Critical Capability Needs for Reduction of Transmission of SARS-CoV-2 Indoors.

Coordination of efforts to assess the challenges and pain points felt by industries from around the globe working to reduce COVID-19 transmission in the indoor environment as well as innovative solutions applied to meet these challenges is mandatory. Indoor infectious viral disease transmission (such as coronavirus, norovirus, influenza) is a complex problem that needs better integration of our current knowledge and intervention strategies. Critical to providing a reduction in transmission is to map the four core technical areas of environmental microbiology, transmission science, building science, and social science. To that end a three-stage science and innovation Summit was held to gather information on current standards, policies and procedures applied to reduce transmission in built spaces, as well as the technical challenges, science needs, and research priorities. The Summit elucidated steps than can be taken to reduce transmission of SARS-CoV-2 indoors and calls for significant investments in research to enhance our knowledge of viral pathogen persistence and transport in the built environment, risk assessment and mitigation strategy such as processes and procedures to reduce the risk of exposure and infection through building systems operations, biosurveillance capacity, communication form leadership, and stakeholder engagement for optimal response. These findings reflect the effective application of existing knowledge and standards, emerging science, and lessons-learned from current efforts to confront SARS-CoV-2.

Lessons learned in managing risk: Tools and strategies for confident operations from the CLEAN 2020 Summit.

Risk mitigation of COVID-19 in the indoor environment requires an articulated strategy for creating a bridge between science and the business community that focuses on knitting together four core capabilities-environmental microbiology, transmission science, building science, and social science-advancing scientific knowledge. The purpose of this article is to share insights from the CLEAN 2020 Summit, which assembled leaders from business, policy, standards development, science, and engineering working to mitigate risk of transmission in the built environment. The Summit worked to assess current challenges and pain points felt by industries from around the globe as well as innovative solutions applied to meet these challenges. Although SARS-CoV-2 and the COVID-19 diseases are unique, the foundation of knowledge to assess and mitigate the risk of viral transmission in the built environment is robust. There are opportunities to improve science and engineering technology solutions, processes, and procedures to better meet the dynamic needs of the evolving pandemic.

The Opioid Epidemic: Moving Toward an Integrated, Holistic Analytical Response.

In many jurisdictions, public safety and public health entities are working together to enhance the timeliness and accuracy of the analytical characterization and toxicology testing of novel synthetic opioids. The improved sharing and early detection of these analytical data are intended to inform surveillance, interdiction efforts, patient intervention and treatment, all of which are critical to curbing the opioid epidemic. Forensic practitioners working to identify novel synthetic opioids struggle to provide timely results when encountering new or unknown substances, such as the fentanyl analogs. These compounds, which mimic heroin in pharmacologic effect but can be far more potent, are inconsistently present in chemical identification libraries, and are currently largely unavailable as reference materials for analytical comparison. Additionally, federal, state and local governments as well as nongovernmental organizations require potency, toxicity and potential-for-abuse data to evaluate the potential health risks of emerging drug threats. Subsequent scheduling efforts and criminal prosecutions also require these thorough drug characterization studies. Pilot programs have demonstrated that early communication of real-time drug toxicity and analytical data significantly impacts the successful response to emerging opioids. High-quality, real-time, national-level data on chemical composition, toxicological test data, drug toxicity and overdoses, and analysis of seized materials by law enforcement are needed to track drug trends. However, the USA still lacks a national system to coordinate and communicate toxicology, medical and medical examiner and coroner data with the broader medical and law enforcement communities. Opportunities to address these gaps as well as recent advancements collected through interagency efforts and technical workshops in the toxicology and analytical chemistry communities are presented here. Opportunities for partnership, increased communication and expanding best practices to move toward an integrated, holistic analytical response are also explored.

Challenging a bioinformatic tool's ability to detect microbial contaminants using in silico whole genome sequencing data.

High sensitivity methods such as next generation sequencing and polymerase chain reaction (PCR) are adversely impacted by organismal and DNA contaminants. Current methods for detecting contaminants in microbial materials (genomic DNA and cultures) are not sensitive enough and require either a known or culturable contaminant. Whole genome sequencing (WGS) is a promising approach for detecting contaminants due to its sensitivity and lack of need for a priori assumptions about the contaminant. Prior to applying WGS, we must first understand its limitations for detecting contaminants and potential for false positives. Herein we demonstrate and characterize a WGS-based approach to detect organismal contaminants using an existing metagenomic taxonomic classification algorithm. Simulated WGS datasets from ten genera as individuals and binary mixtures of eight organisms at varying ratios were analyzed to evaluate the role of contaminant concentration and taxonomy on detection. For the individual genomes the false positive contaminants reported depended on the genus, with Staphylococcus, Escherichia, and Shigella having the highest proportion of false positives. For nearly all binary mixtures the contaminant was detected in the in-silico datasets at the equivalent of 1 in 1,000 cells, though F. tularensis was not detected in any of the simulated contaminant mixtures and Y. pestis was only detected at the equivalent of one in 10 cells. Once a WGS method for detecting contaminants is characterized, it can be applied to evaluate microbial material purity, in efforts to ensure that contaminants are characterized in microbial materials used to validate pathogen detection assays, generate genome assemblies for database submission, and benchmark sequencing methods.

