The Study Of Antimicrobial Resistance And Whole Genome Sequencing For Helicobacter Pylori Isolated From Children

Objectives:Helicobacter pylori(H.pylori)is a common pathogen widely prevalent throughout the world that causes digestive ulcers,gastritis,even gastric cancer if no effective anti-H.pylori therapeutic approaches performed and the H.pylori is persistent in the stomach.Since the irrational drug use in the patients with H.pylori’s infection,the resistance rate of H.pylori to antibiotics has increased year by year worldwide,subsequently leading to decrease the cure rate of triple eradication therapy.Joint ESPGHAN/ NASPGHAN guidelines for the management of Helicobacter pylori in children and adolescents suggest that the use of antibacterial should be under the guidance of drug sensitivity test.Since the harsh conditions for isolation and culture of H.pylori,most hospitals are unable to carry out isolation and drug sensitivity test of H.pylori.so guidelines propose to establish regional centers which exert the special technical conditions and publish resistance information,paving the ways to guide the rational use of antibiotics in adjacent area.To date,the sensitivity rates of H.pylori to antibiotics for children in Southwest China remains largely unknown.To improve the eradication rate of H.pylori infection in children,especially for children with gastritis or duodenal ulcer,this study will perform phenotypic and genotypic resistance tests on clinical isolates of H.pylori in children in Southwest China and conduct whole genome sequencing studies,specifically:1.This study explored H.pylori resistance data in Southwest China,especially H.pylori resistance data in children,analyzed the difference in drug resistance rates between newly diagnosed and retreated children,analyzed the factors including gender,age,ethnicities,diseases related to H.pylori resistance rate,summarized the cure rate of children under empirical eradication therapy,and compared the H.pylori susceptibility between children and adults,aiming at the rational use of drugs to anti-H.pylori eradication treatment in children.2.By Whole-genome sequencing(WGS),we analyzed the H.pylori isolated from children to search for known drug resistance loci,compared the consistency of genotype resistance and phenotypic resistance results;analyzed the virulence gene characteristics of H.pylori strains in children,and the homology of H.pylori in Asia area.Based on these data,by establishing a phylogenetic tree,we try to get a better understanding of the evolution about this strain.3.Using H.pylori culture and drug susceptibility test as the gold standard,the sensitivity and specificity of fluorescence quantitative PCR for H.pylori and drug resistance detection were evaluated.Methods:1.Identification and resistance analysis of H.pylori in childrenThe gastric mucosal specimens were obtained from children who underwent gastroscopy at the Sichuan University West China Second Hospital from September2018 to August 2019 and adults received gastroscopy at Sichuan University West China Hospital from 2018 to 2019.The patients’ gastric mucosa was also cultured to obtain H.pylori,and the clinical data including age,race,sex,clinical symptoms,diagnosis,examination time,underlying diseases,drug selection,gastroscopic results,treatment,adverse reactions,13C-UBT results were included for analysis.After the successful culture of H.pylori,by oxidase test,urease test,hydrogen peroxide test,Gram staining,we confirmed the H.pylori strain.The minimum inhibitory concentration(MIC)values of amoxicillin(AMO),clarithromycin(CLA),metronidazole(MET),levofloxacin(LEV),rifampicin(RFP),tetracycline(TET)against H.pylori were determined by E-test.The MIC values of furazolidone(FRD)against H.pylori were determined by disk diffusion(K-B)method and agar dilution method respectively.We compared the different H.pylori resistance characteristics between children and adult and analyzed the effects of the related factors including gender,age,ethnicities and diseases on H.pylori resistance.We also compared with the resistance rate in the children with primary or secondary anti-H.pylori eradication treatment.In this study,we try to analyze efficacy of anti-H.pylori eradication treatment in children with H.pylori infection.2.Whole-genome sequencing and comparative genomics analysis of H.pylori in childrenFirstly,by WGS(Illumina),the H.pylori cultured