Phenotypic and Genotypic Methods for Detection of Extended Spectrum β Lactamase Producing Escherichia coil and Klebsiella pneumoniae Isolated from Ventilator Associated Pneumonia
Phenotypic and Genotypic Methods for Detection of Extended Spectrum β Lactamase Producing Escherichia coil and Klebsiella pneumoniae Isolated from Ventilator Associated Pneumonia
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DOI：
10.7860/jcdr/2013/6544.3376
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发表时间：
2013-09-01
影响因子：
0.2
通讯作者：
Nagaraj, E. R.
中科院分区：
文献类型：
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作者：
Krishnarnurthy, Veena;Vijaykumar, G. S.;Nagaraj, E. R.
背景资料：呼吸机相关性肺炎（VAP）是常见的医院感染之一，其病原菌耐多药，发病率高。病原菌株对广谱β-内酰胺类抗生素的耐药性迅速蔓延，导致抗生素无效和疾病严重程度增加。CTX-M是世界上许多地区肠杆菌科细菌中最主要的超广谱内酰胺酶（ESBL）。本研究的目的是确定ESBL的发生，并通过传统的聚合酶链反应（PCR）方法检测负责ESBL生产的基因。方法：这项前瞻性研究包括患者，临床诊断为VAP。收集气管内吸出物（EA），采用定量方法进行培养。按照标准方法鉴定细菌分离株。用纸片扩散法和纸片扩散法联合筛选对第三代头孢菌素耐药的ESBL菌株。用常规PCR方法对ESBL阳性菌株进行基因分型。统计学分析：用MS Excel表格进行统计学分析。结果：428例VAP患者中，肠杆菌科细菌144株，其中肺炎克雷伯菌87株，大肠埃希菌57株。用纸片逼近法和双纸片联合法分别检测出66株产ESBL菌（肺炎克雷伯菌28株，大肠埃希菌38株）和63株。在本研究中，用常规PCR方法检测ESBL的共有基因为blaCTX-M，占48.5%，其次为blaSHV和blaTEM，分别占22.22%和14.81%。结论：用特异性PCR扩增耐药基因的基因型方法检测ESBL具有100%的特异性和敏感性，而表型方法缺乏恒定的敏感性。
Background: Ventilator Associated Pneumonia (VAP) is one of the common nosocomial infections associated with high morbidity due to nnultidrug resistant pathogens. Rapid spread of resistance to broad spectrum beta-lactams in pathogenic strains causes antibiotics ineffectiveness and increased severity of illness. The CTX-M is the most dominant Extended Spectrum Lactamase (ESBL) among Enterobacteriaceae in many regions of the world. The aim of the study was to identify the occurrence of ESBL and detect the genes responsible for ESBL production by conventional Polymerase Chain Reaction (PCR) method.Methods: This prospective study included patients, clinically diagnosed as VAP. Endotracheal aspirates (EA) were collected and cultured by quantitative method. The bacterial isolates were identified as per standard methods. Isolates resistant to 3rd generation cephalosporins were screened for ESBL production by disk approximation method and combination disc diffusion method. Isolates confirmed as ESBL producers were subjected to genotyping by conventional PCR.Statistical Analysis: Statistical analysis was done by using MS Excel sheet. Descriptive statistics like percentage was done in the study.Results: Among the isolates from 428 patients who developed VAP, 144 isolates belonged to the Enterobacteriaceae family (Klebsiella pneumoniae 87 and Escherichia coil 57). A total of 66 isolates (28 Klebsiella pneumoniae and 38 Escherichia colt) were confirmed as ESBL producer by disc approximation method and 63 isolates by double disc combination method. In the present study by conventional PCR bla CTX-M was the common gene in 48.5% strains followed by 22.22% bla SHV and 14.81% bla TEM.Conclusion: The genotypic methods using specific PCR amplification of resistance genes seems to have 100% specificity and sensitivity in detection of ESBL when compared to phenotypic methods which lacks the constant sensitivity.