RU2797383C1 - Polyresistant escherichia coli bacterial strain for determining the bactericidal effect of antibacterial drugs in veterinary medicine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Escherichia coli is proposed, isolated from biological material of poultry (litter) when monitoring antibiotic resistance in the Central Federal District of the Russian Federation, deposited in the All-Russian Collection of Microorganism Strains Used in Veterinary Medicine and Animal Husbandry under the following number: VKShM-B-891M.

EFFECT: strain is characterized by phenotypic resistance to ampicillin, amoxicillin, ciprofloxacin, enrofloxacin, tetracycline, gentamycin, streptomycin, chloramphenicol, sulfamethoxazole, trimethoprim and colistin, contains genetic determinants of resistance to the same antimicrobial agents: blaTEM-1, gyrA_D87N, S83L, parC_S80I, parE_S458A , tetB , aadA1, aadA2, aac(3)-IIa, aph(3')-Ia, cmlA1, dfrA14, sul3, mcr-1 and can be used as a control strain to assess the effectiveness of the bactericidal action of the developed therapeutic antibacterial agents in veterinary medicine.

1 cl, 1 dwg, 1 tbl, 2 ex

Description

The invention relates to veterinary microbiology and biotechnology, concerns a new strain of Escherichia coli having phenotypic resistance to ampicillin, amoxicillin, ciprofloxacin, enrofloxacin, tetracycline, gentamicin, streptomycin, chloramphenicol, sulfamethoxazole, trimethoprim, colistin and containing genetic determinants of resistance to the same antimicrobials: bla _TEM-1 , gyrA_D87N, S83L, parC_S80I, parE_S458A , tetB, aadA1, aadA2, aac(3)-IIa, aph(3')-Ia, cmlA1, dfrA14, sul3, mcr-1, which can be used as control strain to determine the effectiveness of the bactericidal action of the developed therapeutic antibacterial drugs in veterinary medicine.

The reason for the emergence of microbial antibiotic resistance, which lies at the intersection of medical and veterinary science, is the use of antibiotics in animal husbandry and the transfer of resistant microorganisms to humans through direct contact with animal carriers and / or through food. This category of microorganisms includes pathogens of food toxic infections (Salmonella, Campylobacter and Listeria) and zooanthroponoses (diseases common to humans and animals) caused by poly- or pan-resistant enterococci, Pseudomonas aeruginosa, E. coli of various types, etc.

The resistance of Escherichia coli isolates to antibacterial drugs is due to both the natural resistance of the microorganism to the main clinically significant antimicrobial substances and the implementation of genetically determined molecular mechanisms of resistance and virulence, acquired mainly due to the horizontal transfer of the carriers of genetic information encoding them [1–4]. At the same time, in order to conduct rational antibacterial therapy in farm animals, it is necessary to study the resistance of Escherichia coli to antimicrobial drugs.

The strain of Escherichia coli proposed by us contains all relevant markers of resistance to antimicrobial drugs: beta-lactams, fluoroquinolones, tetracyclines, aminoglycosides, fenicols, trimethoprim, sulfonamides, polymyxins, therefore, it can be effective for standardizing the evaluation of promising antimicrobial drugs in veterinary medicine.

The closest in essence to the claimed invention is a bacterial strain Acinetobacter baumannii to standardize the evaluation of the effectiveness of developed antimicrobials and disinfectants, however, it does not have resistance to important for veterinary antimicrobial agents of the class of fluoroquinolones (enrofloxacin, ciprofloxacin) and fenicols (chloramphenicol), as the proposed strain Escherichia coli VKShM-B-891M [5].

The invention is known The bacterial strain of Salmonella infantis, used as a positive control for molecular genetic and microbiological studies related to the determination of the sensitivity of microorganisms to antibacterial drugs, however, it does not have resistance to polymyxins and does not contain the genetic determinant mcr-1, as the present invention [6].

Known invention "Strain of bacteria SALMONELLA ENTERICA SBSP. ENTERICA SEROVAR KENTUCKY B-9045 of the international multidrug-resistant clone SALMONELLA KENTUCKY ST198, used as a control strain for phenotypic and molecular studies in the diagnosis of salmonellosis" according to the patent for invention 2744203 C1. The patent describes a strain that can be used as a control strain for quality control in the diagnosis of typhoid fever: at the stages of phenotypic studies (determination of sensitivity to antibiotics: detection of high-level resistance to fluoroquinolones) and molecular studies (determination of the mechanism of resistance to fluoroquinolones by detecting mutations in the gyrA and parC genes, phylogenetic studies using the MLST method (from the English. Multi Locus Sequence Typing, multilocus sequencing), however, the proposed invention is more universal, because it has resistance to a wider range of antibacterial drugs and identical genetic determinants of resistance [7] .

