儿童感染肺炎链球菌的青霉素结合蛋白基因突变分析

Gene mutation in penicillin-binding protein gene of Streptococcus pneumoniae in infected children

目的分析儿童感染肺炎链球菌的青霉素结合蛋白基因突变与青霉素耐药水平之间的关系。方法自2012年1月至2014年12月期间分离的1 317株肺炎链球菌中随机抽取出青霉素MIC=2.0μg/mL、4.0μg/mL、≥8.0μg/mL各20株共60株作为实验菌株,采用PCR方法对实验菌株进行青霉素结合蛋白PBP1a、PBP1b、PBP2a、PBP2b、PBP2x、PBP3的基因扩增,扩增产物进一步纯化和测序,测序结果与青霉素敏感肺炎链球菌R6就国际上公认的PBPs保守序列进行比对分析。结果 60株肺炎链球菌的PBP2b、PBP1a、PBP2x、PBP2a基因的保守区或保守区附件均发现氨基酸突变,未发现PBP3与PBP1b突变。中介与耐药菌株基因突变位点存在重合,主要出现在单一的PBP1a序列的370STMK模体元件内Thr371Ser置换突变或伴有PBP2b序列的Thr451Ala/Ser和Ala624Gly置换突变,同时PBP2a序列的465SLN模体元件前置位发生Ser461Ala的置换突变。结论肺炎链球菌对青霉素中、高水平耐药菌株绝大部分合并有不同PBP序列中4~6个氨基酸的置换突变,但合并多个氨基酸置换突变并非就必然引起耐药水平的相应升高。中、高水平耐药与PBP1a、PBP2b、PBP2a的变异关系密切,其中PBP1a的STMK保守区域Thr371Ser置换是引起耐药的主要因素之一。

Objective To analyze the relationship between the gene mutation in penicillin-binding protein and resistance of the pathogen to penicillin in children infected by Streptococcus pneumoniae.Methods From the1,317 strains of Streptococcus pneumoniae isolated during January 2012 and December 2014,20 strains with MIC of 2.0μg/mL,20 with MIC of 4.0μg/mL and 20 strains with MIC of≥8.0μg/mL were selected as the subject strains.PCR method was used to amplify the PBP1 a,PBP1 b,PBP2 a.PBP2 b,PBP2 xand PBP3 genes in penicillin binding protein;the amplification products were further purified and sequenced.The sequencing results were compared with the sequence of penicillin sensitive Streptococcus pneumoniae R6 based on the internationally recognized conserved sequences of PBPs.Results In the 60 strains of Streptococcus pneumoniae,amino acid mutations were found in the conserved regions or their attachments of PBP2 b,PBP1 a,PBP2 xand PBP2 agenes;no PBP3 and PBP1 bmutations were found.The sites of gene mutation in the intermediate strains and resistant strains were coincident.The mutation was mainly found in the370 STMK of a single PBP1 asequence,which was a substitution mutation of Thr371 Ser,or substitution mutation of Thr451 Ala/Ser and Ala624 Gly with the sequence of PBP2 b.Meanwhile,a substitution mutation of Ser461 Ala occurred in the position of 465 SLN of the PBP2 asequence.Conclusion Most strains of Streptococcus pneumoniae with high or medium level of penicillin resistance often have substitution mutations of 4 to6 amino acids in different PBP sequences,but the multiple amino acid substitutions are not necessarily leading to higher resistance.Moderate and high resistance is closely related to the variation of PBP1 a,PBP2 b and PBP2 a.Among them,the substitution mutation of Thr371 Ser in the STMK conserved region of PBP1 a is one of the major factors causing resistance.