Electromagnetic and acoustic technologies in antibacterial preparation development

Subject and Purpose. The present paper is concerned with the use of wave technologies in the development of antibiotics-alternative approaches for pathogenic microflora suppression. Lactobacilli strains picked in different ecological niches and their activity against pathogenic strains are studied with a focus on a targeted modification of adhesive and antagonistic properties of lactobacilli by exposing them to low-intensity electromagnetic (EM) fields and the ultrasound. Methods and Methodology. Lactobacilli picked in different ecological niches are experimentally studied, including (1) standard strains from probiotic preparations and (2) circulating strains picked in humans and bees. For the ultrasonic and electromagnetic radiation sources, G3-109 and G3-F and G4-141 and G4-142 generators are taken, respectively. The adhesive properties of Lactobacillus spp. strains and their antagonistic activity are estimated against C. diphtheriae, S. aureus and yeast-like fungi of Candida genus in aerobic and microaerophilic culture conditions. Statistical technology is employed in the data processing and analysis. Results. It has been established that L. plantarum strains picked in the gut of healthy bees are most antagonistic towards pathogens. It has been demonstrated that the priority culture conditions for lactobacilli are microaerophilic conditions simulating their stay in vivo. It has been shown that it is possible to modify properties of microorganisms by their exposure to ultrasound and low-intensity electromagnetic fields in narrow bands of the EHF range. The effect efficiency versus frequency has a dispersion character. Individual features of various pathogenic strains have been recognized. Conclusion. The obtained results open up prospects for electromagnetic and acoustic technologies in the development of safe alternative means to antagonize persisting pathogens and increase human body resilience.

Nowadays, environmentally friendly technologies have received extensive attention in medicine, vaccinology, pharmacology, microbiology. A strong need exists in new methods and means based on physical impacts on biological objects of diff erent classes, including microorganisms, to purposefully modify their functional properties [1,2]. Ultrasound and low-intensity electromagnetic (EM) fi elds in the extremely high frequency (EHF) range are considered such impact factors, with environmental requirements and maximum allowable levels and doses of radiation taken into account [1,3,4].
Antibiotics that are widely used in today's medicine can quickly and eff ectively suppress various infections, including particularly dangerous ones. Yet a positive eff ect from antibiotic therapy is accompanied by such functional disorders aff ecting the human body as slower hematopoiesis, inhibited metabolic processes, maldigestion, etc. Under the action of antibiotics, polyresistant strains of the agents increase their diversity, thus causing atypical forms of common infectious diseases, such as pneumonia, tuberculosis, meningitis, typhoid, etc. Th erefore, in non-life threatening conditions, pro-, pre-and synbiotic preparations must be taken to keep the fl ora of the macro-organism in the normal state [5,6].
A search for new sources of pro-, pre-and synbiotic preparations against pathogenic bacteria is very important. Synbiotic preparations based on representatives of normal fl ora and having high colonization capacity and high antagonistic activity towards pathogenic and opportunistic microorganisms [6][7][8][9] hold promise. Probiotics, which include lactobacilli living on the body mucous membranes, are most commonly used to cope with dysbiotic conditions. Th eir species composition is very diverse [8,[10][11][12][13][14]. At present, certain lactobacilli species that persevere in the human body are only studied for their general biological properties. Consequently, it is necessary to fi nd and select candidates for production strains among representatives of alternative econisches.
Th e diff erentiation and identifi cation of probiotic bacteria is a critical point in the main microbiological study. Potential strain candidates should not only be certifi ed according to their basic characteristics, but "probiotic" properties as well, among which are, fi rst of all, adhesive and antagonistic activities. Th e adhesion property determines the bacteria ability to gain a foothold in an econiche and provides colonization resistance to the mucosa. Th e antagonistic properties in biocenosis ensure production of antimicrobial compounds of protein origin (bacteriocins), which exerts a positive eff ect on the human body [15].
We seek to investigate abilities of lactobacilli strains isolated from diff erent econisches in an effort to antagonize pathogenic strains of diphtheria, golden staphylococcus, and yeast-like fungi of the genus Candida under diff erent culture conditions (aerobic and microaerophilic). Of interest is also a targeted modifi cation of adhesive and antagonistic properties of lactobacilli by exposing them to a low-intensity EM fi eld and the ultrasound.
1. Equipment and measurement technique. Th e irradiation of microorganism strains and their exometabolites was carried out on a special experimental bench.
