Highly Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification and Nanopore Sequencing (LAMPore) for Wastewater-Based Surveillance.

Wastewater-based surveillance (WBS) has gained attention as a strategy to monitor and provide an early warning for disease outbreaks. Here, we applied an isothermal gene amplification technique, reverse-transcription loop-mediated isothermal amplification (RT-LAMP), coupled with nanopore sequencing (LAMPore) as a means to detect SARS-CoV-2. Specifically, we combined barcoding using both an RT-LAMP primer and the nanopore rapid barcoding kit to achieve highly multiplexed detection of SARS-CoV-2 in wastewater. RT-LAMP targeting the SARS-CoV-2 N region was conducted on 96 reactions including wastewater RNA extracts and positive and no-target controls. The resulting amplicons were pooled and subjected to nanopore sequencing, followed by demultiplexing based on barcodes that differentiate the source of each SARS-CoV-2 N amplicon derived from the 96 RT-LAMP products. The criteria developed and applied to establish whether SARS-CoV-2 was detected by the LAMPore assay indicated high consistency with polymerase chain reaction-based detection of the SARS-CoV-2 N gene, with a sensitivity of 89% and a specificity of 83%. We further profiled sequence variations on the SARS-CoV-2 N amplicons, revealing a number of mutations on a sample collected after viral variants had emerged. The results demonstrate the potential of the LAMPore assay to facilitate WBS for SARS-CoV-2 and the emergence of viral variants in wastewater.

Investigation of the Influence of Stress on Label-Free Bacterial Surface-Enhanced Raman Spectra.

Label-free surface-enhanced Raman spectroscopy (SERS) has been proposed as a promising bacterial detection technique. However, the quality of the collected bacterial spectra can be affected by the time between sample acquisition and the SERS measurement. This study evaluated how storage stress stimuli influence the label-free SERS spectra of Pseudomonas syringae samples stored in phosphate buffered saline. The results indicate that when faced with nutrient limitations and changes in osmatic pressure, samples at room temperature (25 °C) exhibit more significant spectral changes than those stored at cold temperature (4 °C). At higher temperatures, bacterial communities secrete extracellular biomolecules that induce programmed cell death and result in increases in the supernatant SERS signals. Surviving cells consume cellular components to support their metabolism, thus leading to measurable declines in cell SERS intensity. Two-dimensional correlation spectroscopy analysis suggests that cellular component signatures decline sequentially in the following order: proteins, nucleic acids, and lipids. Extracellular nucleic acids, proteins, and carbohydrates are secreted in turn. After subtracting the SERS changes resulting from storage, we evaluated bacterial response to viral infection. P. syringae SERS profile changes enable accurate bacteriophage Phi6 quantification over the range of 104-1010 PFU/mL. The results indicate that storage conditions impact bacterial label-free SERS signals and that such influences need to be accounted for and if possible avoided when detecting bacteria or evaluating bacterial response to stress stimuli.

Highly porous gold supraparticles as surface-enhanced Raman spectroscopy (SERS) substrates for sensitive detection of environmental contaminants.

Surface-enhanced Raman spectroscopy (SERS) has great potential as an analytical technique for environmental analyses. In this study, we fabricated highly porous gold (Au) supraparticles (i.e., ∼100 µm diameter agglomerates of primary nano-sized particles) and evaluated their applicability as SERS substrates for the sensitive detection of environmental contaminants. Facile supraparticle fabrication was achieved by evaporating a droplet containing an Au and polystyrene (PS) nanoparticle mixture on a superamphiphobic nanofilament substrate. Porous Au supraparticles were obtained through the removal of the PS phase by calcination at 500 °C. The porosity of the Au supraparticles was readily adjusted by varying the volumetric ratios of Au and PS nanoparticles. Six environmental contaminants (malachite green isothiocyanate, rhodamine B, benzenethiol, atrazine, adenine, and gene segment) were successfully adsorbed to the porous Au supraparticles, and their distinct SERS spectra were obtained. The observed linear dependence of the characteristic Raman peak intensity for each environmental contaminant on its aqueous concentration reveals the quantitative SERS detection capability by porous Au supraparticles. The limit of detection (LOD) for the six environmental contaminants ranged from ∼10 nM to ∼10 µM, which depends on analyte affinity to the porous Au supraparticles and analyte intrinsic Raman cross-sections. The porous Au supraparticles enabled multiplex SERS detection and maintained comparable SERS detection sensitivity in wastewater influent. Overall, we envision that the Au supraparticles can potentially serve as practical and sensitive SERS devices for environmental analysis applications.

Nanobiotechnology enabled approaches for wastewater based epidemiology.

