幽门螺杆菌对克拉霉素、甲硝唑和左氧氟沙星的耐药率及其相关耐药基因突变特征

doi:10.13481/j.1671-587X.20220311

Abstract

Abstract:

Objective： To detect the drug resistance rates of Helicobacter pylori （Hp） to three common antibiotics，and to explore the mutation forms of drug resistance genes in order to provide the basis for elucidating the molecular mechanism of drug resistance.

Methods

The gastric mucosa samples from 35 patients with chronic gastritis or peptic ulcer were isolated and cultured to identify Hp.The minimal inhibitory concentrations （MIC） of Hp to clarithromycin， metronidazole and levofloxacin were detected by the agar dilution method and Hp was divided into sensitive group and resistant group according to the results of the drug sensitivity test.The clarithromycin resistance gene 23S rRNA， metronidazole resistance gene rdxA， and levofloxacin resistance gene gyrA were amplified by PCR method. The nucleotide and amino acid mutations were analyzed after the sequencing of PCR purified product.

Results

After cultured for 3－5 d， smooth， translucent， needle-like colonies could be seen on the plate of Hp strain. All 35 Hp strains were resistant to metronidazole and levofloxacin， the resistance rate to clarithromycin was the highest（80%）. Compared with the sensitive strains， all the clarithromycin- resistant strains had gene mutations. The most common mutation site of the 23S rRNA gene was A2143G， followed by mutation sites A2223G and A2142G+A2164G. The rdxA gene of metronidazole resistant strains mainly had missense mutations， followed by frameshift mutations and nonsense mutations. The N87K mutation in the gyrA gene of levofloxacin resistant strains was the highest， followed by the D91N mutation， N87I， D91G， and N87K+D143E mutations，but 17.1% of the resistant strains did not have any site mutations.

Conclusion

The resistance rates of Hp to clarithromycin， metronidazole， and levofloxacin are high， and the mutations of related drug resistance genes are the main cause of the antibiotic resistance. Screening these mutations may help to prevent the antibiotic resistance and select the appropriate antimicrobials to eradicate Hp.

Key words: Helicobacter pylori, Drug resistance, Clarithromycin, Metronidazole, Levofloxacin

CLC Number:

R446.5

Shunjia SONG,Xinying WANG,Feifei JIANG,Huijian JIA,Yuan ZHAO,Zhiping YANG,Liyuan SUN,Yundong ZHAO. Resistance rates of Helicobacter pylori to clarithromycin, metronidazole, and levofloxacin and mutation characteristics of their related drug resistance genes[J].Journal of Jilin University(Medicine Edition), 2022, 48(3): 630-637.

Figures/Tables 13

Tab. 1

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Tab. 5

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Primer	Sequence（5'→3'）	Target fragment size（bp）
16S rRNA	F:ACGCATAGGTCATGTGCCTC	182
16S rRNA	R: GTGTCCGTTCACCCTCTCAG	182
23S rRNA	F: CCACAGCGATGTGGTCTCAG	425
23S rRNA	R: CTCCATAAGAGCCAAAGCCC	425
rdxA	F: TGAAACGCTTGAAAACACCC	851
rdxA	R: ATTTGAGCATGGGGCAGATTT	851
gyrA	F: TAGCTTATTCCATGAGCGTG	530
gyrA	R: TAAGACATGCACTAAAGCGT	530

Mutation form	Number of strains	Mutation rate（η/%）
A2143G	24	85.7
A2223G	3	10.7
A2142G + A2164G	1	3.6

Strain	Nucleotide mutation form	Amino acid mutation form
1	Insertion of T（64,65）	Frameshift mutation in amino acid 23
2	148 C→T	Premature stop codon (50)
4	352 G→T， Deletion of A（356）	118 A→S, frameshift mutation in amino acid 120
5	46 C→T，468 G→T	16 R→C，156 L→F
6	Insertion of G（134）， Insertion of C（135）	Frameshift mutation in amino acid 46
7	352 G→T，497 C→T，526 C→T	118 A→S，166 P→L，176 R→C
8	172 A→G，238 G→A，322 T→G，623 C→T	58 T→A，80 A→T，108 S→A，208 T→I
9	140 A→G	47 Y→C
10	Deletion of T（213）	Frameshift mutation in amino acid 72
11	90 C→A，148 C→T	30 S→R，Premature stop codon (50)
12	322 T→G，614 A→C	108 S→A，205 D→A
13	41 A→G，203 C→T，377 A→G，434 G→A，516 T→C	14 N→S，68 A→V，126 N→S，145 G→E，172 V→I
14	148 C→T	Premature stop codon (50)
16	352 G→A，610 G→A	118 A→T，204 V→I
17	317 C→T，488 G→A，168 G→A，393 A→G	106 P→L，163 G→D，56 M→I，131 R→K
18	8 T→C，47 G→A	3 F→S，16 R→H
19	202 G→A	68 A→T
20	47 G→A，192 A→T，293 G→A	16 R→H，64 K→N，98 G→N
21	419 G→A，536 A→G	140 C→Y，179 K→R
22	199 G→A，577 G→A	67 A→T，193 A→T
23	47 G→A，（61 A→G、62 T→C），89 G→A，462 G→A	16 R→H，21 M→A，30 S→N，154 M→I
24	89 G→A	30 S→N

Mutation form	Number of strains	Mutation rate（η/%）
N87K	23	65.7
N87I D91N D91G	1 3 1	2.9 8.5 2.9
N87K+D143E	1	2.9
—	6	17.1
“—”：No mutation.

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Resistance rates of Helicobacter pylori to clarithromycin, metronidazole, and levofloxacin and mutation characteristics of their related drug resistance genes

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Antibiotic	Number of drug- resistant strains	Drug resistance rate (η/%)
Clarithromycin	28	80
Metronidazole	35	100
Levofloxacin	35	100