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https://doi.org/10.61796/jmgcb.v1i6.601
RIFIZOSTREP – NEW DRUG AGAINST
TUBERCULOSIS
Mamadullaev G.Kh
Doctor of Veterinary Sciences, senior researcher, scientific
supervisor, head of laboratory
Fayziev U.M
Independent Researcher
Received: March 22, 2024; Accepted: Apr 29, 2024; Published: Jun 12, 2024;
Abstract
:
The article presents the results of bacteriological studies of the internal organs of
guinea pigs and rabbits experimentally infected with M. bovis and M. tuberculosis with the
subsequent use of a new anti-tuberculosis drug “Rifisostrep”.
Keywords
:
Rifizostrep, Drug, Antimicrobial, Mycobacterium, M.Bovis, M.Tuberculosis,
Bacteriological, Against- Microbes' Strain, Rezistance, Sensivity.
This is an open-acces article under the
CC-BY 4.
0 license
Introduction
Tuberculosis is a chronic infectious disease found in farm animals, wild animals, ungulates
and poultry. In diseased internal organs and tissues, special nodules are formed - bumps (tubercles).
Tuberculosis is also common among people [1; 9].
Considering the extreme resistance of the pathogen to external environmental factors
(sunlight, temperature, humidity, pressure, pH, disinfectants, etc.), it is necessary to strengthen the
fight and prevention of animal tuberculosis in the republic, as well as diagnostic measures. [4;5].
According to the World Health Organization (WHO), in 2015, 10.4 million new cases of
tuberculosis were registered worldwide, of which 5.9 million (56%) were men, 3.5 million (34%)
were women, and 1. 0 million (10%) children. That same year, 1.4 million people died from
tuberculosis.
According to WHO websites, tuberculosis is currently the deadliest disease. In 2017 alone, 10
million people became infected with tuberculosis and 1.6 million people died
(Source:
https://mir24.tv).
In the modern period, many aspects of the disease have been studied and clarified by scientists
around the world in the direction of studying tuberculosis infection using the science of molecular
biology. The biology of the disease, the main epidemiological and epizootological patterns,
pathogenesis, and diagnosis have been widely studied at the molecular genetic level. At the same
time, many chemotherapy drugs are being discovered to combat this disease. In this regard, scientific
research is widely conducted in leading scientific centers and higher educational institutions of the
world [3; 6; 8].
In recent years, mono-, bi- and polyresistance of pathogenic strains to single-drug therapy for
tuberculosis has begun to increase. To prevent this problem, scientists are conducting large-scale
research to create a set of new combination anti-tuberculosis drugs. In medical physiatry, there is an
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international DOTS program (DOTS - (Directly Observed Treatment, Short-course under direct
observation). The DOTS strategy to combat tuberculosis used a combination (mixture) of 2-3 or 4
different drugs.
As a result of the implementation of the DOTS strategy, it was possible to reduce
morbidity and mortality from the disease [2;7].
Our research has been carried out in this direction: a new combined anti-tuberculosis drug
“Rifizostrep” has been created on the basis of the tuberculosis laboratory of the Veterinary Research
Institute, and a large-scale test of its antibacterial activity against the pathogen is being carried out.
The goal and objectives of our research are to determine the effectiveness of the drug “Rifizostrep”
in the div of laboratory animals against strains of M. tuberculosis and M. bovis using the
bacteriological method.
Methods
Scientific research was carried out in accordance with the methodological guidelines and
instructions “Prevention and control of animal tuberculosis”, approved by the Committee of
Veterinary Medicine and Livestock Development of the Republic of Uzbekistan (M. 1982, 1988,
Tashkent 1998, 2011).
Cultivation and storage of mycobacterial strains in the museum, examination of pathological
samples taken from experimental animals in the laboratory, brought from livestock farms were carried
out according to the instructions “Laboratory diagnosis of animal tuberculosis” (Omsk 1988), the
manual “Laboratory diagnosis of tuberculosis” and the instructions “Diagnostics of animal
tuberculosis "(Tashkent 2011), as well as on the basis of methodological manuals by T.N.
Yashchenko, I.S. Mecheva "Guide to laboratory tests for tuberculosis. - M.: Medicine, 1973" [10;11].
Bacteriological tests of a new drug against strains of M.bovis and M.tuberculosis continued
in order to test the combination of a new tuberculostatic against tuberculosis pathogens - the drug
"Rifizostrep".
