基质辅助激光解析电离飞行时间质谱快速检测ST11型产KPC-2肺炎克雷伯菌

doi:10.3969/j.issn.1673-8640.2018.06.016

摘要/Abstract

摘要：

目的采用基质辅助激光解析电离飞行时间质谱（MALDI-TOF MS）筛选并验证ST11型产KPC-2肺炎克雷伯菌的特异峰,建立快速检测此型别肺炎克雷伯菌的方法。方法收集前期已进行碳青霉烯酶基因检测和多位点序列分型（MLST）的118株肺炎克雷伯菌作为特异峰筛选组,其中ST11型产KPC-2肺炎克雷伯菌67株,非ST11型产KPC-2肺炎克雷伯菌51株,采用Clinpro Tools 3.0软件和Flex analysis 3.0图谱分析软件筛选出ST11型产KPC-2肺炎克雷伯菌的特异峰;再收集前期已进行碳青霉烯酶基因检测和MLST的另外89株肺炎克雷伯菌作为特异峰验证组,严格按照试验条件获得质量图谱,对特异峰的特异性进行验证并进行重复性试验;最后随机收集95株肺炎克雷伯菌临床分离株及其临床鉴定图谱,利用特异峰直接判断菌株是否为ST11型产KPC-2肺炎克雷伯菌,同时对菌株进行碳青霉烯酶基因检测和MLST,以评估特异峰的临床应用价值。结果利用分析软件筛选获得ST11型产KPC-2肺炎克雷伯菌的最佳特异峰为m/z 4 521;在试验条件下,以该峰作为检测此型别细菌的特异峰,其特异性和敏感性分别为98.1%和97.3%,且该特异峰的重复性达97.4%;采用临床鉴定图谱对特异峰的临床应用价值进行评估,其特异性和敏感性分别为95.2%和96.9%。结论 MALDI-TOF MS可检出ST11型产KPC-2肺炎克雷伯菌的特异峰m/z 4 521。可依据细菌鉴定图谱是否存在此特异峰来直接检测ST11型产KPC-2肺炎克雷伯菌,该特异峰具有较高的特异性和敏感性,可快速、准确地为临床合理、有效的治疗和医院感染的控制提供依据。

关键词: 基质辅助激光解析电离飞行时间质谱, 特异峰, 肺炎克雷伯菌, 碳青霉烯酶, 多位点序列分型

Abstract:

Objective To screen and verify the specific peak of KPC-2 producing Klebsiella pneumoniae ST11 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF MS）,and to establish a rapid method for the detection of Klebsiella pneumoniae. Methods A total of 118 isolates of Klebsiella pneumoniae which had been confirmed by carbapenemase gene detection and classified by multilocus sequence typing（MLST） were collected as specific peak screening group,67 isolates of them were KPC-2 producing Klebsiella pneumoniae ST11,and 51 isolates of them were non-KPC-2 producing Klebsiella pneumoniae ST11. Clinpro Tools 3.0 software and Flex analysis 3.0 profile analysis software were used to screen the specific peak. Other 89 isolates of Klebsiella pneumoniae which had detected by carbapenemase gene detection and classified by MLST were selected as specific peak verifying group. Mass spectra were acquired strictly according to experimental conditions,and the specificity and repeatability of specific peak were detected. Finally,95 isolates of Klebsiella pneumoniae and their mass spectra were collected. These isolates were distinguished directly by the specific peak. Meanwhile,carbapenemase gene detection and MLST were performed for these isolates in order to evaluate the efficiency of specific peak. Results The specific peak of KPC-2 producing Klebsiella pneumoniae ST11 was m/z 4 521. In the experimental conditions,the specificity and sensitivity were 98.1% and 97.3%,and the repeatability of specific peak was 97.4%. In the clinical conditions,using mass spectra,the specificity and sensitivity were 95.2% and 96.9%,respectively. Conclusions The specific peak m/z 4 521 on MALDI-TOF MS is the specific peak of KPC-2 producing Klebsiella pneumoniae ST11. KPC-2 producing Klebsiella pneumoniae ST11 can be detected directly according to the specific peak with high specificity and sensitivity,which can provide a reference for clinical treatment and nosocomial infection control rapidly and accurately.

Key words: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Specific peak, Klebsiella pneumoniae, Carbapenemase, Multilocus sequence typing

中图分类号:

R446.5

余佳佳, 刘婧娴, 李媛睿, 俞静, 刘瑛. 基质辅助激光解析电离飞行时间质谱快速检测ST11型产KPC-2肺炎克雷伯菌[J]. 检验医学, 2018, 33(6): 536-542.

YU Jiajia, LIU Jingxian, LI Yuanrui, YU Jing, LIU Ying. Rapid detection of KPC-2 producing Klebsiella pneumoniae ST11 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry[J]. Laboratory Medicine, 2018, 33(6): 536-542.

图/表 6

表1 118株特异峰筛选组菌株的碳青霉烯酶基因类型和MLST

组别	总株数	碳青霉烯酶基因	MLST（ST型）	株数
A组	67	KPC-2	11	67
B组	51	NDM-1	37、48、147、438、520、846	18
		IMP-1	1723	1
		IMP-4	323	1
		Neg	11、15、23、29、323和1 436等19个ST型	31

图1 质量峰m/z 4 521、2 763、4 510、6 514和4 648的ROC曲线m/z 4 510信噪比注：（a）m/z 4 512,AUC=1.000;（b）m/z 2 763,AUC=0.998;（c）m/z 4 510,AUC=0.947;（d）m/z 6 514,AUC=0.925;（e）m/z 4 648,AUC=0.893

图2 ST11型产KPC-2肺炎克雷伯菌和其他型别肺炎克雷伯菌特异峰信噪比分析注：×代表ST11型产KPC-2肺炎克雷伯菌（A组）;ο代表其他类型肺炎克雷伯菌（B组）

图3 不同型别肺炎克雷伯菌的MALDI-TOF MS质量图谱

表2 89株特异峰验证菌株的碳青霉烯酶基因型别和MLST

碳青霉烯酶基因	MLST（ST型）	株数
KPC-2	11	37
KPC-2	423、65	7
NDM-1	37、48、76、478、520、846	20
IMP-4	2168、2169	2
Neg	14、15、17、23、285和1655等17个ST型	23

表3 95株临床菌株碳青霉烯酶基因型别及MLST

特异峰	总株数	碳青霉烯酶基因	MLST（ST型）	株数
无m/z 4 521	61	Neg	15、29、65、322、1326和2180等35个ST型	55
		KPC-2	11	1
		NDM-1	17、337	5
有m/z 4 521	34	KPC-2	11	31
		Neg	11、29	3

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