CN113186136B - Multiple drug-resistant helicobacter pylori and application thereof - Google Patents

Abstract

The invention discloses a multi-drug resistant helicobacter pylori and application thereof. The invention provides helicobacter pylori (helicobacter pylori) GZ6B5 resistant to five antibiotics such as amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline and the like, and the accession number is GDMCC No: 61571. the invention provides a multi-drug resistant targeted strain which definitely carries 2 multi-drug resistant genes hp1181 and hp1184, is completely resistant to 5 antibiotics which are clinically approved at present and used for treating helicobacter pylori, can be used as a targeted strain for screening novel functional microorganisms, drugs and antibacterial materials, and has good application prospect.

Description

A kind of multi-drug resistant Helicobacter pylori and its application

Technical field:

The invention belongs to the fields of microorganism technology and medical technology, in particular to a Helicobacter pylori which carries two drug resistance genes hp1181 and hp1184 at the same time and is resistant to five commonly used clinical antibiotics and its application.

Background technique:

Helicobacter pylori (HP) is a Gram-negative, microaerophilic helical bacterium closely related to human gastrointestinal diseases. Studies have pointed out that Helicobacter pylori infection can cause chronic gastritis and promote the occurrence and development of various digestive tract diseases, such as indigestion, peptic ulcer and gastric cancer. Among them, the most serious consequence of Helicobacter pylori infection is the induction of gastric cancer. Some studies have pointed out that 90% of the incidence of gastric cancer in the world is closely related to Helicobacter pylori infection. According to the World Health Organization, gastric cancer is the fourth most common cancer in the world and the second most deadly cancer, causing nearly 750,000 deaths every year. Therefore, Helicobacter pylori infection seriously threatens human health and causes huge economic and social losses. The prevention and treatment of Helicobacter pylori infection has great applied research value.

The treatment regimen for H. pylori is quite different from other microbial infections. Due to the great difficulty in isolating and culturing the bacteria, doctors mainly rely on clinical experience and the statistical results of large health institutions (such as the World Health Organization) to formulate anti-infection programs. Currently, the World Health Organization recommends that the treatment regimen for Helicobacter pylori infection is clarithromycin-based triple therapy: a proton pump inhibitor, clarithromycin combined with amoxicillin or metronidazole or tinidazole for 14 days. However, the effectiveness of anti-Helicobacter pylori therapy has been declining in recent years, and the success rate of domestic anti-Helicobacter pylori therapy has been lower than 80%. The failure of anti-Helicobacter pylori infection treatment is mainly related to the increase of antibiotic resistance of the bacteria. A study found that the resistance rate of Helicobacter pylori to clarithromycin was 21.5%, the resistance rate to metronidazole was 95.4%, and the resistance rate to levofloxacin was 20.6%. The rate of resistance to commonly used antibiotics is as high as 7.6%. Due to the extensive resistance of Helicobacter pylori to antibiotics in my country, the existing treatment regimens cannot effectively control the infection of Helicobacter pylori in my country. Therefore, in response to the problem of multidrug-resistant Helicobacter pylori, it is of great significance to carry out research and development of new drugs, preparations and materials for improving the health of our country.

The development of drugs, preparations and materials to solve the problem of multidrug-resistant Helicobacter pylori requires targeted strains with multidrug-resistant properties. At present, the standard strains ATCC 26695 and ATCC 43504 are mainly used for the development of drugs and materials against Helicobacter pylori infection. Among them, ATCC 43504 has a resistance phenotype against amoxicillin, and is more used in the research and development of new antibacterial drugs and materials. However, the current prevalence of Helicobacter pylori in my country is mainly resistant to metronidazole, clarithromycin and levofloxacin, and the resistance rate to amoxicillin is only 0.1%. The corresponding standard strains have seriously hindered the research and development of drugs and materials for the drug resistance of Helicobacter pylori in my country. Therefore, it is of great significance to obtain targeted strains with clear genetic background, stable biological traits and multidrug resistance to promote the research and development of anti-Helicobacter pylori infection.

hp1181 and hp1184 are genes known to cause multidrug resistance in Helicobacter pylori. These two genes are both translocases located on the chromosome of Helicobacter pylori, of which hp1181 belongs to the RND (resistance-nodulation-division) efflux pump family, and the encoded protein has the effect of effluxing a variety of antibiotics through proton pump energy supply; While hp1184 belongs to the MATE (multidrug and toxic compound extrusion) efflux pump family, its encoded protein has the function of actively effluxing a variety of toxic compounds and drugs to the outside of the cell. Designing drugs and materials for preventing and reversing the multidrug resistance of Helicobacter pylori based on these two drug resistance genes has high application value.

