Adhesion and removal of E. coli K12 as affected by leafy green produce epicuticular wax composition, surface roughness, produce and bacterial surface hydrophobicity, and sanitizers.

Contaminated leafy vegetables have been associated with high-profile outbreaks causing severe illnesses. A good understanding of the interactions between human pathogen and produce is important for developing improved food safety control strategies. Currently, the role played by produce surface physiochemical characteristics in such interactions is not well-understood. This work was performed to examine the effects of produce physiochemical characteristics, including surface roughness, epicuticular wax composition, and produce and bacteria surface hydrophobicity on attachment and removal of vegetative bacteria. Escherichia coli K12 was used as a model microorganism to evaluate attachment to and removal from five leafy green vegetables after washing with selected sanitizers. A detailed epicuticular wax component analysis was conducted and the changes of wax composition after sanitation were also evaluated. The results showed that E. coli K12 removal is positively correlated with alkanes, ketones, and total wax content on leaf surfaces. Vegetables with high surface wax content had less rough leaf surfaces and more bacterial removal than the low wax produce. Produce surface roughness positively correlated to E. coli K12 adhesion and negatively correlated to removal. The cells preferentially attached to cut vegetable surfaces, with up to 1.49 times more attachment than on leaf adaxial surfaces.

Rapid quantification of pathogenic Salmonella Typhimurium and total bacteria in eggs by nano-flow cytometry.

Rapid quantification of pathogenic Salmonella Typhimurium (S. Typhimurium) and total bacteria in eggs is highly desired for food safety control. However, the complexity of egg matrix presents a significant challenge for sensitive detection of bacteria. In this study, a sample pretreatment protocol, including dilution, fat dissolution, protein degradation, filtration, and washing was developed to circumvent this challenge. A laboratory-built nano-flow cytometer (nFCM) that is hundreds of fold more sensitive than the conventional flow cytometer was employed to analyze individual bacteria upon nucleic acid and immunofluorescent staining. Eggs spiked with pathogenic S. Typhimurium and harmless Escherichia coli K12 (E. coli K12) were used as the model system to optimize the sample pretreatment protocol. S. Typhimurium and total bacteria in eggs can be quantified without cultural enrichment, and the whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1.5 h. The bacterial recovery rate upon sample pretreatment, detection limit, and dynamic range for S. Typhimurium in eggs were 92%, 2 × 103 cells/mL, and from 2 × 103 to 4 × 108 cells/mL, respectively. The as-developed approach can specifically distinguish S. Typhimurium from other bacteria and successful application to bacterial detection in eggs freshly purchased from supermarket and spoiled eggs upon inappropriate storage was demonstrated.

A Nanostructured Gold/Graphene Microfluidic Device for Direct and Plasmonic-Assisted Impedimetric Detection of Bacteria.

Hierarchical 3D gold nano-/microislands (NMIs) are favorably structured for direct and probe-free capture of bacteria in optical and electrochemical sensors. Moreover, their unique plasmonic properties make them a suitable candidate for plasmonic-assisted electrochemical sensors, yet the charge transfer needs to be improved. In the present study, we propose a novel plasmonic-assisted electrochemical impedimetric detection platform based on hybrid structures of 3D gold NMIs and graphene (Gr) nanosheets for probe-free capture and label-free detection of bacteria. The inclusion of Gr nanosheets significantly improves the charge transfer, addressing the central issue of using 3D gold NMIs. Notably, the 3D gold NMIs/Gr detection platform successfully distinguishes between various types of bacteria including Escherichia coli (E. coli) K12, Pseudomonas putida (P. putida), and Staphylococcus epidermidis (S. epidermidis) when electrochemical impedance spectroscopy is applied under visible light. We show that distinguishable and label-free impedimetric detection is due to dissimilar electron charge transfer caused by various sizes, morphologies, and compositions of the cells. In addition, the finite-difference time-domain (FDTD) simulation of the electric field indicates the intensity of charge distribution at the edge of the NMI structures. Furthermore, the wettability studies demonstrated that contact angle is a characteristic feature of each type of captured bacteria on the 3D gold NMIs, which strongly depends on the shape, morphology, and size of the cells. Ultimately, exposing the platform to various dilutions of the three bacteria strains revealed the ability to detect dilutions as low as ∼20 CFU/mL in a wide linear range of detection of 2 × 101-105, 2 × 101-104, and 1 × 102-1 × 105 CFU/mL for E. coli, P. putida, and S. epidermidis, respectively. The proposed hybrid structure of 3D gold NMIs and Gr, combined by novel plasmonic and conventional impedance spectroscopy techniques, opens interesting avenues in ultrasensitive label-free detection of bacteria with low cost and high stability.

