International Laboratory for the Creation of Means of Prevention of Socially Significant Infenctions of Productive Animals

Karlyshev Andrey Vladimirovich

Contract number

075-15-2022-1124

Time span of the project

2022-2024

Laboratory's website

As of 01.12.2023

Number of staff members

scientific publications

Objects of intellectual property

Name of the project:

To create means for the prevention of socially significant infections in productive animals on the basis of modern nutrigenomics methods.

Research directions: Agricultural sciences

Goals of project:

To developed means for the prevention of socially significant infections in productive animals, including probiotic microorganisms, with targeted antagonistic activity with respect to causative agents of the food zooanthroponotic infections Campylobacter jejuni, Salmonella enterica var. Enteritidis, Escherichia coli. The developed complex of probiotic agents will ensure the immune homeostasis, increase the security of cattle, the productivity, safety and quality of meat.

Project objective:

To create an innovative symbiotic consortium on the basis of the most promising candidate strains of probiotic microorganisms demonstrating targeted antagonistic activity with respect to a wide range of pathogenic E. coli and a prebiotic for the prevention of intestinal and extraintestinal infectious diseases caused by pathogenic antibiotic-resistant strains of E. coli, including globally widespread strains of a ST131 clone that produce ESBL in agricultural animals;
To study the influence of the symbiotic consortium on the intestinal microbiota of agricultural animals and its preventive effect against the background of the pathogenic antibiotic-resistant E. Coli stain of the ST 131 clone that produces ESBL;
To conduct a transcriptome analysis of the expression of genes in enterocytes of agricultural animals when they interact with symbiotic consortia of microorganisms aimed at the prevention of intestinal and extraintestinal infectious diseases caused by the pathogenic antibiotic-resistant strains E. coli, including widespread strains of the ST131 clone that produces ESBL;
To create am innovative symbiotic consortium on the basis of the most promising candidate strains of probiotic microorganisms that demonstrate targeted antagonistic activity against S. enterica var. Enteritidis for preventing salmonellosis in agricultural animals.
To study the influence of the symbiotic consortium as a feed supplement on the intestinal microbiota of agricultural animals and its preventive efficiency against the background of the S. Enteritidis pathogen;
To conduct a transcriptome analysis of the expression of genes in enterocytes of agricultural animals when they interact with the symbiotic consortium of microorganisms aimed at the prevention of salmonellosis;
To create an innovative symbiotic consortium based on the most promising candidate strains of probiotic microorganisms demonstrating targeted antagonistic activity against the C. Jejuni pathogen and a prebiotic for the prevention of campilobacteriosis in agricultural animals;
To study the influence of the symbiotic consortium on the intestinal microbiota in agricultural animals and its preventive efficiency against the background of the C. Jejuni pathogen;
To conduct a transcriptome analysis of the expression of genes in enterocytes of agricultural animals when they interact with the symbiotic consortium of microorganisms aimed at the prevention of campilobacteriosis;
Using bioinformatics methods, to conduct a modeling of the role of the intestinal microbiota of the host organism on the regulation of congenital and adaptive immunity for the formation of the immune homeostasis of the digestive system in the light of the development of the general theory of immunity.

Scientific results:

The data on biocompatibility of L.rhamnosus MD IIE 2166 and L.plantarum MD IIE-2165 was obtained. According to the results from the method of co-cultivation on a nutrient-dense medium and the deferred antagonism technique, it was concluded that L.rhamnosus MD IIE 2166 and L.plantarum MD IIE-2165, forming a microbial consortium, do not exhibit any antagonistic activity with respect to each other and are biocompatible.

As a result of the research on antagonistic activity of 7 candidate strains of probiotic cultures: Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus plantarum MD IIE-2165, Lactobacillus fermentum ATCC 9338, Lactobacillus casei, Bacillus subtilis VKPM 10172, Bacillus licheniformis VKPM 10135 d in pure cultures and in various combinations with 5 prebiotic components against two E.coli isolates, it was found that the research results varied depending on the method used to detect antagonistic activity. In this regard, it is essential to conduct an integrated assessment to obtain reliable results and apply all the listed methods and microbial cultivation techniques (i.e., deferred cultivation and co-cultivation) to obtain reliable results. Reliable synergism aimed at enhancing probiotic cultures' antagonistic characteristics against E.coli antibiotic-resistant strains was observed for "lactulose + probiotic" and "probiotic + lactulose with dietary fibers"combinations.

It was found that a microbial consortium of L.rhamnosus IIE 2166 and L.plantarum IIE 2165 has more pronounced enzymatic activity during milk fermentation compared to the individual strains, of which the consortium is comprised. This data indicates that L.rhamnosus IIE 2166 and L.plantarum IIE 2165 in the form of a microbial consortium exhibit a symbiotic relationship. According to the research results, it has been proven that the created innovative consortium of strains is symbiotic.

The symbiotic microbial consortium of L.rhamnosus IIE 2166 and L.plantarum IIE 2165 strains has more pronounced antagonist activity against E.coli antibiotic-resistant strains, including globally widespread strains of a ST131 clone containing extended-spectrum beta-lactamases (ESBLs), which cause intestinal and extraintestinal diseases in animals and humans, compared to the individual lactobacilli strains, of which the consortium is comprised.

The genome sequence was annotated using NCBI GenBank and RAST software [8 Aziz R.K., et al., 2008]. The results of genomic sequencing of L.rhamnosus MD IIE 2166 strain were deposited in DDBJ/EMBL/GenBank under registration number AVFG00000000.

The genome of L.plantarum MD IIE 2165 strain was analyzed, revealing the presence of a large number (412) of genes, which are responsible for sugar metabolism and enzymatic activity. Plantaricin biosynthesis gene cluster was discovered. During L.plantarum 2165 genome annotation using NCBI GenBank software, 3141 genes encoding 2702 proteins were identified. These genes are responsible for resistance to bile salts and other toxic compounds.

The data on the influence of candidate strains of symbiotic consortium, which are aimed at the prevention of intestinal and extraintestinal infectious diseases in farm animals caused by E.coli, on the expression of Toll-like 4;9 (TLR4; TLR9) receptors of innate immunity in an in vitro system using enterocytes was obtained. It was found that the strains consortium (MD IIE-2166 + L. plantarum MD IIE-2165), as well as DNA isolated from its strains, inhibit the expression of TLR4 on Caco-2 enterocytes induced by LPS and stimulate the expression of TLR9.

Implementation of research results:

The innovative symbiotic consortium that is comprised of the most promising candidate strains and probiotics for the prevention of intestinal and extraintestinal infectious diseases in agricultural animals caused by pathogenic antibiotic-resistant strains of E.coli, including globally widespread strains of a ST131 clone that contain ESBL;

The probiotic microbial strains that demonstrate targeted antagonistic activity against pathogenic antibiotic-resistant strains of E.coli, including globally widespread strains of a ST131 clone that contain ESBL, causing intestinal and extraintestinal infectious diseases in animals.

Introducing the strains in poultry farms will provide high quality and bio-safety of their poultry products (e.g., meat, eggs) due to elimination of antibiotics usage in poultry feeds. Thus, resulting poultry products can be placed in higher value categories due to their high quality, which is relatively close to the quality of organic products. Therefore, poultry industry enterprises can receive extra profit, which will enhance poultry farm financial stability.

Organizational and infrastructural changes:

The Laboratory of Genomic Research has been established at VGNKI.

Education and personnel occupational retraining:

Training of members of the research team and junior researchers in practical work with equipment. Development of new educational programs. Establishing a scientific school for junior researchers.

6 members of the research team are currently pursuing post-graduate education at VGNKI.

6 members of the research team completed an internship at the V.M. Gorbatov RAS FSC of food systems.

14 members of the research team underwent advanced training at the FSBEI HE "I. Kant Baltic Federal University".

Cooperation:

SNCO "The National Center for Veterinary, Sanitary and Phytosanitary Lab Services" Food Safety State Service of Ministry of Agriculture of Armenia
FSBEI HE "The I. Kant Baltic Federal University"
LLC "Alltech"
FSBEI HE "The Saint-Petersburg State University of Veterinary Medicine"
FSBEI HE "The K.I. Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology"
JSC "III" - Open Joint Stock Company Institute of Engineering Immunology
The V.M. Gorbatov RAS FSC of Food Systems

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V. M. Abramov, I. V. Kosarev, A. V. Machulin, A.N. Panin, O.E. Ivanova, T.N. Abashina, A.M. Manoyan, A.A. Ahmetzyanova, T.T. Papazyan, I.N. Nikonov

