| 摘要: |
| 【目的】研究鸡源大肠杆菌成簇间隔短回文重复序列（Clustered regularly interspaced short palindromic repeats，CRISPR）与耐药性的关系，并对CRISPR进行生物信息学分析，为进一步研究CRISPR功能及其对细菌耐药的影响奠定基础。【方法】对从安徽省合肥地区养鸡场分离鉴定的130株鸡源大肠杆菌进行耐药性（16种抗菌药物）和CRISPR检测，分析CRISPR与鸡源大肠杆菌多重耐药的关系。挑选扩增出片段长度不同的12株大肠杆菌，对其CRISPR PCR产物进行测序，通过BLAST进行二级结构预测，用CRISPRTarget等对其重复序列和间隔序列进行生物信息学分析。【结果】130株鸡源大肠杆菌对16种抗菌药物的耐药率为2.3%～97.7%，118株大肠杆菌对3种及3种以上药物耐药；共有39株菌检测到CRISPR，阳性率为30%；CRISPR阳性菌株均为多重耐药菌株，其耐药率（100%）显著高于CRISPR阴性菌株（87%），CRISPR序列差异性与耐药性之间无直接相关性。CRISPR序列同源性较低的12株大肠杆菌均包含2条（11号菌除外）长度为29 bp的重复序列，这些序列可分为5类，其RNA二级结构都能形成回文结构，但茎环大小有差异。从这12株大肠杆菌中共发现间隔序列168条，其中77条为新发现序列；同源性比对发现，在112条有比对结果的间隔序列中，72.3%来源于质粒，25.0%来源于噬菌体，2.7%来源于细菌和病毒。【结论】携带CRISPR的鸡源大肠杆菌广泛存在，CRISPR可能与大肠杆菌耐药表型相关，明确了CRISPR的结构特征。 |
| 关键词: 大肠杆菌 CRISPR 耐药性 重复序列 间隔序列 |
| DOI： |
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| 基金项目:国家自然科学基金项目(31772802)；安徽高样自然科学重点项目(KJ2017A132);安徽省优秀青年人才支持计划重点项目(gxyqZD2016041) |
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| Relationship of CRISPR in Escherichia coli isolates from chickens with drug resistance and its characteristics |
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LI Lin,ZHAO Xia,WANG Lei,et al
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| Abstract: |
| 【Objective】The relationship between clustered regularly interspaced short palindromic repeats (CRISPR) in Escherichia coli isolates from chickens and drug resistance was analyzed,and bioinformatics analysis of CRISPR was conducted to provide basis for further study of CPISPR functions and its effect on bacterial resistance.【Method】The relationship between CRISPR and multidrug resistance in chicken-derived Escherichia coli was analyzed through drug resistance (16 antimicrobials) and CRISPR detection in 130 Escherichia coli strains isolated from chicken farms in Hefei,Anhui.Twelve isolates with different lengths were selected and their PCR products were sequenced.The bioinformatics analysis of CRISPR was carried out by BLAST,RNA secondary structure prediction and CRISPRTarget.【Result】The resistance rates of 130 strains of chicken-derived Escherichia coli to 16 antimicrobial agents were 2.3%-97.7% and 118 strains were resistant to 3 and more drugs.CRISPR was detected in 39 strains with positive rate of 30%.CRISPR positive strains were multidrug resistant strains,with resistance rate (100%) significantly higher than the CRISPR negative strains (87%).There was no direct correlation between CRISPR sequence difference and drug resistance.Twelve Escherichia coli strains (except 11 strain) contained two types 29 bp long repeat sequences,and they were divided into five groups.The RNA secondary structure can form a palindromic structure with different stem ring sizes.A total of 168 isolates were found,of which 77 were new.The homology comparison showed that 72.3% of the spacer sequences were homologous to the genomic sequence of plasmid,25.0% were homologous to phage,and 2.7% were from bacteria and virus.【Conclusion】Chicken-derived Escherichia coli carrying CRISPR were widespread,and CRISPR may be associated with drug resistance phenotypes.The biological information of CRISPR structure was also identified. |
| Key words: Escherichia coli CRISPR drug resistance direct repeats spacers |