Shunting microfluidic PCR device for rapid bacterial detection.

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip - fabricated using micro-milling and thermal fusion bonding for sealing of the cover - was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng µl-1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) - for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases.

Fully Automated Microsystem for Unmediated Electrochemical Characterization, Visualization and Monitoring of Bacteria on Solid Media; E. coli K-12: A Case Study.

In this paper, we present a non-fluidic microsystem for the simultaneous visualization and electrochemical evaluation of confined, growing bacteria on solid media. Using a completely automated platform, real-time monitoring of bacterial and image-based computer characterization of growth were performed. Electrochemical tests, using Escherichia coli K-12 as the model microorganism, revealed the development of a faradaic process at the bacteria-microelectrode interface inside the microsystem, as implied by cyclic voltammetry and electrochemical impedance spectrometry measurements. The electrochemical information was used to determine the moment in which bacteria colonized the electrode-enabled area of the microsystem. This microsystem shows potential advantages for long-term electrochemical monitoring of the extracellular environment of cell culture and has been designed using readily available technologies that can be easily integrated in routine protocols. Complementarily, these methods can help elucidate fundamental questions of the electron transfer of bacterial cultures and are potentially feasible to be integrated into current characterization techniques.

Microfluidic smartphone quantitation of Escherichia coli in synthetic urine.

In spite of the clinical need, there is a major gap in rapid diagnostics for identification and quantitation of E. coli and other pathogens, also regarded as the biggest bottleneck in the fight against the spread of antimicrobial resistant bacterial strains. This study reports for the first time an optical, smartphone-based microfluidic fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 min without sample preparation nor concentration. A limit of detection (LoD) up to 240 CFU/mL, comensurate with cut-off for UTIs (103-105 CFUs/mL) was achieved. Replicas of full response curves performed with 100-107 CFUs/mL of E. coli K12 in synthetic urine yielded recovery values in the range 80-120%, assay reproducibility below 30% and precision below 20%, therefore similar to high-performance automated immunoassays. The unrivalled LoD was mainly linked to the 'open fluidics' nature of the 10-bore microfluidic strips used that enabled passing a large volume of sample through the microcapillaries coated with capture antibody. The new smartphone based test has the potential of being as a rapid, point-of-care test for rule-in of E. coli infections that are responsible for around 80% of UTIs, helping to stop the over-prescription of antibiotics and the monitoring of patients with other symptomatic communicable diseases caused by E. coli at global scale.

Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode.

Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of >25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli.

We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E. coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens.

Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with integrated contactless conductivity detection.

This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C4D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C4D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span® 80 were pumped through flow rates fixed at 5 and 60 µL min-1, respectively. The droplets with incorporated cells were monitored in the C4D system applying a 500-kHz sinusoidal wave with 1 Vpp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321 ±â€¯9 µm and presented volume of 17.3 ±â€¯0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 CFU droplet-1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet-1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.

Gold nanoparticles-based multifunctional nanoconjugates for highly sensitive and enzyme-free detection of E.coli K12.

Immobilization of proteins on a biocompatible conductive interface is highly desirable for the fabrication of biosensors. In this study, a nanocomposite has been prepared by assembling well-distributed gold nanoparticles (AuNPs) on the surface of a polypyrrole-reduced graphene oxide (PPy-rGO) composite through electrostatic adsorption. This serves as a platform for immobilization of a capture antibody, which was conjugated onto the ferrocene doped polypyrrole-gold nanoparticles (PPy@Fc/AuNPs) composite. The design and performance of the biosensor was tested against detection of a whole-cell bacteria E. coli K12. This nanocomposite has a high surface area, good conductivity and biocompatibility, which is shown to be very suitable for enzyme-free detection of this bacteria. Results show excellent analytical performance with a linear range from 1.0 × 101 to 1.0 × 107 CFU mL-1 and a low detection limit of 10 CFU mL-1. The sensor has high selectivity, excellent reproducibility, and good stability.

Rapid and selective concentration of bacteria, viruses, and proteins using alternating current signal superimposition on two coplanar electrodes.