Therefore, combined with the prevalence of H. pylori resistance to antibiotics in my country, the development of targeted strains with clear genetic background, clear drug resistance genes and stable multi-drug resistance phenotypes is conducive to the development of anti-Helicobacter pylori infection problems in my country. New antibacterial drugs, preparations and materials can help alleviate the current technical bottlenecks in the prevention and control of Helicobacter pylori in public health and clinical treatment, and help improve the health of our nation.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide Helicobacter pylori (Helicobacter pylori) GZ6B5 resistant to five antibiotics such as amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline, etc., which is preserved in the Guangdong Provincial Microorganism Culture Collection Center ( GDMCC), the deposit number GDMCC No: 61571, the deposit date is March 19, 2021, and the deposit address is the Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

The second object of the present invention is to provide the application of the above-mentioned Helicobacter pylori GZ6B5 in screening functional microorganisms, medicines, preparations or antibacterial materials against Helicobacter pylori infection. This is of great significance for promoting the research and development of anti-Helicobacter pylori infection.

The functional microorganism, drug or antibacterial material is a functional microorganism, drug, preparation or antibacterial material that inhibits Helicobacter pylori resistant to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline.

The third object of the present invention is to provide a nucleotide sequence capable of specifically recognizing Helicobacter pylori GZ6B5, which is shown in SEQ ID NO.6.

The fourth object of the present invention is to provide a primer capable of specifically identifying Helicobacter pylori GZ6B5, which is shown in SEQ ID NO.4 and SEQ ID NO.5.

The fifth object of the present invention is to provide a method that can specifically identify Helicobacter pylori GZ6B5, which is to use the above-mentioned primers to carry out PCR amplification of Helicobacter pylori to be tested. If a band can be generated, the bacteria to be tested is Helicobacter pylori. Bacillus GZ6B5, if no bands can be produced, the bacteria to be tested is not Helicobacter pylori GZ6B5.

Described PCR amplification, PCR reaction system is:

For the PCR amplification, the PCR reaction conditions are: 95°C for 7 min; 95°C for 30s, 65°C for 30s, 72°C for 30s; 30 cycles; 72°C for 10 min.

The Helicobacter pylori (Helicobacter pylori) GZ6B5 of the present invention is a Helicobacter pylori strain resistant to 5 kinds of commonly used clinical antibiotics at the same time, and it carries 2 kinds of drug resistance genes hp1181 and hp1184 at the same time. Helicobacter pylori, which is resistant to five antibiotics at the same time, is an important target strain in the study of the drug resistance mechanism of Helicobacter pylori, and can be used to screen new functional microorganisms, drugs and antibacterial materials.

Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a multi-drug resistant targeting strain clearly carrying two multi-drug resistance genes hp1181 and hp1184, which is currently clinically approved for the treatment of Helicobacter pylori is resistant to all 5 antibiotics, and can be used as a target strain for screening new functional microorganisms, drugs and antibacterial materials, and has good application prospects.

Helicobacter pylori GZ6B5 was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 19, 2021. The preservation number is GDMCC No.61571. : 510070.

Description of drawings

Fig. 1 is the colony shape of Helicobacter pylori (Helicobacter pylori) GZ6B5 on Helicobacter pylori selective medium;

Figure 2 is the morphology of Helicobacter pylori (Helicobacter pylori) GZ6B5 under a microscope after Gram staining;

Fig. 3 is the evolution relationship analysis of Helicobacter pylori (Helicobacter pylori) GZ6B5 based on 16S rRNA gene in Helicobacter;

Figure 4 is the genome-wide ANI analysis of Helicobacter pylori GZ6B5 and different subtypes of Helicobacter pylori in the database. Note: GZ6B5 is compared with the whole genome ANI of East Asian, American and Pecan type Helicobacter pylori at 95.20～ 96.79%, 94.00-94.83% with Asian and European, 92.74-93.87% with African and SJM180;

Figure 5 shows the drug susceptibility detection of Helicobacter pylori GZ6B5 to five antibiotics, Note: A. GZ6B5 grows in the medium of different concentrations of five antibiotics. The medium from top to bottom is: amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline, and the drug concentration is shown in the figure. The upper hole in the far right row is the chromogenic medium without GZ6B5, the lower hole in the far right row is the chromogenic medium without antibiotics but with GZ6B5, B.GZ6B5 in the chromogenic medium with five antibiotics at different concentrations Heat map of detection value of OD _450nm after 72 hours of culture;

Fig. 6 is agarose electrophoresis image of PCR products of Helicobacter pylori GZ6B5 drug resistance genes hp1181 and hp1184;

Fig. 7 is the application of Helicobacter pylori GZ6B5 in the screening of functional microorganisms against multidrug-resistant Helicobacter pylori;

Figure 8 is the agarose electrophoresis verification of the molecular target of Helicobacter pylori (Helicobacter pylori) GZ6B5, Note: M is a DNA marker, and the size of the bands from top to bottom is shown in the figure. 1 is the electrophoresis of GZ6B5 PCR products after molecular target amplification, 2 and 3 are respectively the electrophoresis of PCR products of Helicobacter pylori ATCC 43504 and Sydney SS1 after molecular target amplification, and 4-23 are the different Helicobacter pylori isolates. Electrophoresis of PCR products after molecular target amplification. The results indicated that only GZ6B5 could form a specific PCR product of 359 bp after molecular target amplification, and other Helicobacter pylori could not form similar PCR products.

