Molecular regulatory mechanisms of Escherichia coli O157:H7 in response to ultrasonic stress revealed by proteomic analysis

The antimicrobial effects of ultrasonic filed have been studied for years at the phenotypic level, but there is little research to reveal the molecular regulatory mechanisms underlying the phenotypes. In this study, isobaric tag for relative and absolute quantification (iTRAQ) proteome was applied to analyze the regulatory networks of Escherichia coli O157:H7 in response to ultrasonic stress in whole-genome scale. A total of 1856 differentially expressed proteins were identified, of which 1141 were significant up-regulated and 715 down-regulated compared with live control cells. The comprehensive proteome coverage analysis showed that ultrasonic filed influenced various metabolic pathways in Escherichia coli O157:H7 cells. The ultrasound-induced up-regulation of global stress response regulator RpoS, bacterial mechanosensitive channels and SOS response protein RecA were described from the molecular level for the first time. In addition, we proposed a possible action mechanism that the free radicals produced by acoustic cavitation might enter into cells via the activated mechanosensitive channels, leading to the elevated intracellular ROS level and subsequent cell death. Last but not the least, we illustrated the all-or-nothing phenomenon of power ultrasound might due to the destruction of crucial cell defensive systems, including heat shock proteins and oxidative response regulators. These new findings can let us understand the ultrasonic effects more deeply and will contribute to this area.     

Antibacterial mechanism of ultrasound against Escherichia coli: Alterations in membrane microstructures and properties

This study was aimed at providing new insights on the response of bacterial cell membranes to ultrasound exposure. Escherichia coli (E. coli) O157:H7 cells were exposed to different ultrasound treatments (power intensities of 64, 191, 372, and 573 W/cm², frequency of 20 kHz, pulsed mode of 2 sec: 2 sec) and the dynamic changes in cell viability within 27 min were assessed. With an increase in ultrasonic intensity and prolonged duration, a 0.76–3.52 log CFU/mL reduction in E. coli populations was attained. The alterations in the sensitivity of ultrasound-treated cells to antimicrobial compounds were evaluated by exposure to thyme essential oil nanoemulsion (TEON). The treatment reduced the E. coli population by 2.16–7.10 log CFU/mL, indicating the effects of ultrasonic field on facilitating the antibacterial efficacy of TEON. Ultrasonic-treated E. coli cells also displayed remarkable morphological and ultrastructural damages with destroyed membrane integrity and misshaped cell structures, which was observed by electron microscopy analysis. Significant increase in outer and inner membrane permeability, along with the cytoplasmic leakage and membrane depolarization were assessed utilizing spectrophotometry. For the first time, significant reduction in the membrane fluidity in response to ultrasound exposure were investigated. Additional efforts in exploring the effect of ultrasonic field on some bacterial membrane compositions were performed with infrared spectroscopy. In this study, multiple lines of evidence effectively served to elucidate the alterations on cellular membrane structure and property during exposure to sonication that could extend our understanding of the antimicrobial molecular mechanisms of ultrasound.     

Synergistic inactivation of bacteria based on a combination of low frequency,low-intensity ultrasound and a food grade antioxidant

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.     

Proteomic responses of spores of Bacillus subtilis to thermosonication involve large-scale alterations in metabolic pathways

Thermosonication (TS) impacts numerous characteristics of spores, such as morphology, cell metabolism, and stress resistance. However, relevant mechanisms need to be clarified. In the present study, the effect of TS treatment on Bacillus subtilis spores was investigated at phenotypic and proteomic levels. The results showed that TS treatment induced significant changes to spores in growth kinetics and morphology. A total of 167 differentially expressed proteins (DEPs) were obtained after TS treatment at 6.67 W/mL and 80 °C. Among these proteins, 80 were up-regulated, whereas 87 were down-regulated. These DEPs were classed into 20 functional categories. Enrichment analysis of the proteome revealed that the major categories were associated with metabolic functions, including energy metabolic processes, amino acids biosynthesis and metabolism, translation and ribosomal protein. In summary, B. subtilis spores showed alteration primarily in the proteins that were associated with metabolism under TS treatment. These findings could be applied to the development and optimization of TS-based sporicidal treatment.     

