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Ukrainian Journal of Ecology
Ukrainian Journal ofEcology, 2020,10(3), 12-18, doi: 10.15421/2020_126
RESEARCH ARTICLE
The sensitivity of the strains of the pathogen B. anthracis and
Anthrax-like Bacilli for Antibiotics
H.A. Zaviryukha1, T.B. Vasylieva1, U.M. Yanenko1*, V.A. Synytsyn1, N.I. Kosyanchuk2, N. H. Sorokina2, O.L. Kravtsova3, L.M. Muzykina4, O.O. Marchuk3
1 State Centre of Innovation Biotechnologies, Kyev, 2National University of Life and Environmental Sciences of Ukraine 3Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise, Kyev, Ukraine 4Institute of Veterinary Medicine the National Academy of Agrarian Sciences of Ukraine, Kyev, Ukraine
Corresponding author E-mail: u!vanakuzyk@ukr. net
Received: 10.06.2020. Accepted: 10.07.2020
An important test is the study of sensitivity to antibiotics in certain strains, which is created for the anti-anthrax drug as well as the direction of antibiotic therapy in the treatment of the disease. According to the order N 23 from 04.04.02, on the territory of Ukraine it is forbidden to use drugs which contain the following active substances: nitrofurans, including furazolidone, ronidazole, dapsone, chloramphenicol (chloramphenicol), dimetridazole, colchicine, chlorpromazine, chloroform, metronidazole, due to their harmful effects on the body, in particular mutagenic and teratogenic effects. That is why the introduction of antibiotic therapy to avoid the development of resistance to the pathogen B. anthracis is relevant. The research was conducted on the basis of the State Center for Innovative Biotechnology: B. anthracoides 67, B. subtllis 17, B. cereus 8035, B. anthracis Tsenkovsky II IBM 92 Z, B. anthracss K-79 Z, B. anthracis Stern 34F2, B. anthracis 55, B. anthracis SB, B. anthracis Tsenkovsky I. We determined the sensitivity to the antibiotic by the size of the diameter of the zones of growth retardation of the strain on nutrient agar. We considered the growth retardation of the strain up to 15 mm as a sign of low sensitivity to the antibiotic, a sign of regulatory sensitivity was considered a zone of 15-24 mm, the zone of more than 24 mm - a sign of high sensitivity of the strain to the antibiotic. In the absence of zones of growth retardation of the microorganism, the strain was insensitive to the antibiotic. According to the results of research it is established that benzylpenicillin does not affect the growth of strains B. anthracoides 67, B. subtllis 17, B. cereus8035, B. anthracis Tsenkovsky II IBM 92 Z, B. anthracis 55, B. anthracis SB, B. anthracis Tsenkovsky I. Benzylpenicillin has a weak effect on strains of B. anthracis K-79 Z and B. anthracis Stern 34F2. Vaccine strains of B. anthracis K-79 Z, B. anthracis Stern 34F2, B. anthracis55, B. anthracis SB showed high sensitivity to amoxiclav, tetracycline, norfloxacin. The vaccine strain of B. anthracis Stern 34F2 showed high sensitivity to furazalidone. We plan to continue the study of antibiotic susceptibility of B. anthracis strains in the future, in order to prevent antibiotic resistance in the treatment and prevention of the disease.
Keywords: B. anthracis, Sensitivity; Resistance; Antibiotics_
Introduction
B. anthracis is an acute anthropozoonotic disease that primarily affects herbivorous mammals caused by Bacillus anthracis spores. The disease primarily affects domestic and wild herbivores. This disease is widespread throughout the planet and remains enzootic in many regions of Russia and the world, especially in sub-Saharan Africa, Asia and Central and South America. People are most often infected by infected animals or their products such as meat, animal skins, bones (Shadomy et al., 2016). B. anthracis disease is often associated with agriculture, the disease most often affects young and middle-aged people. Mortality is rare with appropriate therapy, but in the absence of treatment, the mortality rate can be up to 20% (Siddiqui, M. et al., 2012).
