Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

Optimizing Sustainable Energy Generation in Ethanol Fuel Cells: An Exploration of Carrageenan with TiO2 Nanoparticles and Ni/CeO2 Composites.

Based on the search for new biodegradable materials that are low cost and easy to synthesize by environmentally friendly methods, we report the use of carrageenan membranes (mixture of κ and λ carrageenans) with different concentrations of titanium dioxide nanoparticles (TiO2 NPs) and Ni/CeO2 (10 wt % Ni) for the fabrication of a novel fuel cell electrode for the oxidation of ethanol. Each membrane was characterized to determine its physicochemical properties using X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. Using impedance spectroscopy (IS), a maximum value of 2.08 × 10-4 S/cm in ionic conductivity was found for the carrageenan nanocomposite with a concentration of 5 wt % TiO2 NPs (CR5%). Due to its high conductivity values, the CR5% membrane was mixed with Ni/CeO2 to prepare the working electrode for cyclic voltammetry measurements. Using a solution of 1 M ethanol and 1 M KOH, the oxidation of ethanol over CR5% + Ni/CeO2 resulted in peak current density values at forward and reverse scan voltages of 9.52 and 12.22 mA/cm2, respectively. From our results, the CR5% + Ni/CeO2 membrane proves to be more efficient in the oxidation of ethanol compared with commercially available Nafion membranes containing Ni/CeO2.

Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21.

Infections with globally disseminated Shiga toxin-producing Escherichia coli (STEC) of the O113:H21 serotype can progress to severe clinical complications, such as hemolytic uremic syndrome (HUS). Two phylogeographically distinct clonal complexes have been established by multi locus sequence typing (MLST). Infections with ST-820 isolates circulating exclusively in Australia have caused severe human disease, such as HUS. Conversely, ST-223 isolates prevalent in the US and outside Australia seem to rarely cause severe human disease but are frequent contaminants. Following a genomic epidemiology approach, we wanted to gain insights into the underlying cause for this disparity. We examined the plasticity in the genome make-up and Shiga toxin production in a collection of 20 ST-820 and ST-223 strains isolated from produce, the bovine reservoir, and clinical cases. STEC are notorious for assembly into fragmented draft sequences when using short-read sequencing technologies due to the extensive and partly homologous phage complement. The application of long-read technology (LRT) sequencing yielded closed reference chromosomes and plasmids for two representative ST-820 and ST-223 strains. The established high-resolution framework, based on whole genome alignments, single nucleotide polymorphism (SNP)-typing and MLST, includes the chromosomes and plasmids of other publicly available O113:H21 sequences and allowed us to refine the phylogeographical boundaries of ST-820 and ST-223 complex isolates and to further identify a historic non-shigatoxigenic strain from Mexico as a quasi-intermediate. Plasmid comparison revealed strong correlations between the strains' featured pO113 plasmid genotypes and chromosomally inferred ST, which suggests coevolution of the chromosome and virulence plasmids. Our pathogenicity assessment revealed statistically significant differences in the Stx2a-production capabilities of ST-820 as compared to ST-223 strains under RecA-induced Stx phage mobilization, a condition that mimics Stx-phage induction. These observations suggest that ST-820 strains may confer an increased pathogenic potential in line with the strain-associated epidemiological metadata. Still, some of the tested ST-223 cultures sourced from contaminated produce or the bovine reservoir also produced Stx at levels comparable to those of ST-820 isolates, which calls for awareness and for continued surveillance of this lineage.

Delayed lactose utilization among Shiga toxin-producing Escherichia coli of serogroup O121.

Two outbreaks of Shiga toxin-producing Escherichia coli O121:H19 associated with wheat flour, in the United States of America and Canada, involved strains with an unusual phenotype, delayed lactose utilization (DLU). These strains do not ferment lactose when initially cultured on MacConkey agar (MAC), but lactose fermentation occurs following subculture to a second plate of MAC. The prevalence of DLU was determined by examining the ß-galactosidase activity of 49 strains of E. coli O121, and of 37 other strains of E. coli. Twenty four of forty three O121:H19 and one O121:NM displayed DLU. Two strains (O121:NM and O145:H34) did not have detectable ß-galactosidase activity. ß-glucuronidase activity of O121 strains was also determined. All but six DLU strains had normal ß-glucuronidase activity. ß-glucuronidase activity was suppressed on MAC for 17 of 23 O121 non-DLU strains. Genomic analysis found that DLU strains possessed an insertion sequence, IS600 (1267 bp), between lacZ (ß-galactosidase) and lacY (ß-galactoside permease), that was not present in strains exhibiting normal lactose utilization. The insert might reduce the expression of ß-galactoside permease, delaying import of lactose, resulting in the DLU phenotype. The high probability of DLU should be considered when using lactose-containing media for the isolation of STEC O121.