Using fluorescence measurement of zinc ions liberated from ZnS nanoparticle labels in bioassay for <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

In this study, an alternative approach using ZnS nanoparticle biolabels as fluorescence signal transducers is reported for the immunoassay of E. coli O157:H7 in tap water samples. Instead of measuring the fluorescence of ZnS nanoparticles in the assay, the fluorescence signal is generated through the binding of zinc ions released from nanoparticle labels with zinc-ion sensitive fluorescence indicator Fluozin-3. In the assay, ZnS nanoparticles around 50 nm in diameter were synthesized, bioconjugated, and applied for the detection of E. coli O157:H7. The assay shows a detection range over two orders of magnitude and a detection limit around 1000 colony-forming units (cfu) of E. coli O157:H7.     

Lactobacillus plantarum-derived biosurfactant: Ultrasound-induced production and characterization

The aim of the present study was to investigate the effect of ultrasonic treatment (25 kHz) on biosurfactant production by Lactobacillus plantarum ATCC 8014. The impacts of the ultrasonication (with a frequency of 25 kHz and power of 7.4 W for 30 min time duration) were examined at different stages of the fermentation process to obtain the optimum stimulation instant(s). The optimum scenario was found to be one-time sonication at the 12^th hour of fermentation which can be beneficial from an economic point of view (compared with multiple applications of sonication). Ultrasonic treatment at this time resulted in enhancement of the productivities of biomass (4.5 g/L) and biosurfactant (2.01 g/L) which was almost 1.3 times higher than those of the non-sonicated control samples. According to our results, it was clearly observed that glucose consumption increased after ultrasonic treatment representing the improved substrate uptake and progression of the cellular metabolism. Furthermore, the transmission electron microscopic images immediately after sonication clarified the pore formation on the cell surfaces. The results also indicated the enhancement of plasma membrane permeability of the sonicated cells. Fourier transform infrared spectroscopy and scanning electron microscopy coupled with energy dispersive x-ray spectroscopy analyses also disclosed respectively no structural differences before and after ultrasonic exposure in the produced biosurfactant and bacterial cell membrane. The biosurfactant was characterized to be a mixture of carbohydrate (28%), protein (23%) and lipid (specified by gas chromatography-mass spectrometry) known as glycolipoprotein. The sustainable critical micelle concentration and the stability of the synthesized biosurfactant can feature its potential applicability in various processes in the food and pharmaceutical industries.     

Effects of ultrasound-assisted low-concentration chlorine washing on ready-to-eat winter jujube (Zizyphus jujuba Mill. cv. Dongzao): Cross-contamination prevention,decontamination efficacy,and fruit quality

Wash water is circulated for use in the minimal processing industry, and inefficient disinfection methods can lead to pathogen cross-contamination. Moreover, few disinfection strategies are available for ready-to-eat fruits that do not need to be cut. In this study, the use of chlorine and ultrasound, two low-cost disinfection methods, were evaluated to disinfect winter jujube, a delicious, nutritious, and widely sold fruit in China. Ultrasound treatment (28 kHz) alone could not decrease the cross-contamination incidence of Escherichia coli O157:H7, non-O157 E. coli, and Salmonella Typhimurium, and free chlorine treatment at 10 ppm decreased the incidence from 55.00% to 5.00% for E. coli O157:H7, 65.00% to 6.67% for non-157 E. coli, and 70.00% to 6.67% for S. Typhimurium. The cross-contamination incidence was completely reduced (pathogens were not detected in sample) when the treatments were combined. The counts of aerobic mesophiles, aerobic psychrophiles, molds, yeasts, and three pathogens in the group subjected to combination treatment (28 kHz ultrasound + 10 ppm free chlorine) were significantly lower than those in the control, chlorine-treated, and ultrasound-treated groups during storage (0–7 d at 4 °C). Analysis of weight loss, sensory quality (crispness, color, and flavor), instrument color (a*/b*), soluble matter contents (total soluble solids, reducing sugar, total soluble sugar, and titratable acid), and nutritional properties (ascorbic acid and polyphenolic contents) indicated that treatment with ultrasound, chlorine, and their combination did not lead to additional quality loss compared with properties of the control. Additionally, the activities of phenylalanine ammonia-lyase and polyphenol oxidase were not significantly increased in the treatment group, consistent with the quality analysis results. These findings provide insights into disinfection of uncut ready-to-eat fruits using a minimum dose of disinfectant for cross-contamination prevention under ultrasonication. The use of ultrasound alone to decontaminate fresh produce is accompanied by a high risk of pathogen contamination, and the use of sanitizers to decrease cross-contamination incidence is recommended.     

