CN113186136B - Multiple drug-resistant helicobacter pylori and application thereof - Google Patents
Multiple drug-resistant helicobacter pylori and application thereof Download PDFInfo
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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
The technical field is as follows:
the invention belongs to the technical field of microbiology and medicine, and particularly relates to helicobacter pylori which simultaneously carries two drug-resistant genes of hp1181 and hp1184 and is resistant to five common clinical antibiotics and application thereof.
Background art:
helicobacter Pylori (HP) is a gram-negative, microaerophilic helical bacterium and is closely associated with human gastrointestinal diseases. Studies have shown that infection with helicobacter pylori causes chronic gastritis and promotes the occurrence and development of various digestive tract diseases, such as dyspepsia, peptic ulcer and gastric cancer. Among them, the most serious consequence of helicobacter pylori infection is the induction of gastric cancer, and studies have pointed out that 90% of gastric cancer onset in the world is closely related to helicobacter pylori infection. According to the statistics of the world health organization, gastric cancer is the fourth cancer with the worldwide incidence rate and the second mortality rate, and causes nearly 75 million deaths each year. Therefore, the helicobacter pylori infection seriously threatens the health of human beings, causes huge economic and social losses, and has great application and research values in preventing and treating the helicobacter pylori infection.
The treatment regimen for H.pylori is quite different from other microbial infections. Because the isolation and culture difficulty of the bacteria is very high, doctors mainly rely on clinical experience and statistical results of large-scale health organizations (such as the world health organization) to make anti-infection schemes. Currently, the recommended regimen for the treatment of helicobacter pylori infections by the world health organization is a triple therapy based on clarithromycin: treating with proton pump inhibitor, clarithromycin, amoxicillin, metronidazole or tinidazole for 14 days. However, in recent years, the effectiveness of anti-H.pylori treatment has been decreasing, and the success rate of anti-H.pylori treatment in China has been lower than 80%. Failure of therapy against helicobacter pylori infection is mainly associated with increased resistance of the bacterium to antibiotics. Research shows that the helicobacter pylori infected by people in Jiangzhe and Zhejiang areas in China has 21.5 percent of drug resistance to clarithromycin, 95.4 percent of drug resistance to metronidazole and 20.6 percent of drug resistance to levofloxacin, and simultaneously, the drug resistance ratio of the helicobacter pylori to three clinical common antibiotics reaches 7.6 percent. Because helicobacter pylori in China has wide drug resistance to antibiotics, the conventional treatment scheme cannot effectively control the helicobacter pylori infection in China. Therefore, aiming at the problem of multi-drug-resistant helicobacter pylori, research and development work of new drugs, preparations and materials is carried out, and the method has great significance for improving the national health level of China.
The development of drugs, preparations and materials for solving the problem of multidrug-resistant helicobacter pylori all need targeted strains with multidrug-resistant characteristics. At present, the standard strains ATCC 26695 and ATCC43504 are mainly adopted for the development of the anti-helicobacter pylori infection medicines and materials. Among them, ATCC43504 has a drug-resistant phenotype against amoxicillin, and is more used for the development of novel antibacterial drugs and materials. However, currently, helicobacter pylori popular in China mainly shows drug resistance to metronidazole, clarithromycin and levofloxacin, the drug resistance rate to amoxicillin is only 0.1%, and currently, no corresponding standard strain which is resistant to the three antibiotics exists in China, so that the research and development work of drugs and materials aiming at the problem of drug resistance of helicobacter pylori in China is seriously hindered. Therefore, the acquisition of the target strain with clear genetic background, stable biological properties and multi-drug resistance has important significance for promoting research and development work of resisting helicobacter pylori infection.
hp1181 and hp1184 are genes that are currently known to cause multi-drug resistance in helicobacter pylori. The two genes are translocases located on the chromosome of helicobacter pylori, wherein hp1181 belongs to RND (resistance-reduction-division) efflux pump family, and the encoded protein has the function of supplying and efflux a plurality of antibiotics through a proton pump; hp1184 belongs to the MATE (matrix and toxin compound exclusion) efflux pump family, and the encoded protein has the function of actively efflux a plurality of toxic compounds and medicaments to the outside of cells. Aiming at the two drug-resistant genes, the design of drugs and materials for preventing and reversing the multidrug resistance of helicobacter pylori has higher application value.