Resistance of pathogen spores to environmental factors, long-term preservation of biological qualities (more than a hundred years), the presence of lethal, fast-acting toxin created the conditions for the use of B. anthracis as a lethal weapon (Bengis & Frean, 2014, Ahmadi et al., 2015, Baillie, 2005). Anthrax spores are highly resistant to external influences and remain viable under the most adverse external conditions (Hugh-Jones & Blackburn, 2009). Studies of spore-forming bacteria have shown resistance to chemicals and high temperatures. We found that resistance to chemical disinfectants and temperature factors can vary both in different species of microorganisms and between strains of the same species (Melnikova & Panin, 1980).
There are 2,000 to 20,000 cases of Anthrax worldwide each year (Waits et al., 2018; Logvin et al., 2017). There is a need for a rapid and effective response to anthrax outbreaks, given the risk of anthropozoonotic infection. The main means of prevention and control of anthrax is the vaccination of animals and in case of infection - the use of antibiotics (InstruktsIya pro zahodi z profHaktiki ta borotbiz sibIrkoyu tvarin) Instructions on measures to prevent and control anthrax in animals, 2000).
In Ukraine, spore vaccines from Sterne strains, K79Z, UA-07 are used for prophylactic and forced vaccinations of animals against anthrax (Skripnik & Kovalenko, 2011). Modern anthrax vaccines, made from spores of vaccine strains, are spore suspensions preserved with 30% glycerol with or without the addition of appropriate adjuvants. The reliability of such vaccines and the duration of post-vaccination immunity depends primarily on the quality of the anthrax strain used to make the vaccine. An important step in the study of such strains is to determine the sensitivity to antibiotics.
Recently, the problem of antibiotic resistance has become widespread. Irrational use of antibacterial drugs can lead to the emergence of antibiotic-resistant strains in naturally occurring disease (Cavallo et al., 2002). Determination of antibiotic susceptibility is one of the priority areas of research on anthrax and Anthrax-type bacilli strains. The antibiotic is used for treatment in the first hours after contact with Anthrax, despite reports of penicillin resistance in some strains (Mohammed et al., 2002). Therefore, the study of sensitivity to antibiotics is an important test for identifying strains for the design of against anthrax drugs and the direction of antibiotic therapy in the treatment of the disease.
Materials and Methods
The research was conducted on the basis of the State Center for Innovative Biotechnology. Strains of anthrax and anthrax-like bacilli from the collection of microorganisms of the State Center for Innovative Biotechnology were taken for experiments. To assess the sensitivity of the experimental strains were sown on nutrient agar in the amount of: B. anthracoides67 - 5x106 CFU, BB. subtiiis 17 -3 x 106 CFU, B. cereus 8035 -3 x 106 CFU, B. anthracis Tsenkovsky II IBM 92 Z -2 x 106 CFU, B. anthracis K-79 Z - 3 x 106 CFU, B. anthracis Stern 34F2 -2 x 106 CFU, B. anthracis 55 -2 x 106 CFU, B. anthracis SB -5 x 106 CFU, B. anthracis Tsenkovsky I -3 x 106 CFU. We poured 25 cm3 of medium into a cup with a diameter of 100 mm, the thickness of the agar in the cups was not less than 4 mm. We left the cups in the box at room temperature to solidify the agar. Before inoculation of bacterial masses, the plates were dried in a thermostat at a temperature of 35°C with the lid open for 10-20 min, to prevent the formation of condensate. For the study were used standard paper discs with a diameter of 7 mm, saturated with antibiotics, manufactured by Laboratories Pvt. Limited Mombay - 400086 India. Cups with seeding strains and applications of disks in the inverted state were cultured in a thermostat for 24 hours at a temperature of 37°. After incubation, the plates were viewed against a dark background. The diameters of growth retardation of the studied strains of anthrax and anthrax-like bacilli were divided into: weakly sensitive to antibiotics - a zone of growth retardation of 1-15 mm; normatively sensitive - with a growth retardation of 15-24 mm; highly sensitive - with a growth retardation of more than 24 mm. That strain was considered insensitive to antibiotics when growth retardation in the area of application of the disc was absent.
Results and Discussion
The results for determining the sensitivity of vaccine strains of B. anthracis and anthrax-like bacilli are presented in Table 1. Table 1. Susceptibility of anthrax strains and anthrax-like bacilli to antibiotics.