Cyclodextrin metal–organic framework by ultrasound-assisted rapid synthesis for caffeic acid loading and antibacterial application

Cyclodextrin metal–organic framework by ultrasound-assisted rapid synthesis for caffeic acid (CA) loading and antibacterial application (U-CD-MOF) was successfully studied and this method shortened the preparation time to a few minutes. It was found that the ultrasonic power, reaction time and temperature would affect the morphology and size of the obtained crystal. Under the optimal conditions, U-CD-MOF had a cubic structure with uniform size of 8.60 ± 1.95 μm. U-CD-MOF was used to load the antibacterial natural product CA to form the composite (CA@U-CD-MOF) and the loading rate of CA@U-CD-MOF to CA could reach 19.63 ± 2.53%, which was more than twice that of γ-CD. Various techniques were applied to characterize the synthesized crystal, including Powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and N₂ adsorption. In addition, antibacterial tests were performed on the obtained crystal. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CA@U-CD-MOF for Escherichia coli O157: H7 (E. coli O157: H7) were both 25 mg·mL⁻¹, and the MIC for Staphylococcus aureus (S. aureus). was 25 mg·mL⁻¹. The sustained release behavior of CA@U-CD-MOF to CA in ethanol fitted well to Higuchi model and the loading of CA was supported by molecular docking results. In general, U-CD-MOF was successfully achieved by ultrasound-assisted rapid synthesis and the obtained crystal was further evaluated for potential antibacterial application.     

Synergistic antibacterial effects of ultrasound and thyme essential oils nanoemulsion against Escherichia coli O157:H7

Essential oil nanoemulsions have been proven to have stronger antimicrobial effects compared to the essential oil alone or coarse emulsion. Sonoporation could be the promising candidate to trigger a synergistic effect with thyme essential oil nanoemulsion (TEON) and produce a more effective antibacterial efficacy. Therefore, in this study, the bactericidal effects of ultrasound (US) in combination with TEON treatments against Escherichia coli (E. coli) O157:H7 were investigated. The remarkable synergistic effects of US (20 kHz, 255 W/cm², 9 min) and TEON (0.375 mg/mL) treatments at 22 °C reduced E. coli O157:H7 populations by 7.42 ± 0.27 log CFU/mL.The morphological changes of cells exposed to different treatments were observed by scanning electron microscopy and transmission electron microscopy. The results showed that the synergistic effects of the ultrasound and TEON treatments altered the morphology and interior microstructure of organism cells. Laser scanning confocal microscopy (LSCM) images revealed that the combination treatments of ultrasound and TEON altered the permeability of cell membranes, and this affected the integrity of E. coli O157:H7 cells. This was further indicated by the high amounts of nucleic acids and proteins released from these cells following treatment.The results from this study illustrated the mechanisms of the synergistic effects of sonoporation and TEON treatments and provided valuable information for their potential in food pasteurization.     

Sonophotocatalytic inactivation of E. coli using ZnO nanofluids and its mechanism

The present study evaluated inactivation efficiency of a sonophotocatalytic process using ZnO nanofluids including ultrasonic parameters such as power density, frequency and time. The result showed that inactivation efficiency was increased by 20% when ultrasonic irradiation was combined with photocatalytic process in the presence of natural light. Comparison of inactivation efficiency in photocatalytic, ultrasonic and sonocatalytic processes using Escherichia coli as a model bacteria identified that inactivation efficiencies are shown in the following order: ultrasonic irradiation < sonocatalysis < photocatalysis < sonophotocatalysis. Furthermore, inactivation mechanism of sonophotocatalysis was proposed. Studies of reactive oxygen species (ROS) and zinc ions (Zn²⁺) release evaluation revealed that ROS play a key role in bacterial inactivation rather than Zn²⁺. Permeability of outer membrane (OM) and inner membrane (IM) of E. coli bacterial cells were studied and exhibited that sonophotocatalysis increased the permeability of OM and IM significantly. The enhanced bacterial inactivation effect in sonophotocatalytic process contributed to acoustic cavitation, sonocatalysis of ZnO and sonoporation phenomenon.     

