Bulletin "Veterinary biotechnology"

Veterynarna biotekhnolohiia– Veterinary biotechnology, 2023, 42, 98-106 [in Ukrainian]. https://doi.org/10.31073/vet_biotech42-11

TARASOV O.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., BEZYMENNYI M.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.ZAHAROVA O.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS



Introduction. It is known that bacteria of different species form spores that can remain dormant for a long time in the absence of nutrients, but under certain conditions, spores can quickly germinate and form vegetative forms (1-4). According to official data, during the period from 1920 to 1970, the largest number of problematic areas regarding anthrax were recorded in Vinnytsia (396), Kharkiv (338), Chernihiv (336), Ternopil (332), Luhansk (275), Poltava (274), and Kirovohrad (267) regions [5-6]. As of the end of 2021, according to official data, more than 13,000 problematic settlements regarding anthrax have been registered in Ukraine. Infection with anthrax mainly occurs through the soil. Infected chernozem and gray forest soils pose a particular danger. In the widespread chernozem typical of Ukraine, spore germination occurs within the first 24 hours, followed by the multiplication of the pathogen's vegetative forms .

Unlike bacterial spores, which are difficult to destroy, vegetative forms of bacteria do not have high resistance and can be relatively easily destroyed by the most common disinfectants (9-12).

Studying the influence of germinants on the germination of bacterial spores is a relevant task, the accomplishment of which will significantly facilitate the decontamination of substrates of various origins from spores of bacterial pathogens causing infectious diseases in humans and animals.

The goal of the work was to study the effect of germinant dodecylamine on the spores of bacteria of the genus Bacillus germination in experiments in vitro

Materials and methods. We used some strains of representatives of the genus Bacillus, which are kept in the Museum of the Laboratory of Zoonotic Infections and Risk Assessment: the non-pathogenic vaccine strain of B. anthracis (UA 07) and B. cereus (ATCC 10702). Spore concentration was quantified by direct counting of colonies on nutrient meat peptone agar (MPA) (HiMedia, India), which are formed by spore germination. Also, to evaluate spore germination, a SF-42 spectrophotometer was used, which determined the density at a wavelength of λ=600 nm. The value of 1 optical unit of density (OD) of the spore suspension corresponded to a concentration of 5×107 CFU.

Germination was induced by adding a germinant composed of 8.0 mM decylamine (1.48 g) to 1 liter of a buffer solution (10 mM Tris-HCl buffer, pH 7.5, with 10 mM NaCl and 0.1% Tween-80), and changes in optical density were measured every 15 minutes for 2 hours and expressed as a percentage of the starting optical density value. At the same time, a suspension was plated on nutrient meat-peptone agar (MPA) (HiMedia, India).

The research was conducted at different temperatures provided by the design of the experiment to simulate the average temperatures that the soil can acquire during the warm season (spring-summer): 8°С, 15°С, 25°С, 35°С.

Results of research and discussion. At a temperature of 8°C for 60 minutes, a sweet growth dynamic of spores is observed with the addition of germinant (reducing the optical density OD600 by 21-30%). In the absence of germinant control, growth did not exceed 7%. At a temperature of 15°C with the use of germinant, a decrease in optical density by 41-46% was observed, while in control, these values reached only 11-20%. The highest spore growth dynamic was registered at temperatures of 25°C and 35°C, during which all viable spores sprouted within the first 40 minutes, accompanied by a decrease in optical density to maximum values of 21-30% of initial OD600 values. In control variants without germinant addition, spore growth occurred much slower and in fewer numbers, so the initial optical density decreased to 63-55% of initial values (Р≤0.05).

It has been established that with increasing temperature, the time from the beginning of spore germination to complete germination of viable spores significantly decreases (from 35.0 ± 3.6 min at 8°C to 11.3 ± 2.7 min at 35°C). It should be noted that at temperatures from 8°C to 15°C, only a portion of the spores germinate, while at temperatures from 25°C to 35°C, almost all viable spores undergo germination.

Conclusions and prospects for further research.The research findings indicate that the use of 0.8 mM solution of dodecylamine affects the growth of Bacillus spores in vitro, with most inoculated spores germinating and forming vegetative bacterial forms within the first hour of incubation, accompanied by a decrease in optical density of the suspension by 61.7 ± 3.2% (B. anthracis) and 68.3 ± 4.6% (B. cereus).

It has also been established that the optimal temperature for spore germination using the germinant is within the range of 25-35°C, with the interval from the beginning of germination to complete spore germination ranging from 38.8 ± 1.9 to 11.3 ± 2.7 minutes.

The importance of these findings for veterinary medicine lies in the development of new approaches to ensure rapid spore germination in different substrates, which can be used to improve the process of decontamination of soils from the spores of the Siberian plague agent on the territory of Ukraine.

Keywords: germinant, bacteria of the genus Bacillus, B. cereus, B. anthracis, spores.


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