Qiqihar, Китайская Республика
Kaliningrad, Калининградская область, Россия
Kaliningrad, Россия
Kaliningrad, Калининградская область, Россия
Kemerovo, Кемеровская область, Россия
Introduction. Increased resistance of microorganisms to traditional antibiotics has created a practical need for isolating and synthesizing new antibiotics. We aimed to study the antibiotic activity and resistance of bacteriocins produced by lactic acid bacteria and other microorganisms. Study objects and methods. We studied the isolates of the following microorganism strains: Bacillus subtilis, Penicillium glabrum, Penicillium lagena, Pseudomonas koreenis, Penicillium ochrochloron, Leuconostoc lactis, Lactobacillus plantarum, Leuconostoc mesenteroides, Pediococcus acidilactici, Leuconostoc mesenteroides, Pediococcus pentosaceus, Lactobacillus casei, Lactobacillus fermentum, Bacteroides hypermegas, Bacteroides ruminicola, Pediococcus damnosus, Bacteroides paurosaccharolyticus, Halobacillus profundi, Geobacillus stearothermophilus, and Bacillus caldotenax. Pathogenic test strains included Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus mycoides, Alcaligenes faecalis, and Proteus vulgaris. The titer of microorganisms was determined by optical density measurements at 595 nm. Results and discussion. We found that eleven microorganisms out of twenty showed high antimicrobial activity against all test strains of pathogenic and opportunistic microorganisms. All the Bacteroides strains exhibited little antimicrobial activity against Gramnegative test strains, while Halobacillus profundi had an inhibitory effect on Gram-positive species only. The Penicillium strains also displayed a slight antimicrobial effect on pathogenic test strains. Conclusion. The antibiotic resistance of the studied lactic acid bacteria and other bacteriocin-producing microorganisms allows for their use in the production of pharmaceutical antibiotic drugs.
Lactic acid bacteria, bacteriocins, antibiotic properties, antibiotic resistance, natural sources, isolates
INTRODUCTION
New microorganisms that are resistant to traditional
antibiotics have recently become known to medicine [1].
Today, large numbers of people worldwide are dying
from various infections caused by antibiotic-resistant
strains of microorganisms [2]. Therefore, there is an
increasingly important scientific and practical need for
new antimicrobial drugs with a wide spectrum of action.
Modern researchers are actively studying
bacteriocins produced by Gram-positive bacteria,
which are antibiotic proteins [3]. Due to their complex
structure, bacteriocins can be classified as peptides
with different activity, gene control, and biochemical
processes [4, 5]. They do not develop antimicrobial
resistance and therefore are widely used in medicine
and pharmacology [4, 6]. These substances are known for high antibiotic activity against closely related strains
of microorganisms. Lactic acid microorganisms are
among the most effective producers of bacteriocins and
bacteriocin-like agents [3].
Bacteriocinogenesis has apparently evolved as
a result of adaptation and survival in a harmful
environment, having occupied a certain niche in
microbiology [7]. Bacteriocins are produced by lactic
acid bacteria – natural microbiota in the digestive
system of humans and animals, as well as in food
raw materials, products, or animal feed. Bacteriocins
colonize natural and industrial substrates [8–10].
Most often, they do not dominate over saprophytic
microorganisms of spore and non-spore forms, over
cocci, yeasts, molds, and Gram-negative bacteria, which
inhibit antibiotics [11, 12].
Bacteriocin production is a complex process that
requires optimal parameters to affect the system. Not
all bacteria can synthesize bacteriocins. It has been
proved that the ability to synthesize a small amount
of bacteriocinogenic substances by individual strains
is hereditary [13, 14]. However, the synthesis can
be improved by genetic engineering, DNA-tropic
substances ultraviolet rays, peroxides, chemical
mutagens, and other agents [15, 16]. Since mid-20th
century, extensive experiments have been in operation to
create new bacteriocin-producing bacteria.
