Antibacterial and antibiofilm performance of low-frequency ultrasound against Escherichia coli O157:H7 and its application in fresh produce.

Ultrasound technology has been focused on due to its unique advantages in biofilm removal compared with traditional antibacterial methods. Herein, the anti-biofilm properties of low-frequency ultrasound (LFUS) were studied against Enterohemorrhagic Escherichia coli O157: H7 (E. coli O157:H7). After ultrasonication (20 kHz, 300 W) for 5 min, the removal rate of biofilm from polystyrene sheets reached up to 99.999 %. However, the bacterial cells could not be inactivated completely even extending the duration of ultrasonic irradiation to 30 min. Fortunately, this study indicated that LFUS could efficiently weaken the metabolic capacity and biofilm-forming ability of bacterial cells separated from biofilm. It could be associated with the removal of cell surface appendages and damage to cell membrane induced by mechanical vibration and acoustic cavitation. Besides, the genetic analysis proved that the transcription level of genes involved in curli formation was significantly down-regulated during ultrasonic irradiation, thus impeding the process of irreversible adhesion and cells aggregation. Finally, the actual application effect of LFUS was also evaluated in different fresh produces model. The results of this study would provide a theoretical basis for the further application of ultrasound in the food preservation.

Inhibition of Escherichia coli O157:H7 and Salmonella enterica virulence factors by benzyl isothiocyanate.

Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P < 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.

Photothermal enhancement in sensitivity of lateral flow assays for detection of E-coli O157:H7.

Lateral flow immunoassay (LFA) is a well-known point-of-care technology for the detection of various analytes. However, low sensitivity and lack of quantitative results are some of its critical drawbacks. Here we report a photothermal enhanced lateral flow sensor on the basis of the photothermal properties of reduced graphene oxide (rGO) for the detection of E-coli O157:H7 as a model pathogen. The calibration curve of the photothermal method exhibited a linear range from 5 × 105 to 5 × 107 CFU/ml with a correlation coefficient of R2 = 0.96 and a regression equation of y = 8.1x-43 for standard bacteria solutions in phosphate buffer. The limit of detection was ∼5 × 105 CFU/ml for standard bacteria solutions, which was a 10-fold enhancement in sensitivity compared to the qualitative results. Specificity experiments showed that the photothermal method can only detect the target bacteria among 6 types of bacteria strains. It was confirmed that the developed technique could be a highly potential method for the rapid detection field because it can provide fast quantitative results with improved sensitivity.

Inactivation mechanism of E. coli O157:H7 under ultrasonic sterilization.

Ultrasonic sterilization (US), as a promising non-thermal sterilization method, exhibits unique superiorities than traditional sterilization methods. In this study, the inactivation mechanism of E. coli O157:H7 under US was investigated in cucumber and bitter gourd vegetable juices. Results revealed that the US treatment showed good antibacterial ability in countering E. coli O157:H7. Through determinations of conductivity and ß-galactosidase activity, significant augmentation in membrane permeability of the bacteria was confirmed after the US treatment. The morphologies of the US treated E. coli O157:H7 demonstrated that the integrity of the cell membrane was disrupted by US treatment. SDS-PAGE and LSCM data further proved the disruptive action of US, leading to the leakage of proteins and DNA through the breakage on cell membrane. The decrease of metabolic-related enzyme activity was verified through investigation of bacterial metabolism. The antibacterial mechanism analysis indicated that the US can generate free radicals which resulted in the rise of intracellular oxidative stress, attenuation of energy metabolism and inhibition of hexose monophosphate pathway. As the application verification, the US treatment can cause the deprivation of E. coli O157:H7 cell viability in vegetable juices without obvious impact on the sensory quality.

Phase-Dependent Fluorescence Quenching Efficiency of MoS2 Nanosheets and Their Applications in Multiplex Target Biosensing.

Two-dimensional layered transition-metal dichalcogenide nanosheets have shown great potential in biosensors owing to their unique properties. Here, we exfoliated ultrathin metallic and semiconductive MoS2 nanosheets based on a chemical exfoliation method. We compared the difference of fluorescence quenching efficiency between metallic and semiconductive MoS2 nanosheets. We found that the fluorescence quenching efficiency of MoS2 nanosheets is phase-dependent. The ultrathin metallic MoS2 nanosheets with larger contents of a 1T-phase structure show higher fluorescence quenching efficiency than semiconductive MoS2 nanosheets, which can be ascribed to the higher conductivity of metallic MoS2 nanosheets. On the basis of the excellent fluorescence quenching efficiency of metallic MoS2 nanosheets and their discriminative adsorption toward single-strand DNA and double-strand DNA, a fluorescent biosensor for multiplex detection of DNA was developed. This fluorescent biosensing platform allows simultaneous fluorescence quenching of two single-strand DNA probes labeled with different fluorophores, resulting in multiplex detection of different DNA sequences in one homogeneous solution with high sensitivity.

