Population Reductions of Gram-Negative Pathogens Following Treatments with Nisin and Chelators under Various Conditions

Abstract

When used in combination with chelating agents (EDTA, EGTA, citrate, phosphate), the bacteriocin nisin is effective for reducing populations of gram-negative bacteria in vitro. This study examined parameters (buffers, temperature presence of divalent cations) that affect nisin inhibition of Escherichia coli O157:H7 and Salmonella typhimurium. Approximately 7 log10 colony-forming units (CFU) per ml of E. coli and S. typhimurium were treated in PBS or MOPS buffers containing 50 μg/ml of purified nisin, alone or in combination with 500mM lactate, 100mM citrate, 50mM EDTA, and 1% (wt/vol) sodium hexametaphosphate (pH 7.0) at 37°C for 60 min or 5°C for 30 min. Surviving bacterial populations were compared to untreated controls (buffers without nisin). Data indicated that treatments with nisin in buffers resulted in reductions of 4.30 and 2.30 log10 CFU/ml of E. coli and S. typhimurium, respectively, as compared to untreated controls. Population reductions ranging from 2.29 to 5.49 log10 CFU/ml were observed when cells were treated with nisin and chelator combinations at either 37°C for 60 min or 5°C for 30 min. The addition of magnesium and calcium to buffers with nisin decreased inhibition. Data obtained from spectrophotometric experiments indicated that treatments were causing the release of cellular constituents. However, transmission electron microscopy (TEM) analyses were inconclusive, since cellular membranes did not appear to be disrupted.