Lifetime trials of the drug “Rifizostrep” against tuberculosis pathogens were carried out on
guinea pigs. 27 guinea pigs were infected with virulent strains of mycobacterium tuberculosis M.bovis
8-03, which causes disease in cattle, and M.tuberculosis 7880, which causes disease in humans,
subcutaneously at a dose of 0.03 mg/kg.
90 days after the end of the experiment, all animals in the experimental and control groups
were killed for pathological and bacteriological studies. A pathological sample taken from the internal
organs of animals in the experimental and control groups was processed according to the Ghosn-
Levenshtein-Sumiyoshi method and placed on a Levenshtein-Jensen nutrient medium; smears
prepared from a suspension of pathological samples were stained using the Sil-Nielsen method.
method and subjected to microscopy (magnification 12x100). Bacteriological examination of
pathological samples lasted 3 months.
The specific bactericidal activity of the drug “Rifisostrep” against strains of mycobacterium
tuberculosis M.bovis and M.tuberculosis was comparatively studied on 28 rabbits in comparison with
the drug isoniazid [11].
After the end of the experiment, rabbits in all experimental, control and comparative groups
were killed, the internal organs of the animals were examined for pathology, and samples were taken
from the internal organs for bacteriological examination. The resulting pathological sample was
processed according to the Ghosn-Levenshtein-Sumiyoshi method and sown on Levenshtein-Jensen
nutrient medium; smears prepared from pathological samples were stained using the Seel-Neelsen
method and subjected to microscopy. The period of bacteriological examination of pathological
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samples was 3 months.
Based on the results of a bacteriological study of pathological samples, a conclusion was
made about the level of antibacterial action of a new tuberculostatic combination of drugs - the drug
Rifizostrep against tuberculosis pathogens.
Results and Discussion
According to the study design, the results of cultural and microscopic examination of
pathological samples of guinea pigs receiving the drug Rifizostrep after tuberculosis infection are
presented in Table 1. Table 1 shows that after infection with the M. bovis strain of group 1, during
bacteriological examination of pathological samples of 9 guinea pigs that received Rifizostrep once
every 5 days, internal M. bovis was not detected in any of the 9 guinea pigs. the pathogen has not
been isolated. During bacterioscopic examination, no mycobacterial bacilli were found in any smear
stained using the Seal-Neelsen method.
From the entrails of guinea pigs infected with the M. bovis strain in group 2 as a control and
not receiving the drug, tuberculosis pathogens were found in the visceral samples cultures of all 3
guinea pigs on days 18-24 of Löwenstein-Jensen, and typical colonies were formed on the surface of
the nutrient medium. As a control, in all test tubes planted in pure form, tuberculosis colonies grew
and had the following characteristics: - growth rate - on average 18-24 days; Description of the
colonies - uneven shape, lumpy surface, dry R-colonies, cloudy single number, small dewy volume,
straight and curved shape, rough surface, dry and sticky consistency, ivory pigmented, medium
dilution of the colonies. Consistency - crumbles.
Table 1
Results of a cultural study of pathological preparations of guinea pigs receiving the drug
Rifizostrep after tuberculosis infection
№ Kind
of
animal
group
number
of heads
strain name
Name of the drug and
method of use
Bacteriological
research
1
Guinea pig,
experiment
I
9
M.bovis 8-03 Rifisostrep
parenteral
– – – – – – – –
2
Guinea pig,
control
II
3
M.bovis 8-03 Control, no drugs
+ + +
3
Guinea pig,
experiment
III
9
M.tuberculosi
s 7880
Rifisostrep
parenteral
– – – – – – – –
4
Guinea pig,
control
IV
3
M.tuberculosi
s 7880
Control, no drugs
+ + +
5
Guinea pig,
comparative
group
V
3
M.tuberculosi
s 7880
Isoniazid orally
– – +
Jami
27
Reminder: + tuberculosis detected;
- tuberculosis was not detected.
Microscopy of smears stained by the Ziel-Neelsen method of tuberculosis colonies obtained
from the food environment using a bacterial bacillus revealed the following morphological and
tinctorial features: the M. bovis strain absorbed the fuchsin dye in the smear and was colored red-
violet. Polymorphism is clearly expressed in the morphology of M. bovis bacilli. Under the
microscope, short, thick, thin-flat, pointed and somewhat thickened bacterial rods were detected.
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Cocci-like forms were also detected in some visual fields, and granules were expressed in some
senescent cells.