Dielectrophoresis (DEP) is usually effective close to the electrode surface. Several techniques have been developed to overcome its drawbacks and to enhance dielectrophoretic particle capture. Here we present a simple technique of superimposing alternating current DEP (high-frequency signals) and electroosmosis (EO; low-frequency signals) between two coplanar electrodes (gap: 25 µm) using a lab-made voltage adder for rapid and selective concentration of bacteria, viruses, and proteins, where we controlled the voltages and frequencies of DEP and EO separately. This signal superimposition technique enhanced bacterial capture (Escherichia coli K-12 against 1-µm-diameter polystyrene beads) more selectively (>99%) and rapidly (~30 s) at lower DEP (5 Vpp) and EO (1.2 Vpp) potentials than those used in the conventional DEP capture studies. Nanometer-sized MS2 viruses and troponin I antibody proteins were also concentrated using the superimposed signals, and significantly more MS2 and cTnI-Ab were captured using the superimposed signals than the DEP (10 Vpp) or EO (2 Vpp) signals alone (p < 0.035) between the two coplanar electrodes and at a short exposure time (1 min). This technique has several advantages, such as simplicity and low cost of electrode fabrication, rapid and large collection without electrolysis.

Electrochemical Surface-Enhanced Raman Spectroscopy as a Platform for Bacterial Detection and Identification.

The field of bacterial screening is in need of a rapid, easy to use, sensitive, and selective platform for bacterial detection and identification. Current methods of bacterial identification lack time efficiency, resulting in problems for many sectors of society. Surface-enhanced Raman spectroscopy (SERS) has been investigated as a possible candidate for bacterial screening due to its demonstrated ability to detect biological molecules with a high degree of sensitivity. However, the field of bacterial screening using SERS is currently facing limitations such as signal irreproducibility, weak spectra, and difficulty differentiating between strains based on the SERS spectra of bacteria alone. The current study reports on the first ever use of electrochemical surface-enhanced Raman spectroscopy (EC-SERS) for bacterial screening. The results of this study demonstrate the ability of EC-SERS to greatly improve upon the SERS performance for the detection of Gram-positive and Gram-negative bacteria both in terms of improved peak intensities and spectral richness. EC-SERS shows great promise in its ability to advance SERS-based bacterial screening and could potentially be used for more efficient species discrimination at the point-of-need (PON).

AIEC infection triggers modification of gut microbiota composition in genetically predisposed mice, contributing to intestinal inflammation.

A high prevalence of adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease patients has been shown. AIEC colonize the intestine and induce inflammation in genetically predisposed mouse models including CEABAC10 transgenic (Tg) mice expressing human CEACAM6-receptor for AIEC and eif2ak4-/- mice exhibiting autophagy defect in response to AIEC infection. Here, we aimed at investigating whether gut microbiota modification contributes to AIEC-induced intestinal inflammation in these mouse models. For this, eif2ak4+/+ and eif2ak4-/- mice or CEABAC10 Tg mice invalidated for Eif2ak4 gene (Tg/eif2ak4-/-) or not (Tg/eif2ak4+/+) were infected with the AIEC reference strain LF82 or the non-pathogenic E. coli K12 MG1655 strain. In all mouse groups, LF82 colonized the gut better and longer than MG1655. No difference in fecal microbiota composition was observed in eif2ak4+/+ and eif2ak4-/- mice before infection and at day 1 and 4 post-infection. LF82-infected eif2ak4-/- mice exhibited altered fecal microbiota composition at day 14 and 21 post-infection and increased fecal lipocalin-2 level at day 21 post-infection compared to other groups, indicating that intestinal inflammation developed after microbiota modification. Similar results were obtained for LF82-infected Tg/eif2ak4-/- mice. These results suggest that in genetically predisposed hosts, AIEC colonization might induce chronic intestinal inflammation by altering the gut microbiota composition.

Effects of high intensity ultrasound on the inactivation profiles of Escherichia coli K12 and Listeria innocua with salt and salt replacers.

This study investigated the efficacy of power ultrasound (US) for the inactivation of Escherichia coli and Listeria innocua in the presence of sodium salt and salt replacers. Inoculated bacteria suspensions were treated at ultrasonic frequencies of 33 or 20 kHz alone or in combination, and in the presence of 5% NaCl, 5% KCl or 5% NaCl/KCl. Inactivation curves were fitted to the Weibull and the Biphasic models. The goodness of the fit for each model was evaluated based on R2 and RMSE, while AIC and BIC values were used to choose the best model predictor. The Weibull and the biphasic models showed high regression coefficient (R2 > 0.99) and low RMSE (<0.03) values. According to the results, inactivation up to 6 log for E. coli K12 and to 4 log for L. innocua could be achieved within one hour of ultrasound treatment. However, the presence of NaCl, or its substitution with KCl did not affect the degree of inhibition for both microorganisms. The results of this study suggest that power ultrasound treatment may be employed for the inactivation of microorganisms when low salt or salt substitutes are employed.