from children was used for genome sequencing.Then,based on the published paper,the genome sequencing was compared with the known drug resistance loci to get a result which was made comparison with H.pylori resistance data from drug sensitive test.Thirdly,to get all the known sequences of H.pylori virulence genes from the virulence factor database(VFDB).Lastly,compared these sequences with those genome sequences from H.pylori in children to get the virulence genes for each strain and explore its correlation with diseases.3.Methods for detecting H.pylori resistance and their comparisonOne-time detection for H.pylori infection and its resistance to CLA and LEV respectively were achieved by fluorescence PCR.Totally,there were three methods including H.pylori culture,RUT and fluorescent PCR performed to compare the consistency for the detection of H.pylori among the three methods,the sensitivity and specificity of RUT and fluorescent PCR.Further,we analyzed the consistency of the drug resistance test results of the gastric mucosa with the drug sensitivity test results and the genome-wide resistance analysis by fluorescence PCR.Also,based on genome sequences,we evaluated the sensitivity and specificity of H.pylori resistance detected by fluorescence PCR.Results:1.Identification and drug resistance analysis of H.pylori in childrenA total of 142 children who underwent gastroscopy were included in the study.74 strains of H.pylori isolated from children’s gastric mucosa were included in present study.Twenty strains of H.pylori were isolated from Tibetan children,5were isolated from Yi children,and the remaining 49 were isolated from Han children.72 strains of H.pylori were cultured from 102 specimens with RUT positive,thus the culturing positive rate in RUT-positive specimens was 70.6%(72/102)in children and it was 80%(40/50)in adults.Our results have shown that only 2 strains of H.pylori from children are sensitive to all antibiotics while the other 72 strains are resistant to at least one antibiotic,so the resistance rate of H.pylori was 97.3%.The resistance rate to antibiotics which are commonly used was 12.2%,20.3%,55.4%,60.8%,71.6% for AMO,LEV,CLA,RFP and MET,respectively.Surprisingly,all H.pylori were sensitive to FRD and TET.Dual resistance rate was 40.5%,and the dual resistance patterns including RFP+MET(17.6%),CLA+RFP(9.5%),CLA + MET(8.1%),MET + LEV(4%)and RFP + LEV(1.4%).Triple resistance rate was 18.9%,and triple resistance patterns including CLA + MET+RFP(10.8%),AMO +MET+RFP(2.7%),LEV + MET+CLA(2.7%),LEV+CLA+RFP(1.3%),LEV+MET+RFP(1.3%).Four-drug resistance rate was 14.9%,for the four-drug resistance patterns,they were as followed: AMO+MET+RFP+CLA(6.8%),CLA+MET+RFP+LEV(6.8%),AMO+LEV+RFP+CLA(1.3%),and there was a five-fold resistance strain with resistance pattern: AMO+LEV+RFP+CLA+MET(1.3%).Among them,there were 27 strains of H.pylori which were resistant to both CLA and MET,and the double resistance rate to CLA and MET was 36.5%(27/74).In the presence of multi-drug resistance,there were 26 strains resistant to three or more antibiotics(35%)(26/74),H.pylori in retreated children was more resistant to CLA(71.4%vs45.7%)than in the newly diagnosed.The difference was statistically significant.When it came to adults,the resistance rate of H.pylori to antibiotics in 40 patients was 10% for AMO,67.5% for LEV,82.5% for CLA,97.5% for MET,respectively.The RFP resistance rate was 27.5%,and all H.pylori were sensitive to FRD and RFP.Dual resistance rate was 12.5%,and the pattern of dual resistance was CLA + MET(12.5%).Triple resistance rate was 42.5%,the pattern of triple resistance including CLA + MET + LEV(35%),CLA + MET + RFP(5%)and LEV+ MET + RFP(2.5%).The four-drug resistance was 30%,the four-drug resistance patterns were shown as followed: AMO + MET + LEV + CLA(10%),CLA + MET+ RFP + LEV(20%).Among them,33 strains of H.pylori were resistant to both CLA and MET,and the resistance rate to both CLA and MET was 82.5%(33/40),there were 29 strains resistant to three or more antibiotics,and the multi-drug resistance ratio was 72.5%(29/40).The statistical analysis data have demonstrated that the resistance rate of H.pylori to CLA,MET and LEV in adults was higher than children(82.5%vs55.4%,97.5%vs 71.6%,67.5%vs 20.3%)while the RFP in children was higher than adults(60.8%% vs 27.5%).With the empirical treatment,the total eradication rate of children was 66.7%(36/54).For