The technical result of the invention is the expansion of the range of Escherichia coli strains suitable for the development of effective antimicrobial drugs.

The specified technical result of the invention is achieved by isolating a polyresistant strain of Escherichia coli for evaluating the effectiveness of the bactericidal action of the developed antibacterial drugs, deposited in the "All-Russian collection of microorganism strains used in veterinary medicine and animal husbandry" under the number VKShM-B-891M, having phenotypic resistance to ampicillin, amoxicillin, ciprofloxacin , enrofloxacin, tetracycline, gentamicin, streptomycin, chloramphenicol, sulfamethoxazole, trimethoprim, colistin and containing genetic determinants of resistance to the same antimicrobial drugs: bla _TEM-1 gyrA_D87N, S83L, parC_S80I, parE_S458A , tetB, aad A1, aadA2, aac(3) -IIa, aph(3')-Ia, cmlA1, dfrA14, sul3, mcr-1. Escherichia coli strain VKShM-B-891M natural, isolated from the biological material of poultry (litter) when monitoring antibiotic resistance in the Central Federal District of the Russian Federation.

Whole genome sequencing of the Escherichia coli VKShM-B-891M strain was carried out on the Illumina platform (MiSeq), tagging and indexing were performed according to the Illumina instructions “NexteraXT DNA Library Prep Kit. Reference Guide" using the NexteraXT reagent kit. Normalization and denaturation were performed according to the Illumina Denature and Dilute Libraries Guide. The quality of DNA tagmentation and the calculation of the molar concentration of purified PCR products were assessed using a TapeStation 4200 microcassette electrophoresis system. Sample preparation, electrophoretic separation of PCR products were carried out in accordance with the instrument manual and instructions G2991-90030 / G2991-90050. Data analysis and calculation of the molar concentration of purified PCR products is performed automatically using the built-in Agilent TapeStation Analisis software.

For bioinformatic analysis of whole genome sequencing data and de novo genome assembly, the following programs were used: FastQC 0.11.17, Trimmomatic v.0.36, SPAdes 2.11.1, QUAST 4.6.3, MAUVE v.20150226, IntegronFinder v5.

Strain characteristic:

According to modern concepts, the species Escherichia coli belongs to the genus Escherichia , family Enterobacteriaceae , order Enterobacteriales , class Gamma proteobacteria, phylum Proteobacteria, the kingdom of bacteria.

Morphological, cultural, antigenic, enzymatic properties of the strain were studied according to the guidelines for bacteriological diagnosis of colibacillosis (escherichiosis) in animals (approved by the Ministry of Agriculture of the Russian Federation on July 27, 2000 No. 13-7-2/2117).

Morphological properties of the strain - small polymorphic gram-negative rods with rounded ends, spores and capsules do not form. Pathogenic properties: pathogenic strain for humans, pathogenicity group 4 according to SP 1.2.036-95 and SP 1.3.2322-08 (as amended on 06/29/2011).

Cultural properties - uniform turbidity of the medium on the BCH, a small, easily breaking amorphous precipitate; on MPA in Petri dishes - they form round, convex, with a smooth surface colonies in the "S" shape, with a diameter of 2-3 mm; in pancreas - diffuse growth (mobile).

Antigenic structure: the strain is polyagglutinable, agglutinated by polyvalent O-coli agglutinating sera (group 2 and 4)

Enzymatic properties: ferments with the formation of acid and gas glucose, lactose, mannitol, arabinose, sorbitol, xylose, maltose, trehalose, fructose. Forms indole, does not form hydrogen sulfide, does not liquefy gelatin.

The stability of the morphological, cultural, and enzymatic properties of the Escherichia coli VKShM-B-891M strain was tested by a three-fold passage on nutrient media from various manufacturers: FBSI SRC PMB (Russia), NITsF (Russia), Oxoid (Great Britain), HiMedia Laboratories Pvt. Limited (India), BioMerieux (France), Merck (Germany). It was found that the strain gave stable results on nutrient media of all manufacturers.