Combination of ozone and ultrasonic-assisted aerosolization sanitizer as a sanitizing process to disinfect fresh-cut lettuce

Reduction of sanitizer dosage and development of non-immersion disinfection methods have become major focuses of research. Here, we examined the disinfection efficacy of combining gaseous ozone (4 and 8 ppm) with aerosolized oxidizing sanitizer [sodium hypochlorite (SH, 100 and 200 ppm)] and aerosolized organic acid [acetic acid (AA, 1% and 2%) and lactic acid (LA, 1% and 2%)]. Notably, 1% AA and 4 ppm gaseous ozone were ineffective for disinfecting Salmonella Typhimurium, and treatment with 1% AA + 8 ppm ozone caused browning of lettuce leaves and stimulated increases in aerobic mesophilic count (AMC), aerobic psychrotrophic count (APC), S. Typhimurium, and Escherichia coli O157:H7. Treatment with 2% LA + 8 ppm ozone resulted in the lowest S. Typhimurium, E. coli O157:H7, Listeria monocytogenes, AMC, APC, and molds and yeasts during storage (0–7 days at 4 °C). Quality analysis indicates that LA + 8 ppm ozone and SH + 8 ppm ozone did not negatively affect L*, a*, b*, polyphenolic content, weight loss, and sensory properties; however, the levels of two individual phenolic compounds (3,4-dihydroxybenzoic acid and vanillin), responsible for phenylpropanoid synthesis, were significantly increased after treatment with 2% LA + 8 ppm ozone. These findings provided insights into the use of LA combined with gaseous ozone for application in disinfecting fresh produce.     

Combined effect of ultrasound,heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity,and anthocyanins in blueberry (Vaccinium corymbosum) juice

The objective of this study was to evaluate the effect of different treatments—heat treatment (HT), sonication (SC), thermosonication (TS), manosonication (MS), manothermal (MT), and manothermosonication (MTS) on Escherichia coli O157:H7, polyphenol oxidase (PPO), and anthocyanin content in blueberry juice. First, samples were treated at different temperatures (30, 40, 50, 60, 70, and 80 °C) and power intensities (280, 420, 560, and 700 W) for 10 min. Subsequently, samples were treated using combinations of power intensity and mild temperature for 10 min. For further study, samples were treated using HT (80 °C), TS (40 °C, 560 W), MT (350 MPa, 40 °C), MS (560 W, 5 min/350 MPa), or MTS (560 W, 5 min, 40 °C/350 MPa, 40 °C) for 5, 10, 15, 20 min for each treatment, and the results compared between treatments. HT significantly reduced PPO activation (2.05% residual activity after only 5 min), and resulted in a 2.00-log reduction in E. coli O157:H7 and an 85.25% retention of anthocyanin. Escherichia coli O157:H7 was slightly inactivated by TS after 5 min (0.17-log reduction), while residual PPO activity was 23.36% and anthocyanin retention was 98.48%. However, Escherichia coli O157:H7 was rapidly inactivated by MTS (5.85-log reduction) after 5 min, while anthocyanin retention was 97.49% and residual PPO activity dropped to 10.91%. The destruction of E. coli cells as a result of these treatments were confirmed using SEM and TEM. Therefore, a combination of sonication, high pressure, and mild heat allows the safety of blueberry juice to be maintained without compromising the retention of desirable antioxidant compounds.     

Detection of pathogenic microorganisms using biosensor based on multi-walled carbon nanotubes dispersed in DNA solution

The present paper introduces a facile and cost-effective route for the direct dispersion of multi-walled carbon nanotubes (MWCNTs) in DNA solution. Their application in detecting Escherichia coli O157:H7 using DNA biosensor was demonstrated. The dispersion state of the MWCNTs was characterized via UV–Vis spectroscopy, transmission electron microscopy, and field emission scanning electron microscopy. The interaction between DNA sequence and the MWCNTs was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. As-obtained MWCNT solution was used in the preparation of DNA sensor. Results revealed that the developed DNA sensor can detect a DNA target as low as 1 nM in a buffer solution. The sensitivity of the DNA sensor reached approximately 0.19 nM/mV. The effect of dispersion parameters, including pH values, DNA concentration, ion strength, and sonication time, on sensor response was also studied. The DNA sensor can respond well to 120 min of sonication time, a pH value of 9, and 20 μM of DNA sequence concentration. The results of the present study showed a potential application of the DNA sensor in the detection of Escherichia coli O157:H7.     

