A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7
A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7
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DOI：
10.1021/bp060020q
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发表时间：
2006-06-01
影响因子：
2.9
通讯作者：
Tanji, Yasunori
中科院分区：
文献类型：
--
作者：
Awais, Raheela;Fukudomi, Hiroshi;Tanji, Yasunori
用绿色荧光蛋白（GFP）标记的特异于大肠杆菌O 157：H7的PP 01噬菌体构建溶菌酶灭活的GFP标记的PP 01噬菌体（PP 01 e（-）/GFP）。新的重组噬菌体缺乏裂解活性，因为基因e失活，而基因e产生负责细胞裂解的溶菌酶。通过插入琥珀终止密码子使基因e失活。延长E. PP 01 e（-）/GFP在大肠杆菌O 157：H7细胞中的增殖增加了绿色荧光的强度，而PP 01 e（-）/GFP在大肠杆菌O 157：H7细胞中的增殖没有导致细胞裂解。细胞形态的保留和荧光的增加使得能够直接观察和计数E。coli O 157：H7细胞。PP 01 e（-）/GFP系统，当结合营养吸收分析，进一步允许区分检测可培养的，可行的，但不可培养（VBNC），和死细胞在胁迫诱导的水生环境。应力诱导的细胞，保留可培养性，允许噬菌体繁殖，并产生明亮的绿色荧光。不可培养的细胞（VBNC和死亡）只允许噬菌体吸附，但没有增殖，并保持低荧光。低荧光不可培养细胞进一步分化为VBNC和死细胞的基础上的营养摄取分析。在营养培养基中长时间孵育期间通过营养掺入而生长的低荧光细胞被定义为代谢活跃且处于VBNC状态。然后，细长的VBNC细胞很容易从死细胞中识别出来。所提出的测定能够检测和定量VBNC细胞。此外，它揭示了群体中可培养细胞与VBNC细胞的比例，而传统技术将VBNC细胞显示为总活细胞计数和可培养细胞计数的差值。
A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e(-)/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e(-)/GFP did not lead to cell lysis, while the propagation of PP01e(-)/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e(-)/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.