the primary diagnosed patients,the eradication rate was 79.4%(31/39),however,it was 33%(5/15)in the patients who received retreatment,and there was a statistic difference between them(chi-square: 10.38,P < 0.05).Further,the cure rate in the patients received retreatment was lower than those were given treatment for the first time.2.Whole-genome sequencing and comparative genomics analysis of H.pylori in childrenBased on the whole genomic sequencing data from the 64 H.pylori stains conducted in this study,we analyzed the H.pylori virulence genes Combined with these 64 H.pylori stains and 325 Asian stains to establish the phylogenetic tree,Han children can be clustered with Japanese strains and Tibetan children’s isolates clustered with Indian strains.No statistic difference of consistency between the whole genomic sequencing analysis and drug sensitivity test to identify the resistant for CLA and LEV.The genomic analysis for H.pylori resistant to LEV show that the sensitivity was 92.3%(95%CI 64%-99.8%)and the specificity was 100%(95%CI93%-100%).In the presence of CLA,the sensitivity was 100%(95%CI 90%-100%)and the specificity was 100%(95%CI 88.1%-100%).Intriguing,both whole genomic sequencing analysis and drug sensitivity test possessed no well consistency in identification of H.pylori resistant to AMO while exerted a statistic difference to diagnose the resistant to MET.3.Methods for detecting H.pylori resistance and their comparisonIn this study,RUT,bacterial culture and real-time fluorescent PCR were used individually to diagnose H.pylori infection in 40 children with positive samples was29,28,26,respectively.So the consistency of three methods were equal to each other.Importantly,the sensitivity of real-time fluorescent PCR diagnosing H.pylori was 100%(95% CI 86.8%-100%)and the specificity was 78.6%(95% CI49.2%-95.3%).Data from 26 culture-positive specimens were analyzed for evaluating three methods to detect H.pylori resistance to CLA and LEV.The drug sensitivity test and fluorescent PCR were performed individually to analyze the resistance of H.pylori to CLA and there was consistency between the two methods(Kappa= 0.922,P < 0.05).Since the Kappa value ≥ 0.75,it suggested that the consistency was very high.The sensitivity of real-time fluorescent PCR evaluating resistance rate of H.pylori to CLA was100%(95% CI 71.5%-100%),specificity was 93.3%(95% CI68.1%-99.8%),The sensitivity of real-time fluorescent PCR evaluating resistance rate of H.pylori to LEV was100%(95% CI 51.4%-100%),specificity 90%(95% CI68.3%-98.8%).Conclusion:1.H.pylori in children from Southwest China has high resistance rate to clarithromycin,rifampicin and metronidazole,and exhibits highly double and multiple drug resistance rate.It is recommended to rational use of antibiotics with the guidance of drug sensitivity experiments or use bismuth quadruple therapy.2.It is not recommended to use empirical therapy to eradicate H.pylori infection in children as for the cure rate is low.It is recommended to rational use of antibiotics with the guidance of drug sensitivity experiments.3.H.pylori isolated from adults in Southwest China is highly resistant to clarithromycin,levofloxacin and metronidazole.It is not recommended to use clarithromycin,levofloxacin and metronidazole for the empirical treatment of H.pylori.It is recommended to rational use of antibiotics with the guidance of drug sensitivity experiments.4.Genotyping revealed a total of cag A+vac A+ combinations 100 %(64/64)and98.4%(63/64)in H.pylori from 64 children.The isolated strains of Han children can be clustered each other and clustered with Japanese strains.They all located in hp East Asia.Tibetan children’s isolates can be clustered and clustered with Indian strains.5.Genomics analysis of drug resistance genes of H.pylori has a high accuracy rate for finding CLA and LEV resistance rates,consistent with the drug sensitivity test.This method could be used for H.pylori resistance analysis and to guide rational drug use.6.Real-time fluorescent PCR detection of gastric mucosa specimens for H.pylori infection possesses time-saving,efficient,economical,high accuracy.Not only the Real-time fluorescent PCR can detect the presence of H.pylori infection but also obtain data on H.pylori resistance to CLA and LEV,which is recommended as the preferred method for H.pylori resistance analysis in units that do not have H.pylori culture techniques.