Specific implementation plan:

The present invention will be further elaborated below in conjunction with specific embodiments.

The culture medium and reagents involved in the following examples are as follows:

Preparation of antibiotic suspension: vancomycin 10mg/mL, amphotericin B 5mg/mL, trimethoprim 20mg/mL, cefsulodin 10mg/mL, the above antibiotics were dissolved in sterilized water according to their content, well mixed.

Helicobacter pylori selection medium (g/L): yeast extract powder 3.0g/L, caseptone 12.0g/L, animal tissue digest 5.0g/L, beef extract powder 3.0g/L, corn starch 1.0g/L , sodium chloride 5.0g/L, agar 13.5g/L, sterile sheep red blood cells or fetal bovine serum 70mL/L, antibiotic suspension 10mL/L, dissolve the above components in sterilized water according to their contents, and mix well.

Helicobacter pylori proliferation medium (g/L): yeast extract powder 2.02g/L, tryptone 10.52g/L, glucose 1.56g/L, sodium bisulfite 0.10g/L, soluble starch 10.0g/L, chlorine Sodium chloride 5.0g/L, fetal bovine serum 70mL/L, antibiotic suspension 10mL/L, the above components are dissolved in sterilized water according to their contents, and mixed evenly.

Columbia agar blood plate (g/L): casein tryptic digest 10.0g/L, meat stomach enzyme digest 5.0g/L, heart tryptic digest 3.0g/L, yeast extract powder 5.0g/L, corn Starch 1.0g/L, sodium chloride 5.0g/L, agar 15.0g/L, sterile sheep red 70mL/L, the above components were dissolved in sterile water according to their contents, and mixed evenly.

Helicobacter pylori preservation solution (g/L): peptone 4.0g/L, yeast extract 0.8g/L, tryptone 4.0g/L, sodium chloride 2.0g/L, glucose 0.4g/L, hydrogen sulfite Sodium 0.04g/L, Sodium Metabisulfite 0.1g/L, Ferrous Sulfate Heptahydrate 0.1g/L, Sodium Pyruvate 0.1g/L, Glycerol 200mL/L, the above components were dissolved in sterilized water according to their contents, well mixed.

Example 1 Isolation, culture and identification of Helicobacter pylori

1.1. Isolation and culture of Helicobacter pylori

(1) Isolation and preservation of Helicobacter pylori: Helicobacter pylori GZ6B5 was isolated from gastric mucosal tissue of a patient with chronic gastritis who had been ineffective in multiple antibiotic treatments in Guangzhou. In a sterile environment, fresh gastric mucosa samples were spread on Helicobacter pylori selective medium, and then placed in a microaerobic environment (5% O ₂ , 10% CO ₂ , 85% N ₂ ) at 37°C for culture 5 to 7 days. Pick a typical colony on the plate and put it on the Helicobacter pylori selective medium plate for streak purification. After purifying twice, pick a single colony and inoculate it into the Helicobacter pylori proliferation medium, and cultivate it under microaerobic conditions at 37°C for 96 hours. back. Add 400 μL of bacterial solution to 600 μL of Helicobacter pylori preservation solution by pipetting and mixing, and store in -80°C ultra-low temperature refrigerator. Thus, strain GZ6B5 was obtained.

(2) Recovery and cultivation of Helicobacter pylori: Dip 10 μL strain GZ6B5 preservation solution from the storage tube, spread it on the selective medium of Helicobacter pylori, and cultivate it in a microaerobic environment at 37°C for 72-96 hours. Resuscitated H. pylori GZ6B5 was obtained as pin-tip-sized translucent colonies on the plate (Figure 1). Pick a single colony from the plate containing the revived Helicobacter pylori GZ6B5, inoculate it in the Helicobacter pylori proliferation medium, and cultivate it in a microaerobic environment at 37°C for 72 to 96 hours to obtain a proliferated Helicobacter pylori GZ6B5 bacterial solution.

1.2. Identification of Helicobacter pylori

The purified Helicobacter pylori GZ6B5 can be identified by morphological characteristics, molecular biology and protein profiles.

(1) Microscopic examination by staining: The smear of Helicobacter pylori GZ6B5 was gram stained, and the morphology was observed by microscopic examination. The Helicobacter pylori GZ6B5 of the present invention was shown to be Gram-negative, with an S-shaped or arc-curved shape under the microscope (Fig. 2).

