Heteroresistance On Two First-Line Antibiotics And Evolutionary Analysis Of Helicobacter Pylori

Helicobacter pylori(H.pylori)is a gram-negative,spiral pathogen colonized in the stomach,and closely related with chronic gastritis,digestive ulceration,gastric carcinoma and gastric MALT lymphoma;some extragastric diseases,for instance,iron deficiency anemia and idiopathic thrombocytopenic purpura are highly sceptical to be related with H.pylori infection.Over 50%worldwide population are infected with H.pylori,and the infectious rate is 41.7%in China,as reported lately in 2017.Infection of H.pylori usually occurs in childhood,and it can persist lifetime without treatment.Kyoto global consensus report on H.pylori published in 2015 strongly recommended it to be treated even when patients have no symptoms.Recent years,H.pylori antibiotic resistance is becoming severe,especially in developing countries,while H.pylori eradication therapy were usually determined by avoiding the drug that have been used by patient and the high resistant antibiotics in the area.With the increasing of antibiotic resistance,the eradication rate is decreasing annually.As known,H.pylori gastritis usually have few symptoms,and infection is hard to find without detection,therefore,H.pylori can colonize the stomach for decades.Long term infection of gene mutant and recombination leads to the high heterogeneity of H.pylori.Heteroresistance,meaning infected with susceptible and resistant H.pylori to the same antibiotics simultaneously,caused concern because it can lead to possible treatment failure with the resistant isolates undetected.There were few reports on heteroresistance,mostly on its occurrence,virulence factor typing and RAPD fingerprinting,but genetic analysis is still unexplored in H.pylori heteroresistance.Part 1:Heteroresistance on clarithromycin and levofloxacin of H.pylori by twomethodsIn this part,we applied a special made cytology brush in 49 13C urease breath test positive patients to collect endoscopic mucus samples from gastric antrum and corpus,at the meantime stool samples of each patient were collected.We isolated H.pylori clones by serially diluted the mucus sample in culture,confirmed by gram stain microscopy,biochemical reactions(urease,oxidase and catalase tests)and MALDI-TOF MS test.Etest on clarithromycin and levofloxacin were performed on H.pylori isolates to identify the heteroresistance in subjects.Meanwhile,we introduced droplet digital PCR(ddPCR)on gastric mucus samples and stool samples by analyze H.pylori 16S rRNA gene and 23S rRNA genotype to detect H.pylori infection and clarithromycin resistance.Totally,in 49 patients,there were 43,46 and 43 H.pylori positive by culture,gastric ddPCR and stool ddPCR,respectively.Clarithromycin susceptibility test shows that in 43 culture positive subjects,there were 23 susceptible,11 heteroresistant and 9 resistants.Levofloxacin susceptibility test shows that in 15 subjects,there were 8 susceptible,3 heteroresistant and 4 resistants.ddPCR of gastric samples indicated 21 had only wildtype alleles,13 had mixed genotype,and 9 had only mutant alleles.Stool ddPCR detected 17 wildtype,13 mixed,and 9 mutant subjects.Comparing ddPCR assays in gastric and stool samples,there were 75%agreement including 15 wildtype,10 mixed and 8 mutant samples.Comparing ddPCR and Etest methods,we found 86%agreement with 19 susceptible/wildtypes,9 heteroresistance/mixed genotypes and 9 resistant/mutants by 23 S rRNA genotyping.This study for the first time applied gastric mucus sample in culture and compared Etest with ddPCR assays,found out that heteroresistance of clarithromycin and levofloxacin in H.pylori infection is significant,while ddPCR is more sensitive in detecting heteroresistance than culture and Etest.It is also suggested that ddPCR in stool sample is efficient as well,and it is a promising non-invasive way to detect clarithromycin resistance in H.pylori infection.Part 2:Clarithromycin heteroresistance and evolutionary analysis ofHelicobacter pyloriIn this study,we performed evolutionary analysis on 81 H.pylori isolates from 6 clarithromycin heteroresistant samples using multilocus sequence typing(MLST).Minimum inhibitory concentration value between H.pylori isolates in the same sample ranged from 100 to 1000 times.Isolates in each heteroresistant samples were divided into 1 to 6 ST types,and 2 to 8 genotypes when combining ST types and vacA genotype.Phylogenic tree showed H,pylori isolates from the same sample turned out to be a cluster,while sensitive and resistant isolates were divided into different branches in 2 samples,and there was no significant relativity between antimicrobial susceptibility and genotypes in 4 samples,suggesting that H.pylori isolates that were close in evolutionary analysis can be the same susceptibility to clarithromycin,while they can be the opposite susceptibility as well,indicating that antibiotics selection and resistance burden play different roles in H.pylori evolution in different subjects.