Study of disinfectant activity against bee pathogenic enterobacteria in vitro

Dysbiosis is a pathological condition caused by violating the normal coexistence of bacterial organisms in one or more body systems. This disease arises due to the action of many ethological factors, the mechanisms of which arise from each other. Klebsiellosis of bee colonies is a relatively new disease of Apis mellifera , caused by pathogenic enterobacteria, which leads to the mass death of bees with signs of acute dyspepsia, particularly in the spring and autumn periods. To prevent klebsiellosis in bees, it is necessary to carry out veterinary and sanitary measures in apiaries every year, including disinfection. Modern beekeeping encourages the search for new antibacterial drugs to ensure the production technology of safe and high-quality beekeeping products suitable for sale. Preliminary laboratory assessment ( in vitro ) of the effectiveness of disinfectants against specific pathogens of specific diseases of bees will ensure an increase in the effectiveness of such agents in apiaries, which is economically effective for practical beekeeping. The purpose of the research was to determine the activity of different concentrations of the disinfectant (0.25 %, 0.5 %, 1.25 %, 2.5 %) with an active substance of an organic nature with covalent weakly polar bonds against pure cultures of pathogenic enterobacteria of bees of the species Klebsiella pneumoniae and Klebsiella (Enterobacter) aerogenes in vitro . The modified Kirby-Baur method on Mueller-Hinton agar was used for research. A bactericidal effect against a pure culture of Klebsiella pneumoniae species was registered after 24 hours with lysis zone diameters at 8.6 ± 0.27 mm (0.25 % concentration) – 15.0 ± 0.35 mm (2.5 % concentration). On the 3rd day of research, a bacteriostatic effect was registered with the largest diameter of the zone of growth retardation (24.4 ± 0.27 mm) at 2.5 % disinfectant concentration. Bactericidal and bacterio-static effects were recorded on the culture of enterobacteria of bees of the species Klebsiella (Enterobacter) aero-genes for 24 hours of cultivation with zones of lysis – 6.8 ± 0.42 mm (0.25 %) – 11.8 ± 0.22 mm (2.5 %) and inhibition of bacterial growth at the level of – 21.8 ± 0.42 mm (0.25 %) – 25.8 ± 0.42 mm (2.5 %). At the same time, there was no significant difference between the zones of Klebsiella (Enterobacter) aerogenes growth retardation in concentrations of 0.5 % – 2.5 % on the 3rd day of research. The studied disinfectant has bactericidal and bacteriostatic effects on pure cultures of bee enterobacteria of the species Klebsiella pneumoniae and Klebsiella (Enterobacter) aerogenes in laboratory conditions, which is promising for further study of the drug.


Introduction
The mass death of bees in the winter-spring period is one of the problems of world beekeeping (Zhu et al., 2020;Becsi et al., 2021).Such seasonality is due to a significant decrease in the resistance of the insect organism, which in turn directly affects the level of immunity.One of the etiological factors that cause dysbiosis in bee colonies is the accumulation of opportunistic microorganisms in the hive during wintering, which, in turn, affects the sensitivity of the bee organism (Yang et al., 2019;Galatiuk et al., 2020;Zhu et al., 2020).Subsequently, the microflora balance in the midgut of Apis mellifera is disturbed (Wang et al., 2021).Also, the appearance of an increased number of opportunistic bacteria in the apiary reduces the resistance of individual bee families (Braglia et al., 2021;Wang et al., 2021).Among the most common clinical manifestations of bee dysbiosis are acute dyspeptic disorders in the form of diarrhea, which leads to dehydration of the body and loss of the ability to fly, general weakness of bee colonies, and loss of productivity, which leads to economic losses for apiary owners (Meng et al., 2020;Lakhman et al., 2021).
Modern realities of the introduction of beekeeping and growing requirements for the quality of products obtained with the help of this industry encourage the search for new effective disinfectants (Qi et al., 2020).The purpose of conducting veterinary and sanitary measures in apiaries is to inactivate pathogens of infectious diseases.After all, inventory, combs, and the hives themselves can be potential factors in their transmission.Such measures make it possible to interrupt the transmission mechanism of viral and bacterial infectious agents.One of the main requirements for disinfectants is their harmlessness to the environment (Li et al., 2022).Therefore, searching for environmentally safe alter-Citation: Romanishina Т. A., Lakhman A. R., Galatiuk O. Ye., Behas V. L., & Zastulka M. V. (2024).Study of disinfectant activity against bee pathogenic enterobacteria in vitro.Ukrainian Journal of Veterinary and Agricultural Sciences,7(1),[41][42][43][44][45] natives in the field of disinfection is vital for ensuring the safety and quality of beekeeping products and for their successful implementation (Verwold et al., 2021).However, the market of effective drugs for the treatment and prevention of bee colony dysbiosis is minimal and narrow (Yang et al., 2019).It is essential to conduct a preliminary assessment of the effectiveness of environmentally safe, new, modern means against specific pathogens of specific bee diseases in laboratory conditions (in vitro) (Kim et al., 2020;Qi et al., 2020;Li et al., 2022).In our opinion, such methodical approaches will ensure an increase in the efficiency of new means in apiaries, which is economically beneficial for practical beekeeping.
"Brovadez-20" is a disinfectant, where the active substance is a compound of organic nature with covalent weakly polar bonds -alkyldimethylbenzylammonium chloride.This surface active agent (surfactant) is able to exert a bactericidal effect on staphylococci, streptococci, gram-negative bacteria such as Escherichia coli, Proteus, Klebsiella, etc., as well as anaerobic bacteria and fungi (Kim et al., 2020;Denis et al., 2023;Xu et al., 2023;Duarte et al., 2024).
The purpose of the research is to investigate the activity of the drug "Brovadez-20" against pure cultures of bee pathogenic enterobacteria of the species Klebsiella pneumoniae and Klebsiella (Enterobacter) aerogenes in vitro.

