Influence of antibacterial agents on vaccine strains of Anthrax

State Centre of Innovation Biotechnologies, Kyev, National University of Life and Environmental Sciences of Ukraine State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise, Kyiv, Ukraine Institute of Veterinary Medicine The National Academy of Agrarian Sciences of Ukraine, Kyev, Ukraine Corresponding author E-mail: ulyanakuzyk@ukr.net Received: 14.09.2020. Accepted: 14.10.2020


Introduction
Anthrax -an acute zoonotic infectious disease that most often affects herbivores, it is a potential problem for the veterinary and health care system (Counotte et al., 2016). The pathogen is transmitted to humans by contact with sick animals and products of animal origin (Dixon et al., 1999, Hanna, 1998. Bacillus anthracis is the causative agent of anthrax, a gram-positive bacterium that forms spores. Under favorable and extreme environmental conditions, for a long period of time, anthrax spores remain viable for more than a hundred years (Shadomy et al., 2016;Hatami et al., 2010Hatami et al., , 2015Cizauskas et al., 2014). Infection of humans occurs through spores that affect the skin, respiratory system and gastrointestinal tract. Infection through inhalation of anthrax spores without timely treatment is the most lethal to humans, this feature of infection with the pathogen can be used as a biological weapon (Owen et al., 2015;Swartz, 2002;Keim et al., 2000). Inactivation of anthrax spores takes place in two hours with the use of 1% formalin solution. In a few hours, a 0.5% solution of carbolic acid inactivates bacteria. The following solutions have a detrimental effect on the pathogen: formaldehyde (4%), caustic soda (10%), sulfuric carbolic acid (10%), chlorinated lime (containing at least 5% active chlorine). The vegetative form of the anthrax pathogen is unstable to disinfectants (Prokopishyn, 2015). Veterinary facilities are periodically treated against pathogenic microflora with solutions of chemically active compounds. To neutralize the pathogenic microflora use 2-4% solutions of formalin, sodium hydroxide and chlorine-containing drugs (chlorinated lime, chloramine, etc.). Solutions of these disinfectants adversely affect animal and human health and the quality of livestock products. (Yashchenko et al., 2009). The most common are disinfectants developed on the basis of Quaternary ammonium compounds (QAС), aldehydes, phenols and alcohols, as well as oxygen-containing drugs. The antimicrobial spectrum of action of disinfectants can be enhanced by the creation of complex drugs, which include several chemical agents. Manufacturers of disinfectants and antiseptics test drugs on collection strains, which may differ in their properties from field isolates, which can lead to errors in the choice of concentrations and exposure time with disinfectant. We observe the disinfecting effect of hydrogen peroxide and lysoformin on gram-negative bacteria that contaminate food production (Furtat et al., 2004). Spore-forming pathogenic microflora is many times treated with aqueous solutions of disinfectants in industrial enterprises. With repeated use of disinfectants, microorganisms may lose sensitivity to them (Yashchenko et al., 2009). Today, antibacterial agents when used uncontrolled can reduce the sensitivity of microorganisms and as a result cause severe complications. Researchers point to a gradual decrease in the sensitivity of microorganisms to bactericidal agents, while the environment also affects the genetic characteristics of the strains (Cavallo et al., 2002;Salmanov & Mariievskyi, 2011). Of considerable interest is the combined use of antibiotics with antibacterial drugs of plant origin. The use of antimicrobial agents of plant origin is due to their low toxicity, the possibility of long-term use, greater availability and ability to biodegradation, while synthetic drugs: antibiotics, fluoroquinolones, antiseptics have strong activity. The advantages of herbal remedies also include the absence of dysbacteriosis and allergic reactions (Zatylnikova et al., 2010). Plant extracts that are active against strains of microorganisms insensitive to certain antibiotics and synthetic drugs are promising objects of study. Chlorophyllipt according to the PBX classification belongs to disinfectants and antiseptics. A feature of Chlorophylliptum is its active bactericidal action against antibiotic-resistant staphylococci as well as gram-positive bacteria. Its positive effect on the pathogenetic mechanism of inflammation is confirmed (Khadzhyeva et al., 2010;Zylfykarov, 2008). High molecular weight chlorophyll compounds overcome the cell wall of Staphylococcus spp. and vegetative cells of bacteria of the genus Bacillus (Olefir et al., 2015).
Propolis exhibits significant biological activity against pathogenic microflora. This is a unique natural complex -a product of processing resinous substances collected from plants by bees. The use of propolis harmoniously affects the normalization of the body, activates internal self-rehabilitation, restores metabolic disorders, slows down the process of tumors. It does not show toxic effects after long-term use. It is established that the main antibacterial factors of propolis are fatty acids (oleic, palmitic, stearic, linoleic), as well as bioflavonoids, which inhibits the growth of a wide range of bacteria that have pathogenic properties and fungi of the genus Сandida. Propolis solutions inhibit the viability of streptococci (S. sobrinus, S. mutans, S. cricetus). Antimicrobial activity of propolis against strains of B. cereus and S. aureus is registered (Karomatov, 2014;Gunar et al., 2015). The most common antiseptics for surface and hand disinfection are alcohol and sterility. 70% ethyl alcohol has the greatest antimicrobial effect on pathogenic microflora, it has the ability to enhance the properties of other antiseptics. The antimicrobial mechanism is associated with both the dehydrating properties of alcohol and its ability to denature protein (Chekman et al., 2016). Vegetative forms of gram-positive and gram-negative bacteria are sensitive to the action of alcohols. Bacterial spores, fungi and simple viruses are insensitive to alcohols. Cases are described when alcohol solutions served as a factor in the transmission of clostridia spores in anaerobic gas infection (Danyleichenko et al., 2017). Modern combined products based on alcohols -Sterillium, Octeniderm, octenisept and others are widely used in veterinary medicine and medicine. General purpose disinfectants cannot be fully effective and safe. Therefore, only complex disinfectants that have a wide spectrum of antimicrobial action can be promising disinfectants. (Horzheiev, 2013).

