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

Abstract

bAs a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry. With increasing demands for safe, nutritious, and minimally processed products, ultrasound has attracted great interest as an alternative nonthermal technology for microbial inactivation in food preservation (1, 2). Since the 1960s, many studies have been conducted to investigate the bactericidal effects and mechanisms of ultrasound (3-5). At present, acoustic cavitation is most widely accepted as the mechanism of sterilization by highpower ultrasound (frequencies between 18 kHz and 100 kHz). The formation, growth, and collapse of cavitation bubbles in liquid media cause mechanical effects (microstreaming, high shear force, shock waves) and sonochemical reactions (free radicals, hydrogen peroxide), eventually resulting in the impairment or disruption of bacterial cells (6-8). The inhibitory effects of ultrasound on microbial cells are multifactorial, including pore formation, cell wall thinning, cell membrane disruption, release of cytoplasm contents, and damage to DNA structure (9, 10). However, there is still no consensus about the primary effect of ultrasound that causes cell death. 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).There have been numerous studies on the antimicrobial efficacy of ultrasound in the food industry, which have been reviewed by Awad et al. Unfortunately, in order to determine the impact of ultrasound treatment, most of...

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Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

“…Khan et al (2010) provided a useful insight into the magnitude of the VBNC populations present when cells were cultured in a non-nutrient limiting broth medium, showing that > 30% of cells were in the VBNC state while up to ∼5% could be classified as dead. The effects of cell damage on E. coli and S. aureus strains induced by various ultrasound treatments were evaluated by Li et al (2016). These authors compared plate counts with FCM following dual staining with PI and c FDA, the latter stain may reflect cell viability and/or cell vitality.…”

Inactivation of Staphylococcus aureus and Escherichia coli in milk by different processing sequences of ultrasound and heat

“…These treatments can severely impact the properties of food, causing undesirable flavors and decrease in nutritional value (Lacroix et al, ). For this reason, alternative nonthermal sterilization technologies, such as ultrasound (Ashokkumar, ; Jose et al, ; Li, Ahn, et al, ), high pressure carbon dioxide (Li, Xu, Zhao, Wang, & Liao, ; Liao, Zhang, Hu, & Liao, ), pulsed electric field (Zeng, Han, & Zi, ; Zhao, Yang, Shen, Zhang, & Chen, ), plasma (Liao et al, ), and ultraviolet radiation (Kumar, Williams, Sumner, & Eifert, ) have been explored to minimize the processing intensity, maximize the food quality, and ensure the microbiological safety.…”