blaCTX-M-55介导的宋内志贺菌耐药机制研究

Resistance mechanisms of bla_(CTX-M-55) in a clinical Shigella sonnei strain

目的研究bla_(CTX-M-55)介导的宋内志贺菌#1083的耐药机制。方法双纸片协同扩散法验证#1083是否为产超广谱β-内酰胺酶(ESBL)菌株;PCR方法鉴定其耐药基因;接合转移实验验证携带ESBL基因的质粒是否具有可转移性;VITEK 2仪器检测菌株对多种抗生素的MIC值;基因组测序鉴定介导耐药基因转移的移动元件;引物延伸实验鉴定耐药基因转录起始位点。结果 #1083为bla_(CTX-M-55)介导产ESBL的菌株,携带bla_(CTX-M-55)的耐药质粒可通过接合转移的方式进入受体菌EC600,并使受体菌具有相应的耐药谱;介导bla_(CTX-M-55)转移的转座单元为ISEcp1-bla_(CTX-M-55)-Δorf477,且其上游的插入序列ISEcp1为耐药基因提供强启动子区,促进耐药基因表达,且此表达为恒定表达,不受抗生素的诱导作用影响。结论质粒携带的bla_(CTX-M-55)为#1083的主要耐药基因,插入序列ISEcp1介导此基因的表达与传播。

Objective To characterize the resistance mechanisms of a clinical Shigella sonnei strain harboring blaCTX-M-55 .Methods A double-disk synergy test was conducted to detect ESBL.Antibiotic resistance genes were determined by PCR followed by amplicon sequencing.Conjugation experiments were performed to verify the transferability of the plasmids carrying ESBL genes.The minimum inhibitory concentration values were tested using VITEK 2.The transposition unit was confirmed by DNA sequencer,and the transcriptional start site was identified using primer extension assay.Results Strain #1083 produced CTX-M-55,which was encoded by plasmid p1083-CTXM that could be transferred into E.coli through conjugation experiments to confer corresponding antibiotic resistance to the transconjugant #1083-EC600.The transposition unit mediating the transfer of blaCTX-M-55 was ISEcp1-blaCTX-M-55 -Δorf477.ISEcp1 offered strong promoter regions for the resistance genes,facilitating their expressions.Besides,the expressions were constant,not induced by antibiotics.Conclusion BlaCTX-M-55 on plasmids is the major resistance genes for strain #1083.Their expressions and spread are mediated by the insertion sequence ISEcp1.