Therefore, by combining the epidemic situation of helicobacter pylori resistant antibiotics in China, the targeted strain with clear genetic background, definite drug-resistant gene carrying and stable multi-drug resistant phenotype is developed, the novel antibacterial drug, preparation and material aiming at the helicobacter pylori infection problem in China are favorably developed, the technical bottleneck of prevention and control of helicobacter pylori in public health and clinical treatment at present can be helped to be relieved, and the health level of China is favorably improved.
Disclosure of Invention
The first purpose of the invention is to provide Helicobacter pylori (Helicobacter pylori) GZ6B5 which is resistant to five antibiotics such as amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline, and is preserved in Guangdong province microorganism culture Collection (GDMCC), and the accession number is GDMCC No: 61571, the preservation date is 2021, 3 months and 19 days, and the preservation address is Guangdong province microbial strain preservation center of No. 59 building, No.5 building, Guangdong province of No. 100 college of Miehuo, Guangzhou.
The second purpose of the invention is to provide the application of the helicobacter pylori GZ6B5 in screening functional microorganisms, medicines, preparations or antibacterial materials for resisting helicobacter pylori infection. The method has important significance for promoting research and development work of resisting helicobacter pylori infection.
The functional microorganism, medicine or antibacterial material is a functional microorganism, medicine, preparation or antibacterial material for inhibiting helicobacter pylori resistant to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline.
The third purpose of the invention is to provide a nucleotide sequence which can specifically recognize helicobacter pylori GZ6B5 and is shown as SEQ ID NO. 6.
The fourth purpose of the invention is to provide a primer which can specifically identify helicobacter pylori GZ6B5, and the primer is shown as SEQ ID NO.4 and SEQ ID NO. 5.
The fifth purpose of the invention is to provide a method for specifically identifying helicobacter pylori GZ6B5, which is to perform PCR amplification on helicobacter pylori to be detected by adopting the primers, wherein if a band can be generated, the bacterium to be detected is the helicobacter pylori GZ6B5, and if the band cannot be generated, the bacterium to be detected is not the helicobacter pylori GZ6B 5.
The PCR amplification comprises the following reaction systems:
the PCR amplification has the following PCR reaction conditions: 7min at 95 ℃; 30s at 95 ℃, 30s at 65 ℃ and 30s at 72 ℃; 30 cycles; 10min at 72 ℃.
The Helicobacter pylori (Helicobacter pylori) GZ6B5 is a Helicobacter pylori strain which can resist 5 clinical common antibiotics at the same time, and carries 2 drug-resistant genes hp1181 and hp1184 at the same time. The helicobacter pylori capable of tolerating 5 antibiotics is an important target strain in the research of a drug resistance mechanism of the helicobacter pylori, and can be used for screening novel functional microorganisms, drugs and antibacterial materials.
Compared with the prior art, the invention has the beneficial effects that: 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.
Helicobacter pylori GZ6B5 was deposited at 19 days 3 and 2021 in the Guangdong province culture Collection with the deposit number GDMCC No.61571 and the deposit addresses: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070.
