Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

Abstract

Aims: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). Methods and Results: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. Conclusions: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. Significance and Impact of the study: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.

Fundamental and Applied Aspects of Pulsed Electric Fields for Microbial Inactivation

“…The fact that the presence of sodium chloride in the recovery medium prevents the growth of sublethal injured cells after PEF treatment and the demonstration that this damaged cells required the synthesis of lipids for injury repair supports the involvement of the citoplasmatic membrane on the microbial inactivation by PEF [7].…”

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

“…The PBS used in our study had a pH of 7.0, which was around the best growth pH of E. coli, while the pH of citrateephosphate buffer in García et al (2005) study was 4.0. It was reported that the sublethally injured cells had an increased sensitivity to stress conditions such as the presence of acids or other antimicrobial agents (García et al, 2006). Somolinos, García, Cond on, Ma nas, and Pag an (2007) also found that sublethally injured yeast cells by PEF were sensitive to sorbic acid and these cells were progressively inactivated rather than repaired by sorbic acid at a concentration of 2000 ppm in citratephosphate buffer.…”

“…PBS (10 mM potassium phosphate buffer, 8.4 g/L NaCl, pH 7.00), 0.1% peptone water (pH 7.00), TSB (pH 7.20), carrot juice (pH 6.80) were prepared and used to test the influence of different liquid media on the recovery of treated cells. TSB (pH 7.20) with sodium azide (500 mg/ml), chloramphenicol (100 mg/ml), rifampicin (10 mg/ml), or penicillin G (100 mg/ml) added was prepared and used to study the effect of inhibitors of specific metabolic processes on (Chilton et al, 2001;García et al, 2006). TSB was filled in dialysis bags (MD25, Union Carbide Corporation, US) with a molecular weight cutoff of 500, then dialyzed against deionized water (pH 6.0) at 4 C for 12 h to remove the ions.…”

“…Repair of membrane damage can be examined by incubating the sublethally injured cells in broth and monitoring changes in the selective media (Chilton, Isaacs, Ma nas, & Mackey, 2001;García, Ma nas, G omez, Raso, & Pag an, 2006). Meanwhile, it is possible to deduce the biosynthetic requirements for membrane repair by including inhibitors of specific metabolic processes in the broth medium (Chilton et al, 2001).…”

Aplicación de tecnologías no térmicas de conservación, pulsos eléctricos de alta intensidad (PEAI) y altas presiones hidrostáticas (APH), para el control de Cronobacter sakazakii en fórmulas lácteas infantiles: desarrollo de modelos predictivos y evaluación de la exposición

“…Sin embargo publicaciones recientes, describen el proceso de inactivación desde otro punto de vista (Garcia et al, 2003(Garcia et al, , 2005(Garcia et al, , 2006. El efecto de los PEF sobre la membrana celular es bien conocido en la actualidad.…”