Materials and Methods
Vaccine strains of B. anthracis K-79Z, B. anthracis Sterne 34F2 and anthrax-like bacilli B. cereus 8035 were tested according to current documents (Yakubchak et al., 2005). The strains were grown on meat peptone broth at 37°C for 48 hours. To obtain the spore material, the culture was incubated on MPA at a temperature of 37°C for 14 days. The concentration of spore biomass of experimental crops ranged from 30.0 to 35.0 × 106 colony-forming units (CFU), namely: B. anthracis Sterne 34F2 Sterne -30.0 × 10 6 CFU, B. anthracis K-79Z -35, 0 × 10 6 CFU, B. cereus 8035 -32.0 × 10 6 CFU. Determined the bactericidal effect of disinfectants: propolis tincture (Tinctura propolisi), chlorophyllipt alcohol solution (Chlorophyllipt alcohol solution), sterillium classic pur. For each experimental strain, the bactericidal action of the disinfectant was determined in the native and diluted state. To determine the bactericidal effect of disinfectants on spore cultures in the first tube to the suspension of microorganisms with a pipette was added a native disinfectant solution in the amount of 1.0 cm 3 . A disinfectant solution in dilution (50/50 with sterile 0.9% NaCL) in the amount of 1.0 cm 3 was pipetted into the second tube with the spore suspension of experimental cultures. The exposure was maintained for 30, 60, 120 minutes and 24 hours, after which the bactericidal loop was inoculated from each tube on the МPA, followed by cultivation at 37°C for 48 hours. After the end of the incubation period, the presence of the pathogen was determined by visual assessment of the growth of colonies on MPA. Each result was photographed, below is the effect of disinfectants (propolis, Sterillium, chlorophyllipt, 96% ethanol) at different exposure times (30, 60, 120 min) on the culture under study.

Results and Discussion
In control samples, without the addition of disinfectants, in crops on MPA, after 24 hours of incubation at 37 °C, a well-defined growth of strains B. cereus 8035 (Fig. 1a), B. anthracis 34F2 Sterne (Figure 2a) and B. anthracis K-79Z (Fig. 3a) Effects of chlorophyllipt in a native and diluted state; after 30 min, the growth of B. anthracis Sterne 34F2 culture was observed (Fig. 2b). Active disinfecting effect on microbes B. anthracis Sterne 34F2 for 60 minutes exposure revealed sterilium (Fig. 2d). Microbial growth after incubation at 37 °C for 24 hours was absent. Under the action of ethanol 96%, during 120 min of exposure, the growth of strain B. anthracis 34 F2 Sterne was not observed (Figure 2e). Figure 3b shows that the effect of propolis, in the native and diluted state on the culture of B. anthracis K-79Z, for 30 min of exposure is not effective and there was growth of the strain. There is also no effect of chlorophyll on B. anthracis K-79Z. Chlorphyllipt in the native and diluted state did not show a disinfectant effect on B. anthracis K-79Z and after 30 minutes of exposure to this drug -the growth of the experimental culture is registered (Fig. 3c). Exposure to 30 minutes of sterilium was not effective against B. anthracis K-79Z, recorded growth of bacilli (Figure 3d). After incubation at 37°C for 24 hours, the growth of the strain was active. Study of the effect of ethanol 96%, after 30 min. exposure also showed the absence of disinfecting effects. After 24 hours of incubation of the inoculations under the influence of ethanol 96%, the growth of B. anthracis K-79Z culture was established (Fig. 3e). When using a solution of chlorophyllipt and propolis at exposures of 30, 60, 120 minutes and 24 hours, there was a clear growth of all experimental cultures (Table 1). After application of ethanol 96% and sterilium in the native and diluted state relative to the strain B. anthracis Sterne 34F2, even at minimal exposure, the growth of the culture was not observed, which indicates the bactericidal effect of the

Conclusion
Studies have shown that bacteria of the strain B. cereus 8035 were resistant to disinfectants of synthetic origin in the native and diluted state at exposures of 30, 60, 120 minutes and 24 hours of incubation, as evidenced by the intensive growth of the culture on МPA. In the study of the disinfecting effect of bactericidal agents of ethanol 96% and sterillium on the B. anthracis K-79Z, it was found that after exposure of 30, 60, 120 minutes and 24 hours, growth was noted when using the native and diluted agent. Strains B. anthracis K-79Z and B. cereus 8035 were found to be more resistant to disinfectants of plant and nonplant origin. In the future, the continuation of studies of strains of the causative agent of anthrax and anthrax-like bacilli on sensitivity to disinfectants and the relationship between the toxigenic activity of strains.