A number of Gram-positive strains, such as
Lactobacillus, Streptococcus, Bacillus, Mycobacterium,
Staphylococcus, Corynebacterium, Leuconostoc, Sarcina,
Micrococcus, Clostridium and Streptomyces, have been
reported to synthesize bacteriocins [2, 3, 17, 18].
A lot of current research is focused on bacteriocins
produced by lactic acid microorganisms. For example,
diacetin B-1, a bacteriocin isolated from Lactococcus
lactis, consists of 37 amino acid residues and has a
molecular weight of 4300 Da [19–21]. Scientists know of
14 strains of Lactococcus lactis capable of synthesizing
bacteriocins. All bacteriocins inhibit the growth of
S. aureus, P. acidilactici, L. Plantarum, and many
Listeria species [14, 22, 23].
Amylovorin 471, a bacteriocin produced by
Lactobacillus amylovorus D CE 4 71, i s u sed a s a b iopreservative
in food and feed [24].
A purified form of enterocin A obtained from
Enterococcus faecium contains 47 amino acid residues,
including 4 cystine residues, and has a molecular weight
of 4289 Da. Enterocin A has a similar amino acid
sequence to that of nisin, a bacteriocin produced by
lactic acid bacteria [25].
Bacteriocins are also formed by other types of
enterococci. For example, E. faecalis S-48 produces a
80 kDa bacteriocin that is sensitive to proteases and has
an inhibitory effect on E. faecalis [26].
Thus, many infectious diseases can be prevented
and treated by isolating new strains of lactic acid
microorganisms that produce bacteriocins with
antibacterial action [27, 28]. Unlike Lactobacillus
strains, the antimicrobial activity of Lactococcus strains
has not been well studied [2, 14].
Therefore, there is an urgent need for isolating new
antimicrobial and antibiotic-resistant bacteriocins
formed by lactic acid bacteria and other antagonist
microorganisms, as well as studying their properties and
prospects for the pharmaceutical industry [29, 30].
We aimed to study the antibiotic activity and
resistance of bacteriocins produced by lactic acid
bacteria and other antagonist microorganisms isolated
from natural systems in the Kemerovo region.
In particular, we aimed to:
– study the antimicrobial effect of lactic acid
bacteria and other antagonist bacteriocin-producing
microorganisms on pathogenic and opportunistic
microflora that can cause severe infectious diseases in
humans;
– select the isolates of microorganisms with bacteriocin
properties (antimicrobial activity) to determine their
antibiotic resistance; and
– examine the resistance of lactic acid bacteria and other
antagonist microorganisms to the main antibiotics of
various series.
STUDY OBJECTS AND METHODS
Microbial communities in various habitats
(soil, water, animal gastrointestinal tract, animal
products, etc.) were used as natural systems from
which we isolated strains of bacteriocin-producing
microorganisms. The sampling took place in the
Kemerovo region.
Our objects of study included the isolates of
bacteriocin-producing microorganism strains, such
as Bacillus subtilis, Penicillium glabrum, Penicillium
lagena, Pseudomonas koreenis, Penicillium
ochrochloron, Leuconostoc lactis, Lactobacillus
plantarum, Leuconostoc mesenteroides, Pediococcus
acidilactici, Leuconostoc mesenteroides, Pediococcus
pentosaceus, Lactobacillus casei, Lactobacillus
fermentum, Bacteroides hypermegas, Bacteroides
ruminicola, Pediococcus damnosus, Bacteroides
paurosaccharolyticus, Halobacillus profundi,
Geobacillus stearothermophilus, and Bacillus
caldotenax.
Prior to isolation, we incubated microorganisms
on an agar medium melted and poured into Petri
dishes (covering a third or a quarter of the area), then
sterilized and cooled. The incubation lasted 4–5 days
at 30°C (until complete or almost complete sporulation
by vegetative cells). Then, the grown colonies were
suspended in 30 mL of a sterile liquid T3 medium.
