| Objective:In this paper,we explored the characteristics of H.pylori antibiotic resistance in this area,analyzed the mutation of the major drug-resistant genes and screened the mutations affecting antibiotic resistance.This study will reveal the characteristics of H.pylori resistance-related gene mutations in Zhuanghe and provide a theoretical basis for exploring the molecular mechanisms of drug resistance.Methods:1.A total of 100 strains were collected from the primary cultures of 454gastric mucosal tissues obtained during an endoscopic biopsy which were all from the Zhuanghe,started in 1998 to 2017.2.The susceptibility of H.pylori to metronidazole,levofloxacin,clarithromycin,amoxicillin,and tetracycline was tested using an E-test.3.Whole genomic DNA of H.pylori was extracted by phenol-chloroform method.H.pylori virulence factors cagA,vacA?s1,s2,m1a,m1b,m2,i1,and i2?,iceA1,iceA2,babA2,oipA,slyD,hrgA,and cagA-EPIYA motif were amplified using polymerase chain reaction?PCR?.4.23SrRNA V,gyrA,gyrB,rdxA and frxA genes were amplified and sequenced,respectively.The bioinformatics software was used to predict the changes before and after mutations,to screen and identify the genetic changes related to drug resistance.5.All statistical analysis was carried out using SPSS 18.0 software.A binary logistic regression model was used to calculate the odds ratios?OR?and 95%confidence interval?CI?.The Minimum inhibitory concentration?MIC?value differences between the mutants and non-mutants in the resistant strains were tested using Mann–Whitney U or t-tests.The P value of<0.05 was accepted as statistically significant.Result:1.H.pylori resistance rates to metronidazole,levofloxacin,clarithromycin,amoxicillin,and tetracycline were 78.0%,56.0%,31.0%,9.0%,and 15.0%,respectively.Double,triple,quadruple,and quintuple resistance rates were 23%,20%,6%,and 4%,respectively.2.During the three periods,1998-1999,2002-2004 and 2016-2017,the resistance rates to metronidazole,clarithromycin,and tetracycline were not significant over the three decades,but the resistance rates to levofloxacin and amoxicillin were increasing?OR:2.089,95%CI:1.142-3.821,P=0.017;and OR:5.035,95%CI:1.327-19.105,P=0.018,respectively?;The clarithromycin and multidrug resistance rates were significantly higher in males than females?clarithromycin:44.4%?24/54?vs 15.2%?7/46?,respectively,P=0.002;multidrug:68.5%?37/54?vs 34.8%?16/46?,respectively;P<0.001?;The antibiotic resistance rates were unassociated with the host disease state;Metronidazole-resistance rate was lower in the vacAs1m1/m2 group than the vacAs1m1m2group?65%vs85.7%,respectively;P=0.026?.Asfor levofloxacin-resistance rate,it was higher with cagA+than cagA-?60.9%vs 23.1%,respectively;P=0.020?but lower with slyD+than slyD-?41.4%vs 68.5%,respectively;P=0.009?.Clarithromycin had a lower resistance rate with iceA++than iceA-+?19.7%vs52.4%,respectively;P=0.017?.For amoxicillin,the iceA++group had a lower resistance rate than the iceA--group?1.6%vs 27.8%,respectively;P=0.009?.3.In the clarithromycin-resistant strains,the mutation rate on A2143G site of 23SrRNA was74.2%?n=23?.The mutations at sites C1883T,C2131T and T2179G can cause structural changes;In the levofloxacin-resistant strains,the mutation rates of the 87?N to K/I?and91?D to N/Y/G?of GyrA were 28.6%?n=16?and 12.5%?n=7?respectively,with one of 91 mutation accompanied by the change of D99N.In addition,a D143E mutation can be found in one drug-resistant strain.There were changes in tertiary structure before and after these mutations;The mutation type of RdxA protein include premature stop codons leading to protein truncation?n=26,33.3%?,frameshift mutation?n=8,10.3%?,site mutation binding to the flavin mononucleotide cofactor?n=16,20.5%?and others?n=11,14.1%?.Predictive analysis showed that mutations in the first three groups and the A118S in the last group can lead to structural alteration.Conclusion:1.The antibiotic resistance of H.pylori in Zhuanghe area was dominated by metronidazole,levofloxacin and clarithromycin,and the double and multidrug resistance rates were high.2.The changing profile of H.pylori antibiotic resistance in Zhuanghe during the past three decades:Metronidazole,clarithromycin and tetracycline tended to be stable,whereas levofloxacin and amoxicillin increased over time.Factors influencing antibiotic resistance of H.pylori include sex and virulence factors cagA,vacA,slyD,iceA.3.The clarithromycin-resistance sites was mainly in A2143G of 23SrRNA and other C1883T,C2131T and T2179G may also be related to resistance.The levofloxacin resistance was mainly based on the amino acid changes at 87 and 91 sites coded by gyrA.The new sites D99N and D143E may also be related to resistance.Metronidazole resistance is related to the RdxA protein truncation,frameshift,and FMN binding.The new site A118S may also be associated with drug resistance. |
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