drawings
FIG. 1 is a colony pattern of Helicobacter pylori (Helicobacter pylori) GZ6B5 on a Helicobacter pylori selective medium;
FIG. 2 is a gram-stained Helicobacter pylori (Helicobacter pylori) GZ6B5 under a microscope;
FIG. 3 is an analysis of the evolutionary relationship of Helicobacter pylori (Helicobacter pylori) GZ6B5 based on the 16S rRNA gene in Helicobacter;
FIG. 4 is an ANI analysis of H.pylori (Helicobacter pylori) GZ6B5 at the genome-wide level with different subtypes of H.pylori in the database, note: the content of GZ6B5 is 95.20-96.79% compared with the eastern subtype, American type and Pecan type helicobacter pylori whole genome ANI, 94.00-94.83% compared with Asia type and European type, and 92.74-93.87% compared with Africa type and SJM 180;
FIG. 5 shows the drug sensitivity of Helicobacter pylori (Helicobacter pylori) GZ6B5 to five antibiotics, and is given by: GZ6B5 was grown in medium with different concentrations of five antibiotics. The culture medium comprises from top to bottom: amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline, and the drug concentrations are shown in the drawing. The upper hole of the rightmost row is a chromogenic medium without adding GZ6B5, the lower hole of the rightmost row is a chromogenic medium without adding antibiotics but with adding GZ6B5, and the OD of the GZ6B5 is OD after culturing for 72 hours in chromogenic media with adding five antibiotics with different concentrations450nmA detected value heatmap of;
FIG. 6 is an agarose electrophoresis picture of PCR products of Helicobacter pylori (Helicobacter pylori) GZ6B5 drug-resistant genes hp1181 and hp 1184;
FIG. 7 shows the application of Helicobacter pylori (Helicobacter pylori) GZ6B5 in screening functional microorganisms against multidrug-resistant Helicobacter pylori;
FIG. 8 shows the agar electrophoresis validation of the molecular target of Helicobacter pylori (Helicobacter pylori) GZ6B5, note: m is DNA marker, and the sizes of the bands from top to bottom are shown in the figure. 1 is the electrophoresis condition of PCR products of GZ6B5 amplified by molecular targets, 2 and 3 are the electrophoresis conditions of PCR products of helicobacter pylori ATCC43504 and Sydney SS1 amplified by molecular targets, and 4 to 23 are the electrophoresis conditions of PCR products of different helicobacter pylori isolates amplified by molecular targets. The results suggest that 359bp specific PCR product can be formed by GZ6B5 after molecular target amplification, and similar PCR product can not be formed by the rest helicobacter pylori.
The specific implementation scheme is as follows:
the invention is further illustrated with reference to specific examples.
The media and reagents involved in the following examples are as follows:
preparation of antibiotic suspension: 10mg/mL of vancomycin, 5mg/mL of amphotericin B, 20mg/mL of trimethoprim and 10mg/mL of cefsulodin, and the antibiotics are dissolved in sterilized water according to the content and are uniformly mixed.
Helicobacter pylori selection medium (g/L): 3.0g/L of yeast extract powder, 12.0g/L of casein peptone, 5.0g/L of animal tissue digests, 3.0g/L of beef extract powder, 1.0g/L of corn starch, 5.0g/L of sodium chloride, 13.5g/L of agar, 70mL/L of sterile sheep red blood cells or fetal calf serum and 10mL/L of antibiotic suspension, dissolving the components in sterile water according to the content, and uniformly mixing.
Helicobacter pylori proliferation medium (g/L): 2.02g/L yeast extract powder, 10.52g/L tryptone, 1.56g/L glucose, 0.10g/L sodium bisulfite, 10.0g/L soluble starch, 5.0g/L sodium chloride, 70mL/L fetal calf serum and 10mL/L antibiotic suspension, and the components are dissolved in sterilized water according to the content and are uniformly mixed.
Columbia agar blood plates (g/L): 10.0g/L of pancreatic casein digest, 5.0g/L of gastric and meat enzyme digest, 3.0g/L of pancreatic heart digest, 5.0g/L of yeast extract powder, 1.0g/L of corn starch, 5.0g/L of sodium chloride, 15.0g/L of agar and 70mL/L of sterile sheep erythrocyte, and the components are dissolved in sterilized water according to the content and are uniformly mixed.
Helicobacter pylori preservation solution (g/L): 4.0g/L of peptone, 0.8g/L of yeast extract, 4.0g/L of tryptone, 2.0g/L of sodium chloride, 0.4g/L of glucose, 0.04g/L of sodium bisulfite, 0.1g/L of sodium metabisulfite, 0.1g/L of ferrous sulfate heptahydrate, 0.1g/L of sodium pyruvate and 200mL/L of glycerol, and the components are dissolved in sterilized water according to the content and are uniformly mixed.
Example 1 isolation, culture and identification of helicobacter pylori
1.1. Isolation and culture of helicobacter pylori
(1) Separation and preservation of helicobacter pylori: helicobacter pylori GZ6B5 was isolated from gastric mucosal tissue of chronic gastritis patients who were not effective in several antibiotic treatments in Guangzhou. In sterile environment, fresh gastric mucosa sample is spread on helicobacter pylori selective medium, and placed in 37 deg.C microaerophilic environment (5% O)2、10％CO2、85％N2) Culturing for 5-7 days. And (3) picking colonies with typical shapes on the plate to a helicobacter pylori selective medium plate for streak purification, after 2 times of purification, picking a single colony to inoculate into a helicobacter pylori proliferation medium, and culturing for 96 hours under a microaerobic condition at 37 ℃. Adding 400 μ L of the bacterial liquid into 600 μ L of helicobacter pylori preservation liquid, gently blowing, stirring, and storing in an ultra-low temperature refrigerator at-80 deg.C. Thereby obtaining a strain GZ6B 5.
