted obliquely in 12 cassettes attached in front of the video camera, or 2 pcs. with immersion in a vessel with 500 ml of water (ultrasonic bath), also installed in the field of view of the camera.

The study of the action of ultrasound was carried out in two ways:

1) observed the luminescence of bacteria (tubes in cassettes) at the initial stage, the culture tubes of interest were removed, they were sonicated in an ultrasonic bath (500 ml vessel) and re-placed in the cassette for video surveillance. Frames corresponding to the extraction and reinstallation of the tubes were cut out;

2) tubes of 2 pcs. they were placed directly in a 500-ml ultrasonic bath of the apparatus, luminescence was observed at the initial stage (the ultrasound source was turned off), then the ultrasound source was turned on for a fixed time (without turning off the camera), after which luminescence was again observed with the source turned off.

An ultrasonic generator was used as a source of ultrasonic radiation, giving oscillations with a frequency of 44.5 kHz .; average acoustic power of 30 watts.

To study the luminescence of bacteria, we used a VNC-748-H3 black-and-white CCD video camera (EVS, Russia, St. Petersburg) with a sensitivity of up to 0.0004 lux at the facility and manual control functions. An Avenir lens was installed on the camera (relative aperture 1.2, focal length 6 mm). Video recording lasting up to 4 days was carried out with a frequency of 1 frame / sec. Video capture was carried out by the program VDub 1.8.1. Using the same program, a * .avi file was converted into a sequence of images (a series of * .bmp files). The sequence was opened in ImageJ. On one of the frames, the image regions of interest were highlighted - the surface of a liquid medium with a suspension of luminous bacteria in individual test tubes. For each such section, a numerical time series was obtained — the dependence of the average brightness on the frame number by the tools Analyse → Tools → ROI manager → More → MultiMeasure, which is converted by the standard software Exel or Origin into a kinetic curve. A video frame of samples of a culture of luminous bacteria obtained at the initial stage (15 h after sowing) of the formation of a biofilm (before sonication) is shown in Fig. 16. Of the 12 samples observed in the experiment, two were processed with ultrasound (10 min exposure) - No. 8 (at the 16th hour) and 11 (at the 18th hour).

Samples 1, 3, 5, and 12 were prepared using a reduced initial concentration of bacteria in suspension. Luminescence of these samples by 15 hours of incubation was not observed. The nature of the luminescence of the samples that started the glow is parietal-peripheral (along the edges of the meniscus of the fluid) with the formation of luminous ring structures. The luminescence zones visually corresponded to the areas of biofilm formation. By 19 h after sowing (Fig.17-19), an increase in the luminescence of all samples, both untreated and sonicated, was observed (sample No. 7-10 min of treatment at the 16th hour after sowing). The nature of the luminescence of all luminescent samples changed from parietal-peripheral to continuous throughout the entire open surface of the liquid medium, which also corresponded to visually observed biofilms. At the same time, samples 1, 3, 5 at a given time did not enter the phase of the onset of luminescence and the formation of a biofilm. By 22 h after sowing, there was a sharp increase in the luminescence of cultures with a delay in the formation of biofilms, the initial concentration of bacteria in which was reduced (No. 1, 3, 5, and 12). Other cultures with a normal developmental rate reduced the level of luminescence compared to 19 h of incubation, and sample No. 11 decreased the luminosity more rapidly. At the final stage of the experiment, by 26 h after sowing, the maximum brightness remained in samples No. 1, 3, 5, and 12. The cultures in samples No. 2, 4, 6, 7, 9, 10, 11 showed a slight glow, sample 11 showed a minimum glow brightness. There was an increase in the glow of sample No. 8.

Thus, a visual (from the selected video frames) qualitative assessment of the level of luminescence in the experiment indicates that the sample treated with ultrasound at the initial stage of biofilm formation, during subsequent long-term incubation, develops a more powerful luminescence maximum, while the sample processed at a later stage biofilm formation (on the decline of luminescence), has a lower final level of luminescence in comparison with untreated samples. Therefore, under experimental conditions, in order to achieve more effective suppression of film formation, ultrasound treatment of bacterial cultures must be performed at the stage of the formed biofilm.