The flasks with the inoculated medium were placed
on an orbital shaker (220 rpm, 72–80 h, 30°C). The
stage of sporulation was determined by phase contrast
microscopy. At the end of incubation, we found
98–100% of spores and crystals in the liquid medium
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in relation to the vegetative cells. The number of
colony-forming units per ml of culture fluid (CFU/mL)
was determined with a series of dilutions followed
by incubation in Petri dishes with a T3 medium (five
replicates) for 24 h at 30°C. After incubation, we
counted the grown colonies and expressed the results in
CFU/ml, or spores/ml in our case.
We used the following pathogenic test strains:
– Escherichia coli ATCC 25922 – opportunistic bacteria
causing gastroenteritis in humans;
– Salmonella enterica ATCC 14028 – pathogenic
bacteria causing gastroenteritis in humans;
– Staphylococcus aureus ATCC 25923 – pathogenic
bacteria causing pneumonia, meningitis, osteomyelitis,
endocarditis, infectious toxic shock and sepsis in
humans;
– Pseudomonas aeruginosa B6643 – opportunistic
bacteria causing nosocomial infections in humans;
– Bacillus mycoides EMTC (Russian collection of
extremophilic microorganisms and type cultures)
9 – opportunistic bacteria causing foodborne toxic
infections in humans;
– Alcaligenes faecalis EMTC 1882 – opportunistic
bacteria causing septicemia and meningitis in newborns
and intra-abdominal infections in adults;
– Proteus vulgaris ATCC 63 – opportunistic bacteria
causing acute intestinal infections in humans.
Cultivation of microorganism test strains.
Escherichia coli ATCC 25922 was cultivated on a
medium composed of 10 g tryptone, 5 g yeast extract,
10 g sodium chloride, and 1 L water (pH 7.5–8.0, 37°C).
Salmonella enterica ATCC 14028 was cultivated on a
medium composed of 10 g peptic digest of animal tissue,
5 g meat extract, 5 g glucose, 4 g sodium hydrogen
phosphate, 0.3 g iron sulfate, 8 g bismuth sulfite,
0.025 g brilliant green, 20 g agar-agar, and 1 L water
(pH 7.5–7.9, 35°С).
Staphylococcus aureus ATCC 25923 was cultivated
on a medium composed of 10 g casein hydrolysate,
2.5 g yeast extract, 30 g gelatin, 10 g D-mannitol, 55 g
sodium chloride, 75 g ammonium sulfate, 5 g potassium
hydrogen phosphate, 15 g agar-agar, and 1 L water
(рН 6.8–7.2, 30°С).
Pseudomonas aeruginosa B6643 was cultivated on a
medium composed of 1 L meat water, 5 g NaCl, and 10 g
peptone (рН 6.8–7.0, 37°С).
Bacillus mycoides EMTC 9 was cultivated on a
medium composed of 10 g casein hydrolysate, 2.5 g
yeast extract, 5 g glucose, 2.5 g potassium hydrogen
phosphate, 3 g agar-agar, and 1 L water (рН 7.2–7.6,
30°С).
Alcaligenes faecalis EMTC 1882 was cultivated on a
medium composed of 10 g special peptone, 5 g sodium
chloride, 0.3 g sodium azide, 0.06 g chromogenic
mixture, 2 g Tween-80, 1.25 g sodium hydrogen
phosphate, 15 g agar-agar, and 1 L water (рН 7.3–7.5,
37°С).
Proteus vulgaris ATCC 63 was cultivated on a
medium composed of 8 g peptone, 5 g sodium chloride,
1 g sodium deoxycholate, 1.5 g chromogenic mixture,
10.5 g propylene glycol, 15 g agar-agar, and 1 L water
(рН 7.1–7.5, 37°С).
The quantity of microorganisms (titer) in the
suspensions of overnight broth cultures grown on
standard media was determined by optical density
measurements at 595 nm.
Lactic acid bacteria and other antagonist
microorganisms isolated from natural sources in the
Kemerovo region were assessed for their antimicrobial
action in two ways, using the diffusion method and
measuring optical density.