(2) And (3) resuscitation and culture of helicobacter pylori: 10. mu.L of the stock solution of strain GZ6B5 was dipped from the storage tube, spread on a helicobacter pylori selection medium, and cultured in a microaerophilic atmosphere at 37 ℃ for 72 to 96 hours to obtain resuscitated helicobacter pylori GZ6B5 as a needle-tip-sized translucent colony on a plate (FIG. 1). Picking single colony from a plate containing recovered helicobacter pylori GZ6B5, inoculating the single colony in a helicobacter pylori proliferation culture medium, and culturing for 72-96 hours in a microaerophilic environment at 37 ℃ to obtain a proliferated helicobacter pylori GZ6B5 bacterial liquid.
1.2. Identification of helicobacter pylori
The purified helicobacter pylori GZ6B5 can be identified by morphological characteristics, molecular biology, protein mass spectrum and the like.
(1) And (3) dyeing microscopic examination: the helicobacter pylori GZ6B5 is smeared, gram staining is carried out, and the morphology is observed through microscopic examination. The helicobacter pylori GZ6B5 of the present invention is shown to be gram-negative and microscopically S-shaped or arcuately curved (FIG. 2).
(2) Molecular characterization based on 16S rRNA gene sequence: helicobacter pylori DNA was extracted using a bacterial DNA extraction kit (Mabio, CHINA), and then amplified using Polymerase Chain Reaction (PCR) using 2 × PCR mix (Dongshengbio, CHINA). The PCR amplification primer adopts a 16S rRNA gene universal primer, and the sequence of an upstream primer is 27F: 5'-AGAGTT TGATCC TGG CTCAG-3', respectively; the sequence of the downstream primer is 1492R: 5'-CTAC GGC TAC CTT GTTACGA-3' are provided. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ 30s, 56 ℃ 30s and 72 ℃ 1min 30s, and annealing and extending at 72 ℃ for 10 min. The PCR product was recovered by cutting and then subjected to one-generation sequencing (performed by Jinzhi Biotechnology, Inc., Suzhou). The sequence of the obtained 16S rRNA gene is shown in SEQ ID No. 1. This sequence was aligned with the NCBI database (https:// blast. NCBI. nlm. nih. gov), which suggested the highest homology with H.pylori, and was designated H.pylori (Helicobacter pylori) GZ6B 5. A Neighbor-Joining method is adopted to establish a genetic evolutionary tree of Helicobacter pylori (Helicobacter pylori) GZ6B5 and other Helicobacter standard strains in NCBI database, and genetic evolutionary shows that GZ6B5 has a closest genetic relationship with Helicobacter pylori strain ATCC43504, and the similarity is 99.56%, as shown in FIG. 3.
Analysis of average nucleotide identity at the genome level: average Nucleotide Identity (ANI) analysis based on the entire genome of a microorganism is the gold standard that defines prokaryotic species at the genome level, ANI values above 95% being considered as the same species. After extraction of the whole genomic DNA of H.pylori GZ6B5, Library construction was performed using QIAseq FX DNA Library Kit (Qiagen, Germany), and High throughput sequencing was performed on the Illumina Nextseq 550 platform using the High Output v2.5 Kit (Illumina, USA). And (3) assembling the off-line data by using Trimmomatic (v0.39) software quality control and SPAdes (v3.13.1) software. The average nucleotide identity of the whole genome of the helicobacter pylori of different subtypes of GZ6B5 and NCBI database 29 strain was analyzed by ANI calcotor software (http:// ave-omics.ce.gatech.edu/ANI/index), and the results are shown in FIG. 4, which indicates that the helicobacter pylori GZ6B5 is highly homologous with the eastern and American helicobacter pylori, and ANI is above 95%.