The colonies grown on solid nutrient medium were examined using a MALDI-TOF Biotyper Microflex mass spectrometer. For this, proteins were extracted by sequential treatment of the microbial suspension with ethyl alcohol, 70% formic acid, followed by the addition of acetonitrile. A clean MALDI target was covered with 1 µl of the supernatant, followed by drying the samples in a laminar box at room temperature and applying 1 µl of the mass spectrometric matrix HCA (α-cyano-4-hydroxycinnamic acid, 99%), also with drying. The analysis was carried out automatically. The results obtained were analyzed using software by the value of the logarithmic growth, the similarity index was greater than 2.0 - which corresponds to a reliable result of the identification of the microorganism.

The conducted studies allow us to conclude that the studied strain was identified as Escherichia coli with a high degree of certainty and does not contain impurities of other bacterial cultures.

Annotation of genomes was performed using the RAST server on the open platform for comparative analysis of genomes SEED. The high-throughput whole genome sequencing system did not detect any antibiotic resistance gene that did not have the corresponding microbiological trait of antibiotic resistance (Table 1). The following biochemical mechanisms of resistance to antibiotics of various classes were revealed in the strain:

- modification of the target of action - due to the dfrA14, mcr-1 genes or mutations in the gyrA_D87N, S83L, parC _S80I, parE_S458A genes;

- antibiotic inactivation - is determined by the aadA1, aadA2, aac(3)-IIa, aph(3')-Ia, bla _{TEM genes.}

- active removal of the antibiotic from the microbial cell (efflux) - is determined by the tetB, cmlA genes.

Using the IntegronFinder program, a class 1 integron was found (Fig. 1) with the following composition Int, attI, sat2, attC, aadA2b, attC, cmlA1, attC, aadA1, attC, qacE, attC. Virulence factors were identified - a fim cluster with fimbriae 1- and 2- types B, C, D, E, F, G, I was identified.

The stability of the presence of genetic resistance factors of the proposed strain was confirmed by repeated sequencing and analysis of the nucleotide sequence after storage of the strain for 6 months in lyophilized form in ampoules under vacuum at a temperature of 2-8°C.

The invention is implemented as follows:

Example 1

Quality control in the determination of sensitivity to antimicrobial drugs by the method of serial dilutions. Guided by the following guidelines: MUK 4.2.1890-04 "Determination of the sensitivity of microorganisms to antibacterial drugs"; VET01-A4 Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals, Approved Standard - Fourth Edition.

The choice of reference (control) strains is determined by the type of microorganism. A typical strain with well-studied phenotypic characteristics, including sensitivity to ABP, characterized by genetic stability of Escherichia coli VKShM-B-891M, was used as a control test strain.

Testing was performed by the micromethod using 96-well plates. An inoculum with a turbidity of 0.5 McFarland standard (10 ⁸ , then diluted 100 times) was prepared for the study. For this, a daily culture was used, 3-5 colonies were selected and dissolved in 0.9% physiological saline, dissolved in broth and added to 96-well plates, in a ratio of 1:1 with an antibiotic solution (final concentration 10 ⁵ ). The plates were then incubated for 24 hours at 37°C. The results were recorded visually. The first well, in which no visible growth of the microorganism was observed (turbidity of the medium), was taken as the minimum inhibitory concentration of the antibacterial drug.

The control strain showed pronounced resistance to ampicillin, amoxicillin, ciprofloxacin, enrofloxacin, tetracycline, gentamicin, streptomycin, chloramphenicol, sulfamethoxazole, trimethoprim, colistin.

Example 2

Evaluation of the effectiveness of the bactericidal action of commercial antibacterial drugs used in veterinary medicine was carried out using the inoculum of the reference strain Escherichia coli VKShM-B-891M .

To prepare the inoculum, a 5-6-hour broth culture of the microorganism was used. To do this, several single-type isolated colonies were selected from a daily culture of microorganisms grown on MPA, a small amount of material was transferred by a loop into a test tube with 4.0-5.0 ml of MBA. Incubated at 37°C. After 5-6 hours of incubation, the density of the microbial suspension approximately corresponded to the required one, and it was exactly adjusted to 0.5 according to McFarland by adding sterile MPB so that its concentration was 1.5⋅10 ⁸ CFU/ml.

For the preparation of ABP solutions, ABP substances with known activity were used. To weigh the ABP, an electronic laboratory balance was used with an accuracy of 4 digits. In test tubes containing 1.0 ml of MPB, 1.0 g of an antibacterial preparation was added, shaken until it was completely dissolved.

0.5 ml of the inoculum was added to each tube containing 1.0 ml of the corresponding antibacterial drug, and to one tube with 1.0 ml of BCH without ABP ("negative" control).

The tubes were closed with sterile cotton-gauze stoppers and incubated at 37°C for 16-20 hours, except for the "negative" control tube. The "negative" control tube was placed in a refrigerator at 4°C, where it was stored until the results were recorded.

