The Resistance Of Helicobacter Pylori And The Comparison Of Metabolic Phenotypes Between Sensitive And Resistant Strains

Background and ObjectivesHelicobacter pylori(H.pylori)has an infection rate of more than half of the population,and it is closely related to the occurrence of gastric diseases.In clinic,antibiotics are often used in combination to eradicate H.pylori,but with the widespread use of antibiotics,the resistance rate of H.pylori is gradually increasing.At present,the research on the mechanism of multidrug resistance of H.pylori has been reported,but the difference in metabolic phenotype between H.pylori multidrug-resistant strains and sensitive strains has not been studied yet.In this study,the H.pylori resistance in the Marshall Medical Laboratory of Zhengzhou University was measured by the disc diffusion method to understand the resistance of H.pylori to clarithromycin,levofloxacin,amoxicillin,furazolidone,tetracycline and metronidazole in Henan.Then analyze the factors that may affect the resistance rate of H.pylori(gender,age,collection site,number of treatments),and use drug resistance gene detection to analyze common gene mutation modes of drug resistance.At the same time,the evidence-based medicine(Meta analysis)method was used to compare H.pylori resistance in foreign and domestic regions to guide clinicians in the rational use of antibiotics.Finally,in order to explore the resistance mechanism of H.pylori from the perspective of bacterial metabolic phenotypes,the study adopted the Biolog phenotype chip technology and used the PM1-PM4 metabolism plate to determine the metabolic phenotypes of 190 carbon sources,95 nitrogen sources,59 sulfur sources and 35 phosphorus sources of H.pylori P12 sensitive strain and 2 multi-drug resistant strains.The metabolic characteristics of H.pylori provide an information basis for the research of its function and its interaction with the host,and lay a theoretical foundation for the study of multidrug resistance of H.pylori in the clinic.Methods1.Strain cultivation: From January 2019 to June 2020,the gastric mucosal tissue blocks of patients with gastrointestinal diseases were collected by the conventional method of endoscopy in the Marshall Medical Endoscopy Room of Zhengzhou University,and inoculated on solid medium by smearing method.Cultivation and passage in a 37℃ constant temperature incubator in a micro-aerobic environment.Antimicrobial resistance was measured using the drug sensitive paper method to measure the size of the antibacterial ring.The detection of drug resistance genes is by extracting H.pylori DNA,using PCR to amplify fragments in the 23 S r RNA,gyr A and rdx A genes related to drug resistance and sequencing,and using restriction fragment length polymorphism(PCR-RFLP)to detect resistant strains Point mutations.2.Meta analysis: systematically searched 6 Chinese and English databases,selected studies related to H.pylori resistance from January 2018 to the present,and after screening the literature,the two independently extracted data.Finally,the data was statistically analyzed by Rev Man 5.3 software.3.Determination of PM metabolic phenotype: Use 96-well microplates PM1,PM2 A,PM3B and PM4 A for PM determination to determine the metabolic curves of the 3 types of H.pylori.The experiment was carried out according to the procedure developed by the manufacturer,using a sterile cotton swab to transfer H.pylori into the mixed inoculum,and load the bacterial suspension into each well of the PM microplate.All plates and anaerobic bags are sealed together in an airtight bag,and placed in an Omni Log thermostat at 37℃.Cultivate and record for 72-96 hours,and use Omni Log PM software to evaluate the data.4.Statistical analysis: The statistical software SPSS was used for statistical analysis in the data processing of this study.The chi-square test was used for correlation analysis,and P<0.05 was regarded as statistically significant.Results 1.Among 249 H.pylori strains,the drug resistance rates of clarithromycin,levofloxacin,amoxicillin,furazolidone,tetracycline,and metronidazole were 46.18%,50.60%,5.62%,8.44%,1.61%,89.96%,respectively.The multi-drug resistance rate of H.pylori was 38.15%,among which triple-resistance to clarithromycin plus levofloxacin plus metronidazole was the most common,which was 