The data obtained confirm the conclusion drawn from the results of the previous experiment that the luminescence decay phase (mature biofilm) is the more preferable period of ultrasound exposure to the bacterial biofilm.

Example 4. The dynamics of the luminescence of samples subjected to a 60-minute treatment with ultrasound outside the installation under conditions of continuous registration of luminescence

The kinetic luminescence curve of bacterial cultures subjected to a 60-minute sonication directly in the video recording apparatus is shown in FIG. Sample 1 - suspension culture of bacteria in a liquid medium (in the luminescence decay phase), sample 2 - wet bacterial biofilm on the glass surface (luminescence growth phase). The inclusion of the ultrasound generator led to a rapid (1-2 min) decrease in luminescence in both samples. However, the sample in a liquid medium (No. 1) reduced luminescence by 9%, while the sample No. 2 - by 92%. For both samples, a period of rapid decay of luminescence was followed by a period of its slow growth with a maximum 20 minutes after the inclusion of ultrasound, and sample 2 showed more pronounced stimulation. The predominance of the stimulating effect over the inhibitory effect for sample 2 is apparently determined with a lower intensity of exposure to ultrasound due to the fact that this sample is immersed in liquid, while sample 1 is immobilized on the glass surface. Similar results were obtained for samples processed at lower ultrasound intensity (Fig. 20). In order to reduce it at the same power of the ultrasonic generator, a larger bath was used - 1 l. Sample 1 corresponds to a bacterial suspension in a liquid - a biofilm is formed on the surface of a liquid medium. Sample 2 - wet biofilm on the glass surface. Ultrasonic treatment of lower power in the experiment (compared with the previous one) showed an almost complete absence of the initial rapid decline in luminosity and a more powerful slow growth with a 20-minute maximum. Under experimental conditions, a sample in a liquid medium and a sample immobilized on a glass surface showed similar kinetics (Fig. 21).

Example 5. The dynamics of the luminescence of samples subjected to the combined effects of ultrasound and subthreshold doses of antibiotic

The dose of the antibiotic used in the experiment was preliminarily selected as a subthreshold one for the effect on the kinetics of luminescence so that its effect did not affect the level of luminescence at any stage of the control experiment without ultrasound. An antibiotic (chloramphenicol, 0.25 μg / ml) was added 1-2 hours before exposure to ultrasound in test tubes with samples of bacterial suspensions, the glow of which was previously recorded with a video camera. The sample tube was removed from the experimental setup, an antibiotic solution was added, mixed and returned to the setup to observe the luminescence kinetics. Ultrasound exposure was carried out with the camera turned on. The experiment, the results of which are shown in Figs. 22 and 23, was carried out with the ultrasound dividing in - from 18 hours after the start of incubation and until the end of observation - more than 8 hours. Observation began after 15 hours of incubation, after the initial phase of biofilm formation was detected by a weak increase in luminescence . Sample a - without antibiotic, sample b - 0.25 μg / ml chloramphenicol. Before the inclusion of ultrasound, the studied samples showed a sharp increase in luminescence, which regains its level after mixing the sample at the time of administration of the antibiotic. After the inclusion of ultrasound, the luminescence of the studied samples increased for 30 min, and in the sample with antibiotic this increase was more weakly expressed. Further kinetics of the luminescence of the samples showed a decline to the background values without subsequent recovery. Thus, the introduction of an antibiotic into the samples reduces the stimulation of luminescence induced by ultrasound.

