Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

Abstract

Aims: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. Method and Results: According to plate count results, E. coli (D-value 8AE3 min) was far more sensitive than L. rhamnosus (D-value 18AE1 min) in their response to the ultrasound intensity applied (20 kHz, 17AE6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. Conclusions:The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. Significance and Impact of the Study: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.

Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

“…Given the existence of the outer membrane, no considerable changes in the PI-positive population were observed at first for E. coli compared to S. aureus, indicating that the E. coli cytoplasmic membrane was not severely affected under those circumstances. This phenomenon was in agreement with a published study (13) proving that under certain processing conditions, ultrasound could be selective, merely destabilizing the E. coli outer membrane without rupturing the cytoplasmic membrane. Thus, the outer membrane of an E. coli cell is an important target site for ultrasound treatment.…”

“…Most researchers have argued that the primary target of ultrasound is the cytoplasmic membrane, which consists of lipoprotein layers (11,12). Ananta et al, on the other hand, suggested that ultrasound-induced cell death might not be related to cytoplasmic membrane damage (13).…”

Low-frequency ultrasound increases outer membrane permeability of Pseudomonas aeruginosa

“…Our current work strengthens the hypothesis that ultrasound creates holes or perturbations in the outer membrane lipid bilayer sufficiently large for the passage of relatively large hydrophilic compounds, including antibiotics. A more recent publication investigating the ultrasonic-enhanced permeability of E. coli and Lactobacillus rhamnosus toward fluorescent dyes also supported the hypothesis that transient holes in cell membranes are formed (Ananta et al, 2005).…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Ananta and colleagues (32), found primarily the OM of E. coli affected by ultrasound when certain processing conditions (i.e., continuous removing of the resulting heat) were applied. E. coli cells with an intact OM and stained with cFDA showed only weak green fluorescence, whereas ultrasound treated cells presented an increased fluorescent intensity.…”