Size-Dependent Electrical Transport Properties in Conducting Diamond Nanostripes.

With the advances in nanofabrication technology, horizontally aligned and well-defined nitrogen-doped ultrananocrystalline diamond nanostripes can be fabricated with widths in the order of tens of nanometers. The study of the size-dependent electron transport properties of these nanostructures is crucial to novel electronic and electrochemical applications. In this paper, 100 nm thick n-type ultrananocrystalline diamond thin films were synthesized by microwave plasma-enhanced chemical vapor deposition method with 5% N2 gas in the plasma during the growth process. Then the nanostripes were fabricated using standard electron beam lithography and reactive ion etching techniques. The electrical transport properties of the free-standing single nanostripes of different widths from 75 to 150 nm and lengths from 1 to 128 µm were investigated. The study showed that the electrical resistivity of the n-type ultrananocrystalline diamond nanostripes increased dramatically with the decrease in the nanostripe width. The nanostripe resistivity was nearly doubted when the width was reduced from 150 nm to 75 nm. The size-dependent variability in conductivity could originate from the imposed diffusive scattering of the nanostripe surfaces which had a further compounding effect to reinforce the grain boundary scattering.

Ultrananocrystalline Diamond Nanowires: Fabrication, Characterization, and Sensor Applications.

The aim of this review is to provide a survey of the recent advances and the main remaining challenges related to the ultrananocrystalline diamond (UNCD) nanowires and other nanostructures which exhibit excellent capability as the core components for many diverse novel sensing devices, due to the unique material properties and geometry advantages. The boron or nitrogen doping introduced in the gas phase during deposition promotes p-type or n-type conductivity. With the establishment of the UNCD nanofabrication techniques, more and more nanostructure-based devices are being explored in measuring basic physical and chemical parameters via classic and quantum methods, as exemplified by gas sensors, ultraviolet photodetectors, piezoresistance effect-based devices, biological applications and biosensors, and nitrogen-vacancy color center-based magnetic field quantum sensors. Highlighted finally are some of the remaining challenges and the future outlook in this area.

Effect of Tungsten Oxide Nanostructures on Sensitivity and Selectivity of Pollution Gases.

We report on the different surface structures of tungsten oxides which have been synthesized using a simple post-annealing-free hot-filament CVD technique, including 0D nanoparticles (NPs), 1D nanorods (NRs), and 2D nanosheet assemblies of 3D hierarchical nanoflowers (NFs). The surface morphologies, crystalline structures, and material compositions have been characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Raman spectroscopy, respectively. The sensor performances based on the synthesized samples of various surface morphologies have been investigated, as well as the influences of operating temperature and applied bias. The sensing property depends closely on the surface morphology, and the 3D hierarchical nanoflowers-based gas sensor offers the best sensitivity and fastest response time to NH3 and CH3 gases when operated at room temperature.

Bandgap-Tuned 2D Boron Nitride/Tungsten Nitride Nanocomposites for Development of High-Performance Deep Ultraviolet Selective Photodetectors.

This study presents a fast and effective method to synthesize 2D boron nitride/tungsten nitride (BN-WN) nanocomposites for tunable bandgap structures and devices. A few minutes of synthesis yielded a large quantity of high-quality 2D nanocomposites, with which a simple, low-cost deep UV photo-detector (DUV-PD) was fabricated and tested. The new device was demonstrated to have very good performance. High responsivity up to 1.17 A/W, fast response-time of lower than two milliseconds and highly stable repeatability were obtained. Furthermore, the influences of operating temperature and applied bias voltage on the properties of DUV-PD as well as its band structure shift were investigated.

