Phage-based forensic tool for spatial visualization of bacterial contaminants in cheese

Abstract

Traditional procedures for microbial testing typically involve a homogenizing step. These methods give valuable information on the presence or enumeration of a bacterial contaminant, but not where the contaminant was in the original sample. Spatial information could be useful in troubleshooting sources of bacterial contamination in a processing plant. For example, if the contaminant was localized on the top of a food such as cheese, this might indicate dripping condensate along a specific processing line as its source. The objective of this proof-of-concept study was to evaluate the use of a genetically engineered phage to detect bacterial contaminants on cheese to be able to visualize the contaminants without the use of magnification. In this study, a T7 bacteriophage engineered to overexpress the luciferase NanoLuc (Promega, Madison, WI) was utilized to reveal the spatial location of Escherichia coli on lysogeny broth (LB) agar and queso fresco (QF). Four scenarios were tested to explore how phage may be applied, with a blue bioluminescent signal revealing the spatial location of contaminants: (1) phage applied topically via molten soft agar to E. coli-inoculated (a) LB agar or (b) QF; and (2) phage incorporated within (a) LB agar or (b) QF and then inoculated with E. coli. Each was tested in triplicate. Cultures of E. coli BL21 grown for 18 h were serially diluted in phosphate-buffered saline and inoculated onto 8 ± 0.5 g of LB agar or QF in 6-well plates. Plates were incubated at 37°C for 8 h for condition 1a, 24 h for 1b and 2b, and 22 h for 2a. For 1a and 1b, stock phage was added to molten soft agar, applied topically, and incubated for 2 additional hours to allow for E. coli infection. After incubation, the substrate NanoGlo (Promega) was added to cover the surface of the agar or cheese and imaged immediately in a dark box using a digital camera and long exposure to capture the bioluminescent signal. Photographs captured small blue spots where the incubated colony-forming units were located. The lowest inoculum level of E. coli detected for each scenario was 1.43 × 10¹ ± 9.94, 1.18 × 10¹ ± 7.07, 5.48 × 10¹ ± 1.19 × 10¹, and 2.37 × 10¹ ± 1.40 × 10¹ cfu/well, for 1a, 1b, 2a, and 2b, respectively. These data demonstrate that the reporter phage proof-of-concept could be used as a forensic tool to visualize the spatial location of bacteria in a cheese matrix. Future work will translate this concept to dairy-relevant phage-pathogen systems.

Keywords: NanoLuc; cheese; luciferase; phage-based forensics.