The impacts of the ongoing coronavirus pandemic highlight the importance of environmental monitoring to inform public health safety. Wastewater based epidemiology (WBE) has drawn interest as a tool for analysis of biomarkers in wastewater networks. Wide scale implementation of WBE requires a variety of field deployable analytical tools for real-time monitoring. Nanobiotechnology enabled sensing platforms offer potential as biosensors capable of highly efficient and sensitive detection of target analytes. This review provides an overview of the design and working principles of nanobiotechnology enabled biosensors and recent progress on the use of biosensors in detection of biomarkers. In addition, applications of biosensors for analysis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus are highlighted as they relate to the potential expanded use of biosensors for WBE-based monitoring. Finally, we discuss the opportunities and challenges in future applications of biosensors in WBE for effective monitoring and investigation of public health threats.

Discriminatory Detection of ssDNA by Surface-Enhanced Raman Spectroscopy (SERS) and Tree-Based Support Vector Machine (Tr-SVM).

We report label-free detection of 86-base single-stranded DNA (ssDNA) gene segments by surface-enhanced Raman spectroscopy (SERS). The use of a slippery liquid infused porous (SLIP) membrane induced aggregation of 43 nm gold nanoparticles and ssDNA upon pin-free droplet evaporation. The combined SLIPSERS approach generates significant numbers of SERS hot-spots and enabled detection at the 100 nM level of mecA and intI1 gene segments-two genes of interest in the context of antibiotic resistance. Tree-based multiclass support vector machine (Tr-SVM) classifiers were built to discriminate SERS spectra of 12 different gene sequences obtained by SLIPSERS: mecA, intI1, as well as analogues of mecA and intI1, respectively, with 2-10 base mismatches, and two random sequences. The trained predictive Tr-SVM classifiers correctly identified each gene sequence with a prediction accuracy of ∼90%. This study illustrates a novel means for discriminatory label-free SERS detection of ssDNA enabled by Tr-SVM.

Surface-Enhanced Raman Spectroscopy of Bacterial Metabolites for Bacterial Growth Monitoring and Diagnosis of Viral Infection.

Bacterial metabolites are intermediate products of bacterial metabolism and their production reflects metabolic activity. Herein, we report the use of surface-enhanced Raman spectroscopy (SERS) for detection of both volatile and nonvolatile metabolites and the application of this approach for bacterial growth quantification and diagnosis of viral infection. The time-dependent SERS signal of the volatile metabolite dimethyl disulfide in the headspace above bacteria growing on an agar plate was detected and quantified. In addition, SERS signals arising from the plate reflected nutrient consumption and production of nonvolatile metabolites. The measurement of metabolite accumulation can be used for bacterial quantification. In the presence of bacteriophage virus, bacterial metabolism is suppressed, and the relative decrease in SERS intensity reflects the initial virus concentration. Using multivariate analysis, we detect viral infection with a prediction accuracy of 93%. Our SERS-based approach for metabolite production monitoring provides new insights toward viral infection diagnosis.

Plasmonic Electronic Raman Scattering as Internal Standard for Spatial and Temporal Calibration in Quantitative Surface-Enhanced Raman Spectroscopy.

Ultrasensitive surface-enhanced Raman spectroscopy (SERS) still faces difficulties in quantitative analysis because of its susceptibility to local optical field variations at plasmonic hotspots in metallo-dielectric nanostructures. Current SERS calibration approaches using Raman tags have inherent limitations due to spatial occupation competition with analyte molecules, spectral interference with analyte Raman peaks, and photodegradation. Herein, we report that plasmon-enhanced electronic Raman scattering (ERS) signals from metal can serve as an internal standard for spatial and temporal calibration of molecular Raman scattering (MRS) signals from analyte molecules at the same hotspots, enabling rigorous quantitative SERS analysis. We observe a linear dependence between ERS and MRS signal intensities upon spatial and temporal variations of excitation optical fields, manifesting the |E|4 enhancements for both ERS and MRS processes at the same hotspots in agreement with our theoretical prediction. Furthermore, we find that the ERS calibration's performance limit can result from orientation variations of analyte molecules at hotspots.

Synthesis and SERS application of gold and iron oxide functionalized bacterial cellulose nanocrystals (Au@Fe3O4@BCNCs).