The appearance, morphology, gram staining, 16S rRNA gene comparison and genome ANI analysis result of the strain are combined, and the GZ6B5 is the helicobacter pylori. It is named as Helicobacter pylori (Helicobacter pylori) GZ6B5, and is deposited in Guangdong province microorganism culture Collection (GDMCC), and the deposition number is GDMCC No: 61571, the preservation date is 2021, 3 months and 19 days, and the preservation address is Guangdong province microbial strain preservation center of No. 59 building, No.5 building, Guangdong province of No. 100 college of Miehuo, Guangzhou.
Example 2 drug susceptibility profiling of multiple antibiotic resistant helicobacter pylori GZ6B5
The helicobacter pylori GZ6B5 is subjected to drug sensitivity detection by adopting an agar dilution method, and the helicobacter pylori ATCC43504 is adopted as a quality control strain. Assays were performed according to methods and Standards established by The European Committee for Antimicrobial drug Susceptibility Testing (EUCAST) and The American society for Clinical and Laboratory Standards Institute (CLSI).
2.1. Agar dilution method
Dissolving powders of five antibiotics of amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline in corresponding solutes according to a CLSI recommended mode to prepare an antibiotic stock solution, and then respectively adding the antibiotic stock solution into a helicobacter pylori selective culture medium according to the drug sensitivity Concentration to be detected to prepare a helicobacter pylori drug sensitivity detection plate containing the corresponding antibiotic Concentration so as to detect the Minimum Inhibitory Concentration (MIC) of the antibiotic to helicobacter pylori GZ6B 5. Adjusting the concentration of the suspension of helicobacter pylori GZ6B5 to be detected to 1 × 107cfu/mL, inoculating 10 μ L of the bacterial suspension to a detection plate containing antibiotics with corresponding concentration, culturing for 72 hours in a microaerophilic environment at 37 ℃, and observing whether the detection plate containing the antibiotics with corresponding concentration has the colony formation of helicobacter pylori. The MIC of this antibiotic for H.pylori was taken as the concentration in the lowest concentration drug plate that failed to form colonies. Each drug concentration was run in 3 replicates and the mean MIC was obtained.
Experiments prove that the minimum inhibitory concentrations of the helicobacter pylori GZ6B5 to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline are respectively 1mg/L, 2mg/L, 32mg/L and 8mg/L which are higher than the drug resistance standard of the EUCAST standard, so that the helicobacter pylori is judged to be the multi-drug resistant helicobacter pylori.
2.2. Broth dilution method
Dissolving powders of five antibiotics of amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline in corresponding solutes according to a CLSI recommended mode to prepare antibiotic stock solutions, then respectively adding the antibiotic stock solutions into a helicobacter pylori chromogenic medium (Yangguangbio, CHINA) according to the drug sensitivity concentration to be detected to prepare the antibiotic-containing chromogenic medium with the corresponding detection concentration, and preparing the antibiotic-containing chromogenic medium for use at present. 100. mu.L of chromogenic medium containing different concentrations of drug was added to a 96-well plate, and then 100. mu.L of 10-concentration chromogenic medium was added to each well5cfu/mL helicobacter pylori GZ6B 5. Culturing 96-well plate in microaerophilic environment at 37 deg.C for 72 hr, observing color change of different drug wells, and detecting OD of each well by using protease microplate reader (BIOTEK, USA)450nmThe value is obtained. By color and OD450nmThe drug concentration in the lowest concentration drug well with no change was the minimum inhibitory concentration of this antibiotic for GZ6B5, as shown in fig. 5. Each drug concentration was subjected to 3-time parallel operations to obtain the average of the minimum inhibitory concentration. The drug sensitivity of helicobacter pylori GZ6B5 to amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline is shown in Table 1, and the helicobacter pylori GZ6B5 is known to be resistant to the five antibiotics.