Single cell immunodetection of Escherichia coli O157:H7 on an indium-tin-oxide electrode by using an electrochemical label with an organic-inorganic nanostructure.

A rapid and highly sensitive method is described for the detection of enterohemorrhagic Escherichia coli O157:H7. An organic-inorganic nanostructure in which numerous gold nanoparticles (AuNPs) are enclosed with polyaniline (PANI) was utilized as an electrochemical label. The nanostructure showed (a) strong light scattering intensity due to the coupling effect of the surface plasmon resonance based on the presence of AuNPs, and (b) high electrochemical response due to the redox activity of PANI. To achieve selectivity, antibody against E. coli O157:H7 was immobilized on the surface of the nanostructure. The method exploits the combination of strong adsorption of bacterial cells onto the indium-tin-oxide (ITO) glass electrode without any special processing and specific binding of the nanostructured label to E. coli O157:H7. This enables the electrochemical detection of a single cell on the ITO electrode. The electrochemical response to E. coli O157:H7 was 30-fold higher than that to other types of bacteria. This procedure can be applied to the determination of E. coli O157:H7 even in the presence of other bacteria. Graphical abstract Schematic of a voltammetric immunoassay for Escherichia coli O157:H7 by using a nanocomposite consisting of gold nanoparticles and polyaniline on an ITO electrode.

Engineering of chitosan-derived nanoparticles to enhance antimicrobial activity against foodborne pathogen Escherichia coli O157:H7.

Chitosan is an abundant and natural polymer derived from chitin, which presents a wide variety of properties, including antimicrobial activity. The raising of antibiotic resistant bacteria has increased the interest in finding alternatives to traditional antibiotics. Many studies have assessed the antimicrobial activity of chitosan itself, but a few have performed comparisons among different chitosan nanoparticle synthesis, which will be of particular interest for further applications. In this study, the effects of two types of cross-linking agents, sodium sulfate vs. tripolyphosphate, along with molecular weight (Mw) of chitosan, low vs. high Mw, and different sonication treatments, time and power, were assessed to determine the optimal conditions to enhance antimicrobial activity against bacterial pathogens. Physiochemical characteristics of the engineered chitosan nanoparticles were determined. It was observed that 20 min sonication time, low Mw of chitosan, Sodium sulfate as cross-linker, and particle size smaller than <300 nm, showed the greatest antimicrobial activity. Chitosan nanoparticles generated at this condition completely killed pathogenic E. coli O157:H7 without raising resistant bacteria, providing great insights into potential use as alternative antimicrobial agents.

Binding Constant of the Cell-shaped Cavity Formed on a Polymer for Escherichia coli O157.

The binding constant of receptors for small molecules, proteins, or antibodies is usually determined based on the concentrations of the ligand, receptor, and their complexes. The binding constant is used as a measure of the affinity between the ligand and the receptor. In the present study, we introduce a procedure to determine the binding constant of a cell-shaped cavity formed on a polymer by molecular imprinting for a whole cell. To determine the binding constant, we clarified the numbers of cavities and cells, based on the fluorescence of a single cell, and defined their concentrations. We successfully determined the binding constant of the complementary cavity for a whole cell (1.1 × 105 M-1). This is the first report to describe the binding constant of a complementary cavity for a whole cell.

Inactivation of Escherichia coli and Staphylococcus aureus on contaminated perilla leaves by Dielectric Barrier Discharge (DBD) plasma treatment.

This study focused on sterilization methods for the reduction of microorganisms on perilla leaves by cylinder type Dielectric Barrier Discharge (DBD) plasma with underwater bubbler treatment. S. aureus and E. coli in a suspension were reduced to less than 3.4 and 0.5 log CFU/ml after the plasma treatment for 3 min, respectively. On the perilla leaves, they were also reduced to 4.8 and 1.6 log CFU/ml after the plasma treatment, respectively. The S. aureus and E. coli bacterial cell wall was damaged by the plasma treatment evident by scanning electron microscopic analysis. The observed infrared bands of the FTIR spectra demonstrated changes in protein, lipid, polysaccharide, polyphosphate group and other carbohydrate functionalities of plasma treated bacteria and untreated bacterial cell membranes. The degradation of the constituent bonds of the bacterial cell membrane by RONS generated from plasma destroys the DNA, RNA, and proteins within the cell, and may eventually cause cell death. In this study, H2O2 (13.68 µM) and NO3 (138 µM), which are the main factors generated by plasma, proved to have a bactericidal effect by inducing lipid peroxidation of bacterial cell membranes. In conclusion, cylinder type DBD plasma with underwater bubbler can be used as an environmentally friendly food disinfection device in cleaning processes of the food industry.

DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method.

Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen. In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases.

Effect of immobilization on the antimicrobial activity of a cysteine-terminated antimicrobial Peptide Cecropin P1 tethered to silica nanoparticle against E. coli O157:H7 EDL933.

Antimicrobial peptides (AMPs) have the ability to penetrate the cell membrane, form pores which eventually lead to cell death. Immobilization of AMP on nanoparticles can play a major role in antimicrobial materials, biosensors for pathogen detection and in food safety. The minimum inhibitory concentration (MIC) of free Cecropin P1 (CP1, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPR) and adsorbed on silica nanoparticle against E. coli O157:H7 EDL933 were 0.78µg/ml. This was found to be consistent with preservation of α-helical secondary structure of CP1 upon adsorption as indicated by circular dichroism (CD). Cysteine-terminus modified Cecropin P1 (CP1C, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPRC) was chemically immobilized onto silica nanoparticles with maleimide-PEG-NHS ester cross-linkers of different PEG chain lengths. The antimicrobial activity of CP1C in solution and adsorbed on silica nanoparticles against E. coli O157:H7 EDL933 were found to be the same as those for CP1. However, tethered CP1C exhibited much higher MIC of 24.38, 37.55 and 109.82µg/ml for (PEG)20, (PEG)6 and (PEG)2 linkers respectively. The antimicrobial activity of CP1C tethered to silica nanoparticles with (PEG)20 linker was found to be lower for lower surface coverage with MIC values being 86.06, 36.89, 24.38 and 17.84µg/ml for surface coverage of 12.3%, 24.4%, 52.8% and 83.8% respectively. All atom MD simulation of 1:3 DOPG/DOPC mixed membrane interacting with free and PEGlyated CP1C indicated that presence of PEG linker prevented CP1C from interacting with the bilayer which may explain the loss of antimicrobial activity of tethered CP1C.

Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli O157 under cryopreservation.

As major food-borne pathogens worldwide, Escherichia coli are capable of toxin production directly causing severe human disease. However, routine methods are incapable of detecting viable but non-culturable (VBNC) bacteria in food products and raw materials, leading to false-negative identification. In this study, VBNC E. coli O157 strains were acquired after cryopreservation at -20 °C, with and without freeze-thawing; morphology was observed to be of shorter rod-shape, and toxin expression remained at relatively high levels. PMA-PCR assay for VBNC detection was also validated. Therefore, these results suggest that VBNC E. coli O157 strains may represent a strong threat to public health and food safety.

An integrated microfluidic PCR system with immunomagnetic nanoparticles for the detection of bacterial pathogens.

There is growing interest in rapid microbial pre-concentration methods to lower the detection limit of bacterial pathogens of low abundance in samples. Here, we report an integrated microfluidic PCR system that enables bacterial cells of interest in samples to be concentrated prior to PCR. It consists of two major compartments: a preconcentration chamber for the immunomagnetic separation of bacterial cells, and a PCR chamber for the DNA amplification of the concentrated cells. We demonstrate the feasibility of the system for the detection of microbial pathogens by preconcentrating the human pathogen Escherichia coli O157:H7, and also amplifying its DNA. The detection limit of E. coli O157:H7 in the PCR system is 1 × 103 CFU (colony forming unit)/mL. On-chip processing steps, including preconcentration and PCR steps, take less than two hours. Our system can serve as a rapid, specific, and quantitative platform for the detection of microbial pathogens in samples of large volume.

A new analytical platform based on field-flow fractionation and olfactory sensor to improve the detection of viable and non-viable bacteria in food.

An integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

Specific Single-Cell Isolation of Escherichia coli O157 from Environmental Water Samples by Using Flow Cytometry and Fluorescence-Activated Cell Sorting.

Contamination of food and water with pathogenic bacteria is of concern. Although culture-independent detection and quantification of pathogens is useful, isolation of pathogenic bacteria is still important when identifying the sources of pathogens. Here, we report the use of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) to specifically detect and isolate individual Escherichia coli O157:H7 cells from water samples. When present at >10 cells/mL water, target pathogen was specifically detected and isolated. The FACS-sorted E. coli O157:H7 population reflected the original population diversity, in contrast to the populations obtained by immunomagnetic separation. Relative abundance of multiple pathogenic strains is important when performing source-tracking studies; therefore, single-cell isolation with FCM-FACS can be a useful tool to obtain pathogenic bacteria for source tracking purpose.

