INFLUENCE OF LED RADIATION ON THE STAPHYLOCOCCUS AUREUS SENSITIVITY TO ANTIBIOTICS

Hz; 3000 Hz; 8000 Hz. The dependence of radiation on the studied objects depending on exposure, wavelength and frequency were defined. The result of photomodification on the state of microorganisms was increase of their sensitivity to some of the tested antibiotics. Based on the data obtained, we developed an algorithm and clinical recommendations for the use of LED radiation in the complex therapy of purulent-inflammatory diseases. LED emission of blue-IR range led to the most pronounced increase of sensitivity to ampicillin and oxacillin, while at red and infrared radiation — to levofloxacin, tetracycline and meroponem.


Introduction
The problem of resistance to antimicrobial drugs continues to deteriorate, this is one of the greatest threats to health protection and for its solution active actions in different fields are required [1,2]. Antibacterial drugs currently occupy one of the most important parts of the arsenal of drugs of medical institutions. From 25 % to 35 % of all hospitalized patients receive antibiotics according to various indications. At the same time, according to the WHO 50 % of antibiotics are appointed without reason [1,3].
S. aureus is one of the main pathogens of skin and soft tissue diseases -boils, abscesses, cellulitis, phlebitis, mastitis, causing pneumonia, osteomyelitis, meningitis, endocarditis and others. Clinical strains of S. aureus are the most common microorganisms which are found in surgical patients [4,5,6].
Despite the natural sensitivity of Staphylococcus aureus to most antibiotics (beta-lactams, aminoglycosides, fluoroquinolones, macrolides, lincosamides, glycopeptides, rifampicin, etc.), its resistance to them grows intensively and substantially extend. Consequently, results of infections treatment caused by Staphylococcus aureus complicated and worsen [5,7]. A significant concern evokes the emergence and spread of methicillin-resistant strains of Staphylococcus aureus (MRSA) that cause outbreaks of nosocomial and community-acquired infections [8].
Among the solutions of the problem, special attention deserves the use of non-drug methods, in particular radiation of the optical range, to increase sensitivity of microorganisms to existing drugs [9][10][11][12][13][14]. Due to proven positive influence on the human's body and almost complete absence of side effects and contraindications [10], LED light application is a promising method. It was also established that the microflora of the mouth can be affected by the light from halogen and LED sources, polarized and unpolarized. This reduces the number of bacterial colonies and their sizes, indicating to suppression or destruction of microbes [11]. But these data do not explain all the mechanisms of antimicrobial effects of light, which depend on its spectral characteristics, power density, frequency of impulses and polarization. Also important is to clarify possibilities of changes of some of the properties of microorganisms, in particular their sensitivity to damaging factors, such as antibiotics.
Purpose of the work is to investigate direct effect and determine the optimum parameters of action of LED radiation of blue-red and red-infrared ranges on sensitivity to antibiotics of clinical isolates Staphylococcus aureus and test strain Staphylococcus aureus ATCC 25923.

Materials and methods
We studied the effect of LED radiation of red and infra-red and blue and infrared ranges on sensitivity to antibiotics of clinical isolates of Staphylococcus aureus (n = 5) that were isolated from foci of inflammatory processes from surgical department patients of Uzhgorod CRH and collection test strain Staphylococcus aureus ATCC 25923.
Determining the impact of LED radiation on the sensitivity of microorganisms to antibiotics we used disco-diffusion method, according to current requirements of the Ministry of Health of Ukraine [15]. For this purpose, we took a pure 16-24-hour agar culture of microorganisms in a standardized liquid nutrient medium with concentration of 1.5 × 10 8 colony forming units (CFU) per 1 ml (it meets the turbidity matching 0.5 Mcfarland standard) and reseeded on nutrient dense Mueller-Hinton medium in Petri dishes. Then we carried out exposure of microorganisms to LED radiation from a distance of 1 cm (Fig. 1), and then we superimposed discs with antibiotics and cultivated them in the incubator at 37 °C. Results were determined by measuring zones of microbial growth delay around the discs.
In order to exclude the impact of radiation on the growth properties of the nutrient media we carried LED radiation exposure of cultures that were in sterile Petri dishes separately and then replanted them in the culture medium and superimposed discs with antibiotics.
The results were compared with control series of not irradiated cultures. Also, in order to control the quality of microbiology investigations, in each series with clinical strains we used a collection test strain Staphylococcus aureus ATCC 25923 with known range values of microbial growth delays in areas of antibiotics influence.
We tested sensitivity of the research object to antibiotics of beta-lactam group, macrolides, fluoroquinolones, aminoglycosides, and vancomycin, which are included in the list of recommended antibiotics to determine sensitivity of Staphylococcus spp. [15].

