Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor

Abstract

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-Dual^TM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Keywords: HEK-dual hTLR 5; Salmonella; cell-based sensor; detection; flagella; food safety; immunomagnetic separation.

Figure 1

Cell-based assay for detection of Salmonella using HEK dual TLR5 sensor. (a,b) Schematic representation: this panel illustrates the flow diagram of the cell-based assay. The process begins by preparing the Quantiblue solution to track the TLR5 response. HEK cells engineered to express the TLR5 response are then exposed to varying concentrations of Salmonella bacteria, leading to an observable color change. The schematic details the steps in the assay, including cell culture, interaction with the bacteria, and the TLR5 response mechanism triggered by the bacterial flagella. Image created using Biorender.com. (c) This panel represents the analysis of the HEK dual TLR5 sensor’s response to various foodborne bacterial pathogens tested at MOI of 1 for 4 h and 6 h. (d) These panels present the analysis of the signal response from the HEK dual TLR5 sensor cells at different multiplicities of infection (MOI), examining the ratio of HEK cells to bacteria. The MOI ratios analyzed include various levels to determine the sensitivity and dynamic range of the assay. The left panel shows the signal response after 4 h of incubation, while the right panel depicts the response after 6 h. Data in (c,d) represent the average results from three independent experiments presented as mean ± SEM. *, p < 0.05; ***, p < 0.0001; ****, p < 0.00001.

Figure 2

Specificity analysis of the HEK dual TLR5 sensor’s response to various Salmonella serovars. (a) Specificity analysis of the HEK dual TLR5 sensor’s response to various Salmonella serovars. The assay was conducted at a multiplicity of infection (MOI) of 1, with 4 and 6 h exposure times. The data illustrate the sensor’s ability to detect different Salmonella serovars specifically. E. coli O157:H¯493-89, a non-motile strain, was used as a negative control. (b) Specificity analysis of the HEK dual TLR5 sensor’s response against non-Salmonella bacteria: this panel shows the analysis of the HEK dual TLR5 sensor’s response to a range of non-Salmonella bacteria. Like panel (a), the assay was performed at an MOI of 1, with signal responses measured after 4 and 6 h of exposure. Salmonella PT21 was used as a positive control. (c) Cytotoxicity was determined by using an LDH release assay. The assay was conducted at a multiplicity of infection (MOI) of 1, with 6-h exposure times. A 1% Triton-X100 was used as a positive control (100% cytotoxicity). Data in (a–c) represent the average results from three independent experiments presented as mean ± SEM. ***, p < 0.0001; ****, p < 0.00001.

Figure 3

Analysis of the HEK Dual TLR5 Sensor’s Response in the presence of stressors. (a) Response to live and dead bacteria: this panel illustrates the HEK dual TLR5 sensor’s response to both live and dead bacteria after 6 h of exposure at a multiplicity of infection (MOI) 1. Panels on the top right depict representative color changes of the HEK dual TLR5 sensors between live (purple) and dead (pink) tested samples. (b,c) Effects of stressors on TLR5 response: these panels investigate how different stressors affect the TLR5 response of Salmonella PT21. Panel (b) shows the TLR5 response after 4 h of exposure, while panel (c) presents the response after 6 h of exposure, both tested at an MOI of 1. Data in (a–c) represent the average results from three independent experiments presented as mean ± SEM. ****, p < 0.00001.

Figure 4

Limit of detection analysis of HEK dual TLR5 sensors to detect Salmonella from spiked chicken skin and peanut butter samples. (a) TLR 5 sensors’ response after 4 and 6 h incubation of spiked chicken samples. Plate image representative showing color development at 6 h (below). (b) TLR 5 sensors’ response after 4 and 6 h incubation of spiked peanut butter samples. Data in (a,b) represent the average results from three independent experiments presented as mean ± SEM. **, p < 0.001; ***, p < 0.0001; ****, p < 0.00001.

Figure 5

Detection of Salmonella using HEKdualTLR 5 sensor from spiked food samples. (a) TLR 5 sensor response after 6 h incubation of spiked food samples. Spiked food samples were subjected to 4-h pre-enrichment and 4-h selective enrichment (initial contamination level was ~100 CFU/25 g food sample). Salmonella was captured and concentrated using immunomagnetic beads (IMB) in tests with IMS. Data represent the average results from three independent experiments presented as mean ± SEM. ***, p < 0.0001 (b) Representative microscopic images show an intact cell monolayer treated with bacterial suspension concentrated from all food samples.