№
Antibiotics
(A
J* (A
TJ
n
0 n
01 it
re
t o
The name of the strain
<N
vo
n
I «
§ CO
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•SS
1
in f)
0 00
1
CO
N
-1 2 hH
i *
M .¡2
g
I «
§ CO
>
o
n
e s
<t f)
I *
I «
§ CO
in
in
*
I «
§ CO
CO
V) *
I «
§ CO
s
o
J*
n
e s
1 Benzylpenicillin 100 P100 - - - - 8 11 - - -
2 Amoxiclav 10 AC10 15 - 7 - 34 41 30 24 8
3 Streptomycin 10 S10 17 10 7 14 17 22 22 15 18
4 Erythromycin 10 E10 25 18 19 15 18 24 24 17 15
5 Ceftriaxone 10 Ci10 8 9 9 - 8 8 7 10 -
6 Tetracycline 10 T10 29 25 21 15 26 35 30 24 19
7 Norfloxacin 10 Nx10 29 17 18 22 25 30 27 25 19
8 Niroxalin 30 No30 24 18 15 11 15 20 14 18 7
9 Furazalidone 50 Fr50 21 15 12 18 24 30 22 20 12
10 Chloramphenicol 30 ^eB 30 22 21 18 20 25 18 18 14
The results of the effect of antibacterial drugs on anthrax-like bacilli showed that B. anthracoides 67 is sensitive to most antibiotics taken in the experiment. Diameters of growth retardation are in the range from 8 to 20 mm. B. anthracoides was insensitive to benzylpenicillin and weakly sensitive to ceftriaxone. When comparing the zones of growth retardation, it was found that B. anthracoides is sensitive to 40% of antibiotics (amoxiclav, streptomycin, furazalidone, niroxalin) (Figure 1).
■ no growth retardation
■ from 1 to 15 mm
Figure 1. Antibiotic sensitivity of B. anthrax-like strain 67.
B. anthracoides 67 is highly sensitive to erythromycin, tetracycline, norfloxacin and chloramphenicol. Strain B. subtilis 17 was insensitive to 20% of antibiotics (benzylpenicillin and amoxiclav) (Figure 2).
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 2. Antibiotic sensitivity of B. Subtilis 17.
Weak growth of B. subtiiis 17 to streptomycin and ceftriaxone is established in the zones of growth retardation. The strain was sensitive to up to 50% of the studied antibiotics - niroxalin, tetracycline, erythromycin, streptomycin and chloramphenicol. B. subtilis 17 showed high sensitivity to tetracycline.
The sensitivity of B. cereus 8035 to antibacterial drugs is shown in Figure 3.
Strain B. cereus8035 was insensitive to benzylpenicillin, as well as insensitive to 40% of the studied antibiotics (amoxiclav, streptomycin, ceftriaxone, furazalidone). B. cereus8035 showed sensitivity to 50% of antibiotics (erythromycin, norfloxacin, tetracycline, chloramphenicol, niroxalin).
Analysis of the effect of antibacterial drugs on vaccine cultures of anthrax shows that the strain of B. anthracis Tsenkovsky II is insensitive to benzylpenicillin and amoxiclav and weakly sensitive to niroxalin and streptomycin (Figure 4).
The pathogenic strain of B. anthracis Tsenkovsky II showed sensitivity to 50% of the studied antibiotics (niroxalin, tetracycline, erythromycin, streptomycin, chloramphenicol). He was not sensitive to antibiotics such as benzylpenicillin, amoxiclav, ceftriaxone. A study of the vaccine strain of B. anthracis K-79 Z showed that the strain showed weak sensitivity to benzylpecillin and ceftriaxone (Figure 5).
This strain is sensitive to 40% of the studied antibiotics - streptomycin, erythromycin, niroxalin, chloramphenicol, however, B. anthracis K-79 Z showed high sensitivity to amoxiclav, tetracycline, norfloxacin, furazalidone, which also accounted for 40% of the experimental drugs. Insensitivity to the studied antibiotics has not been established.
