摘要目的 分析无抗生素压力下连续传代90次的4株志贺菌耐药表型及成簇规律间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)基因变化.方法 对临床分离的4株耐药谱不同的志贺菌进行无抗生素压力连续传代90次,传代结束后用琼脂稀释法检测传代前、后志贺菌最小抑菌浓度;用PCR对CRISPR位点进行扩增并测序,CRISPR Finder和Clustal X 2.1分析CRISPR位点的变化.结果 经无抗生素压力传代90次后,4株志贺菌对某些抗生素的敏感性有不同程度的增加:mel-sf1998024/zz对氨苄西林、头孢氨苄、头孢噻肟、氯霉素的耐药性降低,mel-s2014026/sx对诺氟沙星、甲氧苄啶的耐药性降低,mel-sf2004004/sx对氨苄西林、头孢呋辛、头孢噻肟、氯霉素、甲氧苄啶的耐药性降低,mel-sf2013004/bj对氯霉素的耐药性降低;志贺菌mel-sf1998024/zz和mel-sf2013004/bj经无抗生素压力传代后,CRISPR3位点3′的重复-间隔序列丢失,其中间隔序列匹配基因的编码产物是Cas蛋白.结论 志贺菌在无抗生素压力下,可降低或丢失对某些抗生素的耐药性.部分志贺菌CRISPR3位点的结构发生了变化,CRISPR3位点与cas基因可能存在共进化.

abstractsObjective To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics.Methods Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics.Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains.After sequence analysis with PCR,CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains.Results After the consecutive transfer of 90 generations,sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased.Mel-sf1998024/zz resistance to ampicillin,cephalexin,cefotaxime,chloramphenicol decreased,mel-s2014026/sx resistance to norfloxacin,trimethoprim decreased,mel-sf2004004/sx drug resistance to ampicillin,cefuroxime,cefotaxime,chloramphenicol,trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased.The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj.Conclusions Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers,without the interference of antibiotics.CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.