Bacterial Lighthouses-Real-Time Detection of Yersinia enterocolitica by Quorum Sensing

Abstract

Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.

Keywords: N-acyl homoserine lactones; Yersinia enterocolitica; autoinducer; co-cultivation bioassay; food safety; foodborne pathogens; plasmid-based biosensor.

Figure 1

Schematic illustration of whole cell biosensor approach for Y. enterocolitica detection based on QS. OHHL produced and secreted by Y. enterocolitica induces the lux operon in recombinant E. coli biosensor resulting in luminescent signal response.

Figure 2

(a) Kinetic measurement of luminescence with the pMA biosensor and different standard concentrations of synthetic OHHL AI ranging from 200 nM to 0.3 nM (mean, n = 3). (b) Averaged calibration curves with corresponding regression functions at different times of luminescence measurement, indicating the relation between the signal generated by the pMA biosensor and OHHL standard concentrations (mean ± SD, n = 3).

Figure 3

Luminescence signal height after 120 min (mean ± SD, n = 3) with the pMA biosensor and culture supernatants plotted against the corresponding growth curve (average of two independent experiments ± SD, n = 4, including specific growth rate µ and doubling time t_D).

Figure 4

(a) Quantification of AI concentrations in culture supernatants of Y. enterocolitica by LC-MS/MS (OHHL: gray bars; HHL: blue bars; mean ± SD, n = 2) and by pMA biosensor (OHHL: green bars; mean ± SD, n = 3 calibration curves at 120 min assay time) in relation to the corresponding growth curve (average of two independent experiments ± SD, n = 4; including specific growth rate µ and doubling time t_D). (b) Zoomed section showing cultivation time 0 h to 6 h of cultivation.

Figure 5

Kinetic measurement of luminescence recorded in intervals of 20 min during the co-cultivation of Y. enterocolitica and the pMA biosensor with readout direction from the top (detection plate without cover). Theoretical initial cell numbers (n = 3) were estimated using the OD₆₀₀ and specific Yersinia correlation factor. The blank signal was obtained from cell medium without Y. enterocolitica (n = 11).

Figure 6

(a) Kinetic measurement of luminescence recorded in intervals of 20 min during the co-cultivation of Y. enterocolitica and the pMA biosensor according to the optimized co-cultivation approach with readout direction from the bottom (covered detection plate). A threshold was determined on the basis of the averaged blank in relation to the whole assay time and Equation (3). Exceeding the threshold indicates a positive, qualitative signal. Theoretical initial cell numbers (n = 3) were estimated using the OD₆₀₀ and specific Yersinia correlation factor. The blank signal was obtained from cell medium without Y. enterocolitica (n = 11). (b) Correlation between initial cell number and time until threshold is exceeded fitted with Hill1 equation (R² = 0.998, n = 3), enabling a quantitative estimation of the initial cell number.