Mammalian cell-based immunoassay for detection of viable bacterial pathogens

Authors: XU, LUPING - Purdue University; BAI, XINGJIAN - Purdue University; TENGURIA, SHIVENDRA - Purdue University; LIU, YI - Purdue University; DROLIA, RISHI - Purdue University; BHUNIA, ARUN - Purdue University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2020
Publication Date: 11/23/2020
Citation: Xu, L., Bai, X., Tenguria, S., Liu, Y., Drolia, R., Bhunia, A.K. 2020. Mammalian cell-based immunoassay for detection of viable bacterial pathogens. Frontiers in Microbiology. Available in: Volume 11.Article 57615.
DOI: https://doi.org/10.3389/fmicb.2020.575615

Interpretive Summary: Rapid detection of live pathogens is paramount to ensure food safety. At present, DNA-based (PCR) and antibody-based (lateral flow assays) methods are the primary choices for rapid foodborne pathogen detection, but these methods are prone to interference from inhibitors and resident microbes. In contrast, mammalian cell-based biosensor assays detect pathogen interaction with the host cells and are responsive to only live pathogens. Unfortunately, the short shelf-life of the mammalian cells is a major impediment to its widespread application. This study reports an innovative approach to prolong the shelf-life of mammalian cells using formalin. This assay provides sensitive and specific detection of Salmonella in 10-12 hours, while the formalin-fixed stable cell monolayers provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate. This assay provides a food safety testing protocol that can distinguish live pathogenic bacteria from dead cells and represents an additional tool for microbial testing of the food supply.

Technical Abstract: Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.