Reduction of Escherichia coli O157:H7 on beef surfaces using low-voltage direct electric current and the impact on sensory properties

Low-voltage direct current was applied to beef, inoculated with Escherichia coli O157:H7 on the surface covered with a thin film of 0.15 M NaCl solution. Experiments were conducted with 15, 30, and 45 mA/cm² currents; 1, 10 and 100 kHz frequencies; 30, 50 and 70% duty cycles, and 2, 8 and 16 min treatment durations. Increase in current intensity, frequency, duty cycle, and treatment duration increased the % reduction of E. coli. A maximum reduction of 98.9% was achieved. Sensory color analysis showed significant differences between treated and untreated beef. The maximum temperature rise of NaCl solution was 31.9 °C.     

Characterization of self-assembled monolayers (SAMs) on silicon substrate comparative with polymer substrate for Escherichia coli O157:H7 detection

This article presents the characterization of two substrates, silicon and polymer coated with gold, that are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the anti-Escherichia coli O157:H7 polyclonal purified antibody.A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed. Immobilization of goat anti-E. coli O157:H7 antibody was performed by covalently bonding of thiolate mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon coated with gold. The F(ab′)₂ fragments of the antibodies have been used for eliminating nonspecific bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best results.These experimental results revealed the necessity to investigate each stage of the immobilization process taking into account in the same time the factors that influence the chemistry of the surface and the further interactions as well and also provide a solid basis for further studies aiming at elaborating sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.     

Microbial inactivation in cloudy apple juice by multi-frequency Dynashock power ultrasound

The study determined the efficacy of Dynashock wave power ultrasound as an alternative processing technique for apple juice against a number of pathogenic and spoilage microorganisms. The effects of several implicit, intrinsic and extrinsic properties on the Dynashock wave inactivation of the microorganisms were also investigated. Results showed that acid adaptation increased the resistance of Escherichia coli O157:H7 and Salmonella spp. but decreased that of Listeria monocytogenes. Spoilage yeast mixed inoculum composed of Debaryomyces hansenii, Torulaspora delbrueckii, Clavispora lusitaniae, Pichia fermentans and Saccharomyces cerevisiae was found to be more resistant than any of the adapted or non-adapted pathogens. Among the individual, acid-adapted E. coli O157:H7, the MN-28 isolate was found most resistant; while three other individual isolates had greater resistance than the composited E. coli inoculum. Increased in pulp content decreased the efficacy of Dynashock waves, but co-treatment with ultraviolet-C rays significantly enhanced inactivation in the cloudy apple juice. The results demonstrated the potential of Dynashock wave technology, together with other antimicrobial hurdles as alternative juice processing technique/s.     

An immunomagnetic separator for concentration of pathogenic micro-organisms from large volume samples

The standard method of immunomagnetic separation of pathogenic bacteria from food and environmental matrices processes 1 ml volumes. Pathogens present at low levels (<1 pathogenic bacteria per ml) will not be consistently detected by this method. Here a flow through immunomagnetic separator (FTIMS) has been designed and tested to process large volume samples (>50 ml). Preliminary results show that between 70 and 113 times more Escherchia coli O157 are recovered compared with the standard 1 ml method.     

Ultrasonic emulsification of food-grade nanoemulsion formulation and evaluation of its bactericidal activity

Basil oil (Ocimum basilicum) nanoemulsion was formulated using non-ionic surfactant Tween80 and water by ultrasonic emulsification method. Process of nanoemulsion development was optimized for parameters such as surfactant concentration and emulsification time to achieve minimum droplet diameter with high physical stability. Surfactant concentration was found to have a negative correlation with droplet diameter, whereas emulsification time had a positive correlation with droplet diameter and also with intrinsic stability of the emulsion. Stable basil oil nanoemulsion with droplet diameter 29.3 nm was formulated by ultrasonic emulsification for 15 min. Formulated nanoemulsion was evaluated for antibacterial activity against Escherichia coli by kinetics of killing experiment. Fluorescence microscopy and FT-IR results showed that nanoemulsion treatment resulted alteration in permeability and surface features of bacterial cell membrane.