(2) Molecular identification based on 16S rRNA gene sequence: The DNA of Helicobacter pylori was extracted by bacterial DNA extraction kit (Mabio, CHINA), and then 2×PCR mix (Dongshengbio, CHINA) was used for polymerase chain reaction (Polymerase Chain Reaction) , PCR) amplification. PCR amplification primers used 16S rRNA gene universal primers, the upstream primer sequence was 27F: 5'-AGAGTT TGATCC TGG CTCAG-3'; the downstream primer sequence was 1492R: 5'-CTAC GGCTAC CTT GTTACGA-3'. PCR reaction conditions were: pre-denaturation at 95°C for 5 min; 35 cycles of 95°C for 30s, 56°C for 30s, 72°C for 1min30s, and annealing and extension at 72°C for 10min. The PCR products were recovered by gel cutting, and then subjected to next-generation sequencing (completed by Suzhou Jinweizhi Biotechnology Co., Ltd.). The obtained 16S rRNA gene sequence is shown in SEQ ID No.1. The sequence was compared with the NCBI database (https://blast.ncbi.nlm.nih.gov), and the results suggested that it had the highest homology with Helicobacter pylori, and was named Helicobacter pylori GZ6B5. The phylogenetic tree of Helicobacter pylori GZ6B5 and other standard strains of Helicobacter in NCBI database was established by Neighbor-Joining method. The phylogenetic tree showed that GZ6B5 and Helicobacter pylori strain ATCC 43504 had the closest genetic relationship, with a similarity of 99.56%, as shown in Figure 3 shown.

③Average Nucleotide Identity Analysis at the Genome Level: Average Nucleotide Identity (ANI) analysis based on the whole microbial genome is the gold standard for defining prokaryotic species at the genome level, and the ANI value is above 95% recognized as the same species. The whole genome DNA of Helicobacter pylori GZ6B5 was extracted and the library was constructed using the QIAseq FX DNA Library Kit (Qiagen, Germany), and the High Output v2.5 kit (Illumina, USA) was used for high-throughput sequencing on the Illumina Nextseq 550 platform. The disembarkation data were assembled using Trimmomatic (v0.39) software quality control and SPAdes (v3.13.1) software. The ANI calculator software (http://enve-omics.ce.gatech.edu/ani/index) was used to analyze the average nucleotide identity of the whole genome of GZ6B5 and 29 different subtypes of Helicobacter pylori in the NCBI database. The results are shown in the figure 4 shows that Helicobacter pylori GZ6B5 is highly homologous to East Asian type and American type Helicobacter pylori, and the ANI is above 95%.

Based on the appearance, morphology, Gram staining, 16S rRNA gene alignment and genome ANI analysis of the bacteria, it was suggested that GZ6B5 was Helicobacter pylori. It is named as Helicobacter pylori GZ6B5, and it is deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), the deposit number is GDMCC No: 61571, the deposit date is March 19, 2021, and the deposit address is Guangzhou City, Guangdong Province. Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road.

Example 2 Analysis of drug susceptibility characteristics of Helicobacter pylori GZ6B5 resistant to multiple antibiotics

The drug susceptibility test of Helicobacter pylori GZ6B5 was carried out by agar dilution method, and Helicobacter pylori ATCC43504 was used as the quality control strain. Testing was performed according to the methods and standards established by The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and The Clinical & Laboratory Standards Institute (CLSI).

2.1. Agar dilution method

The powders of five antibiotics, amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline, were dissolved in the corresponding solute according to the method recommended by CLSI, and prepared into an antibiotic storage solution. Into Helicobacter pylori selection medium, a Helicobacter pylori drug susceptibility detection plate containing corresponding concentrations of antibiotics was prepared to detect the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of antibiotics against Helicobacter pylori GZ6B5. Adjust the concentration of the bacterial suspension of Helicobacter pylori GZ6B5 to be tested to 1×10 ⁷ cfu/mL, inoculate 10 μL of the bacterial suspension to the detection plate containing the corresponding concentration of antibiotics, and cultivate it in a microaerobic environment at 37°C for 72 hours. Whether Helicobacter pylori colonies are formed on the detection plate of the corresponding concentration of antibiotics. The MIC of this antibiotic against Helicobacter pylori was taken as the concentration in the lowest concentration drug plate that could not form colonies. Three parallel operations were performed for each drug concentration, and the average value of MIC was obtained.

Through experiments, the minimum inhibitory concentrations of Helicobacter pylori GZ6B5 of the present invention to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline are 1mg/L, 1mg/L, 2mg/L, 32mg/L, 8mg respectively. /L, which were higher than the EUCAST standard of drug resistance, so the bacteria were determined to be multidrug-resistant Helicobacter pylori.