TABLE 1 susceptibility of pyloric screw rod GZ6B5 to different antibiotics
Example 3 detection of drug-resistant Gene of helicobacter pylori GZ6B5 that is resistant to various antibiotics
Aiming at the multi-drug resistance phenotype characteristics of the helicobacter pylori GZ6B5, two common multi-drug resistance genes of the helicobacter pylori, namely hp1181 and hp1184, are selected to carry out PCR detection on the helicobacter pylori. The primer sequences and PCR amplification conditions were as described in the reference (Falsafi T, 2016). The primer sequences, the lengths of the target gene fragments and the annealing temperatures are shown in Table 2, and the PCR conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ for 30s, annealing temperature for 30s and 72 ℃ for 1min for 30 s; annealing and extending for 10min at 72 ℃. After the PCR was completed, 8. mu.L PCR product was taken out and electrophoresed on 1.5% agarose gel (120V, 25min), and the 100bp DNA Ladder (Dongshengbio, CHINA) was aligned and observed for the presence or absence of the formation of the objective band. For the PCR products with target bands, cut gel recovery was performed for one-generation sequencing (completed by Jinzhi Biotechnology, Inc., Suzhou). The sequences of hp1181 and hp1184 obtained are shown in SEQ ID NO.2 and SEQ ID NO.3, and the sequences are compared with NCBI database (https:// blast. NCBI. nlm. nih. gov), and the results indicate that the homology with the genes hp1181 and hp1184 is highest.
TABLE 2 primer sequences of multiple drug-resistant genes hp1181 and hp1184, fragment lengths and annealing temperatures
Experiments prove that the multi-drug resistant helicobacter pylori GZ6B5 simultaneously carries two multi-drug resistant genes hp1181 and hp1184 (shown in figure 6), and the nucleotide sequences of the multi-drug resistant helicobacter pylori GZ6B5 are shown as SEQ ID NO.2 and SEQ ID NO. 3.
Example 4 use of multiple antibiotic resistant strains of helicobacter pylori GZ6B5
The specific application of the helicobacter pylori GZ6B5 is mainly embodied in two aspects: (1) quality control product: the strain can be used as a positive control for detecting the drug-resistant phenotype and the drug-resistant gene expression of common antibiotics of helicobacter pylori; (2) drug screening of targeted strains: the strain can be used for screening functional microorganisms/medicines/antibacterial materials. The specific application method is as follows:
(1) positive quality control of drug sensitive test:
when the antibiotic agar dilution method or broth dilution method is adopted to detect the clinical separated helicobacter pylori antibiotic drug sensitivity, the GZ6B5 is adjusted to the same McLeod turbidity or OD as the strain to be detected600nmAdding to the test systemGZ6B5 served as a positive control. The method of susceptibility testing was the same as in example 2. And synchronously reading the drug sensitivity result of GZ6B5 when the detection is finished so as to judge whether the whole drug sensitivity detection system (including a culture medium, a culture environment and the concentration of antibiotics) is normal.
(2) Drug screening of targeted strains:
the method is used for screening the target strains of the microorganisms with the antibacterial function:
inoculating the lactobacillus frozen stock solution with bacteriostatic potential to be detected on a lactobacillus culture plate (such as an MRS plate), performing anaerobic culture at 37 ℃ for 48 hours, then selecting a single colony, inoculating the single colony into lactobacillus culture broth (such as MRS broth), and performing anaerobic culture at 37 ℃ for 24 hours. The lactic acid bacteria to be tested were inoculated into the lactic acid bacteria culture broth at an inoculum size of 1% (v/v), and anaerobically cultured at 37 ℃ for 48 hours. And after the culture is finished, taking the bacterial liquid, centrifuging the bacterial liquid at 4 ℃ of 10000g multiplied by 15 minutes to obtain a fermentation supernatant, filtering the fermentation supernatant by adopting a 0.22 mu m filter membrane to obtain a sterilized probiotic fermentation supernatant, and freezing and storing the probiotic fermentation supernatant in a refrigerator at-80 ℃ for later use.
The stored GZ6B5 was frozen at-80 ℃ or in liquid nitrogen and cultured under an enlarged scale, and the recovery and culture method were the same as in example 1. Prepared at a concentration of 1X 108100. mu.L of a GZ6B5 bacterial suspension was applied evenly to Columbia agar blood plates at cfu/mL. And (3) placing the sterilized oxford cup on a blood flat plate uniformly coated with GZ6B5, adding a standby fermentation supernatant of the bacteria-removing probiotics to be detected into the oxford cup, and taking the equal volume of lactobacillus culture broth as a negative control. And (3) placing the blood plate added with the probiotic fermentation liquor into a sealed culture tank of a microaerophilic gas production bag, and placing the sealed culture tank into an incubator at 4 ℃ for standing for 12 hours. The culture tank in the microaerophilic state was then transferred to an incubator at 37 ℃ for 72 hours. And (3) after the culture is finished, taking the oxford cup away, and measuring the diameter of the bacteriostatic ring generated by the probiotic fermentation liquor (figure 7).
