Use of Interactive Laser-Scanning Imaging Cytometry (ILIC) for Real-Time Cytotoxicity Assessment of Bacteriocins Against Cultured Mammalian Cells

by Shelton E. Murinda, PhD, Robert F. Roberts, PhD, Elaine M. Kunze, and Kamal A. Rashid, PhD
Volume 13, Issue 4 (Winter 2014/2015)

The evaluation of bacteriocin cytotoxicity is a critical first step in guaranteeing its safe use in food and therapeutic applications. In this study, the bacteriocins nisin, pediocin, and colicin ([Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured mammalian cells. Cellular membrane potential (ΔΨ) changes of bacteriocin-treated cells were used as an index of cytotoxicity. Simian virus 40-transfected human colon (SV40-HC) cells and African green monkey kidney epithelial cells (Vero) cells were cultured (35°C, 10% CO2, humidified air) as monolayers on tissue culture plates. Log phase cells (~104 cells/mL) were treated with partially purified, individual bacteriocin preparations at 170, 350, and 700 activity units (AU)/mL. Perturbations in cellular ΔΨ were measured using interactive laser-scanning imaging cytometry (ILIC) with 3,3’-dipentyloxacarbocyanine iodide (DiOC5[3]), a potential-sensitive, fluorescent dye. Cellular ΔΨ studies indicated significant levels of dose-dependent toxicity with nisin, pediocin, and colicins E6, E7, and K. In contrast, Col E1 and E3 had no definitive toxic impact on SV40-HC cells. The only colicin to significantly impact Vero cells negatively was the E6. Overall, SV40-HC cells demonstrated greater sensitivity to bacteriocins than Vero cells. Among all the fluorescence parameters and peak statistics measured, the integrated area under the curve appeared to correlate best with the prediction of cytotoxic events. Consequences were quickly apparent with maximum changes occurring within 3–5 minutes following the addition of test substances. Refinements in the ILIC protocol could potentially afford a unique tool for rapid, real-time cytotoxicity testing of bacteriocins in food preservation, and in human and animal health applications...

Citation:
Murinda SE, Roberts RF, Kunze EM, Rashid KA. Use of interactive laser-scanning imaging cytometry (ILIC) for real-time cytotoxicity assessment of bacteriocins against cultured mammalian cells. BioProcess J, 2015; 13(4): 21–32. http://dx.doi.org/10.12665/J134.Murinda.

Posted online January 20, 2015.