To implement the EM fi eld eff ect in a narrow frequency band of the EHF range, generators G4-141 ( f  37.5…53.57 GHz) and G4-142 ( f  53.57…78.33 GHz) were used for Р  5 mW power signals. During the irradiation, the generator waveguide outputs were loaded with horn an-tennas of 6.0 × 5.0 cm 2 and 8.5 × 6.5 cm 2 apertures. Th e examined objects were placed 5…7 cm away from the horn mouth, i.e. in the antenna near zone. Th e power fl ux density (PFD) was 0.1 mW/cm 2 with an irradiation non-uniformity of no more than 3 dB in the object location. Th e non-uniformity is connected with the near zone specifi cs, finite sizes of the aperture and irradiated objects and with a low impedance of the load.
To implement the ultrasound eff ect, generators G3-109 ( f  60 kHz, P  5 W) and G3-F ( f  18 kHz, P  16 W) under the load of circular piezoceramic converters-radiators of PZT type (radiators based on synthetic Plumbum Zirconate Titanate ceramics) were used. When irradiated with G3-109 generator, the excitation amplitude of the signal was U  15 V at a load of R  50  (P  5 W). Th e coeffi cient of the electric into acoustic power transformation was ŋ ≈ 5%, i.e. the average power of acoustic vibrations in the biological object location reached (0.25…0.50) W. Th e bacterial suspension tubes were located in the near zone of the radiator. Th e irradiation took place in an aqueous medium.
Th e conditions for microaerophilic bacteria culture were created in microanathestates using Generator GENbox microaer gas generating packs (bio-Merieux, France) or an industrially manufactured gas mixture consisting of О 2 (5%), СО 2 (10%), and N 2 (85%). Th e examination was given to strains of Lactobacillus spp. picked in diff erent ecological niches, including: 1) probiotic preparations: strain L. acidophilus from preparation "Symbilact" produced by Vivo, Ukraine, strain L. rhamnosus from preparation "PREMA®" produced by Delta Medical Promotions, Switzerland, 2) L. plantarum strain picked in the gut of bees (19 bee families) with the participation of NPC "IEKVM" NAAN, and 3) strain from the mucosa of the upper respiratory tract and the contents of the intestine of people aged 17 to 23.
For pathogenic bacteria, we use circulating and museum strains Corynebacterium diphtheriae and S. aureus obtained by the City Student Hospital of Kharkov. Th e reference strain is S. aureus 209 P (ATCC 6538-R) from the laboratory of medical microbiology in cooperation with the Museum of Microorganisms of SA "IMI NAMN".
Th e electromagnetic exposure was carried out in discrete 42.2 and 61.0 GHz bands for 3 hours. Th e ultrasonic exposure was carried out in the 18.0 and 60.0 kHz bands for 1 hour. Standard probiotic strains of lactobacilli: L. rhamnosus, L. acidophilus and strains of L. plantarum picked in the gut of bees, were exposed to the EM fi eld and the ultrasound.
Th e evaluation was given to: 1) adhesive activity of lactobacilli using the mean adhesion index (MAI), adhesion coeffi cient (AC) and the microorganism adhesion index (MOAI) [16,17] and 2) antagonistic activity of lactobacilli and their exometabolites towards pathogenic strains of C. diphtheria, of S. aureus and yeast-like fungi of the genus Candida. Th e method of delayed antagonism on the spectrum of action was used, i.e. it was considered the ability to suppress the vital activity of diff erent number of test cultures with the indication of growth retardation zones and determination of their biofi lm formation during inter-microbial interaction [18,19].
Th e results were processed according to the rules of variation statistics [20] using standard programs.
2. Discussion of the exposure results. Preliminarily potential strain-candidates of lactobacilli were selected from alternative econiches, specifically from practically healthy people aged 17 to 23 and among representatives of normocenosis of bees. Nineteen bee families were examined to pick 10 5 …10 6 CFU/g of lactobacilli in the gut of healthy bees.
Th e cultivation of bacteria was held under aerobic and microaerophilic conditions. Th e main reason is that in the biological niches of the human body in vivo, the culture conditions of the bacteria diff er signifi cantly when extracted in vitro. One of important parameters of the existence and development of microorganisms is the gas composition of the incubation atmosphere. Th e atmosphere of reduced partial pressure of oxygen and increased partial pressure of carbon dioxide (the microaerophilic conditions) reproduces, to a certain extent, the conditions of lactobacilli in vivo.
