title: A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7.
abstract: 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.
authors.affiliation: Department of Bioengineering, Tokyo Institute of Technology, 4259 J2-15 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Raheela
authors.initials: R
authors.lastname: Awais
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fukudomi
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Miyanaga
authors.forename: Hajime
authors.initials: H
authors.lastname: Unno
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Tanji
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D004926:Escherichia coli / Q000166:cytology* / Q000254:growth & development* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D009113:Muramidase