2.2. Broth dilution method

The powders of five antibiotics, amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline, were dissolved in the corresponding solute according to the method recommended by CLSI, and prepared into an antibiotic storage solution. Into the Helicobacter pylori chromogenic medium (Yangguangbio, CHINA), the antibiotic-containing chromogenic medium of the corresponding detection concentration is prepared, which is now prepared and used. 100 μL of chromogenic medium containing different drug concentrations was added to a 96-well plate, and then 100 μL of Helicobacter pylori GZ6B5 at a concentration of 10 ⁵ cfu/mL was added to each well. The 96-well plate was incubated in a microaerophilic environment at 37°C for 72 hours, and the color changes of the wells of different drugs were observed and the OD _450nm value of each well was detected by a protease marker (BIOTEK, USA). The drug concentration in the lowest concentration drug well with no change in color and OD _450nm was taken as the minimum inhibitory concentration of this antibiotic on GZ6B5, as shown in Figure 5. Three parallel operations were performed for each drug concentration, and the average value of the minimum inhibitory concentration was obtained. The drug susceptibility of Helicobacter pylori GZ6B5 to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline is shown in Table 1. It can be seen that Helicobacter pylori GZ6B5 is resistant to the five antibiotics.

Table 1 Sensitivity of Helicobacter pylori GZ6B5 to different antibiotics

Example 3 Detection of Helicobacter pylori GZ6B5 drug resistance gene resistant to multiple antibiotics

According to the multidrug resistance phenotype characteristics of Helicobacter pylori GZ6B5, two common multidrug resistance genes of Helicobacter pylori, hp1181 and hp1184, were selected for PCR detection of the bacteria. The primer sequences and PCR amplification conditions were the same as the reference (Falsafi T, 2016). The primer sequences used, the length of the target gene fragment, and the annealing temperature are shown in Table 2. The PCR reaction conditions are: pre-denaturation at 95°C for 5min; Extend for 10min. After PCR, 8 μL of PCR products were taken and separated by electrophoresis in 1.5% agarose gel (120V, 25min), compared with 100bp DNA Ladder (Dongshengbio, CHINA) to observe whether the target band was formed. For PCR products with target bands, the gel was cut and recovered and sent to next-generation sequencing (completed by Suzhou Jinweizhi Biotechnology Co., Ltd.). The obtained sequences of hp1181 and hp1184 are shown in SEQ ID NO.2 and SEQ ID NO.3. The sequences are compared with the NCBI database (https://blast.ncbi.nlm.nih.gov), and the results suggest that they are related to the gene hp1181 and hp1184 have the highest homology.

Table 2 Primer sequences, fragment lengths and annealing temperatures of multidrug-resistant genes hp1181 and hp1184

It was verified by experiments that the multidrug-resistant Helicobacter pylori GZ6B5 simultaneously carries two multidrug-resistant genes hp1181 and hp1184 (Fig. 6), the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3.

Example 4 Application of Helicobacter pylori GZ6B5 strain resistant to multiple antibiotics

The specific application of Helicobacter pylori GZ6B5 is mainly reflected in two aspects: (1) quality control material: the bacteria can be used as a positive control for the detection of common antibiotic resistance phenotypes and resistance gene expression of Helicobacter pylori; (2) drug screening Targeted strain: This strain can be used to screen functional microorganisms/drugs/antibacterial materials. Its specific application method is as follows:

(1) Positive quality control for drug susceptibility test:

When using the antibiotic agar dilution method or broth dilution method to detect the antibiotic susceptibility of clinically isolated Helicobacter pylori, adjust GZ6B5 to the same McFarland turbidity or OD _600nm as the strain to be tested, and add GZ6B5 to the detection system as a positive control. The drug susceptibility test method was the same as that in Example 2. At the end of the test, the drug susceptibility results of GZ6B5 were read synchronously to judge whether the entire drug susceptibility testing system (including culture medium, culture environment and antibiotic concentration) was normal.

(2) Targeted strains for drug screening:

①As a target strain for screening microorganisms with antibacterial function:

Take the lactic acid bacteria cryopreserved solution with bacteriostatic potential to be tested and inoculate it on a lactic acid bacteria culture plate (such as MRS plate), cultivate it anaerobically at 37°C for 48 hours, and then pick a single colony and inoculate it into a lactic acid bacteria culture broth (such as MRS broth). , 37 ℃ anaerobic culture for 24 hours. The lactic acid bacteria to be tested were inoculated into the lactic acid bacteria culture broth at 1% (v/v) inoculum amount, and cultured anaerobically at 37°C for 48 hours. After the cultivation, the bacterial solution was taken and centrifuged at 10000g × for 15 minutes at 4°C to obtain the fermentation supernatant, which was filtered with a 0.22 μm filter membrane to obtain the sterilized probiotic fermentation supernatant, which was frozen in a -80°C refrigerator for later use.

