Study On The Mechanisms Of Resistance And Persistence Of Helicobacter Pylori To Amoxicillin Based On Genome And Transcriptome

Objective:To understand whether there is Amoxicillin(AMX)-persistent Helicobacter pylori(H.pylori)strain in clinical strains;to understand the role of antibiotic persistence in the evolution of H.pylori resistance;and to explore possible mechanisms for the evolution of an unstable resistance phenotype of H.pylori AMX into a stable resistance phenotype.Methods:1.Screening and validation of H.pylori AMX-persistent strainsAMX persistence were screened from clinically isolated H.pylori using a modified agar dilution method(MADM),and persistence was further determined by the time–kill curve.2.The role of persistence in the evolution of H.pylori resistance to AMXThe MIC of AMX were determined again after 11 times of exposure of increasing concentration of AMX in vitro using the H443(AMX-persistent)and the H568(AMX-susceptible)with the same minimal inhibitory concentration(MIC)as the parent strains.The obtained AMX-resistant clone H443 R and the parent strain H443 were sequenced by whole genome and transcripome,and the results of transcripome sequencing were verified by RT-q PCR of vac A,atp D,hop B,hop C,met Q and the content of ATP in bacterial cells,and the reasons for the increased resistance of strain H443 R were analyzed.3.Evolution of stable high level resistance phenotype of H.pylori AMX and detection and analysis of gene mutationsFour strains of H.pylori whose MIC against AMX decreased after been freezing at-80℃(unstable drug resistance)were taken as the starting strains.The stable and high level AMX-resistant clones were screened for 20～55 generations in the medium with increasing AMX concentration,and the MIC of the AMX-resistant clones were detected.After cryopreservation at-80℃ for 3 months,the resistance was stable according to whether the MIC decreased after cryopreservation.Genome sequencing and analysis were performed on stable AMX-resistant clone H390 r and starter strain H390.Finally,efflux pump inhibition test was used to detect the MIC of H390 r and H390 on AMX with efflux pump inhibitors.Results :1.Screening and validation of H.pylori AMX-persistent strainsOne strain of AMX-persistent strain(H443)was screened from clinical strains of H.pylori by MADM.The time–kill curve of the strain H443 showed bipeak bactericidal characteristics,which confirmed that H443 was a persister.2.The role of persistence in the evolution of H.pylori resistance to AMXThe AMX-sensitive strain H568 was used as the parent strain,and the resistance increased slowly on the AGAR plate with increasing AMX concentration for 11 generations.The H568S(MIC 0.016mg/L)clones were obtained in 11 th generation,and its sensitivity was similar to that of the parent strain H568,the AMX-persistent strain H443,which had the same MIC as H568,was passed on the AGAR plate with increasing AMX concentration for 11 successive generations,and the resistance increased rapidly.The 11 th generation evolved low level resistant strain H443R(MIC0.5mg/L),and its AMX MIC was more than 31 times higher than that of the parent strain.According to genetic analysis,compared with the parent strain H443,H443 R involved 28 mutations in 16 genes,including pbp1 a,fts I,hef C and hof H genes related to AMX resistance,and mod gene with high frequency reversible ON/OFF switch.Transcriptome analysis showed that H443 R and H443 had significant differences in several KEGG enrichment pathways,in which H443 R down-regulated gene was significantly enriched in oxidative phosphorylation(related to ATP production)and ribosome.However,H443 R up-regulated genes were significantly enriched in flagellar assembly,ABC transport,homologous recombination and mismatch repair,the latter two involved in DNA mismatch repair programs.The expression of vac A,atp D,hop B,hop C and met Q genes in H443 R was down-regulated by RT-q PCR,which was consistent with the results of transcriptome sequencing.The intracellular ATP level of H443 R was significantly lower than that of H443.3.Evolution of stable high level resistance phenotype of H.pylori AMX and detection and analysis of gene mutationsFour H.pylori clones with high and stable resistance to AMX(MIC 12 ～256mg/L)were obtained by AMX resistance screening,which were denoted as H390 r,H443r,H574 r and H576 r,respectively.Compared with H390,H390 r had mutations in several genes,including the genes hef C,hop B,hop C,and fts I,which were associated with AMX resistance.The MIC of H390 r on AMX was ≥256mg/L without efflux pump inhibitor,and decreased to 12mg/L with efflux pump inhibitor.The MIC of H390 on AMX was 0.5mg/L with or without efflux pump inhibitor.Conclusions:There are present AMX-persistent H.pylori in clinical.The persistence of H.pylori is not resistance,but it can rapidly evolve AMX resistance through continuous AMX exposure in vitro.The resistance genes involved in the evolution of resistance in the persister are similar to those obtained from clinical isolates.H.pylori AMX unstable resistance phenotype evolved a stable and high level of resistance phenotype through continuous AMX exposure in vitro,and the mechanism of the evolution of stable and high level of resistance phenotype the mechanism mainly involves drug efflux.