Materials and methods
The experiments were carried out in the conditions of the educational laboratory of the Department of Microbiology, Pharmacology, and Veterinary Epidemiology of the Faculty of Veterinary Medicine of the Polissia National University.Pure cultures of bee enterobacteria of the species Klebsiella pneumoniae and Klebsiella (Enterobacter) aerogenes, which were isolated from apiaries of Zhytomyr, Kyiv, and Khmelnytsky regions from bees with signs of intestinal dysbiosis, were used for research.Bacterial cultures were identified with the participation of the "Zhytomyr Regional State Laboratory of the State Service of Ukraine for Food Safety and Consumer Protection" and the State Institution "Zhytomyr Regional Laboratory Center of the Ministry of Health of Ukraine" (Galatiuk et al., 2020).To determine the effect of the drug "Brovadez-20" (https://brovapharma.ua/brovadez-20_100-ml) on the abovementioned pure cultures of enterobacteria of bees in vitro, the modified Kirby-Bauer method for the beekeeping industry was used (the exposure of impregnation of disks was 30 minutes) on MHA medium (Muller-Hinton agar) (Balázs et al., 2021)."Brovadez-20" was used in the following concentrations: 0.25 %; 0.5 %; 1.25 %; 2.5 %.This drug was diluted with distilled water.The study was performed on five Petri dishes for each culture, which were observed for 72 hours.Statistical processing of the results was carried out using the Statistica software package, numerical values were characterized according to the Student's criterion.

Results and discussion
Carrying out veterinary and sanitary measures in apiaries is a primary link for its well-being (Verwold et al., 2021;Li et al., 2022).After all, disease prevention is the key to prevention before its occurrence.The effectiveness of the use of one or another means for therapeutic or prophylactic purposes is previously determined in laboratory conditions.For example, to determine the required concentration of a pharmacological agent, or to specify the required solvent for a specific and specific pathogen.That is why in vitro studies occupy an important link in testing a disinfectant or other drug.Currently, bacterial diseases of animals, including bees, namely, those that disrupt the biotic balance in the middle intestine of insects, are becoming widespread in private apiaries not only in Ukraine but also in European and American countries (Yang et al., 2019;Galatiuk et al., 2020;Meng et al., 2020).In addition to the quantitative advantage of the pathogenic microbiota in the "gut" of bees, its qualitative composition is also disturbed.Metabolites released by virulent causative agents klebsiellosis of bees suppress the physiological functioning of the entire insect organism (Lakhman et al., 2021;Wang et al., 2021).Therefore, it is important to search for means that have an effect not only on "internal" pathogens, but also that could neutralize them in the environment for the purpose of disinfection.
According to Table 1, the bactericidal effect on the bacterial test culture of the Klebsiella pneumoniae species was registered after 24 hours at the level of 8.6 ± 0.27 mm -15 ± 0.35 mm.Moreover, the zones of lysis at similar concentrations in different terms of the research did not reliably differ from each other, while maintaining a directly proportional against of changes in the lysis zone and the concentration of "Brovadez-20" (Fig. 1 A).