Example 6. The dynamics of the luminescence of samples subjected to the combined effects of ultrasound (60-minute exposure) and subthreshold doses of the antibiotic

On Fig and 25 presents data from a study of the effect of a 60-minute sonication on the luminescence kinetics of three samples:

1) a wet bacterial film immobilized on the surface of the glass wall of the tube and treated with an antibiotic; 2) a bacterial suspension in a liquid nutrient medium without an antibiotic, and 3) a bacterial suspension in a liquid nutrient medium with an antibiotic. Sample 1 is most rapidly restored after a decrease in the luminescence intensity caused by ultrasound to the initial level. Samples of bacterial suspensions in a liquid nutrient medium exhibit a slow and incomplete restoration of the level of luminescence, and this recovery occurs faster in the sample with an antibiotic (No. 3). In the process of research, it was found that the combined action of subthreshold doses of antibiotics and ultrasound leads to the development of an antimicrobial effect.

As can be seen from example 1, as a result of processing a biofilm grown from Vibrio fischeri bioluminescent bacteria with different concentrations of antibiotics, the luminescence intensity of the bacterial biofilm changed depending on the doses of antibiotics, which coincides with the prototype data. In addition, the luminescence dynamics of samples subjected to the combined effects of antibiotic and ultrasound, as in the prototype, showed an improvement in the antimicrobial effect (examples 2-4). This confirms the possibility of using biofilm grown from bioluminescent bacteria Vibrio fischer in preclinical experiments.

The method will allow to work out the treatment of chronic bacterial infections complicated by the formation of bacterial biofilms on a simulation model of a biofilm grown from bioluminescent bacteria Vibrio fischeri that mimic morphological, physiological and biochemical properties similar to pathogenic microorganisms. Moreover, to record the effects of physical and chemical factors on bacterial biofilms, the effect of suppression and induction of luminescence is used, which can be detected by both luminometers and ultra-sensitive cameras. At the same time, there are no manipulations with animals causing them surgical injuries.

Information sources

1. Bartley, J. Ultrasound as a treatment for chronic rhinosinusitis. [Text] / J.Bartley, D.Young // Med. Hypotheses. - 2009 .-- V.73. No. 1. - P.15-17.

2. Ensing, G.T. The combination of ultrasound with antibiotics released from bone cement decreases the viability of planktonic and biofilm bacteria: an in vitro study with clinical strains [Text] / GTEnsing, D.Neut, JRvan Horn, HCvan der Met, HJBusscher // J. Antimicrob. Chemother. - 2006. - V.58. - No. 6. P.1287-1290.

3. RU 2218886 IPC ⁷ A61B 17/56, A61N 7/00; publ. 2003.12.20

4. Pitt, W.G. Ultrasonic enhancement of antibiotic action on gram-negative bacteria [Text] / W.G. Pitt, M.O. McBride, J.K. Lunceford, R.J. Roper, R.D..Sagers // Antimicrob. Agents Chemother. - 1994. - V.38. - No. 11. - P.2577-2582.

5. RU 2119769, IPC ⁶ A61B 17/00, A61N 7/00, A61K 31/505, A61K 38/55; publ. 1998.10.10.

6. RU No. 2087166 IPC ⁶ A61N 7/00; publ. 08/08/20.

7. Rediske. A.M. Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: an in vivo model [Text] / A.M. Rediske, B.L. Roeder, M.K.Brown, J.L. Nelson, R.L. Robison, D.O. Draper, G. B. Schalalje, R.A. Robobison, W.G. Pitt // Antimicrobents. - 1999. - V.43. - No. 5. - P.1211-1214 (prototype).

Claims (1)

A method for evaluating the effectiveness of the antimicrobial effect of antibiotics and ultrasonic radiation on pathogenic bacteria existing in the form of a biofilm, which consists in creating a model of a bacterial biofilm, selecting doses of the effect of antibiotics and ultrasonic radiation on a bacterial biofilm, recording and evaluating the antimicrobial effect, characterized in that as a model of bacterial biofilms use a biofilm of bioluminescent bacteria, which is grown from bioluminescent bacteria Vibrio fischeri, while istratsiyu effects of antibiotics and the ultrasonic radiation on the biofilm is carried out by measuring the intensity of luminescence of bioluminescent bacteria Vibrio fischeri, a antimicrobial effect is evaluated by the degree compared to control emission intensity suppression.

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