Ligilactobacillus salivarius 7247 Strain: Probiotic Properties and Anti- Salmonella Effect with Prebiotics / V. M. Abramov, I. V. Kosarev, A. V. Machulin [et al.] // Antibiotics. – 2023. – Vol. 12, No. 10. – P. 1535. – DOI 10.3390/antibiotics12101535. – EDN HIYEPS. Impact-factor-4,8, Q-1 ISSN 2079-6382

V. M. Abramov, I. V. Kosarev, A. V. Machulin, A.N. Panin, T.N. Abashina, A.A. Ahmetzyanova, I.N. NikonovA.V. Karlyshev

Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 Against Candida albicans Infections / V. M. Abramov, I. V. Kosarev, A. V. Machulin [et al.] // Biomolecules. – 2023. – Vol. 13, No. 12. – P. 1740. – DOI 10.3390/biom13121740. – EDN GZFCIC. Импакт-фактор-5.5, Q1, ISSN 2218-273X

V. M. Abramov, I. V. Kosarev, A. V. Machulin, A.N. Panin, T.N. Abashina, A.A. Ahmetzyanova, I.N. NikonovA.V. Karlyshev, M.V. Selina

Limosilactobacillus fermentum 3872 That Produces Class III Bacteriocin Forms Co-Aggregates with the Antibiotic-Resistant Staphylococcus aureus Strains and Induces Their Lethal Damage / V. M. Abramov, I. V. Kosarev, A. V. Machulin [et al.] // Antibiotics. – 2023. – Vol. 12, No. 3. – P. 471. – DOI 10.3390/antibiotics12030471. – EDN GZLBGY. Импакт-фактор-4,8, Q-1 ISSN 2079-6382

A.N. Panin, L.A. Gnezdilova, I.N. Nikonov, M.V. Selina

Effects of the Strain Lactobacillus plantarum ATCC 8014 on the Zootechnical Indicators of the Broiler Chickens Growth / A. Panin, S. Pozyabin, L. Gnezdilova [et al.] // International Transaction Journal of Engineering, Management and Applied Sciences and Technologies. – 2023. – Vol. 14, No. 1. – P. 141. – DOI 10.14456/ITJEMAST.2023.4. – EDN KGAOJN. Импакт-фактор-1,3, Q-4 ISSN: 2228-9860 (Print), 1906-9642 (Electronic)

V. M. Abramov, I. V. Kosarev, A. V. Machulin [et al.]

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes/ V. M. Abramov, I. V. Kosarev, A. V. Machulin [et al.] // Antibiotics. – 2024. – Vol. 13, No. 1. doi.org/10.3390/antibiotics13010030

Patent for invention: 2821556 Bacterial strain: Levilactobacillu brevis ВКШМ-Г-07ПД Date of state registration Registered in The State Register of Inventions on 25th June 2024

Researchers: Panin A.N., Karlyshev A.V., Abramov V.M., Nikonov I.N., Kosarev I.V., Abashina T.N., Machulin A.V., Papazyan T.T., Manoyan M.G., Ivanova O.E., Blumenkranz D.A., Ivanova A.N., Manoyan A.M., Khlebnikov V.S., Sakulin V.K.

Hosting organization	Field of studies	City	Invited researcher	Time span of the project
Laboratory of livestock genomic and post-genomic technologies (10)	Agriculture, forestry and fisheries	Ufa	Gusev Oleg Aleksandrovich Russia	2024-2028
Laboratory of Agricultural Ecotoxicology Siberian Federal Research Center of Agrobiotechnologies of the RAS - (SFSCA RAS)	Agriculture, forestry and fisheries	Krasnoobsk	Tsatsakis M. Aristides Greece	2022-2024
Grain Quality Assessment Laboratory Omsk State Agrarian University named after P. A. Stolypin - (Omsk SAU)	Agriculture, forestry and fisheries	Omsk	Koksel Hamit Turkey Shamanin Vladimir Petrovich Russia	2021-2023

Hosting organization

Field of studies

City

Invited researcher

Time span of the project

Laboratory of livestock genomic and post-genomic technologies (10)

Agriculture, forestry and fisheries

Ufa

Gusev Oleg Aleksandrovich

Russia

2024-2028

Laboratory of Agricultural Ecotoxicology