After infection with the M. tuberculosis 7880 strain in the 3rd group, during bacteriological
examination of pathological samples from 9 guinea pigs receiving rifisostrep once every 5 days, the
M. tuberculosis 7880 strain was not isolated. In smears stained using the Seal-Nielsen method during
bacterioscopic examination, mycobacterial bacilli were not detected.
As a control, in group 4, internal organs of guinea pigs infected with strain M. tuberculosis
7880 and not receiving the drug were grown from the internal organs of all 3 guinea pigs grown on
the surface of the medium, forming typical colonies. As a control, colonies of tubercles grew in all
tubes grown alone and had the following characteristics:
- growth rate – on average 26-30 days; Description of colonies - smooth, bumpy surface, dry
- R-colonies, single and numerous, small volume, moist color, regular shape, rough surface, dry and
sticky consistency, ivory pigmented, medium dilution of colonies. Consistency - crumbles.
Microscopy of tuberculosis colonies obtained from a food environment using a bacterial
bacillus revealed the following morphological and tinctorial characteristics: in the smear, the M.
tuberculosis 7880 strain was stained red-violet, and polymorphism was expressed in the morphology
of the bacterial bacillus. Under the microscope, long and short, thick, thin, flat-ended and some
thickened bacterial rods were found. In some fields of view, coccoid forms are also found; granules
are formed in some old cells; granules are not expressed in young cells.
After infection with the M. tuberculosis strain in the 5th group, during a bacteriological study
of pathological samples of guinea pigs from the comparative control group receiving the drug
Isoniazid, colonies of tuberculosis grew from the internal organs of one of the 3 guinea pigs; in 2, no
tuberculosis colonies were produced.
Thus, we can conclude that based on the results of a comparative study of the antibacterial
activity of the drug Rifizostrep against strains of tuberculosis in guinea pigs with the drug isoniazid,
the drug Rifizostrep has a more active antibacterial effect on tuberculosis pathogens than the drug
isoniazid. During bacterioscopy of smears stained using the Ziel-Neelsen method, tuberculosis bacilli
with typical morphological and tinctorial properties were identified from the bacterial mass formed
during cultural examination.
The specific antibacterial activity of the drug "Rifisostrep" against the M.bovis and
M.tuberculosis strains of Mycobacterium tuberculosis was studied in 28 rabbits. The activity of the
new drug against pathogens has been comparatively studied in comparison with the medical drug
isoniazid.
Table 2.
Results of a cultural study of pathological samples of rabbits treated with the drug Rifizostrep
after tuberculosis infection
№ Kind
of
animal
group
numb
er of
heads
Name of the
infected
strain
Infecti
ous
dose
Dosage
of
the
drug
Name of the
drug
and
method
of
administration
Result
1 Rabbits,
experience
I
4
4
M.bovis
8-03
0,03
mg/kg
0,5 ml
1,0 ml
Rifizostrep
parenteral
70 %
100 %
2 Rabbits,
control
II
3
M.bovis
8-03
0,03
mg/kg
Control,
no
drugs
+
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3 Rabbits,
experience
III
4
4
M.tuber
culosis 7880
0,03
mg/kg
0,5 ml
1,0 ml
Rifizostrep
parenteral
70%
90 %
4 Rabbits,
control
IV
3
M.tuber
culosis 7880
0,03
mg/kg
Control,
no
drugs
+
5 rabbits,
comparison
group
V
3
M.tuber
culosis 7880
0,03
mg/kg
10 mg/kg Isoniazid,
orally
50 %
6 rabbits,
comparison
group
VI
3
M.bovis
8-03
0,03
mg/kg
10 mg/kg Isoniazid,
orally
60 %
Total
28
Reminder: + tuberculosis detected;
- tuberculosis was not detected.
As can be seen from the results of Table 4, after infection with the M. bovis strain of group
1, during a bacteriological examination of pathological samples of rabbits that received Rifizostrep
once every 5 days, none of the 8 rabbits were found to have M. bovis isolated from their internal
organs. During bacterioscopic examination, no mycobacterial bacilli were found in any smear
stained using the Seal-Neelsen method.