Magnetic Nanoparticle-Based Platform for Characterization of Shiga-like Toxin 1 from Complex Samples.

Foodborne illness outbreaks resulting from contamination of Escherichia coli O157:H7 remain a serious concern in food safety. E. coli O157:H7 can cause bloody diarrhea, hemolytic uremic syndrome, or even death. The pathogenicity of E. coli O157:H7 is mainly caused by the expression of Shiga-like toxins (SLTs), i.e., SLT-1 and SLT-2. SLTs are pentamers composed of a single A and five B subunits. In this study, we propose a magnetic nanoparticle (MNP)-based platform to rapidly identify SLT-1 from the complex cell lysate of E. coli O157:H7. The core of the MNPs is made of iron oxide, whereas the surface of the core is coated with a thin layer of alumina (Fe3O4@Al2O3 MNPs). The Fe3O4@Al2O3 MNPs are functionalized with pigeon ovalbumin (POA), which contains Gal-α(1→4)-Gal-ß(1→4)-GlcNAc termini that can bind SLT-1B selectively. Furthermore, POA is a phosphate protein. Thus, it can be easily immobilized on the surface of the Fe3O4@Al2O3 MNPs through aluminum phosphate chelation under microwave heating within 1.5 min. The generated POA-Fe3O4@Al2O3 MNPs are capable of effectively enriching SLT-1B from complex cell lysates simply by pipetting 20 µL of the sample in and out of the tip in a vial for ∼1 min. To release SLT-1 from the MNPs, Gal-α(1→4)-Gal disaccharides were used for displacement. The released target species are sufficient to be identified by matrix-assisted laser desorption/ionization mass spectrometry. Although the sample volume used in this approach is small (20 µL) and the enrichment time is short (1 min), the selectivity of this approach toward SLT-1B is quite good. We have demonstrated the effectiveness of this approach for rapid determination of the presence of SLT-1 from complex cell lysates and ham/juice samples based on the detection of SLT-1B.

Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.

In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 108 guanine tags per secondary bead (7.5 x 106 biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)3(2+)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.

Label-free detection of pathogenic bacteria via immobilized antimicrobial peptides.

A novel label-free strategy for the detection of bacteria was developed by using a specific antimicrobial peptide (AMP)-functionalized quartz crystal microbalance (QCM) electrode. This electrode interface was successfully applied to detect pathogenic Escherichia coli O157:H7 based on the specific affinity between the small synthetic antimicrobial peptide and the bacterial cell of pathogenic E. coli O157:H7. The concentrations of pathogenic E. coli O157:H7 were sensitively measured by the frequency response of the QCM with a detection limit of 0.4 cfu µL(-1). The detection can be fulfilled within 10 min because it does not require germiculture process. On the other hand, if the specific antimicrobial peptides were immobilized on a gold electrode, this label-free strategy can also be performed by electrochemical impedance spectroscopy (EIS). Compared with QCM technique, the EIS measurement gives a lower sensitivity and needs a longer assay time. The combination of antimicrobial peptides with the real-time responses of QCM, as well as electronic read-out monitoring of EIS, may open a new way for the direct detection of bacteria.

Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

Synthesis, characterization, and evaluation of antibacterial effect of Ag nanoparticles against Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA).

Silver nanoparticles (AgNPs) have been shown great interest because of their potential antibacterial effect. Recently, this has been increased due to resistance in some pathogenic bacteria strains to conventional antibiotics, which has initiated new studies to search for more effective treatments against resistant microorganisms. For these reasons, AgNPs have become an important approach for applications in nanobiotechnology in the development of antibiotic treatment of different bacterial infections. This study was aimed at synthesizing AgNPs using cysteine as a reducer agent and cetyl-tri-methyl-ammonium bromide as a stabilizer in order to obtain more efficient treatment against the pathogen bacteria Escherichia coli O157:H7. These AgNPs were characterized through UV-Vis spectroscopy, transmission electron microscopy, and dynamic light scattering. From these analyses, formation of spherical nanoparticles with an average size of 55 nm was confirmed. Finally, minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) of these AgNPs against pathogenic strains E. coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) were determined in both solid and liquid media. MIC and MBC values were around 0.25 µg/mL and 1 µg/mL, respectively. These parameters were comparable to those reported in the literature and were even more effective than other synthesized AgNPs.