② the targeted bacterial strain for drug screening:
the drug to be tested is precisely weighed and dissolved in a suitable solvent (such as double distilled water, ethanol, 5% DMSO, etc.) to prepare a stock solution of the drug to be tested. A working solution of the drug to be tested is prepared by diluting with a suitable solvent (such as sterile double distilled water), is used for determining the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the drug, and is frozen and stored at 4 ℃ for later use.
The stored GZ6B5 was frozen at-80 ℃ or in liquid nitrogen and cultured under an enlarged scale, and the recovery and culture method were the same as in example 1. Prepared at a concentration of 1X 108100. mu.L of a GZ6B5 bacterial suspension was applied evenly to Columbia agar blood plates at cfu/mL. And (3) placing the sterilized oxford cup on a blood plate uniformly coated with GZ6B5, adding the prepared standby liquid medicine to be detected into the oxford cup, and taking the medicine solvent with the same volume as the negative control. The blood plate added with the drug is placed in a sealed culture tank of a microaerophilic gas production bag, and the culture tank is placed in an incubator at 4 ℃ and kept stand for 12 hours. Then, the culture tank in the microaerophilic state was transferred to an incubator at 37 ℃ for 72 hours. And (5) taking the oxford cup away after the culture is finished, and measuring the diameter of the bacteriostatic ring generated by the medicament.
Selecting a medicament to be detected which is sensitive to GZ6B5 (the diameter of the inhibition zone is more than or equal to 10mm), and determining the MIC value of GZ6B5 to the medicament according to a micro liquid-based dilution method recommended by CLSI. 100 μ L of the H.pylori multiplication medium was added to the 1 st to 10 th wells of each row of the sterile 96-well flat-bottom microplate. Adding 100 mu L of the drug solution to be tested with the highest concentration into the 1 st hole of each row, blowing, beating and uniformly mixing, then taking 100 mu L of the diluted drug solution from the 1 st hole, adding into the 2 nd hole, blowing, beating and uniformly mixing, and then taking 100 mu L of the diluted drug solution from the 2 nd hole, and adding into the 3 rd hole. The same procedure is repeated until reaching the 10 th well, and 100. mu.L of the diluted drug solution is taken out from the 10 th well and discarded. Reference example 1 preparation of 1X 10 concentration8cfu/mL GZ6B5 bacterial suspension, 100 mu LGZ6B5 bacterial suspension is added into each micro-culture hole added with different concentrations of drugs, 100 mu LGZ6B5 bacterial suspension and 100 mu L helicobacter pylori proliferation culture medium without drugs are added into the 11 th hole as positive controls, 200 mu L helicobacter pylori proliferation culture medium without drugs is added into the 12 th hole as negative controls, and the mixture is gently shaken and evenly mixed. The 96-well plate was incubated in a microaerophilic environment at 37 ℃ for 72 hours. Taking out after 72 hours, carrying out visual observation and detecting OD450nmClear and bright as solution, OD450nmLowest concentration drug wells significantly lower than positive control, similar to negative controlThe drug concentration in (c) was taken as the MIC value. MIC values were determined for each drug in three biological replicates.
After MIC is determined, mixed cultures containing GZ6B5 in 3-5 holes before the MIC value are respectively transferred to a Columbia agar blood plate, the mixed cultures are cultured for 72 hours in a microaerophilic environment at 37 ℃, the plate is taken out after 72 hours for observation, and the lowest drug concentration at which the mixture does not form a colony is taken as the MBC value of the drug.
③ the targeted strains screened as the antibacterial materials:
according to GB15979 appendix C-product sterilization performance and bacteriostatic performance stability test method, the antibacterial efficiency of the antibacterial material to be tested on multidrug-resistant helicobacter pylori is tested. The specific method comprises the following steps: reference example 1 preparation of 1X 10 concentration8cfu/mL GZ6B5 bacterial suspension. 4 pieces of each of a test sample (2.0 cm. times.3.0 cm) or a sample solution (5 ml) and a control sample (the same material as the test sample, the same size, but containing no antibacterial material, and subjected to sterilization treatment) were taken, and 4 feces were put in 4 sterilized dishes. 100 mu L of LGZ6B5 bacterial suspension is dripped on each sample to be tested and each control sample, the sample is evenly coated, after 2, 5, 10 and 20 minutes of action, the sample is thrown into a test tube containing 5mL of corresponding neutralizer by using sterile forceps, the sample is fully and evenly mixed and diluted properly, then 2-3 dilutions are taken out to be diluted and coated on a Columbia blood plate, the plate is placed in a microaerophilic environment at 37 ℃ to be cultured for 72 hours, and the plate is taken out for 72 hours to count colonies.