Table 1
Peculiarities of the interaction of "Brovadez-20" and Klebsiella pneumoniae culture on MHA medium (M ± m, n = 5) The bacteriostatic effect was observed 72 hours after the interaction of "Brovadez-20" and bacteria of the Klebsiella pneumoniae species at the level of 18.8 ± 0.42 mm (0.5 %concentration) -24.4 ± 0.27 mm (2.5 % -concentration).The largest diameter of the zone of growth retardation was registered on the third day of the experiment at a concentration of "Brovadez-20" of 2.5 % (24.4 ± 0.27 mm) (Fig. 1 B).This effect (manifest of bacteriostatic action after 72hour contact of the culture with the pharmacological drug), in our opinion, is caused by the presence of a hypermucoid capsule, which is formed due to the factor rmpA/A2, localized in the chromosomal operon cps.(Chang et al., 2019;Huang et al., 2023;Wantuch et al., 2023).Thus, bacteria of this species have a pronounced resistance to phagocytosis and the complement system in the host's body (Xu et al., 2021;Wang et al., 2023).The composition of the cps cluster locus includes conservative genetic sequences with pronounced specific functionality.Six genes are responsible for the processing and transportation of capsule componentsorf 2, galF, wzi, wza, wzb, wzc; formation and polymerization is ensured by a highly variable middle section; direct synthesis of monosaccharides is determined by the 3' site (Walker & Miller, 2020;Wang et al., 2023).Probably, "Brovadez-20" affects one of the links of the intracellular mechanism of capsule formation of young cells of Klebsiella pneumoniae, described above (Dunstan et al., 2023).The bacteriostatic effect of the drug is recorded on the third day of the experiment (Fig. 1 B).
Modern studies indicate that the synthesis of Klebsiella pneumoniae capsules is additionally influenced by such regulatory elements as KvrA/KvrB, CRP RmpA/RmpA2, and RcsAB (Walker & Miller, 2020;Wantuch et al., 2023).In addition, the large plasmid of enterobacteria contains salmochelin (iroBCDN)/aerobactin (iucABCDiutA), thanks to which microorganisms are able to maintain viability in unfavorable conditions for them (Huang et al., 2023;Wang et al., 2023).Since the mechanism of action of the main active substance "Brovadez-20" in the form of alkyldimethylbenzylammonium chloride consists in the denaturation of the protein structures of microorganisms, the activity of bacterial transferases, which enable the formation of a mucous capsule, is probably blocked.In contrast to bacteria of the species Klebsiella (Enterobacter) aerogenes, which does not have a capsule, bactericidal and bacteriostatic effects were registered already at 24 hours of cultivation (Fig. 2 A) with zones of lysis -6.8 ± 0.42 mm (0.25 %) -11.8 ± 0.22 mm (2.5 %) and inhibition of bacterial growth at the level of -21.8 ± 0.42 mm (0.25 %) -25.8 ± 0.42 mm (2.5 %) (Fig. 2 A).Moreover, the manifestations of the interaction of "Brovadez-20" with microorganisms of the species Klebsiella (Enterobacter) aerogenes, recorded by us, did not reliably differ in different periods of recording the results.
Such effects of the disinfectant "Brovadez-20" on pure test cultures of enterobacteria of bees of the species Klebsiella pneumoniae and Klebsiella (Enterobacter) aerogenes are also associated with the active substance alkyldimethylbenzylammonium chloride -a compound of organic nature with covalent weakly polar bonds.Probably, the manifestation of the bactericidal action of this surface active agent (surfactant) against microorganisms included two stages: 1. Stopping growth; 2. Destruction of cells (Denis et al., 2023;Duarte et al., 2024).The mechanism of action of the active substance "Brovadez-20" does not depend on the change in the level of the hydrogen indicator (pH), like other phenol derivatives (Tahir et al., 2021).Therefore, bactericidal and bacteriostatic effects were registered for the two test cultures in the studied concentrations.The localization of the bactericidal effect of the disinfectant directly near the disk is associated with a higher concentration in the location of the impregnated disk.That is, the bacteriostatic effect on bacteria, in our opinion, is caused by a decrease in the concentration of the drug in the agar layer and, therefore, a lower destructive effect on microorganisms.

Conclusions
The studied disinfection solution "Brovadez-20" in concentrations of 0.25 %, 0.5 %, 1.25 % and 2.5 % exhibits bacteriostatic and bactericidal effects against all studied cultures of pathogenic enterobacteria of bees on the MHA medium in vitro.

Fig. 1 .
Fig. 1.Visual changes during bacteriological studies of the activity of "Brovadez-20" against Klebsiella pneumoniae culture on MHA medium: A -manifestation of bactericidal effect on 1 day of the experiment (2.5 %); B -manifestation of bactericidal (a) and bacteriostatic (b) effects on the 3rd day of the experiment

Fig. 2 .
Fig. 2. Visual changes during bacteriological studies of the activity of "Brovadez-20" against Klebsiella (Enterobacter) aerogenes culture on MHA medium: A -manifestation of bactericidal (a) and bacteriostatic (b) effects on 1 day of the experiment; B -a manifestation of bactericidal (a) and bacteriostatic (b) effects on the 3rd day of the experiment

Table 2
Peculiarities of the interaction of "Brovadez-20" in the environment of MHA against culture Klebsiella (Enterobacter) aerogenes (M ± m, n = 5)