Solexa测序法研究肺炎克雷伯菌质粒基因组的耐药基因

Solexa sequencing and resistant analysis of the metagenome of plasmid in Klebsiella pneumoniae

目的通过Solexa高通量测序法研究肺炎克雷伯菌的质粒与耐药性的关系。方法菌株为7年临床分离的206株肺炎克雷伯菌。提取所有菌的全部质粒DNA，Solexa高通量测序获得大规模的短序列。SOAP软件对质粒基因进行分析拼接，分析结果与相关数据库进行比对。MAQ软件分析质粒基因组包含的超广谱β-内酰胺酶（ESBL）多样性及单核苷酸多态性（SNP）情况。结果肺炎克雷伯菌质粒基因组中已知的直接与耐药相关的基因就有13种。质粒基因组中存在多个ABC主动外排转运系统。发现4种编码β-内酰胺酶的ORF，其中SHV型ESBLs分布最广。系统分析了206株肺炎克雷伯菌质粒基因组中SHV型ESBLs的SNPs位点，发现存在着大量的非同义替换SNPs位点。结论发现质粒中SHV型ESBLs基因可能受到选择压力，存在着大量的非同义替换SNPs位点。肺炎克雷伯菌质粒存在外排药物耐药方式，从而形成低水平的非特异多重耐药。

Objective High-throughout sequencing of all plasmid of 206 strains of Klebsiella pneumoniae using Solexa/lllumina sequencing technology in order to investigate the resistance for plasmids in Klebsiella pneumoniae. Methods Bacterial isolates were obtained over the years 2002-2008. Solexa/Illumina sequencing technology was used to sequence both samples （S1 and S2） to a depth of between 10-560 fold coverage. We used SOAP provided by BGI to find SNPS and use velvet package to assemble these sequences and gained some long sequences, and MAQ programs developed in the laboratory were used to annotate SNPs and compare lineage-specific mutations in SHV-ESBLs. Results The Metagenome of plasmid encodes a 13 variety of resistance-related genes with exceptionally high copy numbers, including ABC-type efflux pumps and 4 variety of β-1actamases, SHV-ESBLs is abroad presence. We systematically investigated single nucleotide substitutions in plasmids metagenome, and found an amount of nonsynonymous mutations in the SHV-ESBLs genes. Conclusion Probabily in press of positive selection, we can clearly see these nonsynonymous changes predominantly occurred in plasmid SHV-ESBLs genes. And our findings indicate a unspecial lowlevel resistance contribute to antimicrobial efflux in the metagenome of plasmid in Klebsiella pneumoniae.