Diffusion method. Test strain bacteria inoculated
onto an agar medium using the spread plate technique
were immediately covered with paper disks impregnated
with the metabolites of microorganisms under study
(10 μL/disk). A disc with a nutrient medium was used
as a control, and a disc with ciprofloxacin (a standard
antibiotic) was used as a reference drug. The plates were
incubated for 24 h at a temperature optimal for each test
strain. The quantity of microorganisms was determined
by measuring the size (mm) of a transparent zone around
the disc, indicating the absence of microbial growth [31].
Optical density measurement. Test strain bacteria
were incubated with the metabolites in 96-well culture
plates [32]. We resuspended broth cultures aged for
12 h in a medium corresponding to the species of
microorganisms to inoculate, bringing their amount
to ~ 105 CFU/mL. At the same time, we added the
cell suspension and the metabolites under study to the
wells in an amount of 1/10 of the total volume. A liquid
nutrient medium was used as a control and ciprofloxacin
was used as a reference drug (10 μg/mL). The total
volume of the suspension in the well was 200 μL. The
experiments were performed in duplicate. Incubation
was carried out on a shaker at 580 rpm at a temperature
optimal for each test strain. After 24 h, we measured
the optical density on a PICO01 spectrophotometer
(Picodrop Limited, UK) at 595 nm. The bactericidal
activity was determined by changes in the optical
density compared to the control. In the wells where cell
growth stopped or slowed down, the optical density was
lower than in those with normal growth.
Microbial spores were stained according to the
Schaeffer-Fulton method. The method uses a combined
effect of a concentrated brilliant green solution and
temperature on the impermeable spore membrane with
further decolorization of the cytoplasm of a vegetative
cell and its contrast staining with safranin. Microscopic
examination showed that the spores were stained green
and the cells, red. To establish the presence of flagella,
we studied the mobility of cultures in the “squashed
straw” preparations [33].
The antibiotic resistance was determined by the
zones of growth inhibition for the isolates with antibiotic
discs. For this, we inoculated isolate cells onto a
temporary medium using the spread plate technique,
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with antibiotic discs on the agar. The experimental
results were recorded after 24 hours of cultivation in the
incubator at 28°C [31].
RESULTS AND DISCUSSION
Table 1 shows the results of using the diffusion
method to assess the antimicrobial properties of lactic
acid bacteria and other microorganisms isolated from
natural sources in the Kemerovo region.
Of the twenty microorganism strains under study,
eleven exhibited high antimicrobial activity against
all test strains of pathogenic and opportunistic
microorganisms (Bacillus subtilis, Leuconostoc
lactis, Lactobacillus plantarum, Leuconostoc
Table 2 Antibiotic resistance of Bacillus subtilis isolate
Antibiotic Diameter of a growth inhibition zone, mm
Content of bacteria in 1 ml of strain culture
1×107 5×107 1×108 5×108 1×109 5×109
Ampicillin 0 0 0 0 0 0
