Three Dimensional Vero Cell-Platform for Rapid and Sensitive Screening of Shiga-Toxin Producing Escherichia coli

Abstract

Shiga-toxin producing Escherichia coli (STEC) is a serious public health concern. Current Vero cell assay, although sensitive, is lengthy and requires 48–72 h to assess STEC presence in a sample. In this study, we investigated if Vero cells in a three-dimensional (3D) platform would provide improved sensitivity for rapid screening of STEC. Vero cells (epithelial kidney cell line) were grown as a monolayer (2D) or in a collagen-matrix (3D) and exposed to Shiga-toxin (Stx) preparation or STEC cells that were pre-exposed to antibiotics (mitomycin C, ciprofloxacin, or polymyxin B) for toxin induction. Lactate dehydrogenase (LDH) release from Vero cells was used as a biomarker for cytotoxicity. Modified tryptic soy broth (mTSB) as enrichment broth containing mitomycin C (2 μg/ml) or ciprofloxacin (100 ng/ml) significantly induced Stx production, which was further confirmed by the dot-immunoblot assay. The 3D Vero platform detected STEC after 6 h post-infection with cytotoxicity values ranging from 33 to 79%, which is considerably faster than the traditional 2D platform, when tested with STEC. The cytotoxicity for non-Stx producing bacteria, Salmonella , Listeria , Citrobacter , Serratia , and Hafnia was found to be below the cytotoxicity cutoff value of 15%. The detection limit for the 3D Vero cell assay was estimated to be 10 7 CFU/ml for bacteria and about 32 ng/ml for Stx in 6 h. STEC-inoculated ground beef samples ( n = 27) resulted in 38–46% cytotoxicity, and the bacterial isolates ( n = 42) from ground beef samples were further confirmed to be stx1 and stx2 positive in a multiplex PCR yielding a very low false-positive result. This 3D cell-based screening assay relies on mammalian cell pathogen interaction that can complement other molecular techniques for the detection of cell-free Stx or STEC cells from food samples for early detection and prevention.

Current State of Development of Biosensors and Their Application in Foodborne Pathogen Detection

“…The limitation of previously mentioned sensors is that none of these tests can confirm the functionality of the targeted toxin and pathogen, as antibody only picks up a specific region on the surface, and aptamers only recognize a partial sequence of the analyte. (13,14,43,96).…”

“…and Bhunia(96) recently proposed a three-dimensional Vero cellbased sensor to detect Shiga-toxin-producing E. coli cells or Shiga toxin (Stx). When exposed to STEC/Stx, the cytotoxicity values are increased, which are then used as an indicator of the STEC presence in the food sample(96).…”

“…and Bhunia(96) recently proposed a three-dimensional Vero cellbased sensor to detect Shiga-toxin-producing E. coli cells or Shiga toxin (Stx). When exposed to STEC/Stx, the cytotoxicity values are increased, which are then used as an indicator of the STEC presence in the food sample(96). The sensor's detection limit is estimated to be 10 7 CFU/mL for bacteria and about 32 ng/mL for Stx in 6 h. Other cellular activities, such as cell viability, apoptosis, and intracellular calcium could also be monitored as alternative approaches for pathogen/toxin detection because the exposure to different triggers influences those activities.Jiang et al (42) recently reported a mast cell-based electrochemical sensor for detecting N-acyl-homoserine-lactones (AHL), a pathogenic bacterial quorum signaling molecule.…”

Isolation and characterization of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy Indian cattle

“…Five representative colonies were picked from each plate and further purified by three passages on SMAC agar. The isolates were subjected to Gram staining and IMViC (Indole, Methyl Red, Voges-Proskauer test, and Citrate utilization test) [15]. The presumptive positive E. coli were further confirmed by PCR.…”

Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

“…Later, this cell line was used in a collagen-encapsulated 3-D platform to detect Listeria monocytogenes cells and its toxins (Banerjee et al, 2008) and several other toxin-secreting foodborne pathogens (Banerjee and Bhunia, 2010). Most recently, a 3-D Vero cell-platform was made to screen Shiga-toxin producing Escherichia coli (STEC) by measuring lactate dehydrogenase (LDH) release (To and Bhunia, 2019). Although these studies demonstrate the versatility of CBBs in detecting foodborne pathogens and toxins, the specificity of CBBs cannot be guaranteed when the detection solely relies on cytotoxicity measurement because cytosolic proteins/enzymes could be released from cells in response to more than one type of triggers.…”