Th e application effi ciency of the agents is determined also by the ability of lactobacilli probiotic strains to gain a foothold in human epithelial cells and propagate themselves before mucosal layer cells are renewed. For the experimental investigation results on adhesive properties of Lactobacillus spp., see Table 1.
According to the obtained data, the strains L. plantarum and L. rhamnosus under aerobic conditions of cultivation have average adhesive abilities. Th e adhesive abilities of the strain L. acidophilus are worse (the MOAI is under 2.5).
Under microaerophilic culture conditions, the adhesive activity of all lactobacilli strains gets better. Th e MAI increases by 1.2 to 1.3 times (p  0.05), the AC for L. rhamnosus and L. plantarum strains increases, on the average, by 1.2 times (p  0.05) (Fig. 1).
Th us, a reduced partial pressure of oxygen stimulates the lactobacilli ability to gain a foothold in eukaryotic cells.
According to the evaluation results on the antagonistic activity of lactobacilli picked in diff erent econiches, all the studied lactobacilli strains cannot suppress the growth of yeast-like fungi of Candida genus under both aerobic and microaerophilic cultivation conditions. Th e amount of antagonistic properties of Lactobacillus spp. towards C. diphtheriae and S. aureus depends on the primary residence range of the antagonistic strains. In 100 percent of cases, the greatest anti-diphtheria activity is off ered by L. plantarum strain picked in the gut of bees. Lactobacilli strains picked in humans mostly show moderate antagonistic properties. On the average, 24.7% (aerobic) and 76.7% (microaerophilic) of the pathogens and the standard probiotic strains (L. rhamnosus and L. acidophilus) did not aff ect the growth of the test cultures in all the aerobic cultivation conditions and were 86.96% ineff ective.
Th e ability to suppress the growth of golden staphylococcus strains under the aerobic and microaerophilic cultivation conditions is shown by all the lactobacilli strains examined. Circulating strains of lactobacilli, no matter whether picked in humans or bees, suppress the growth of about a third of the S. aureus crops studied, and the probiotic strains do not possess antagonistic properties towards more than 90% of the test crops. As with diphtheria, an increase in the antagonistic activity was observed under microaerophilic culture conditions.
Th e reason is that lactobacilli are microaerophils, i.e. the environment with an increased partial pressure of carbon dioxide and reduced oxygen is more favourable for their growth.
For the further research, L. plantarum strain picked in the gut of bees is chosen as having pronounced antagonistic properties towards C. diphtheriae and S. aureus strains. Th e investigation results are shown in Table 2.
As a result, the diameter of the growth retardation zone of C. diphtheriae strain under the microaerophilic cultivation conditions increased significantly: by 4.8 times (p  0.05) as compared to the aerobic conditions, and in S. aureus strain from 0 to 2.67 mm, respectively (p  0.05).
Among the most signifi cant factors that infl uence the nature of inter-microbial relationships, namely the development of other members of biocenosis, are the eff ects of bacteria-produced substances. Th e eff ect of exometabolites of L. plantarum strain on the growth dynamics and biofi lm formation of C. diphtheriae and S. aureus is illustrated in Table 3.
A statistically signifi cant inhibitory eff ect on the growth properties of pathogenic bacteria is exerted by the addition of exometabolites of strain L. plantarum in the amount of 0.1 and 0.3 ml per 1.0 ml of the nutrient medium. Th e exometabolites of the candidate strain in the amount of 0.1 ml suppresses, on the average, the growth dynamics of patho-   (Fig. 2).
It should be noted that the inhibition of the corynebacteria ability to form biofi lms is directly proportional to the concentration of exometabolites in the medium.
According to the WHO recommendations with regard to the identifi cation of candidate strains to be put into production, they should not inhibit the host normal fl ora. Investigations were carried out on the eff ect exometabolites of candidate strain L. plantarum exert on the bioplane formation ability in clinical strains Lactobacillus spp. Th e result is a moderately decreased biofi lm formation as opposed to pathogenic bacteria with suppression signifi cant.
In addition, the eff ect of exometabolites of L. plantarum strain on enzymatic and phagocytic activity was investigated. Th e inhibition of invasion enzymes in S. aureus in vitro plasmocoagulase and lecithinase strains [21] has been established. A stimulation of phagocytosis values in neutrophilic leukocytes of donors was noted, including phagocytic activity, oxygen metabolism of neutrophils and opsonization of bacteria [22].