The test was repeated 3 times, and the bactericidal/bacteriostatic rate was calculated according to the following formula:
wherein chi is the sterilization rate/bacteriostasis rate (%), AcontrolAverage number of colonies for control, AtestThe average number of colonies of the test sample, A0hThe average number of colonies at the start.
According to the chemical composition of the antibacterial agent in the antibacterial material, and referring to GB15979 appendix C, the antibacterial performance of the material is evaluated according to the sterilization/bacteriostasis rate of the antibacterial material to be tested.
Example 5 specific molecular target recognition of multiple drug-resistant helicobacter pylori GZ6B5
5.1. Excavation of specific molecular target of multi-drug-resistant helicobacter pylori GZ6B5
Pangenomic analysis was performed on the whole genome of multiple drug resistant helicobacter pylori (helicobacter pylori) GZ6B5 and other 1840 strains of helicobacter pylori in the NCBI database using Prokka (v1.11), Roary (v3.11.2) software. After obtaining the core genome, Gubbins (v2.4.1) was used to identify genes containing a higher density of base substitutions. The specific sequence of helicobacter pylori (helicobacter pylori) GZ6B5 obtained based on pan-genomic analysis is different from that of other helicobacter pylori. And (2) adopting Oligo (v7) software to carry out primer design aiming at the specific sequence, obtaining a specific molecular target sequence SEQ.ID No.6 for identifying the bacterium, and designing an amplification primer thereof: SEQ.ID No.4 and SEQ.ID No. 5.
5.2. Validity verification of helicobacter pylori specific molecule recognition target
The effectiveness of the target sequence recognition of the GZ6B5 specific molecule of helicobacter pylori (helicobacter pylori) is verified by Polymerase Chain Reaction (PCR) and agarose electrophoresis. The detection template is the DNA of the helicobacter pylori, and the DNA extraction method is the same as the method.
The PCR reaction system is configured as follows:
the PCR reaction conditions were as follows:
after the PCR is finished, 5-10. mu.L of the PCR product is subjected to 1.5% agarose electrophoresis. The fact that the helicobacter pylori (helicobacter pylori) GZ6B5 can form a single specific band at 359bp, and the other helicobacter pylori can not form a single band of 359bp indicates that the target pair has the efficacy of well recognizing the helicobacter pylori (helicobacter pylori) GZ6B 5. The PCR product was subjected to one-generation sequencing (performed by Jinzhi Biotechnology Ltd., Suzhou) to obtain SEQ. ID No.6, which is a specific recognition sequence of helicobacter pylori GZ6B 5.
As shown in FIG. 8, no specific amplification product was formed in any of the remaining H.pylori isolates, except that the DNA of H.pylori (helicobacter pylori) GZ6B5 was amplified to 359bp specific amplification products according to SEQ.ID No.4 and SEQ.ID No. 5. The results indicate that the molecular target sequence SEQ.ID No.6 and the amplification primers SEQ.ID No.4 and SEQ ID No.5 can specifically identify the helicobacter pylori (helicobacter pylori) GZ6B5 from other helicobacter pylori.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)
GUANGDONG HUANKAI BIOTECHNOLOGY Co.,Ltd.
<120> multi-drug resistant helicobacter pylori and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1502
<212> DNA
<213> helicobacter pylori GZ6B5 (helicobacter 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 (helicobacter 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 (helicobacter 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 tttttttagg catgactttg ttgtgtgcgg tgtttttcca 420
atccatccaa cgcaccaaaa gttc 444
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 359
<212> DNA
<213> helicobacter pylori GZ6B5 (helicobacter 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 (H.pylori: (Helicobacter pylori) GZ6B5, accession number GDMCC No: 61571.
2. a nucleotide sequence capable of specifically recognizing helicobacter pylori GZ6B5 as claimed in claim 1, wherein the sequence is shown as SEQ ID No. 6.
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