The antibiotic susceptibility study of B. anthracis strain Stern 34F2 showed that it is sensitive to all studied antibiotics (Figure 6). It is weakly sensitive to ceftriaxone and benzylpenicillin (20%). He has shown sensitivity to streptomycin and niroxalin (20%). High sensitivity in B. anthracis Stern 34F2 was detected to chloramphenicol, furazalidone, norfloxacin, erythromycin, tetracycline, amoxiclav (60%) (Figure 6).
no growth retardation from 1 to 15 mm from 15 to 24 mm I more 24 mm
Figure 3. Antibiotic sensitivity of B. cereus8035.
0% ^
r 30%
50%
[ I
L 20%
no growth retardation from 1 to 15 mm from 15 to 24 mm more 24 mm
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 5. Antibiotic sensitivity of B. anthracis K-79 Z.
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 6. Antibiotic sensitivity of B. anthracis Stern 34F2.
A study of the strain B. anthracis 55 showed that the strain is not sensitive to benzylpenicillin (Figure 7). It is weakly sensitive to ceftriaxone and niroxalin and sensitive to streptomycin, furazalidone and chloramphenicol (30%). This strain showed high sensitivity to amoxiclav, erythromycin, tetracycline and norfloxacin (40%).
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 7. Antibiotic sensitivity of B. anthracis 55.
The vaccine strain of B. anthracis SB was insensitive to benzylpenicillin (Figure 8).
This strain showed low sensitivity to ceftriaxone. B. anthracis SB is sensitive to streptomycin, erythromycin, niroxalin and chloramphenicol (50%). Also up to 30% of antibiotics were highly sensitive - amoxiclav, tetracycline and norfloxacin. According to the results of research, it was found that the strain of B. anthracis Tsenkovsky I was insensitive to 20% of antibiotics (benzylpenicillin and ceftriaxone) (Figure 9).
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 8. Antibiotic sensitivity of B. anthracis CB.
■ no growth retardation
■ from 1 to 15 mm
■ from 15 to 24 mm
■ more 24 mm
Figure 9. Antibiotic sensitivity of B. anthracisTsenkovsky I.
B. anthracis Tsenkovsky I showed low sensitivity to chloramphenicol, furazalidone, niroxalin and amoxiclav and is sensitive to 40% of antibiotics: erythromycin, streptomycin, tetracycline and norfloxacin.
According to the order N 23 from 04.04.02, on the territory of Ukraine it is forbidden to use drugs which contain the following active substances: nitrofurans, including furazolidone, ronidazole, dapsone, chloramphenicol (chloramphenicol), dimetridazole, colchicine, chlorpromazine, chloroform, metronidazole, due to their harmful effects on the body, including mutagenic and teratogenic effects. A significant part of the experimental antibacterial drugs that had a positive effect on vaccine strains of anthrax and anthrax-like bacilli belong to the above list (chloramphenicol, furazolidone, norfloxacin). Their long-term use does not cause resistance in the experimental pathogen. It is this ban on their use that is the reason for our search for new effective antibiotics for anthrax.
Conclusion
According to the results of research it is established that benzylpenicillin does not affect the growth of experimental strains of anthrax-like bacilli and anthrax, namely: B. anthracoides 67, B. subttilis 17, B. cereus 8035, B. anthracis Tsenkovsky II IBM 92 Z, B. anthracis 55, B. anthracis SB, B. anthracis Tsenkovsky I. Benzylpenicillin has a weak effect on strains of B. anthracis K-79 Z and B. anthracis Stern 34F2. Vaccine strains of B. anthracis K-79 Z, B. anthracis Stern 34F2, B. anthracis 55, B. anthracis SB showed high sensitivity to amoxiclav, tetracycline, norfloxacin. The vaccine strain of B. anthracis Stern 34F2 showed high sensitivity to furazalidone. We are currently working on our prospects for further antibiotic susceptibility studies of anthrax strains, in order to prevent antibiotic resistance in treatment and prevention of the disease.
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Citation:
Zaviryukha, H.A., Vasylieva, T.B., Yanenko, U.M., Synytsyn, V.A., Kosyanchuk, N.I., Sorokina, N.H., Kravtsova, O.L., Muzykina, L.M., Marchuk, O.O. (2020). The sensitivity of the strains of the pathogen B. anthracis and Anthrax-like Bacilli for Antibiotics. Ukrainian Journal of Ecology, 10(3), 12-18.
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