It has been shown that exometabolites of L. plantarum strain can inactivate or partially degrade an enzyme of aggression, such as diphtheria toxin. Th e determination of the toxicity of diphtheria toxin was carried out on laboratory animals (guinea pigs weighing 350…400 g) [23].
Th e analysis of the obtained results suggests that the candidate strain L. plantarum picked in the  gut of bees can be recommended as a production strain in the development of potential methabiotic complexes for therapeutic and prophylactic purposes. Th e suppression of the functional properties of pathogenic bacteria by co-culturing them with lactobacilli is the basis of the subsequent search for alternative and safe ways to suppress persistent pathogens.
Next, the use of EM and ultrasonic technologies for targeted modifi cation of the functional indices of lactobacilli will be considered.
Microwave radiation is known to aff ect the electrostatic relationships in the bacterial agent-host cell system and to change the adhesive properties on the surface of bacteria. Th e previous experimental works revealed a possibility to modify adhesive properties of pathogenic bacteria C. diphtheriae [24]. In the present research, normophlora representatives are exposed to EM fi elds and the ultrasound.
Th e electromagnetic fi eld exposure of lactobacteria L. plantarum, L. rhamnosus, and L. acidophilus was held in the 42.2 and 61.0 GHz frequency bands under aerobic cultivation conditions for 3 hours. Table 4 illustrates the corresponding changes in the adhesive activity.
As a result, for L. plantarum and L. rhamnosus strains, the average number of microbes attached to one erythrocyte decreases on the average by 1.7 and 3.8 times (p  0.05), respectively, when irradiated in the 42.2 GHz band. Th e percentage of erythrocytes having adhesive microorganisms on their surface decreases on the average by 1.3 and 2.0 times (p  0.05), and the adhesion index of the microorganisms decreases by 1.3 and 1.9 times (p  0.05) ( Table 4). Th e L. acidophilus strain, in contrast, exhibits an increase in the MAI and MOAI, on the average, by 1.4 and 1.2 times, respectively (p  0.05), whereas the AC tends to be suppressed.
Th e use of the millimeter waves in the 61.0 GHz band leads to the stimulation of the adhesive process in L. plantarum and L. acidophilus strains. Th e MAI increases on the average by 1.2 (p  0.05) and 2.1 times (p < 0.05) and the AC -on the average, by 1.3 times (p  0.05) in both cases. As for the MOAI, L. acidophilus shows its average increase by 1.6 times (p  0.05), whereas L. plantarum shows little change in the adhesion index. L. rhamnosus strain irradiation in the 61.0 GHz band results in the MAI suppression by 2.2 times (p  0.05) on the average and the MOAI -by 2.0 times (p  0.05) (Fig. 3).
Th us, the frequency dependence of electromagnetic eff ect effi ciency on the functional state of probiotic strains of lactobacilli cultured under aerobic conditions is observed. In most cases, the exposure in the 42.2 GHz band inhibits the adhesive properties, and in 61.0 GHz band stimulates them.
Th e exposure to the ultrasound was held in the 18.0 and 60.0 kHz frequency bands for 1 hour. In the result, the lactobacilli adhesion signifi cantly decreases in all probiotic strains (Table 4). At the 18 kHz frequency band exposure, the MAI decreases by 1.8…7.5 times (p  0.05), the ACby 1.2…2.9 times (p  0.05), and the MOAIby 1.3…2.5 times (p  0.05). Th e exposure to the 60 kHz band ultrasound reduces the average number of microbes attached to one erythrocyte (MAI) Table 4

. Eff ect of low-intensity EM fi eld and ultrasound on the functional indices of the adhesion of probiotic strains at the cultivation in aerobic conditions
Lactobacillus spp. . Th e percentage of erythrocytes having adhesive lactobacilli (AC) on their surface decreases by 1.2…1.9 times (p  0.05) and the adhesion index of microorganisms decreases by 1.4…2.0 times (p  0.05) (Fig. 4). Th us, the ultrasound exposure is a factor that can lead to a change in the state of the objects under study, which is evident, in particular, in the suppression of the adhesive properties of Lactobacillus spp. under aerobic conditions of the cultivation. Also, the frequency dispersion dependence can be seen in the eff ect magnitude, with general behavioral trends retained.
One of the tasks was to increase the ability of Lactobacillus spp. strains to suppress the growth of C. diphtheriae and S. aureus test cultures due to the EM fi eld and ultrasound exposures.