Pathogenomes of Atypical Non-shigatoxigenic Escherichia coli NSF/SF O157:H7/NM: Comprehensive Phylogenomic Analysis Using Closed Genomes.

The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (-) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (-) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains' featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.

Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses - A literature review.

Many countries use Escherichia coli and coliforms as indicators of sanitary quality of foods and have set limits for cheeses, including raw-milk cheeses. This paper reviewed the scientific literature for E. coli and coliform levels that are found in different types of raw milk, the fate of indicators during the manufacturing and ripening of different cheeses and the indicator levels that have been found in the finished cheeses. These studies from worldwide showed that E. coli and coliforms are found in different types of raw milk but usually at <100 CFU/ml or not found. Instances where raw milk contained indicator levels >1000 CFU/ml have mostly been attributed to unsanitary conditions/production. During cheese-making, indicators present in raw milk will often increase in numbers, but the levels decline as the acidity from lactose fermentation decreases the pH. Except for fresh cheeses that are not aged, indicator levels are further reduced by 2-3 log10 CFU/g or more, during the ripening process. As a result, indicator levels in finished cheeses are often low and within the limits of <10 or <100 CFU/g set by many countries. The cited studies also show that raw milk cheeses that are made with quality raw milk, under hygienic conditions and properly aged, should not contain high levels of indicator bacteria in the final product.

Nanoplasmonic 1D Diamond UV Photodetectors with High Performance.

Diamond nanowires have recently drawn substantial attention because of their unique physical and chemical properties for electrochemical sensors, optoelectronics, and nanophotonics applications. However, diamond nanowire-based ultraviolet photodetectors have not been reported because of the challenges involved in synthesizing crystalline diamond nanowires with controllable morphologies and, more fundamentally, the material's high carrier concentration with low mobilities that limits the obtainable photoresponsivity. The synergetic integration of ultrananocrystalline diamond (UNCD) nanowires with nanoplasmonic enhancement by noble metal nanoparticles is a very promising approach to overcome these shortcomings. Here we report the fabrication of boron-doped ultrananocrystalline diamond nanowires functionalized with the platinum nanoparticles to form self-powered ultraviolet photodetectors that exhibit an ultrahigh photoresponsivity of 388 Amp/Watt at 300 nm wavelength, a fast response time around 20 ms, and a good UV/visible rejection ratio of about 5 orders of magnitude under zero-bias condition.

Synthesis, Characterization and Fabrication of Graphene/Boron Nitride Nanosheets Heterostructure Tunneling Devices.

Various types of 2D/2D prototype devices based on graphene (G) and boron nitride nanosheets (BNNS) were fabricated to study the charge tunneling phenomenon pertinent to vertical transistors for digital and high frequency electronics. Specifically, G/BNNS/metal, G/SiO2, and G/BNNS/SiO2 heterostructures were investigated under direct current (DC-bias) conditions at room temperature. Bilayer graphene and BNNS were grown separately and transferred subsequently onto the substrates to fabricate 2D device architectures. High-resolution transmission electron microscopy confirmed the bilayer graphene structure and few layer BNNS sheets having a hexagonal B3-N3 lattice. The current vs voltage I(V) data for the G/BNNS/Metal devices show Schottky barrier characteristics with very low forward voltage drop, Fowler-Nordheim behavior, and 10-4 Ω/sq. sheet resistance. This result is ascribed to the combination of fast electron transport within graphene grains and out-of-plane tunneling in BNNS that circumvents grain boundary resistance. A theoretical model based on electron tunneling is used to qualitatively describe the behavior of the 2D G/BNNS/metal devices.

Interlaboratory Evaluation of the U.S. Food and Drug Administration Escherichia coli Identification Microarray for Profiling Shiga Toxin-Producing Escherichia coli.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2d subtypes.

A compact design of a characterization station for far UV photodetectors.

A newly fabricated characterization station is presented. It is a compact, cost-effective, and easily adjustable apparatus. Each part including 4-pin probe, manipulators, operating temperature, and applied bias can be independently controlled. The station can provide highly reliable, reproducible, and economical methods to quickly conduct and complete the characterizations of a large amount of sensing materials within a short period of time. It is particularly suitable for studies of various nanostructured materials and their related thermal effect, polarization effect, sensitivity, and electrical and electronic properties.