Benzylpenicillin 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5
Carbenicillin 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0
Polymyxin 0 0 0 0 0 0
Streptomycin 13.5 ± 2.5 13.5 ± 2.5 13.5 ± 2.5 13.5 ± 2.5 13.5 ± 2.5 13.5 ± 2.5
Gentamicin 0 0 0 0 0 0
Clotrimazole 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0
Levomycitin 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0 19.0 ± 1.0
Tetracycline 0 0 0 0 0 0
Monomycin 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5
Neomycin 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5
Ceporin 17.5 ± 1.5 17.5 ± 1.5 17.5 ± 1.5 17.5 ± 1.5 17.5 ± 1.5 17.5 ± 1.5
Kanamycin 18.5 ± 2.5 18.5 ± 2.5 18.5 ± 2.5 18.5 ± 2.5 18.5 ± 2.5 18.5 ± 2.5
Novogramon 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5
Table 1 Antimicrobial activity of natural microorganism isolates by the diffusion method (solid nutrient medium)
Microorganism isolates Lysis zone diameter, mm
Escherichia
coli ATCC
25922–
Salmonella
enterica
ATCC
14028–
Staphylococcus
aureus
ATCC
25923+
Pseudomonas
aeruginosa
B6643–
Bacillus
mycoides
EMTC 9+
Alcaligenes
faecalis
EMTC
1882–
Proteus
vulgaris
ATCC 63–
Control 0 0 0 0 0 0 0
Ciprofloxacin (C) 21.0 ± 1.1 24.0 ± 1.2 19.0 ± 1.0 22.0 ± 1.1 25.0 ± 1.3 23.0 ± 1.2 20.0 ± 1.0
Bacillus subtilis 18.0 ± 0.9 20.0 ± 1.0 17.0 ± 0.9 20.0 ± 1.0 22.0 ± 1.1 20.0 ± 1.0 17.0 ± 0.9
Penicillium glabrum 0 0 5.0 ± 0.3 0 0 0 7.0 ± 0.4
Penicillium lagena 6.0 ± 0.3 10.0 ± 0.5 0 0 0 0 0
Pseudomonas koreenis 12.0 ± 0.6 5.0 ± 0.3 18.0 ± 0.9 0 0 17.0 ± 0.9 15.0 ± 0.8
Penicillium ochrochloron 0 0 0 0 0 6.0 ± 0.3 0
Leuconostoc lactis 20.0 ± 1.0 22.0 ± 1.1 17.0 ± 0.9 21.0 ± 1.1 24.0 ± 1.2 21.0 ± 1.1 18.0 ± 0.9
Lactobacillus plantarum 19.0 ± 1.0 18.0 ± 0.9 15.0 ± 0.8 19.0 ± 1.0 22.0 ± 1.1 21.0 ± 1.1 17.0 ± 0.9
Leuconostoc mesenteroides 17.0 ± 0.9 20.0 ± 1.0 16.0 ± 0.8 19.0 ± 1.0 22.0 ± 1.1 20.0 ± 1.0 18.0 ± 0.9
Pediococcus acidilactici 20.0 ± 1.0 21.0 ± 1.1 17.0 ± 0.9 19.0 ± 1.0 23.0 ± 1.2 21.0 ± 1.1 18.0 ± 0.9
Leuconostoc mesenteroides 15.0 ± 0.8 18.0 ± 0.9 14.0 ± 0.7 17.0 ± 0.9 20.0 ± 1.0 17.0 ± 0.9 15.0 ± 0.8
Pediococcus pentosaceus 21.0 ± 1.1 20.0 ± 1.0 17.0 ± 0.9 19.0 ± 1.0 24.0 ± 1.2 22.0 ± 1.1 18.0 ± 0.9
Lactobacillus casei 18.0 ± 0.9 19.0 ± 1.0 15.0 ± 0.8 10.0 ± 0.5 20.0 ± 1.0 17.0 ± 0.9 15.0 ± 0.8
Lactobacillus fermentum 15.0 ± 0.8 18.0 ± 0.9 14.0 ± 0.7 19.0 ± 1.0 21.0 ± 1.1 17.0 ± 0.9 15.0 ± 0.8
Bacteroides hypermegas 12.0 ± 0.6 10.0 ± 0.5 0 14.0 ± 0.7 0 9.0 ± 0.5 11.0 ± 0.6
Bacteroides ruminicola 7.0 ± 0.4 11.0 ± 0.6 0 12.0 ± 0.6 0 7.0 ± 0.4 10.0 ± 0.5
Pediococcus damnosus 17.0 ± 0.9 22.0 ± 1.1 16.0 ± 0.8 20.0 ± 1.0 23.0 ± 1.2 19.0 ± 1.0 18.0 ± 0.9
Bacteroides paurosaccharolyticus 15.0 ± 0.8 13.0 ± 0.7 0 16.0 ± 0.8 0 12.0 ± 0.6 14.0 ± 0.7
Halobacillus profundi 0 0 11.0 ± 0.6 0 14.0 ± 0.7 0 0
Geobacillus stearothermophilus 20.0 ± 1.0 22.0 ± 1.1 18.0 ± 0.9 19.0 ± 1.0 22.0 ± 1.1 20.0 ± 1.0 18.0 ± 0.9
Bacillus caldotenax 18.0 ± 0.9 23.0 ± 1.2 17.0 ± 0.9 20.0 ± 1.0 21.0 ± 1.1 22.0 ± 1.1 19.0 ± 1.0
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mesenteroides, Pediococcus acidilactici, Pediococcus
pentosaceus, Lactobacillus casei, Lactobacillus
fermentum, Pediococcus damnosus, Geobacillus
stearothermophilus, Bacillus caldotenax).