33.33%.However,the drug resistance of clarithromycin,levofloxacin,amoxicillin and metronidazole was not significantly related to the patient’s gender,age,collection location,and number of treatments(all P>0.05).The detection of drug resistance genes showed that:(1)In the23 S r RNA gene of 243 H.pylori strains,the mutation of this gene was related to clarithromycin resistance(P=0.0073),and 85.60% of the strains had A2143 G mutation;(2)In the gyr A gene of 243 H.pylori strains,the mutation of this gene is related to the resistance of levofloxacin(P=0.0019),of which 47.33% of the strains have 87 site mutations,and 23.05% of the strains have 91 site mutations.(3)Among 51 strains of H.pylori strains,the mutation of the rdx A gene is related to metronidazole resistance(P=0.04).Among them,92.16% of strains have A610 G mutation,90.20% of strains have G392 A mutation.2.Meta analysis: 38 studies were selected from 689 researches,with a total of7285 H.pylori strains.(1)The resistance of H.pylori strains in China to clarithromycin(38%:25%),levofloxacin(33%:30%),and metronidazole(78%:55%)are all higher than those in foreign countries.(2)In China,the western and remote areas have the most severe resistance to the three common antibiotics,followed by the eastern coastal areas(clarithromycin: 47%:42%:24%;levofloxacin: 69%:31%:17%;metronidazole:83%:80%:69%);but the eastern region has the highest resistance to amoxicillin,followed by the central region,and the western region has the lowest(14%:11%:4%);the central region has the lowest resistance to tetracycline The drug resistance is the highest,followed by the western region,and the lowest in the eastern region(10%:2%:1%).(3)In foreign countries,Asia has the highest resistance to clarithromycin(29%:18%:16%),levofloxacin(38%:25%:13%),amoxicillin(21%:11%)and tetracycline(16%:14%:2%).The Americas are second,and Europe is the lowest;the Americas have the highest resistance to metronidazole,followed by Asia,and Europe is the lowest(75%:55%:26%).The heterogeneity is considerable,which may be due to the grouping of resistance rates by country/region.3.Among 190 different carbon sources,3 H.pylori strains were able to metabolize 33(17.37%)of the tested carbon sources.There are 10 most obvious metabolic,including pentose(L-arabinose,D-xylose,D-ribose,L-lyxose,D-arabinose,2-deoxy-D-ribose),hexose(D-Glucosamine,2-keto-D-gluconic acid)and carbohydrates(sorbic acid,dihydroxyacetone).Among the 95 nitrogen sources,only 3compounds are used as the core metabolic nutrient sources of H.pylori,namely Lasparagine,L-aspartic acid and L-cysteine.Among 59 kinds of sulfur sources and 35 kinds of phosphorus sources,all H.pylori do not have much metabolized phosphorus and sulfur sources.It is worth noting that the two H.pylori multi-resistant strains metabolize significantly more L-rhamnose,L-asparagine and L-aspartic acid than the H.pylori P12 sensitive strain.It shows that these three metabolites may be one of the mechanisms of H.pylori resistance,and the mechanism needs to be further explored.Conclusions1.The resistance rate of H.pylori to clarithromycin,levofloxacin and metronidazole in Henan area is higher than the national level,but the resistance rate to amoxicillin and tetracycline is still relatively low.The multi-drug resistance rate of H.pylori is high,mainly clarithromycin,levofloxacin and metronidazole.2.The rate of H.pylori resistance in my country is significantly higher than that in foreign countries.In China,the remote areas in the west are the most serious,followed by the eastern coastal areas;while abroad,Asia is the most serious,the Americas are the second,and Europe is the lightest.Therefore,effective antibacterial drugs should be selected according to the results of drug sensitivity testing in the treatment plan.3.H.pylori can metabolize 17.37% of the tested carbon sources,and the most used carbon sources are pentose,hexose and carbohydrates.There are only 3 compounds in the nitrogen source as the core metabolic nutrient sources of H.pylori,and no substances in the phosphorus and sulfur sources are identified as core nutrients.Among them,H.pylori multidrug-resistant strains metabolize more L-rhamnose,L-asparagine and L-aspartic acid than sensitive strains,so these three metabolites may be one of the mechanisms of H.pylori resistance.