Bacterial cellulose nanocrystals (BCNCs) are biocompatible cellulose nanomaterials that can host guest nanoparticles to form hybrid nanocomposites with a wide range of applications. Herein, we report the synthesis of a hybrid nanocomposite that consists of plasmonic gold nanoparticles (AuNPs) and superparamagnetic iron oxide (Fe3O4) nanoparticles supported on BCNCs. As a proof of concept, the hybrid nanocomposites were employed to isolate and detect malachite green isothiocyanate (MGITC) via magnetic separation and surface-enhanced Raman scattering (SERS). Different initial gold precursor (Au3+) concentrations altered the size and morphology of the AuNPs formed on the nanocomposites. The use of 5 and 10 mM Au3+ led to a heterogenous mix of spherical and nanoplate AuNPs with increased SERS enhancements, as compared to the more uniform AuNPs formed using 1 mM Au3+. Rapid and sensitive detection of MGITC at concentrations as low as 10-10 M was achieved. The SERS intensity of the normalized Raman peak at 1175 cm-1 exhibited a log-linear relationship for MGITC concentrations between 2 × 10-10 and 2 × 10-5 M for Au@Fe3O4@BCNCs. These results suggest the potential of these hybrid nanocomposites for application in a broad range of analyte detection strategies.

Differential Drivers of Antimicrobial Resistance across the World.

Antimicrobial resistance (AMR) is one of the greatest threats faced by humankind. The development of resistance in clinical and hospital settings has been well documented ever since the initial discovery of penicillin and the subsequent introduction of sulfonamides as clinical antibiotics. In contrast, the environmental (i.e., community-acquired) dimensions of resistance dissemination have been only more recently delineated. The global spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) between air, water, soil, and food is now well documented, while the factors that affect ARB and ARG dissemination (e.g., water and air quality, antibiotic fluxes, urbanization, sanitation practices) in these and other environmental matrices are just now beginning to be more fully appreciated. In this Account, we discuss how the global perpetuation of resistance is dictated by highly interconnected socioeconomic risk factors and illustrate that development status should be more fully considered when developing global strategies to address AMR. We first differentiate low to middle income countries (LMICs) and high-income countries (HICs), then we summarize the modes of action of commercially available antibiotics, and then discuss the four primary mechanisms by which bacteria develop resistance to those antibiotics. Resistance is disseminated via both vertical gene transfer (VGT; parent to offspring) as well as by horizontal gene transfer (HGT; cell to cell transference of genetic material). A key challenge hindering attempts to control resistance dissemination is the presence of native, environmental bacteria that can harbor ARGs. Such environmental "resistomes" have potential to transfer resistance to pathogens via HGT. Of particular concern is the development of resistance to antibiotics of last-resort such as the cephalosporins, carbapenems, and polymyxins. We then illustrate how antibiotic use differs in LMICs relative to HICs in terms of the volumes of antibiotics used and their fate within local environments. Antibiotic use in HICs has remained flat over the past 15 years, while in LMICs use over the same period has increased substantially as a result of economic improvements and changes in diet. These use and fate differences impact local citizens and thus the local dissemination of AMR. Various physical, social, and economic circumstances within LMICs potentially favor AMR dissemination. We focus on three physical factors: changing population density, sanitation infrastructure, and solid-waste disposal. We show that high population densities in cities within LMICs that suffer from poor sanitation and solid-waste disposal can potentially impact the dissemination of resistance. In the final section, we discuss potential monitoring approaches to quantify the spread of resistance both within LMICs as well as in HICs. We posit that culture-based approaches, molecular approaches, and cutting-edge nanotechnology-based methods for monitoring ARB and ARGs should be considered both within HICs and, as appropriate, within LMICs.

Effect of using powdered biochar and surfactant on desorption and biodegradability of phenanthrene sorbed to biochar.

The present study aimed to investigate the relationship between the desorption and biodegradability of phenanthrene sorbed to biochars by employing two approaches that may change the desorption and biodegradability: the use of powdered biochars and nonionic surfactants. Biochars derived from two feedstocks (rice husk and sewage sludge; pyrolyzed at 500 °C but showing different aromaticity) were used. When the biochars were powdered to obtain particles <250 µm the mass fractions of the desorbed phenanthrene at ∼80 days (fdes) increased from 0.303 to 0.431 for sewage sludge biochars. On the other hand, fdes for rice husk biochars remained virtually unchanged (from 0.264 to 0.255). The mass fractions of the biodegraded phenanthrene (fbio) increased from 0.191 to 0.306 for rice husk biochars and from 0.077 to 0.168 for sewage sludge biochars. When a nonionic surfactant was added at the sub-critical micelle concentration (CMC), fbio increased by 4.7 times and 8.3 times for rice husk and sewage sludge biochars. For both types of biochars, fbio was larger than fdes when the surfactant was added. This study suggests that the addition of nonionic surfactants can be considered if the inhibition of microbial activity is of concern in soils and sediments treated by biochar.

The fates of microbial populations on pig carcasses during slaughtering process, on retail cuts after slaughter, and intervention efficiency of lactic acid spraying.