Resuscitate the GZ6B5 cryopreserved at -80°C or in liquid nitrogen and carry out expanded culture. The methods of resuscitation and expanded culture are the same as those in Example 1. A GZ6B5 bacterial suspension with a concentration of 1×10 ⁸ cfu/mL was prepared, and 100 μL was spread evenly on a Columbia agar blood plate. The sterilized Oxford cup was placed on the blood plate evenly coated with GZ6B5, and the standby sterilized probiotic fermentation supernatant to be tested was added to the Oxford cup, and an equal volume of lactic acid bacteria culture broth was taken as a negative control. Put the blood plate with the probiotic fermentation broth added into the sealed culture tank of the microaerobic gas generating bag, and put the sealed culture tank into a 4°C incubator for 12 hours. Then, the culture tank in the microaerobic state was transferred to a 37°C incubator for 72 hours. After the cultivation, the Oxford cup was removed, and the diameter of the bacteriostatic ring produced by the probiotic fermentation broth was measured (Fig. 7).

②As target strains for drug screening:

Precisely weigh the drug to be tested and dissolve it in a suitable solvent (such as double distilled water, ethanol, 5% DMSO, etc.) to prepare a storage solution of the drug to be tested. Use a suitable solvent (such as sterile double-distilled water) to dilute the working solution of the drug to be tested to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the drug, and cryopreserved Reserve at 4°C.

Resuscitate the GZ6B5 cryopreserved at -80°C or in liquid nitrogen and carry out expanded culture. The methods of resuscitation and expanded culture are the same as those in Example 1. A GZ6B5 bacterial suspension with a concentration of 1×10 ⁸ cfu/mL was prepared, and 100 μL was spread evenly on a Columbia agar blood plate. Place the sterilized Oxford cup on the blood plate evenly coated with GZ6B5, add the prepared medicinal solution to be tested in the Oxford cup, and take an equal volume of medicinal solvent as a negative control. Put the blood plate with the drug added into the sealed culture tank of the microaerobic gas production bag, and put the culture tank into a 4°C incubator for 12 hours. Then, the culture tank in the microaerobic state was transferred to a 37°C incubator for 72 hours. After the incubation, the Oxford cup was removed, and the diameter of the antibacterial ring produced by the drug was measured.

Select the drug to be tested that is sensitive to GZ6B5 (diameter of inhibition zone ≥10 mm), and determine the MIC value of GZ6B5 to the drug according to the micro liquid-based dilution method recommended by CLSI. Add 100 μL of H. pylori proliferation medium to wells 1-10 of each row of a sterile 96-well flat-bottom microplate. Add 100 μL of the drug solution with the highest concentration to the first well of each row, mix by pipetting, and then add 100 μL of the diluted drug solution from the first well to the second well, pipetting and mixing, and then take it from the second well. 100 μL of the diluted drug solution was added to the third well. And so on until the 10th well, take 100 μL of the diluted drug solution from the 10th well and discard. Referring to the method of Example 1, a GZ6B5 bacterial suspension with a concentration of 1×10 ⁸ cfu/mL was prepared, and 100 μL of GZ6B5 bacterial suspension was added to each microculture well with drugs of different concentrations, and 100 μL of GZ6B5 bacterial suspension was added to the 11th well. 100 μL of Helicobacter pylori proliferation medium without drugs was used as a positive control, and 200 μL of Helicobacter pylori proliferation medium without drugs was added to the 12th well as a negative control, and the mixture was gently shaken and mixed. The 96-well plate was cultured in a microaerophilic environment at 37°C for 72 hours. After 72 hours, the drug was taken out for visual observation and OD _450nm detection, and the drug concentration in the lowest concentration drug hole with the solution clear and translucent, OD _450nm significantly lower than that of the positive control and similar to the negative control was taken as the MIC value. Three biological replicates were performed for each drug to determine MIC values.

After the MIC was determined, the mixed culture containing GZ6B5 in 3 to 5 wells before the MIC value was taken, and then transferred to Columbia agar blood plates, cultured at 37°C for 72 hours in a microaerophilic environment, and taken out after 72 hours. Plate observation, the lowest drug concentration at which the mixture does not form colonies is the MBC value of the drug.