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda.

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ∼38 kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01-5 µg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2 × 104 -2 × 109 CFU/ml, and exhibited a lower detection limit of 2 × 104 CFU/ml.

Microfluidic curved-channel centrifuge for solution exchange of target microparticles and their simultaneous separation from bacteria.

One of the common operations in sample preparation is to separate specific particles (e.g. target cells, embryos or microparticles) from non-target substances (e.g. bacteria) in a fluid and to wash them into clean buffers for further processing like detection (called solution exchange in this paper). For instance, solution exchange is widely needed in preparing fluidic samples for biosensing at the point-of-care and point-of-use, but still conducted via the use of cumbersome and time-consuming off-chip analyte washing and purification techniques. Existing small-scale and handheld active and passive devices for washing particles are often limited to very low throughputs or require external sources of energy. Here, we integrated Dean flow recirculation of two fluids in curved microchannels with selective inertial focusing of target particles to develop a microfluidic centrifuge device that can isolate specific particles (as surrogates for target analytes) from bacteria and wash them into a clean buffer at high throughput and efficiency. We could process micron-size particles at a flow rate of 1 mL min-1 and achieve throughputs higher than 104 particles per second. Our results reveal that the device is capable of singleplex solution exchange of 11 µm and 19 µm particles with efficiencies of 86 ± 2% and 93 ± 0.7%, respectively. A purity of 96 ± 2% was achieved in the duplex experiments where 11 µm particles were isolated from 4 µm particles. Application of our device in biological assays was shown by performing duplex experiments where 11 µm or 19 µm particles were isolated from an Escherichia coli bacterial suspension with purities of 91-98%. We envision that our technique will have applications in point-of-care devices for simultaneous purification and solution exchange of cells and embryos from smaller substances in high-volume suspensions at high throughput and efficiency.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

Addition of Hydrogen Peroxide to Groundwater with Natural Iron Induces Water Disinfection by Photo-Fenton at Circumneutral pH and other Photochemical Events.

Samples of natural groundwater (with low turbidity, neutral pH and 0.3 mg L-1 iron concentration) inoculated with Escherichia coli K-12 were exposed to simulated solar light both in the presence and in the absence 10 mg L-1 of H2 O2. Results demonstrated that the viability of E. coli (by DVC-FISH) was grounded to zero after 360 min of irradiation. This abatement could be caused by the oxidative stress induced by ·OH radicals or another photo-induced reactive oxygen species. Two 23 factorial experimental designs enabled the evaluation of the effects of chemical factors on the inactivation of E. coli. The first experimental design considered the pH, iron and H2 O2 , while the second evaluated the ions fluoride, carbonate and phosphate found in groundwater. pH was found to play a key role in the inactivation of E. coli. The best reduction in viability was obtained at the lower pH (6.75), while a nonsignificant effect was observed when iron or H2 O2 concentrations were raised. At higher concentrations, anions, such as carbonate and phosphate, negatively affected the E. coli abatement. However, a higher concentration of fluoride accelerated it. In all experiments, the pH was observed to rise to values higher than 8.0 units after 360 min of treatment.

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

A convenient purification method for silver nanoclusters and its applications in fluorescent pH sensors for bacterial monitoring.

Herein, we report a convenient approach to purify water-soluble dihydrolipoic acid (DHLA)-capped Ag nanoclusters (Ag NCs) by pH-induced precipitation under acidic conditions. The fluorescence of Ag NCs could be completely recovered by re-dispersing the precipitate into a basic solution using DHLA and NaBH4 as stabilizing ligands and etching reagent. DHLA-Ag NCs-doped agarose hydrogels have been prepared to monitor pH with a wide range from 8.0 to 4.0. When pH decreased, the fluorescence of the hydrogels under a UV lamp decreased and completely disappeared after pH 5. The DHLA-Ag NCs-doped agarose hydrogels biosensor showed low cytotoxicity and long stability. Accordingly, a fluorescent pH sensor for bacterial monitoring has been employed based on the "OFF-ON" signal switch of the Ag NCs-agarose hydrogel.