Effect Of Cryopreservation On Amoxicillin-resistant Phenotype Of Helicobacter Pylori

Helicobacterpylori(H.pylori)is a kind of microaerobic Gram-negative bacilli,which is spiral in shape and requires strict growth conditions.It has been proved that H.pylori is closely related to gastric diseases,such as gastritis,peptic ulcer,gastric cancer,gastric MALT lymphoma,and even some extragastric diseases.According to the Center for Disease Control and Prevention of the United States,about 50%of the world's population is infected with H.pylori.H.pylori infection rates range from 25%to 86%in different regions all over the world,especially in developing countries with poor health conditions.If not been treated,the bacteria are usually carried for the whole life.The Kyoto Global Consensus on H.pylori gastritis reached in Kyoto,Japan,in 2015 further emphasized that chronic gastric disease caused by H.pylori infection is an infectious disease and recommended the eradication of asymptomatic infections.In recent years,however,the success rate of eradication has been seriously reduced.The main reason is the increase in drug resistance.Because of colonization in the stomach,the contact between H.pylori and oral antibiotics makes H.pylori more susceptible to antibiotic resistance.Nowadays,because of the unreasonable use of antibiotics,especially in developing countries,bacterial drug resistance has become more and more serious.Bacterial drug resistance can be divided into genetic resistance and phenotypic resistance.Genetic resistance is characterized by changes in the genome.However,phenotypic resistance was unstable and drug resistance could be lost due to the change of environment,which resulted in cryopreservation for a period of time.As a first-line drug for H.pylori eradication therapy,it has been reported that amoxicillin used to appear phenotypic resistance in clinical strains.It is supposed to existing amoxicillin resistance rates may not be true and some phenotypic resistant strains have not been detected.Failure of many amoxicillin-containing eradicate therapies also points to this question.In this study,the international standard strain ATCC 43504 and 11 Chinese clinical isolates were used as starting strains.AMXR,was successfully induced to observe the changes of H.pylori under pressure screening after 30 and 60 passages.The model of H.pylori of resistance to AMX was established by selecting monoclonal subclone,and the minimal inhibitory concentration(MIC)of the model was detected at the same time.Except AMX,MICs of azithromycin(AZM),clarithromycin(CLA),levofloxacin(LVX),metronidazole(MNZ),gentamicin(GEN),moxifloxacin(MFX),rifampicin(RIF),and tetracycline(TET)were also detected to observe the trend of another 8 antibiotics with the increase in AMX resistance level.The resistant subclones were stored in the refrigerator at-80 ? for 1-3 months and then were revived again.Compare the change of MICs before and after cryopreservation.Drug sensitivity tests were repeated two or three times.Meanwhile,AMXR H.pylori was revived on AMX resistant plate and non AMX plate at the same time.In addition,the nucleic acid sequences of php 1(HP 0597),ftsI(HP 1556),pbp 3(HP 1565),pbp 4(HP 0160)and pbp 5(HP 1542)related to AMX resistance were sequenced.In order to explore the possible mechanism of AMX resistance.The results showed that the models of AMXR H.pylori composed of 42 AMXR clones and 46 AMXR subclones(MIC>0.5 ?g/ml).After-80? cryopreservation,drug resistance level of 19(41.3%)subclones dropped significantly and those with the biggest changes in MIC decreased by more than 98%.The decrease is universal among subclones from different regional sources but has differences among subclones in different drug resistance levels.Twenty clones(43.5%)induced from 5 strains showed 10 types of amino acid substitutions exclusively in PBP 1,2,and 4.Sequence alignment of PBP1 in AMXR subclones revealed multiple amino acid substitutions on sites S414R,T438M,R427K,T541I,N555K,T556S and T593P.In PBP 2,the K376N and K572E substitutions were observed.Furthermore,one substitution T134A was detected in PBP 4.No mutation was observed in PBP3 and PBP5.This study found 10 amino acid changes in AMXR clones.However,in 57.5%clones,there were no detection of mutations of pbpl-5.After cryopreservation,the AMX resistance phenotype of H.pylori was unstable and drug resistance level has declined.It is suggested that there may be a underestimation of AMX resistance rate and resistance level in the isolation and culture of strains carried out by frozen specimens in clinic.A total of 46 AMXR subclones were induced,but only 43.5%of them observed missense mutation in the penicillin-binding protein 1-5,which indicated mutation in PBP 1-5 is not the main cause of AMX resistance in H.pylori.Further detailed molecular and genetic studies are required to shed more light on the AMX resistance mechanisms.