③ Target strains for screening as antibacterial materials:

According to GB15979 Appendix C - Test Method for Stability of Bactericidal Properties and Antibacterial Properties of Products, the antibacterial efficacy of the antibacterial materials to be tested against multidrug-resistant Helicobacter pylori was tested. The specific method is as follows: with reference to the method of Example 1, a GZ6B5 bacterial suspension with a concentration of 1×10 ⁸ cfu/mL is prepared. Take the test sample (2.0cm×3.0cm) or 5ml of sample solution and the control sample (the same material and size as the sample, but without antibacterial material, and sterilized), 4 pieces each, and 4 sets of feces. in 4 sterile petri dishes. Take 100 μL of GZ6B5 bacterial suspension and drop 100 μL on each test piece and control sample, spread evenly, and put the sample into a test tube containing 5 mL of the corresponding neutralizer with sterile tweezers after 2, 5, 10, and 20 minutes of action. Then, take 2 to 3 dilutions, dilute and spread them on Columbia blood plate, place the plate in a microaerophilic environment at 37°C for 72 hours, and take it out after 72 hours. Plates for colony counts.

The test was repeated 3 times, and the bactericidal rate/bacteriostatic rate was calculated according to the following formula:

In the formula, χ is the bactericidal rate/bacteriostatic rate (%), A _control is the average number of colonies of the control substance, A _test is the average number of colonies of the tested sample, and A _0h is the average number of colonies at the beginning.

According to the chemical composition of the antibacterial agent in the antibacterial material, refer to Appendix C of GB15979, and evaluate the antibacterial performance of the material according to the bactericidal/bacteriostatic rate of the antibacterial material to be tested.

Example 5 Specific molecular target recognition of multidrug-resistant Helicobacter pylori GZ6B5

5.1. Specific molecular target mining of multidrug-resistant Helicobacter pylori GZ6B5

Prokka (v1.11) and Roary (v3.11.2) software were used to perform pan-genome analysis on the whole genomes of multidrug-resistant Helicobactor pylori GZ6B5 and other 1840 strains of Helicobacter pylori in the NCBI database. After obtaining the core genome, Gubbins (v2.4.1) was used to identify genes containing higher density of base substitutions. The specific sequence of Helicobactor pylori GZ6B5 obtained based on pan-genome analysis is different from other Helicobacter pylori. Oligo (v7) software was used to design primers for the specific sequence, and the specific molecular target sequence SEQ.ID No.6 for identifying the bacteria was obtained, and the amplification primers were designed: SEQ.ID No.4 and SEQ.ID No.5.

5.2. Validation of Helicobacter pylori-specific molecular recognition targets

Polymerase chain reaction (PCR) and agarose electrophoresis were used to verify the validity of Helicobactor pylori GZ6B5 specific molecules in recognizing target sequences. The detection template is the DNA of Helicobacter pylori, and the DNA extraction method is the same as that described above.

The PCR reaction system configuration is as follows:

The following are the PCR reaction conditions:

After PCR, 5-10 μL PCR products were taken for 1.5% agarose electrophoresis. Helicobactor pylori GZ6B5 can form a single specific band at 359 bp, while other Helicobacter pylori cannot form a single band of 359 bp, indicating that this pair of targets has a good ability to recognize Helicobactor pylori GZ6B5. The PCR product was sent to next-generation sequencing (completed by Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain SEQ.ID No.6, which is the specific recognition sequence of Helicobacter pylori GZ6B5.

As shown in Fig. 8, except that the DNA of Helicobactor pylori GZ6B5 was amplified by SEQ.ID No.4 and SEQ.ID No.5 to form a specific amplification product of 359bp, the remaining Helicobacter pylori isolates No specific amplification product was formed. The results suggest that the molecular target sequence SEQ.ID No.6, and its amplification primers SEQ.ID No.4 and SEQ.ID No.5 can specifically identify Helicobactor pylori GZ6B5 from other Helicobacter pylori.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that , the technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

<110> Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbiological Analysis and Testing Center)

Guangdong Huankai Biotechnology Co., Ltd.

<120> A multi-drug resistant Helicobacter pylori and its application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1502

<212> DNA

<213> Helicobacter pylori GZ6B5 (Helicobactor pylori)