Results and discussion
We established that the LED radiation increased the sensitivity of clinical isolates of S. aureus and collection of test strain S. aureus ATCC 25923 to some antibiotics (compared with non-irradiated cultures). In this case, the effect of influence depended on the wavelength, exposure duration and frequency of radiation. The most pronounced sensitivity increase was observed when irradiation exposure of microorganisms lasted for 5 minutes. Fig. 2 illustrates the effect of 5-minute exposure to MEDOLIGHT BluDoc LED device at different frequencies on sensitivity of S. aureus ATCC 25923 to ampicillin and other antibiotics.
Thus, 5-minute exposure test strain of Staphylococcus aureus ATCC 25923 to LED emission of blue + infrared range with frequency of 0 Hz (continuous emission) increased its sensitivity to ampicillin, in average, for 15.5 %. Application of LED MEDOLI-GHT BluDoc device radiation with exposure of 5 minutes and frequencies of 10, 600, 3000 and 8000 Hz lead to increased sensitivity of Staphylococcus aureus ATCC 25923 to ampicillin for 12-13,5 %. Fig. 3 shows increased sensitivity of Staphylococcus aureus ATCC 25923 to oxacillin after application of blue infrared range LED radiation.
Irradiation of Staphylococcus aureus ATCC 25923 by LED emission of blue + IR range with frequency of 0 Hz for 5 minutes increased its sensitivity to oxacillin by an average of 23.5 %. After similar exposure with frequencies of 10, 600, 3000 and 8000 Hz we observed increase of sensitivity of these strains to oxacillin -by 17-22 %. Fig. 4 illustrates graphically the rate of sensitivity increase to ampicillin and oxacillin of Staphylococcus aureus ATCC 25923 under the influence of LED radiation of Medolight BluDoc device of different frequencies with 5 minutes exposure. Table 1 shows statistically processed results of the impact of LED blue infrared radiation on sensitivity of S. aureus ATCC 25923 to ampicillin and oxacillin.
Using the LED irradiation of red + IR range led to a significant increase in sensitivity of studied strains S. aureus to levofloxacin (group fluoroquinolones), meroponem (group carbapenems) and tetracycline (group of the same name) (Fig. 5).
Thus, LED red + IR irradiation with exposure of 5 minutes at a frequency of 0 Hz resulted in increased sensitivity of S. aureus ATCC 25923 to levofloxacin by an average of 17%, tetracyclin -by 27 % and meroponem -by 22.47 %. After similar exposure with frequencies of 10, 600, 3000 and 8000 Hz we observed an increase of sensitivity of S. aureus ATCC 25923 to levofloxacin by an average of 11-12,5 %, to tetracycline -by 21-23 % and to meroponem -by 14-16 % ( Fig. 6).
In Table 2 we present statistically processed results of the effects of LED red + IR radiation on sensitivity of S. aureus ATCC 25923 to levofloxacin, tetracycline and meroponem.
It should also be noted that after application of LED radiation with 5 minutes' exposition, we observed increased sensitivity of the examined microflora also to such antibiotics as cefotaxime, rifampicin and meroponem for 6.5-12 %, depending on the wavelength and frequency of light. This regularity also concerned clinical isolates. 5-minute exposure to LED radiation increased sensitivity of all strains of S. aureus, cultured from locus of inflammatory diseases to ampicillin, oxacillin, cefotaxime, meroponem and rifampicin, and in some cases also to gentamicin and tetracycline.
Assessing the effects of irradiation of microorganisms in sterile cups, we noted the same regularities as after irradiation of bacteria in Petri caps with Hinton-Mueller agar, which allows to eliminate the impact of light on the properties of nutrient medium. Thus, the obtained results are related with the direct impact of photo modifying LED radiation on the examined flora.
Prolonged exposure of microorganisms to irradiation did not lead to the increase of their sensitivity, compared with 5-minute exposure. On the contrary, when irradiation lasted more than 15 minutes, sensitivity of all the investigated strains was within the statistical error compared with the control.
Taking in consideration almost complete absence of contraindications to the use of LED radiation, it can be used in the treatment of superficial suppurative inflammatory diseases caused by Staphylococcus aureus. This will allow to increase the effectiveness of antibiotics therapy for these infections.

Conclusions
LED radiation of red+IR (640±30 and 880 ± 30 nm) and blue + IR (470 ± 30 and 880 ± 30 nm) ranges makes photo modifying impact on the investigated strains of Staphylococcus aureus, which manifests itself in increase of their sensitivity to most tested antibiotics.
The degree of influence depends on the wavelength, duration and frequency of radiation impulses -the most pronounced increase of sensitivity was noted at the exposure of 5 minutes at frequency of 0 Hz (continuous radiation).