Abstract:

Objective    To compare pbp2b，ply and lytA genes PCR with cerebral spinal fluid culture in diagnosis of Streptococcus pneumoniae meningitis and prediction of its susceptibility. Methods    A nested PCR targeting pbp2b and another two S.pneumoniae specific PCR targeting the genes of pneumolysin（ply） and autolysin（lytA） were developed for detection of S.pneumoniae in cerebral spinal fluid from bacterial meningitis patients. The three PCR results and culture were compared. The consistency of penicillin susceptibility PCR （using resistant and susceptible primers respectively），sequencing and culture-based phenotypic penicillin resistant results were compared. Results    Of the 161 specimens studied，there were 25 cases of S.pneumoniae infection confirmed by different methods（16 by pbp2b PCR，16 by lytA PCR，14 by ply PCR and 9 by cerebrospinal fluid culture）. Of the 16 pbp2b positive specimens，penicillin sensitive and resistant sequence types accounted for half，respectively. Four of the 16 pbp2b positive specimens had culture-based phenotypic penicillin-resistant result. Three of 4 were consistent with penicillin susceptibility PCR result. The results of susceptibility PCR targeting pbp2b was consistent with sequencing result. New sequence types were found but there were no new point mutations in these strains when compared with GenBank （http：//blast.ncbi.nlm.nih.gov/Blast.cgi）. Penicillin resistance in pneumococcal meningitis was 66.67%（6/9） by culture phenotype and 50%（8/16） by PCR and sequencing when culture was negative. Conclusion    Pbp2b can serve as a good target gene to detect S.pneumoniae and predict its penicillin susceptibility，which is especially important when culture is negative.

Key words: streptococcus pneumoniae, penicillin resistance, penicillin binding protein 2B gene （pbp2b）, bacterial meningitis

摘要：

目的    比较青霉素结合蛋白2b（pbp2b）与自溶素（ply）、溶血素（lytA）基因和脑脊液细菌培养在诊断国内儿童化脓性脑膜炎中肺炎链球菌感染及青霉素耐药性方面的情况，并了解肺炎链球菌青霉素结合蛋白基因型特征。方法　2006年1月至2009年9月在北京儿童医院感染内科临床严格诊断为细菌性脑膜炎患儿161例，用pbp2b及其他2个肺炎链球菌种特异的基因（ply、lytA）巢式PCR法来检测细菌性脑膜炎患儿脑脊液中的肺炎链球菌，对不同方法结果进行比较。对其中pbp2b阳性的标本进行青霉素敏感性PCR（分别用耐药、敏感的引物进行PCR检测）、测序，并与青霉素耐药表型进行比较和评估。结果　161例中不同方法确诊的肺炎链球菌共有25例，3种基因检测均阳性的共有11例。pbp2b检测阳性16例，lytA检测阳性16例，ply检测阳性14例。脑脊液培养阳性9例。pbp2b阳性的16例标本中，青霉素敏感和青霉素耐药基因型各占一半。16例中4例有培养结果，其中3例基因型和耐药型相符合。敏感性测定PCR和测序结果相一致。测序结果与NCBI基因库http：//blast.ncbi.nlm.nih.gov/Blast.cgi）相比，有2例为新的氨基酸序列型，但是无新的氨基酸点突变发生。由培养结果检测的肺炎链球菌脑膜炎的青霉素耐药率为66.67%（6/9），敏感性PCR检测的肺炎链球菌脑膜炎的青霉素耐药率为50%（8/16），包括脑脊液培养阴性的病例。结论　3种基因检测的敏感度均高于传统的脑脊液培养，其中pbp2b可以作为一个推荐的基因位点筛查肺炎链球菌感染，并在一定程度上预测青霉素耐药性情况。

关键词: 肺炎链球菌, 青霉素耐药, 青霉素结合蛋白2B基因, 细菌性脑膜炎