Quantification of viable but nonculturable Escherichia coli O157:H7 by targeting the rpoS mRNA.
Escherichia coli O157:H7 easily becomes viable but nonculturable (VBNC) under environmental stresses and escapes detection by current methods. Here, we report a unique method enabling the quantification of VBNC E. coli O157:H7 using a selective marker within the rpoS gene. A nucleotide at position +543 within the rpoS gene open reading frame was identified to be unique to E. coli O157:H7. Specifically designed primers and probe combinations were able to differentiate E. coli O157:H7 from closely related bacteria and other common bacteria. The application of this strategy correctly identified 36 clinical and bovine isolates of E. coli O157:H7. A one-step quantification method combining reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR) was developed to provide a linear relationship (R(2) > 0.99) of copies of RNA with threshold cycles (Ct) and the capability of detecting a single copy of rpoS RNA standards. This technique was used to determine the copies of the rpoS mRNA in culturable cells at different growth phases (mid-log, late-log, and stationary phase) to be 1.57, 0.56, and 0.41 copies/CFU, respectively. VBNC E. coli O157:H7 was determined to have one copy of the rpoS mRNA for every 10 cells, and no rpoS mRNA was detected in 10(6) dead cells and negative controls. This technique had a linear dynamic range over 6 orders of magnitude and >90% amplification efficiency for tap and river water samples. It was able to selectively quantify as few as 7 E. coli O157:H7 cells in pure culture, 9 culturable cells in tap water and river water, and 23 VBNC cells in river water, demonstrating the best quantification limits for culturable and VBNC E. coli O157:H7 in environmental water.
Liu
Yanming
Y
Wang
Chuan
C
Fung
Christina
C
Li
Xing-Fang
XF
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002417:Cattle; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012703:Serotyping; D012808:Sigma Factor / Q000235:genetics*; D014871:Water Microbiology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria