A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases
A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases
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DOI：
10.1111/j.1469-0691.2012.03986.x
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发表时间：
2013-01-01
影响因子：
14.2
通讯作者：
Pina-Vaz, C.
中科院分区：
文献类型：
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作者：
Faria-Ramos, I.;Espinar, M. J.;Pina-Vaz, C.
超广谱 β-内酰胺酶 (ESBL) 的快速检测对大多数临床微生物学实验室来说是一个挑战，因为 ESBL 产生者的不准确识别对抗生素治疗和感染控制具有重要的临床意义。我们研究的目的是开发一种基于流式细胞分析的 ESBL 产生者快速检测方法。对临床分离株（41 个 ESBL 阳性和 20 个 ESBL 阴性）和表达充分表征的 β-内酰胺酶的分离株（包括 ESBL (n = 13)、质粒 AmpCs (n = 3)、苯唑西林酶 (n = 5) 和碳青霉烯酶）进行抗菌药物敏感性测试，然后对 bla(TEM)、bla(SHV) 或 bla(CTX-M) 基因进行分子表征（n = 3）。此外，还使用 CLSI 推荐用于药敏试验的两种 ATCC 菌株作为对照。流式细胞术分析方案包括在存在和不存在克拉维酸的情况下，将细菌细胞与不同浓度的头孢他啶（1、2和4 mg/L）和头孢噻肟（4、8和16 mg/L）一起孵育1和2小时；随后，细胞用荧光染料双-(1,3-二丁基巴比妥酸)三甲川氧杂菁[DiBAC(4)(3)]染色，这是一种能够扩散穿过去极化膜的亲脂性阴离子。此外，还进行了 CFU 计数。孵育 1 小时后，敏感分离株显示出增强的荧光；相反，只有在 ESBL 生产者中与头孢他啶或头孢噻肟相关的克拉维酸一起孵育后，才能观察到去极化群体的增加。通过流式细胞术和传统 CFU 测定定量的非去极化细菌数量之间获得了极好的相关性。一种新颖、准确且快速的流式细胞术可用于检测 ESBL 的存在。
The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla(TEM), bla(SHV) or bla(CTX-M) genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.