This study was conducted to detect and identify microbial populations on pig carcasses at different slaughtering stages and on retail pork cuts at 24 h after slaughter as well as to evaluate the intervention efficiency of sprays containing different concentrations (2% and 4%) of lactic acid. The sprays were applied to the carcass surfaces at the end of the slaughter line. Microbial samples were collected from carcass surfaces after bleeding and after eviscerating, and from retail cuts at 24 h after chilling/spraying. The detected microorganisms were identified through using a Microflex identification instrument and 16S rRNA gene sequencing. The diversity of the bacterial genera; Staphylococcus, Salmonella, Shigella, Enterococci, Escherichia, Acinetobacter and Corynebacterium spp. showed counts ranging from 2.70 to 4.91 log10 cfu/100 cm2 on the carcasses during slaughter. Most of these genera were also detected on the carcasses after 24 h of chilling. Three species (Staphylococcus hyicus, Acinetobacter albensis, and Corynebacterium xerosis) were also found on the retail cuts of non-sprayed carcasses but not on those of the sprayed groups. Significantly greater reductions in all bacterial species were observed on the carcasses and retail cuts that were sprayed with lactic acid, particularly at the 4% level. Thus, spraying with 4% lactic acid may be an effective intervention for controlling bacterial contamination on pig carcasses to improve the microbiological safety of pork meat.

Effect of biochar particle size on hydrophobic organic compound sorption kinetics: Applicability of using representative size.

Particle size of biochar may strongly affect the kinetics of hydrophobic organic compound (HOC) sorption. However, challenges exist in characterizing the effect of biochar particle size on the sorption kinetics because of the wide size range of biochar. The present study suggests a novel method to determine a representative value that can be used to show the dependence of HOC sorption kinetics to biochar particle size on the basis of an intra-particle diffusion model. Biochars derived from three different feedstocks are ground and sieved to obtain three daughter products each having different size distributions. Phenanthrene sorption kinetics to the biochars are well described by the intra-particle diffusion model with significantly greater sorption rates observed for finer grained biochars. The time to reach 95% of equilibrium for phenanthrene sorption to biochar is reduced from 4.6-17.9days for the original biochars to <1-4.6days for the powdered biochars with <125µm in size. A moderate linear correlation is found between the inverse square of the representative biochar particle radius obtained using particle size distribution analysis and the apparent phenanthrene sorption rates determined by the sorption kinetics experiments and normalized to account for the variation of the sorption rate-determining factors other than the biochar particle radius. The results suggest that the representative biochar particle radius reasonably describes the dependence of HOC sorption rates on biochar particle size.

The effects of pre-and post-slaughter spray application with organic acids on microbial population reductions on beef carcasses.

As an attempt to control bacterial cross-contamination of beef carcasses, in the present investigation acetic acid and lactic acid (3% v/v) were used for bacterial decontamination. For the decontamination, cows were sprayed with each above acid at two different stages; (i) on live animal's hides, (ii) on carcass surfaces immediately after slaughter. Microbiological samples were taken on different hide areas of animals before spraying and on carcass surfaces at 24h after spraying. Meat quality traits were also analyzed on the sprayed animals. The detected microorganisms were identified using 16SrRNA gene sequencing. A diversity of bacterial species such as Staphylococcus, Shigella, Bacillus, Escherichia and Salmonella etc. were found on both external hide and carcass surface samples. The decontamination sprays significantly reduced the numbers (2-5 log unit) of all aforementioned bacterial species on carcass surfaces as compared with non-sprayed control. Thus, the two times-spray applications with the acid could be an effective tool for reducing bacterial cross-contaminations of beef carcass without adverse effect on meat quality.

Non-equilibrium passive sampling of hydrophobic organic contaminants in sediment pore-water: PCB exchange kinetics.

This study investigates the isotropic exchange kinetics of PCBs for polyethylene (PE) passive samplers in quiescent sediment and develops a novel non-equilibrium passive sampling method using PE with multiple thicknesses. The release and uptake kinetics of PCBs in quiescent sediment are reproduced by a 1-D diffusion model using sediment diffusion parameters fitted with the data from actual measurements. From the sediment diffusion parameters observed for uptake and release kinetics, it is seen that the uptake kinetics are distinctly slower than the release kinetics, most likely because of the sorption-desorption hysteresis of PCBs in the study sediment. Despite the presence of the anisotropic PCB exchange kinetics, a performance reference compound (PRC)-based method, which is grounded on the assumption of isotropic exchange kinetics, estimated the freely dissolved aqueous concentrations (Cfree) of PCBs in sediment pore-water with less than a factor of two error for the study sediment. The novel method developed in this study using PE with multiple thicknesses also gives reasonable estimates of Cfree, demonstrating its potential as another option for non-equilibrium passive sampling for hydrophobic organic contaminants in sediment pore-water.