摘要
背景世界上50%以上的人口感染幽门螺杆菌(Helicobacter pylori,H.pylori).随着抗生素的广泛使用,抗生素耐药成为H.pylori感染患者根除失败的主要原因.目前有不少研究报道外排泵基因过表达和耐药的产生相关.目的为了未来的H.pylori感染治疗有更多方法,探索外排泵基因的变异与双重耐药菌株耐药性产生的内在联系.方法使用^(13)C呼气试验及药物敏感试验进行双重耐药菌株及敏感菌株的筛选,用特异性PCR的常规方法进行耐药基因突变位点的验证,基于MiSeq平台,对10例临床菌株进行全基因组测序,以鉴定双重耐药表型与敏感表型的外排泵基因的单核苷酸变体(single nucleotide variants,SNV)并分析,参考菌株使用ATCC26695.结果H.pylori药敏试验结果显示,丽水地区H.pylori对克拉霉素及左氧氟沙星都具有较高的耐药率.采用特异性PCR法在5株临床双重耐药菌株中检测到23S rRNA基因及gyrA的点突变,在5株临床敏感菌株中未检测到.全基因测序检测了涉及多耐药性的TolC同源物的四个基因簇(HP0605-HP0607,HP0971-HP0969,HP1327-HP1329和HP1489-HP1487)的遗传变异.在双重耐药性H.pylori菌株中发现一个突变的SNV.结论丽水地区抗生素使用应严格监控,避免滥用.TolC同源基因的基因簇与H.pylori菌株的抗生素耐药性相关.全基因组测序对基因型与表型的关系有了新的认识.
BACKGROUND More than 50%of the world’s population are infected with Helicobacter pylori(H.pylori).With the widespread use of antibiotics,antibiotic resistance has become the main reason for the failure of H.pylori eradication.At present,many studies have reported that the overexpression of efflux pump genes is related to the development of drug resistance.AIM To explore the internal connection between the mutation of efflux pump gene and the resistance of double drug resistant strains.METHODS ^(13)C-breath test and drug susceptibility test were used to screen double drug resistant strains and sensitive strains,and conventional methods of specific PCR were used to verify the mutation sites of drug resistance related genes.Based on the MiSeq platform,the whole genome sequence of ten clinical strains was performed.The single nucleotide variants(SNV)of the efflux pump gene of the double drug resistant phenotype and the sensitive phenotype were then identified and analyzed.The reference strain was ATCC26695.RESULTS The results of the H.pylori susceptibility test showed that H.pylori in Lishui area had a high resistance rate to clarithromycin and levofloxacin.Specific PCR detected 23S rRNA gene and gyrA point mutations in five clinically double drug resistant strains,but not in the five clinically sensitive strains.Whole genome sequencing detected the genetic variation of four gene clusters(HP0605-HP0607,HP0971-HP0969,HP1327-HP1329,and HP1489-HP1487)involved in multi-drug resistance TolC homologs.A mutant SNV was found in double drug resistant H.pylori strains.CONCLUSION The use of antibiotics in Lishui area should be strictly monitored to avoid abuse.The gene cluster of TolC homologous genes is related to the antibiotic resistance of H.pylori strains.Whole genome sequencing can help provide a new understanding of the relationship between genotype and phenotype.
作者
叶淑芳
张剑美
代飞
蓝陈菊
章小君
周丽珍
汤清清
孟飞
Shu-Fang Ye;Jian-Mei Zhang;Fei Dai;Chen-Ju Lan;Xiao-Jun Zhang;Li-Zhen Zhou;Qing-Qing Tang;Fei Meng(Department of Gastroenterology,Lishui City People’s Hospital,Lishui 323000,Zhejiang Province,China)
出处
《世界华人消化杂志》
CAS
2021年第9期455-460,共6页
World Chinese Journal of Digestology
基金
浙江省科学技术厅2020年省公益技术研究项目,No.LGF20H030005
丽水市消化内镜临床医学研究科技创新团队,No.2018cxtd05.
关键词
双重耐药菌株
幽门螺杆菌
全基因组测序
耐药性
外排泵基因
Multi-drug resistant strains
Helicobacter pylori
Whole genome sequencing
Drug resistance
Efflux pump genes