<400> 1

tatggagagt ttgatcctgg ctcagagtga acgctggcgg cgtgcctaat acatgcaagt 60

cgaacgatga agctttctag cttgctagaa ggctgattag tggcgcacgg gtgagtaacg 120

cataggtcat gtgcctctta gtttgggata gccattggaa acgatgatta ataccagata 180

ctccctacgg gggaaagatt tatcgctaag agatcagcct atgtcctatc agcttgttgg 240

taaggtaatg gcttaccaag gctatgacgg gtatccggcc tgagagggtg aacggacaca 300

ctggaactga gacacggtcc agactcctac gggaggcagc agtagggaat attgctcaat 360

gggggaaacc ctgaagcagc aacgccgcgt ggaggatgaa ggttttagga ttgtaaactc 420

cttttgttag agaagataat gacggtatct aacgaataag caccggctaa ctccgtgcca 480

gcagccgcgg taatacggag ggtgcaagcg ttactcggaa tcactgggcg taaagagcgc 540

gtaggcggga tagtcagtca ggtgtgaaat cctatggctt aaccatagaa ctgcatttga 600

aactactatt ctagagtgtg ggagaggtag gtggaattct tggtgtaggg gtaaaatccg 660

tagagatcaa gaggaatact cattgcgaag gcgacctgct ggaacattac tgacgctgat 720

tgcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccctaaacga 780

tggatgctag ttgttggagg gcttagtctc tccagtaatg cagctaacgc attaagcatc 840

ccgcctgggg agtacggtcg caagattaaa actcaaagga atagacgggg acccgcacaa 900

gcggtggagc atgtggttta attcgaagat acacgaagaa ccttacctag gcttgacatt 960

gagagaatcc gctagaaata gtggagtgtc tggcttgcca gaccttgaaa acaggtgctg 1020

cacggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080

ccctttctta gttgctaaca ggttatgctg agaactctaa ggatactgcc tccgtaagga 1140

ggaggaaggt ggggacgacg tcaagtcatc atggccctta cgcctagggc tacacacgtg 1200

ctacaatggg gtgcacaaag agaagcaata ctgcgaagtg gagccaatct tcaaaacacc 1260

tctcagttcg gattgtaggc tgcaactcgc ctgcatgaag ctggaatcgc tagtaatcgc 1320

aaatcagcca tgttgcggtg aatacgttcc cgggtcttgt actcaccgcc cgtcacacca 1380

tgggagttgt gtttgcctta agtcaggatg ctaaattggc tactgcccac ggcacacaca 1440

gcgactgggg tgaagtcgta acaaggtaac cgtaggtgaa cctgcggttg gatcacctcc 1500

tt 1502

<210> 2

<211> 527

<212> DNA

<213> Helicobacter pylori GZ6B5 (Helicobactor pylori)

<400> 2

caaagcgatg gcaatcatgg gagcgtttat tttcattagc ttcactataa gcatggcgat 60

tggccctggg gttgtggcgt tttttggggg agcaaaatgg ctctttttac tcactgcgat 120

cttaacttta ttgagtttat tgatgctttt aaaagtcaaa gacgccccta aaatttctta 180

ccagatcaaa aacataaaag cttatcaacc caactctaaa gccttgtatc ttttatatct 240

aagctctttt tttgaaaaag cgttcatgac gcttattttt gtgctgatcc ctttagcctt 300

agtgaatgaa ttccataaag atgaaagttt tttgatcttg gtgtatgtgc ctggtgcctt 360

attaggggtt ttaagcatgg gagtagcgag cgttatggct gaaaaataca acaagcctaa 420

aggagtgatg ctttctggtg cgttgttgtt tattgtgagt tatttgtgct tgtttttagc 480

cgactctagc tttttaggaa aatatttgtg gctctttatt cttgggg 527

<210> 3

<211> 444

<212> DNA

<213> Helicobacter pylori GZ6B5 (Helicobactor pylori)

<400> 3

tcaaagcacg gcagaattta gcgcttctgt tatgattttg ttgtttaata ccgctatcat 60

gcacactgca ggggaaaggt ttgtgagcat gtatgggatc gttatgtata atgcgattat 120

cttttttacg actttgtttg cgatttctca aggcatccaa ccgattgcga gttttagcta 180

tggggctaga aatttagagc gcgtgaaaga ggtgtttgtc tttggtttga aagtggcgtt 240

ttgtataggg attgttttct atggcgctta ttatttctta gatgaatttt taatcaagct 300

ttatttgcag ccaagcgaac aagatgcact ctttatgcaa gagactaaaa gagcgatgaa 360

tatttattat gttggctatg ttttttttagg catgactttg ttgtgtgcgg tgtttttcca 420

atccatccaa cgcaccaaaa gttc 444

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

tgcgatgaca acgaacaagc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

cctttcggtg gtgctgtgta 20

<210> 6

<211> 359

<212> DNA

<213> Helicobacter pylori GZ6B5 (Helicobactor pylori)

<400> 6

cctttcggtg gtgctgtgta tcctttgtat tcatatatta aattagcggt tggcaatccg 60

gggtttgcta atggataagg catccataac tttcccttat gtttgcctat ctcatctatt 120

ctggtaaatc tctttttaat atattcttga gcatgttttg attgtttttc tttaataatt 180

tttattttat aattctctgt attagcataa agcaaaatac tatcatacgc cgtccctaac 240

ctttttgttt catttgcatt tccaaatttt cgtttccata ctatacaatt cacaaaattc 300

tccctaagaa aaatctcatc catcaacact ttcaagtaag cttgttcgtt gtcatcgca 359

Claims (2)

1. Helicobacter pylori GZ6B5, deposit number GDMCC No: 61571. 2 . A nucleotide sequence capable of specifically recognizing Helicobacter pylori GZ6B5 according to claim 1 , wherein the sequence is shown in SEQ ID NO.6. 3 .

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