Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Abstract

Aims: The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral. Methods and Results: The occurrence of sublethal injury in the outer membrane was measured using selective recovery media containing bile salts. Loss of membrane integrity was measured by the increased uptake of the fluorescent dye propidium iodide (PI). PEF caused nonpermanent and permanent envelope permeabilization of Ent. sakazakii at pH 4·0. After PEF, most surviving cells showed transient cell permeabilization and sublethal injury in their outer membranes. The simultaneous application of a mild PEF treatment (100 pulses, 25 kV cm−1) and 200 μl l−1 of citral to cells suspended in pH 4·0 buffer at a final concentration of 107 cells per ml showed an outstanding synergistic lethal effect, causing the inactivation of more than two extra log10 cycles. Conclusions: Our results confirm that the detection of sublethal injury in the outer membrane after PEF may contribute to the identification of the treatment conditions under which PEF may act synergistically with hydrophobic compounds such as citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by PEF will aid the establishment of successful combined preservation treatments.

The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation

“…The synergistic effect was probably due to the damages inflicted on the outer membrane of E. coli by heat. Similar results have been observed when inactivating Enterobacter sakazakii by means of PEF and citral treatments (Arroyo, Somolinos, Cebrián, Condón, & Pagán, 2010). Since the outer membrane acts as an impermeable barrier to hydrophobic compounds, damage in the cell structure might represent an interesting opportunity to design combined processes that facilitate the action of antimicrobials.…”

“…The occurrence of sublethal injury after heat, especially on the outer membranes of Gram-negative bacteria, demonstrates the effectiveness of combined treatments with hydrophobic antimicrobials (Arroyo et al, 2010;Mackey, 2000;Somolinos et al, 2010). Our results showed that almost the entire surviving population was sublethally damaged after heat treatment at pH 4.0 and were likely susceptible to the presence of most antimicrobials.…”

Sublethal injury and recovery of Escherichia coli O157:H7 by high pressure carbon dioxide

“…Chilton et al (2001) reported that E. coli cells treated by high pressure (400 MPa/ 2 min) took about 3 h of incubation in TSB at 37 C to repair and regain the salt tolerance. Arroyo et al (2010) found more than 99.5% of Enterobacter sakazakii cells were sublethally injured after 100 pulses at 25 kV/cm and these cells completed repair in 2 h of incubation in TSB supplemented with 0.6% of yeast extract (TSBYE) at room temperature. Different results were obtained by Yuk et al (2010), who found that although 84% of E. coli cells were sublethally injured by HPCD at 7.6 MPa, 8% (CO 2 /sample) and 38 C, no re-growth or survival of E. coli was observed in HPCD processed apple cider during storage for 28 days at 4, 8, and 20 C. The authors attributed the observed phenomena to the acidic environment of apple cider, of which the pH was approximately 3.8, acting as a hurdle for the repair of the sublethally injured cells during storage (Yuk et al, 2010).…”

Sublethal injury and virulence changes in Listeria monocytogenes and Listeria innocua treated with antimicrobials carvacrol and citral

“…After each interval of time, 0.1 mL of L. innocua and L. monocytogenes samples adequately diluted in sterile 0.1% (w/v) peptone water (Scharlau Chemie S.A., Barcelona, Spain) was pourplated onto Tryptone Soya Agar (TSA; Scharlab Chemie S.A., Barcelona, Spain) supplemented with 0.6% yeast extract (TSA-YE) and onto TSA-YE selective medium with 5% (w/v) of sodium chloride added (TSA-YE-SC), in accordance with Arroyo et al (2010).…”

“…Sublethal injury is an important aspect that should be considered in evaluating the efficacy of any food preservation method because the presence of injured bacterial cells could be as dangerous as the presence of fully viable ones. Studies have shown that sublethally injured pathogens could proliferate to microbiologically hazardous levels when water and nutrients are plentiful (Busch and Donnelly, 1992;Ariefdjohan et al, 2004) and they may acquire new or modified characteristics (Lado and Yousef, 2002).…”