To determine the presence of microorganism growth, tubes with cultures were viewed visually in transmitted light. Culture growth in the presence of ABP was compared with a reference tube ("negative" control) containing the original inoculum and stored in a refrigerator.

The results are presented in table 1.

Table 1
The effectiveness of the bactericidal action of commercial therapeutic antibacterial drugs for veterinary use. No.
p/n Name
Antibacterial drug Name
active ingredient Quantity
Active ingredient Escherichia coli strain VKShM-B-891M Sensitivity 1. Gentamicin 100 powder Gentamycin sulfate 100 mg/g microorganism growth R 2. Terravitin-500 Oxytetracycline hydrochloride 500 mg/g microorganism growth R 3. Solamox® Amoxilin in the form of trihydrate 700 mg/g microorganism growth R 4. Doxatib® doxycycline hyclate 500 mg/g microorganism growth R 5. Colimixol® 12 million IU Colistin sulfate 12000000 ME/g microorganism growth R 6. Enrofloxacin 100 powder Enrofloxacin 100 mg/g microorganism growth R 7. Amoxi VL80 Amoxicillin trihydrate 800 mg/g microorganism growth R 8. Thiocefur® Ceftiofur
(as sodium salt) 1 g No growth S 9. Amoxigor 80% Amoxicillin trihydrate 800 mg/g microorganism growth R 10. Ceftiofur MZ Ceftiofur sodium 800 mg/g No growth S Note: R (Resistance) - stable; S (sensitive) - sensitive.

According to the data indicated in the table, the studied strain had resistance to drugs of the following groups: aminoglycosides, tetracyclines, penicillins, polymyxins, fluoroquinolones and is sensitive to drugs of the cephalosporin group.

The results obtained confirm the possibility of using the Escherichia coli VKShM-B-891M strain to determine the bactericidal effect of antibacterial drugs in veterinary medicine.

Literary sources:

1. Reshadi P., Heydari F., Ghanbarpour R., Bagheri M., Jajarmi M., Amiri M., et al. Molecular characterization and antimicrobial resistance of potentially human-pathogenic Escherichia coli strains isolated from riding horses. BMC Vet. Res. 2021; 17(1):131. DOI: 10.1186/s12917-021-02832-x.

2. Poirel L., Madec J.Y., Lupo A., Schink A. K., Kieffer N., Nordmann P., SchwarzS. Antimicrobial resistance in Escherichia coli. microbiol. Spectr. 2018; 6(4). DOI: 10.1128/microbiolspec.ARBA-0026-2017.

3. Al'-KhammashN. M., Ignatenko A.V. Analiz antibiotikorezistentnosti mikroorganizmov E. coli =ARM analysis of E. coli. Proceedings of BSTU. Chemistry, organic substances technology and biotechnology. 2012; 4(151):173-175. eLIBRARYID:22002362.

4. Brettin T. [et al] RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes / T. Brettin [et al] // Scientific reports. - 2015. - T. 5. - S. 8365;

5. Kataeva L.V., Kolotova O.N., Stepanova T.F., Bogun A.G., Kislichkina A.A. Multidrug-resistant strain of Acinetobacter baumannii bacteria for standardization of evaluation of the effectiveness of developed antimicrobials and disinfectants // Patent for invention 2711922 C1, 01/23/2020. Application No. 2019119531 dated 06/21/2019.

6. Kish L.K., Ivanova O.E., Soltynskaya I.V., Lenev S.V., Prasolova O.V., Bogomazova A.N., “Salmonella infantis bacterial strain used as a positive control for molecular genetic, as well as microbiological studies related to the determination of the sensitivity of microorganisms to antibacterial drugs. Application 2021139600 dated 12/29/2021.

7. Egorova S.A., Kaftyreva L.A. The bacterial strain SALMONELLA ENTERICA SBSP. ENTERICA SEROVAR KENTUCKY B-9045 of the international multi-resistant clone SALMONELLA KENTUCKY ST198, used as a control strain for phenotypic and molecular studies in the diagnosis of salmonellosis // Patent for invention 2744203 C1, 03.03.2021. Application No. 2020122129 dated 06/29/2020.

Claims (1)

A polyresistant Escherichia coli bacterial strain used as a control strain to determine the effectiveness of the bactericidal action of the developed therapeutic antibacterial veterinary drugs, deposited in the All-Russian Collection of Microorganism Strains Used in Veterinary Medicine and Animal Husbandry under the number VKShM-B-891M.

Images