3D Cell-Based Assay to Detect Shiga-Toxin Producing Escherichia coli
Shiga-like toxin producing Escherichia coli (STEC), a foodborne pathogen, poses a serious public health risk. Rapid detection of STEC in the food supply remains to be a challenge because current detection methods lack the ability to differentiate viable and non-viable STEC or active and inactive Shiga-like toxins (Stx) in less than 24 h. Here, our goal was to develop a mammalian cell-based assay (CARD) for rapid high-throughput detection of Shiga-toxin producing Escherichia coli using Vero (monkey kidney epithelial cells) or THP-1 (human monocyte cell) cell lines. Initially, optimal mammalian cell concentration needed to show a maximum cytotoxicity towards a membrane active detergent, Triton-X 100 (2%) or crude Stx was determined by using different dilutions of Vero cells (8 ×103 – 2.6 ×10 5/well). Cytotoxicity was assayed by measuring the lactate dehydrogenase (LDH) release in a spectrophotometer and by Trypan blue uptake in a light microscope. Then, the effect of STEC growth media (Luria Bertani (LB) broth, modified tryptic soy broth (mTSB), or E. coli (EC) broth), and antibiotics (mitomycin C, 2 µg/ml, ciprofloxacin, 100 ng/ml, and polymyxin B, 1 mg/ml) on assay interference using Vero and THP-1 were examined. Stx production after UV and antibiotic treatments were quantified by dot-immunoblot using anti-Stx antibodies. Next, both cell lines were grown and immobilized in a collagen gel matrix to create a 3-dimension (3D) platform, CARD, which was further evaluated to determine the limit of detection (LOD), and optimal time (2 – 16 h) required to obtain a positive response against crude or purified toxin preparations. Finally, the CARD was tested for specificity against a panel of pathogenic and commensal bacteria in a buffer or in a model food system, where 27 raw ground beef samples (32 ± 3.2 g) were inoculated at 4 CFU/g. Bacterial isolates in food were further verified by stx1 and stx2-specific multiplex PCR and by plating on selective agar media, sorbitol MacConkey agar (SMAC). Optimal mammalian seeding cell number was determined to be 3.2 × 104 cells/well, which showed maximum cytotoxicity after exposure to a STEC O157:H7 strain 204P for 16 h. LB mixed with Dulbecco’s modified Eagles’ Medium (DMEM) showed the least interference with Vero cell cytotoxicity than mTSB or EC broth. Likewise, mitomycin C and ciprofloxacin had no negative effect on Vero cells while polymyxin B did. Ciprofloxacin induced 1.2-fold Stx1 production while mitomycin C induced a 3.2-fold Stx2 production, compared to the polymyxin B and UV light induction. Analysis of cytotoxicity of viable STEC cells on 3D Vero gave the highest LDH release as opposed to Vero and THP-1 cells in their natural monolayer or suspension state, referred to as 2D, and the LOD was estimated to be 107–8 CFU/ml or 31.25 ng toxin/ml in 6 h with a cut off value of 12–15% i.e., three standard deviations above the mean of all negative controls. Crude or purified Stx preparations showed low but similar cytotoxicity towards both cell lines either in 2D or 3D configuration, but at 16 h, toxins induced the highest cytotoxicity from 3D Vero than the 2D Vero, 2D THP-1, or 3D THP-1. STEC or toxin-induced Vero/THP-1 cell damages were also confirmed by Trypan blue staining and Cryo-SEM. 3D-CARD was highly specific for STEC cells and did not yield any positive response from Salmonella enterica, Listeria monocytogenes, Citrobacter freundii, Hafnia alvei, Serratia marcescens, and non-pathogenic E. coli. Finally, 3D-CARD successfully detected STEC from artificially contaminated ground beef samples (n=27) following a 15-h enrichment (~10 8 CFU/ml) in mTSB and 6-h cytotoxicity assay, with confirmation of STEC from positive food samples on SMAC plate and stx1 and stx2 specific PCR. This study investigated and compared the cytotoxicity of STEC or cell-free Stx preparation on THP-1 and Vero cell line in 2D and 3D platforms and the study is the first of a kind for such analysis. The results demonstrated that 3D Vero cell-based CARD is suitable for detection of STEC or Stx after a 15-h enrichment in mTSB followed by a 6-h cytotoxicity assay providing results much faster than the traditional Vero cell assay that requires 72 h or longer.
Bhunia, Purdue University.
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