Synergetic Inactivation Mechanism of Protocatechuic Acid and High Hydrostatic Pressure against Escherichia coli O157:H7

Abstract

With the wide application of high hydrostatic pressure (HHP) technology in the food industry, safety issues regarding food products, resulting in potential food safety hazards, have arisen. To address such problems, this study explored the synergetic bactericidal effects and mechanisms of protocatechuic acid (PCA) and HHP against Escherichia coli O157:H7. At greater than 200 MPa, PCA (1.25 mg/mL for 60 min) plus HHP treatments had significant synergetic bactericidal effects that positively correlated with pressure. After a combined treatment at 500 MPa for 5 min, an approximate 9.0 log CFU/mL colony decline occurred, whereas the individual HHP and PCA treatments caused 4.48 and 1.06 log CFU/mL colony decreases, respectively. Mechanistically, membrane integrity and morphology were damaged, and the permeability increased when E. coli O157: H7 was exposed to the synergetic stress of PCA plus HHP. Inside cells, the synergetic treatment additionally targeted the activities of enzymes such as superoxide dismutase, catalase and ATPase, which were inhibited significantly (p ≤ 0.05) when exposed to high pressure. Moreover, an analysis of circular dichroism spectra indicated that the synergetic treatment caused a change in DNA structure, which was expressed as the redshift of the characteristic absorption peak. Thus, the synergetic treatment of PCA plus HHP may be used as a decontamination method owing to the good bactericidal effects on multiple targets.

Keywords: Escherichia coli O157:H7; high hydrostatic pressure; mechanism; protocatechuic acid; synergetic bactericidal.

Figure 1

The determination of membrane permeability of sample after different treatments by propidium iodide (PI) uptake method. The results were presented in cross quadrant images. (a) and (A), untreated sample and sample with single 1/2 MIC PCA treatment (1.25 mg/mL for 60 min at 37 °C), respectively; (b) to (d) and (B) to (D), HHP group and PCA + HHP group samples with 100, 300, 500 MPa pressure treatments for 5 min, respectively. Percentage of cells in each quadrant was labelled on the top right-hand corner. UL, upper left region; UR, upper right region; LL, lower left region; LR, lower right region. The vertical axis and abscissa axis represented the relative fluorescence intensity at 488 nm and 530 nm.

Figure 2

The effect on the amounts of intracellular protein (A), nucleic acid (B), K⁺ (C) and Mg²⁺ (D) in E. coli O157:H7 after different treatments. Different letters a–f and A–E mean differ significantly within the HHP group and PCA + HHP group (p ≤ 0.05, Duncan), respectively. Symbol * and ** mean the difference is significant at the 0.05 and 0.01 level, respectively, between the HHP group and PCA + HHP group (t-test). The HHP group was E. coli O157:H7 treated with 0 (untreated cells), 100, 200, 300, 400 and 500 MPa for 5 min, respectively. The PCA + HHP group was E. coli O157:H7 treated with 1/2 MIC PCA treatment (1.25 mg/mL for 60 min at 37 °C) combined with 0 (single PCA treated cells), 100, 200, 300, 400 and 500 MPa for 5 min, respectively.

Figure 3

The relative activity of CAT (A), SOD (B), ATPase (C, Na⁺ K⁺-ATPase, Ca²⁺ Mg²⁺-ATPase and total- ATPase activity) and the change of membrane potential (D) of E. coli O157:H7 after different treatments. Different letters a–d and A,B mean differ significantly within the HHP group and PCA + HHP group (p ≤ 0.05, Duncan), respectively. * p ≤ 0.05 and ** p ≤ 0.01 mean differ significantly within HHP and PCA + HHP group.

Figure 4

Circular dichroism of intracellular protein (A) and nucleic acid (B) after different treatments. UT (red line), untreated control; HHP (black line), sample with 300 MPa pressure for 5 min treatment; PCA (blue line), sample with 1/2 MIC PCA treatment (1.25 mg/mL for 60 min at 37 °C); PCA + HHP (green line), sample treated with 300 MPa pressure combined with 1/2 MIC PCA treatment.

Figure 5

Morphological changes of E. coli O157:H7 caused by different treatments. A1, A2, A3 and A4 were atomic force microscopy (AFM) height images of untreated group, HHP group, PCA group and PCA + HHP group, respectively. Red arrowheads indicated cells with shrunken surface and blue arrowheads holes or depressions. (B) was the average bacterial surface RMS in nm obtained on 605.1 × 605.1 nm² areas of surfaces of cells with different treatments. For each group, 10 cells were plotted, respectively. a–b mean statistically significant between different groups (p ≤ 0.05, Duncan).