Phytic Acid and Sodium Chloride Show Marked Synergistic Bactericidal Effects against Nonadapted and Acid-Adapted Escherichia coli O157:H7 Strains

Abstract

The synergistic antimicrobial effects of phytic acid (PA), a natural extract from rice bran, plus sodium chloride against Escherichia coli O157:H7 were examined. Exposure to NaCl alone at concentrations up to 36% (wt/wt) for 5 min did not reduce bacterial populations. The bactericidal effects of PA alone were much greater than those of other organic acids (acetic, citric, lactic, and malic acids) under the same experimental conditions (P < 0.05). Combining PA and NaCl under conditions that yielded negligible effects when each was used alone led to marked synergistic effects. For example, whereas 0.4% PA or 3 or 4% NaCl alone had little or no effect on cell viability, combining the two completely inactivated both nonadapted and acid-adapted cells, reducing their numbers to unrecoverable levels (>7-log CFU/ml reduction). Flow cytometry confirmed that PA disrupted the cell membrane to a greater extent than did other organic acids, although the cells remained viable. The combination of PA and NaCl induced complete disintegration of the cell membrane. By comparison, none of the other organic acids acted synergistically with NaCl, and neither did NaCl-HCl solutions at the same pH values as the test solutions of PA plus NaCl. These results suggest that PA has great potential as an effective bacterial membrane-permeabilizing agent, and we show that the combination is a promising alternative to conventional chemical disinfectants. These findings provide new insight into the utility of natural compounds as novel antimicrobial agents and increase our understanding of the mechanisms underlying the antibacterial activity of PA.

FIG 1

Chemical structure of phytic acid (2,3,4,5,6-pentaphosphonooxycyclohexyl dihydrogen phosphate).

FIG 2

Inactivation of nonadapted (a) and acid-adapted (b) Escherichia coli O157:H7 cells treated with 0.05 to 0.4% phytic acid alone, 2 to 4% sodium chloride alone, a combination of the two, or combinations of sodium chloride plus other organic acids (acetic, citric, lactic, and malic acid) at 0.4% (wt/wt). The gray panels represent combinations of sodium chloride and phytic acid. Initial nonadapted and acid-adapted E. coli O157:H7 populations were 7.13 ± 0.02 log CFU/ml and 7.02 ± 0.06 log CFU/ml, respectively. Values in the same panel but denoted by different capital letters (A to H) are significantly different (P < 0.05).

FIG 3

Flow cytometric analysis of nonadapted Escherichia coli O157:H7 cells treated with 0.2 to 1.6% acetic acid (a), citric acid (b), lactic acid (c), malic acid (d), or phytic acid (PA) (e) or with NaCl-HCl solutions at pH values equivalent to those of 0.2 to 1.6% PA solutions (f).

FIG 4

Flow cytometric analysis of acid-adapted Escherichia coli O157:H7 cells treated with 0.2 to 1.6% acetic acid (a), citric acid (b), lactic acid (c), malic acid (d), phytic acid (PA) (e), or NaCl-HCl solutions with pH values equivalent to those of 0.2 to 1.6% PA solutions (f).

FIG 5

Flow cytometric analysis of nonadapted Escherichia coli O157:H7 cells that were untreated (a) or treated with 2 to 4% NaCl (b to d), 0.2% PA (e), 0.4% PA (i), combinations of 0.2% PA (f to h) or 0.4% PA (j to l) plus 2 to 4% NaCl, a solution with a pH equivalent to that of 0.4% PA (o), or 2 to 4% NaCl solutions with pH values equivalent to those of 0.4% PA plus 2 to 4% NaCl (p to r).

FIG 6

Flow cytometric analysis of acid-adapted Escherichia coli O157:H7 cells that were untreated (a) or treated with 2 to 4% NaCl (b to d), 0.2% PA (e), 0.4% PA (i), combinations of 0.2% PA (f to h) or 0.4% PA (j to l) plus 2 to 4% NaCl, a solution with a pH equivalent to that of 0.4% PA (o), or 2 to 4% NaCl solutions with pH values equivalent to those of 0.4% PA plus 2 to 4% NaCl (p to r).