The use of a novel nanoLuc-based reporter phage for the detection of Escherichia coli O157:H7

Authors: ZHANG, DANDAN - Purdue University; CORONEL-AGUILERA, CLAUDIA - Purdue University; ROMERO, PATRICIA - Purdue University; PERRY, LYNDA - Purdue University; MINOCHA, UDIT - Purdue University; ROSENFIELD, CARLA - Purdue University; Gehring, Andrew; Paoli, George; BHUNIA, ARUN - Purdue University; APPLEGATE, BRUCE - Purdue University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/23/2016
Publication Date: 9/14/2016
Citation: Zhang, D., Coronel-Aguilera, C., Romero, P., Perry, L., Minocha, U., Rosenfield, C., Gehring, A.G., Paoli, G., Bhunia, A.K., Applegate, B. 2016. The use of a novel nanoLuc-based reporter phage for the detection of Escherichia coli O157:H7. Scientific Reports. doi: 10.1038/srep33235.

Interpretive Summary: Harmful E. coli bacteria are major foodborne pathogens, causing more than 200,000 illnesses, 4,000 hospitalizations, and numerous deaths in the US each year, at an economic burden of nearly $300,000,000. More rapid and accurate methods for the detection of this deadly bacterium will prevent the consumption of contaminated foods and reduce the current economic and public health burdens. Scientists from the Purdue University Center for Food Safety Engineering, in collaboration with ARS scientists in Wyndmoor, Pennsylvania, report here the development of a novel method for the detection of pathogenic E. coli using a bacteriophage (a.k.a., phage; a virus that only infects bacteria) that specifically infects the most common type of foodborne pathogenic E. coli. The phage was modified so that when it infects the E. coli it produces light that can be seen by the naked eye or detected using sensitive light sensors. This phage-based method is very sensitive, highly accurate and specific, and more rapid than the current regulatory methods used for detection of pathogenic E. coli. Food producers and regulatory agencies may employ this method to detect the pathogen and prevent the distribution and consumption of potentially harmful food.

Technical Abstract: Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food, as they are host-specific and able to differentiate live cells from dead ones. Upon infection by the reporter phage, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage FV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep- sea shrimp Oplophorus gracilirostris. Once infected by the FV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo®). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76×10E2 pfu ml-1 are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23×10E3 pfu/ml of phage using in selective culture enrichments of ground beef as a representative food matrix within 9 hours using 9.23×10E3 pfu/ml of phage. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.