Bacteroides hypermegas, Bacteroides ruminicola,
and Bacteroides paurosaccharolyticus showed
insignificant antimicrobial activity against Gramnegative
test strains, while Halobacillus profundi had
an inhibitory effect on Gram-positive species only.
Penicillium glabrum had a slight antimicrobial effect on
Staphylococcus aureus, Proteus vulgaris, and Shigella
flexneri; Penicillium lagena, on the test strains of
Escherichia coli, Salmonella enterica, Shigella flexneri,
Aspergillus flavus, and Penicillium citrinum; Penicillium
ochrochloron, on the test strains of Alcaligenes faecalis
and Listeria monocytogenes.
For further studies of antibiotic resistance, we
selected four isolates with maximum antimicrobial
activity against pathogenic and opportunistic test
strains, namely Bacillus subtilis, Leuconostoc
lactis, Lactobacillus plantarum, and Leuconostoc
mesenteroides.
These isolates were tested for antibiotic resistance,
i.e. resistance of a strain to one or more antibacterial
drugs, or decreased sensitivity (immunity) of a culture
to the action of an antibacterial substance.
Antibiotic resistance can develop as a result of
natural selection through random mutations and/
or antibiotic exposure. Microorganisms are able to
transmit genetic information about antibiotic resistance
through horizontal gene transfer. In addition, antibiotic
resistance can be induced artificially by genetic
transformation, for example, by introducing artificial
genes into the genome of a microorganism [13].
Tables 2–5 show the results of studying the antibiotic
resistance of microorganisms isolated from natural
sources in the Kemerovo region.
Table 3 Antibiotic resistance of Leuconostoc lactis isolate
Antibiotic Diameter of a growth inhibition zone, mm
Content of bacteria in 1 ml of strain culture
1×107 5×107 1×108 5×108 1×109 5×109
Ampicillin 29.0 ± 1.0 29.0 ± 1.0 29.0 ± 1.0 29.0 ± 1.0 29.0 ± 1.0 29.0 ± 1.0
Benzylpenicillin 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5
Carbenicillin 19.0 ± 2.0 19.0 ± 2.0 19.0 ± 2.0 19.0 ± 2.0 19.0 ± 2.0 19.0 ± 2.0
Polymyxin 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5
Streptomycin 0 0 0 0 0 0
Gentamicin 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5 22.5 ± 2.5
Clotrimazole 4.0 ± 1.0 4.0 ± 1.0 4.0 ± 1.0 4.0 ± 1.0 4.0 ± 1.0 4.0 ± 1.0
Levomycitin 18.0 ± 2.0 18.0 ± 2.0 18.0 ± 2.0 18.0 ± 2.0 18.0 ± 2.0 18.0 ± 2.0
Tetracycline 0 0 0 0 0 0
Monomycin 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0 6.0 ± 1.0
Neomycin 11.0 ± 1.0 11.0 ± 1.0 11.0 ± 1.0 11.0 ± 1.0 11.0 ± 1.0 11.0 ± 1.0
Ceporin 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0
Kanamycin 0 0 0 0 0 0
Novogramon 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5
Table 4 Antibiotic resistance of Lactobacillus plantarum isolate
Antibiotic Diameter of a growth inhibition zone, mm
Content of bacteria in 1 ml of strain culture
1×107 5×107 1×108 5×108 1×109 5×109
Ampicillin 25.0 ± 2.0 25.0 ± 2.0 25.0 ± 2.0 25.0 ± 2.0 25.0 ± 2.0 25.0 ± 2.0
Benzylpenicillin 28.5 ± 1.5 28.5 ± 1.5 28.5 ± 1.5 28.5 ± 1.5 28.5 ± 1.5 28.5 ± 1.5
Carbenicillin 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0 20.0 ± 2.0
Polymyxin 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5 21.5 ± 1.5
Streptomycin 0 0 0 0 0 0
Gentamicin 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5 16.5 ± 1.5
Clotrimazole 0 0 0 0 0 0
Levomycitin 27.0 ± 1.0 27.0 ± 1.0 27.0 ± 1.0 27.0 ± 1.0 27.0 ± 1.0 27.0 ± 1.0
Tetracycline 30.0 ± 2.0 30.0 ± 2.0 30.0 ± 2.0 30.0 ± 2.0 30.0 ± 2.0 30.0 ± 2.0
Monomycin 15.0 ± 2.0 15.0 ± 2.0 15.0 ± 2.0 15.0 ± 2.0 15.0 ± 2.0 15.0 ± 2.0
Neomycin 10.0 ± 2.0 10.0 ± 2.0 10.0 ± 2.0 10.0 ± 2.0 10.0 ± 2.0 10.0 ± 2.0
Ceporin 0 0 0 0 0 0
Kanamycin 0 0 0 0 0 0
Novogramon 10.0 ± 1.0 10.0 ± 1.0 10.0 ± 1.0 10.0 ± 1.0 10.0 ± 1.0 10.0 ± 1.0
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As we can see, Bacillus subtilis proved to be
resistant to ampicillin, gentamicin, and tetracycline. It
exhibited high sensitivity to neomycin, novogramon,
kanamycin, carbenicillin, levomycitin, and ceporin,
but low sensitivity to benzylpenicillin, monomycin and
clotrimazole.
Leuconostoc lactis was highly sensitive to
ampicillin, gentamicin, benzylpenicillin, and
novogramon, insensitive to clotrimazole and
monomycin, and resistant to streptomycin, tetracycline
and kanamycin.
Lactobacillus plantarum showed resistance to
streptomycin, clotrimazole, ceporin and kanamycin,
high sensitivity to tetracycline, benzylpenicillin, and
levomycitin, and low sensitivity to neomycin and
novogramon.
Leuconostoc mesenteroides was resistant to
streptomycin, tetracycline, and kanamycin, insensitive
to clotrimazole and monomycin, and highly sensitive
to ampicillin, ceporin, benzylpenicillin, gentamicin,
levomycitin, and novogramon.
We found that the isolates with different
concentrations of microorganisms displayed the same
antibiotic resistance. The diameter of the growth
inhibition zone was the same for all concentrations of
microorganisms.
CONCLUSION
Thus, we studied the antibiotic activity and
resistance of lactic acid bacteria and other antagonist
microorganisms isolated from natural sources in the
Kemerovo region. We established a correlation between
the type of isolate and the type of antibiotic. According
to the study, eleven microorganisms out of twenty
exhibited high antimicrobial activity, while the rest of
the strains had an insignificant effect on the test strains
and opportunistic microorganisms.
We found that all the isolates showed some degree
of resistance to the following antibiotics used to treat
infectious diseases: ampicillin, benzylpenicillin,
carbenicillin, polymyxin, streptomycin, gentamicin,
clotrimazole, levomycitin, tetracycline, monomycin,
neomycin, ceporin, kanamycin, and novogramon.
The progressive resistance of the studied bacteriocinproducing
microorganisms to antibiotics allows for their
use in the production of pharmaceutical antibiotic drugs.
CONTRIBUTION
The authors were equally involved in the writing
of the manuscript and are equally responsible for
plagiarism.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
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