As a control in the 2nd group, tuberculosis pathogens grew by forming typical colonies on the
surface of the Lowenstein-Jensen nutrient medium in culture samples from internal organ samples of
3 rabbits infected with the M. bovis strain and not receiving the drug. As a control, colonies of
tubercles grew in all tubes grown in pure form, and they had the following characteristics:
- growth rate - 26-30 days on average; colony description - uneven shape, bumpy surface, dry
R-colonies, single, small dewy volume, regular shape, rough surface, dry and sticky consistency,
ivory pigmented, colony thinning level is average. Consistency - crumbles.
Microscopy of smears of mycobacterial species stained by the Siel-Neelsen method from
tuberculosis colonies obtained from the food environment using a bacterial bacillus revealed the
following morphological and tinctorial features: the M. bovis strain was stained red-violet.
Polymorphism is expressed in the morphology of M. bovis bacilli. Under the microscope, short, thick,
thin-flat-pointed and somewhat thickened bacterial rods were discovered. In some fields of view,
cocci-like forms were also revealed; granules were expressed in the composition of old cells.
After infection with strain M. tuberculosis 7880 in the 3rd group, pathological samples from
8 rabbits that received rifizostrep once every 5 days were bacteriologically examined. No
mycobacteria bacilli were found in smears stained using the Seal-Nielsen method during
bacterioscopic examination.
As a control, in the 4th group, the M. tuberculosis strain 7880 was infected and did not receive
the drug in culture studies from samples of internal organs of rabbits; tuberculosis pathogens grew on
the surface of the Lowenstein-Jensen nutrient medium, forming typical colonies. After 26-28 days,
typical tuberculosis colonies grew rapidly in the control tubes in the form of small growths. Colonies
of pathogens formed R-colonies on the surface of the nutrient medium, pigmented ivory-colored, in
the form of a dewy form, in fragments or connected to each other. The consistency of the colonies is
dry and crumbly, diffusely scattered, some have a slightly sticky consistency.
Microscopy of tuberculosis colonies obtained from a nutrient medium using bacterial bacilli
revealed the following morphological and tinctorial features: in the smear, the M. tuberculosis 7880
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strain was stained red-violet, and polymorphism was expressed in the morphology of the bacterial
bacilli. Under the microscope, long and short, thick, thin, flat-ended and some thickened bacterial
rods were found. In some fields of view, coccus-like forms are also found; granules are formed in
some old cells; granules are not expressed in young cells.
-
During a bacteriological study of pathological samples of rabbits from a comparative control
group that received the drug Isoniazid after infection with strains of M. tuberculosis and M. bovis in
groups 5 and 6, colonies of tuberculosis were found in the internal organs of two of them. 6 rabbits
grew up in the 5th and 6th groups, 4 of them did not form colonies. Smears were prepared from the
bacterial mass formed during cultural examination and stained using the Seel-Neelsen method.
Bacterioscopy revealed tuberculosis bacilli with typical morphological and tinctorial features in
smears.
-
Thus, we can conclude that based on the results of a comparative study of the antibacterial
activity of the drug Rifizostrep against strains of tuberculosis in rabbits with the drug isoniazid, the
drug Rifizostrep has an active antibacterial effect on tuberculosis pathogens compared to the drug
isoniazid.
Conclusion
1.
Bacteriological studies have established that the drug Rifizostrep has a more active
antibacterial effect on tuberculosis pathogens compared to the drug Isoniazid;
2.
During cultural examination and bacterioscopy of smears in samples from guinea pigs of
the experimental group that received the drug, no tuberculosis was detected, and in pathological
samples from animals in the control group that did not receive the drug, tubercle bacilli with typical
morphological and tinctorial characteristics were revealed;
3.
According to the results of a comparative study of the antibacterial activity of the drug
Rifizostrep against strains of tuberculosis in rabbits with the drug isoniazid, the drug Rifizostrep
showed an active antibacterial effect on tuberculosis pathogens;
4.
The drug "Rifisostrep" - exhibits synergism (increasing the effect of one drug by another)
and prolongation (increasing the duration of action of the drug);
5.
The pharmacokinetics of the drug "Rifizostrep" against pathogenic and atypical
mycobacteria, leprosy, gram-negative (Escherichia coli, salmonella, Klebsiella, tularemia, etc.) and
some gram-positive (staphylococci, pneumococci, streptococci, anthrax, rickettsia) has a bactericidal
and bacteriostatic effect on microorganisms, also has a virucidal effect.
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Donchenko N.A. Improvement of means and methods for diagnosing and preventing tuberculosis
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p. 36
[3]
Lysenko A.P. Development and implementation of new methods for diagnosing and preventing
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