High-Performance and Self-Powered Deep UV Photodetectors Based on High Quality 2D Boron Nitride Nanosheets.

High-quality two-dimensional (2D) crystalline boron nitride nanosheets (BNNSs) were grown on silicon wafers by using pulsed plasma beam deposition techniques. Self-powered deep ultraviolet (DUV) photodetectors (PDs) based on BNNSs with Schottky contact structures are designed and fabricated. By connecting the fabricated DUV photodetector to an ammeter, the response strength, response time and recovery time to different DUV wavelengths at different intensities have been characterized using the output short circuit photocurrent without a power supply. Furthermore, effects of temperature and plasma treatment on the induced photocurrent response of detectors have also been investigated. The experimental data clearly indicate that plasma treatment would significantly improve both induced photocurrent and response time. The BNNS-based DUV photodetector is demonstrated to possess excellent performance at a temperature up to 400 °C, including high sensitivity, high signal-to-noise ratio, high spectral selectivity, high speed, and high stability, which is better than almost all reported semiconducting nanomaterial-based self-powered photodetectors.

Exploring the Effects of Argon Plasma Treatment on Plasmon Frequency and the Chemiresistive Properties of Polymer-Carbon Nanotube Metacomposite.

Metacomposites, composite materials exhibiting negative permittivity, represent an opportunity to create materials with depressed plasmon frequency without the need to create complex structural geometries. Although many reports exist on the synthesis and characterizations of metacomposites, very few have ventured into exploring possible applications that could take advantage of the unique electrical properties of these materials. In this article, we report on the chemiresistive properties of a polymer-CNT metacomposite and explore how these are affected by Argon plasma treatment.

Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples.

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse.IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

High Operating Temperature and Low Power Consumption Boron Nitride Nanosheets Based Broadband UV Photodetector.

We extend our work on the use of digitally controlled pulsed laser plasma deposition (PLPD) technique to synthesize high quality, 2-dimensional single crystalline boron nitride nanosheets (BNNSs) at a low substrate temperature for applications in high-performance deep UV photodetectors. The obtained sample consists of a large amount of BNNSs partially overlapping one another with random orientations. Each sheet is composed of a few (from 2 to 10) stacked atomic layers exhibiting high transparency due to its highly ordered hBN crystallinity. Deep UV detectors based on the obtained BNNSs were designed, fabricated, and tested. The bias and temperature effects on the photocurrent strength and the signal-to-noise ratio have been carefully characterized and discussed. A significant shift in the cut off wavelength of the BNNSs based photodetectors was observed suggesting a band gap reduction as a result of the BNNSs' collective structure. The newly designed photodetector presented exceptional properties: a high sensitivity to weak intensities of radiation in both UVC and UVB range while remaining visible-blind, and a high signal-to-noise ratio operation even at temperatures as high as 400 °C. In addition, the BNNSs based photodetector exhibited potential for self-powered operation.

Characterization and Virulence Potential of Serogroup O113 Shiga Toxin-Producing Escherichia coli Strains Isolated from Beef and Cattle in the United States.

Shiga toxin-producing Escherichia coli (STEC) of serotype O113:H21 have caused severe diseases but are unusual in that they do not produce the intimin protein required for adherence to intestinal epithelial cells. Strains of serogroup O113 are one of the most common STEC found in ground beef and beef products in the United States, but their virulence potential is unknown. We used a microarray to characterize 65 O113 strains isolated in the United States from ground beef, beef trim, cattle feces, and fresh spinach. Most were O113:H21 strains, but there were also nine strains of O113:H4 serotype. Although strains within the same serotype had similar profiles for the genes that were tested on the array, the profiles were distinct between the two serotypes, and the strains belonged to different clonal groups. Analysis by clustered regularly interspaced short palindromic repeat analysis showed that O113:H4 strains are conserved genetically, but the O113:H21 strains showed considerable polymorphism and genetic diversity. In comparison to the O113:H21 strains from Australia that were implicated in severe disease, the U.S. isolates showed similar genetic profiles to the known pathogens from Australia, suggesting that these may also have the potential to cause infections.