For this purpose, L. plantarum strain was exposed to the EM fi eld in the 42.2 and 61.0 GHz frequency bands for 3 hours. Th e antagonistic activity of lactobacilli aft er the co-cultivation in aerobic and microaerophilic conditions was determined by the change of the growth retardation zone diameter of pathogenic strains.
Th e obtained data analysis (Table 5) suggests a signifi cant increase in the diameter of the growth retardation zones of the test crops when they are co-cultured with irradiated L. plantarum strains compared to the control (non-irradiated) ones.
Aft er the L. plantarum treatment in the 61.0 GHz frequency band, the antagonism of lactobacilli rises by 3.3 times ( p  0.05) compared to the strains of C. diphtheriae. For S. aureus, the diameter of the growth inhibition zone increases from 0 to  Under the microaerophilic conditions, the EM fi eld exposure of L. plantarum in the 61.0 GHz band increases the antagonistic properties towards C. diphtheriae by 1.5 times ( p  0.05) and towards S. aureus -by 2 times ( p  0.05). Th e irradiation in the 42.2 GHz frequency band yields an opposite effect. Th e growth retardation zone of S. aureus gets 2.4 times less ( p  0.05). For C. diphtheriae, this effect is not observed (Fig. 5).
Th us, the irradiation of L. plantarum strains changes their antagonistic properties towards the C. diphtheriae and S. aureus test cultures. Th is eff ect exhibits the radiation frequency dispersion. Namely, the 61.0 GHz exposure raises the antagonism of lactobacilli in all the cases. Yet, the exposure to the 42.2 GHz fi eld brings an opposite result, which in turn depends on pathogenic bacteria strains and conditions of their co-cultivation.
Th ere is a dependence of the antagonistic activity of the irradiated lactobacilli with non-irradiated pathogenic strains on conditions of the co-cultivation. Under the microaerophilic (in vivo) and aerobic (in vitro) co-cultivation conditions, a biological response to the EM fi eld exposure may have an opposite result.
Th e ultrasound exposure was in the 18 and 60 kHz bands and lasted for 1 hour.
Th e results of the lactobacilli antagonistic activity towards pathogens aft er the ultrasound exposure and in view of diff erent cultivation conditions are shown in Table 6.
Under the aerobic culture conditions, the ultrasound exposure at 18 kHz is eff ective, with the growth retardation zone diameter increase from 0 to 1.27 mm for S. aureus. For the microaerophilic conditions, no signifi cant changes are observed (Fig. 6).
Conclusions. It has been found that among lactobacilli strains taken from standard probiotic preparations and picked in alternative econiches, i.e. from humans and bees, those selected from the gut of healthy bees, L. plantarum, have the greatest antagonistic activity. Exometabolites of candidate  strain L. plantarum are able to suppress enzymes of aggression and invasion of pathogenic corynebacteria and golden staphylococcus, reduce their growth potential and ability to form biofi lm, and do not inhibit human normal fl ora. Th e use of L. plantarum strain is promising as a production one in the development of potential methabiotic complexes for therapeutic and prophylactic purposes. Th e eff ect of the environmental atmosphere gas composition on the viability of microorganisms when co-cultivated with other strains under aerobic and microaerophilic conditions has been shown. Microaerophilic cultivation conditions that imitating the in vivo residence of bacteria have been prioritized.
A possibility has been found to modify properties of microorganisms by their irradiation with the ultrasound and a low-intensity EM fi eld in the UHF range, implying, in particular, the adhesive and antagonistic properties of lactobacilli towards C. diphtheriae and S. aureus test cultures.
Th ere is a dispersion dependence of the eff ect on the radiation frequency of the EM fi eld and the ultrasound. Th e exposure in the 61.0 GHz frequency band increases adhesiveness and activates the lactobacilli antagonism. For the exposure in the 42.2 GHz band, the adhesion is suppressed, and the antagonistic abilities weaken. Th e ultrasound exposure in the 18.0 and 60.0 kHz bands, leads to the suppression of the adhesive properties. A comparison of the two physical factors, i.e. the EM fi eld and the ultrasound, shows that the electromagnetic fi eld exposure exerts more eff ect.
Individual features of diff erent pathogenic strains have been studied to fi nd out that the C. diphtheriae test cultures are more susceptible to the irradiated antagonistic strains than those of S. aureus ones.