Best practices for evaluating single nucleotide variant calling methods for microbial genomics.

Innovations in sequencing technologies have allowed biologists to make incredible advances in understanding biological systems. As experience grows, researchers increasingly recognize that analyzing the wealth of data provided by these new sequencing platforms requires careful attention to detail for robust results. Thus far, much of the scientific Communit's focus for use in bacterial genomics has been on evaluating genome assembly algorithms and rigorously validating assembly program performance. Missing, however, is a focus on critical evaluation of variant callers for these genomes. Variant calling is essential for comparative genomics as it yields insights into nucleotide-level organismal differences. Variant calling is a multistep process with a host of potential error sources that may lead to incorrect variant calls. Identifying and resolving these incorrect calls is critical for bacterial genomics to advance. The goal of this review is to provide guidance on validating algorithms and pipelines used in variant calling for bacterial genomics. First, we will provide an overview of the variant calling procedures and the potential sources of error associated with the methods. We will then identify appropriate datasets for use in evaluating algorithms and describe statistical methods for evaluating algorithm performance. As variant calling moves from basic research to the applied setting, standardized methods for performance evaluation and reporting are required; it is our hope that this review provides the groundwork for the development of these standards.

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

DNA extract characterization process for microbial detection methods development and validation.

BACKGROUND: Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. RESULTS: DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/µL. Forty-seven extracts had concentrations greater than 17.5 ng/µL, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ µL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/µL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/µL and 0.25 ng/µL respectively. The qPCR LOQ varied by cell type (5.72 × 10-3 ng/µL for E. coli, 2.66 × 10-3 ng/µL, for B. cereus, 3.78 × 10-3 ng/µL for B. thailandensis, and 7.67 × 10-4 ng/µL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ. CONCLUSION: The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

Impact of processing method on recovery of bacteria from wipes used in biological surface sampling.

Environmental sampling for microbiological contaminants is a key component of hygiene monitoring and risk characterization practices utilized across diverse fields of application. However, confidence in surface sampling results, both in the field and in controlled laboratory studies, has been undermined by large variation in sampling performance results. Sources of variation include controlled parameters, such as sampling materials and processing methods, which often differ among studies, as well as random and systematic errors; however, the relative contributions of these factors remain unclear. The objective of this study was to determine the relative impacts of sample processing methods, including extraction solution and physical dissociation method (vortexing and sonication), on recovery of Gram-positive (Bacillus cereus) and Gram-negative (Burkholderia thailandensis and Escherichia coli) bacteria from directly inoculated wipes. This work showed that target organism had the largest impact on extraction efficiency and recovery precision, as measured by traditional colony counts. The physical dissociation method (PDM) had negligible impact, while the effect of the extraction solution was organism dependent. Overall, however, extraction of organisms from wipes using phosphate-buffered saline with 0.04% Tween 80 (PBST) resulted in the highest mean recovery across all three organisms. The results from this study contribute to a better understanding of the factors that influence sampling performance, which is critical to the development of efficient and reliable sampling methodologies relevant to public health and biodefense.

Parameters affecting spore recovery from wipes used in biological surface sampling.

The need for the precise and reliable collection of potential biothreat contaminants has motivated research in developing a better understanding of the variability in biological surface sampling methods. In this context, the objective of this work was to determine parameters affecting the efficiency of extracting Bacillus anthracis Sterne spores from commonly used wipe sampling materials and to describe performance using the interfacial energy concept. In addition, surface thermodynamics was applied to understand and predict surface sampling performance. Wipe materials were directly inoculated with known concentrations of B. anthracis spores and placed into extraction solutions, followed by sonication or vortexing. Experimental factors investigated included wipe material (polyester, cotton, and polyester-rayon), extraction solution (sterile deionized water [H(2)O], deionized water with 0.04% Tween 80 [H(2)O-T], phosphate-buffered saline [PBS], and PBS with 0.04% Tween 80 [PBST]), and physical dissociation method (vortexing or sonication). The most efficient extraction from wipes was observed for solutions containing the nonionic surfactant Tween 80. The increase in extraction efficiency due to surfactant addition was attributed to an attractive interfacial energy between Tween 80 and the centrifuge tube wall, which prevented spore adhesion. Extraction solution significantly impacted the extraction efficiency, as determined by statistical analysis (P < 0.05). Moreover, the extraction solution was the most important factor in extraction performance, followed by the wipe material. Polyester-rayon was the most efficient wipe material for releasing spores into solution by rank; however, no statistically significant difference between polyester-rayon and cotton was observed (P > 0.05). Vortexing provided higher spore recovery in H(2)O and H(2)O-T than sonication, when all three wipe materials and the reference control were considered (P < 0.05).

Association of quantum dot nanoparticles with Pseudomonas aeruginosa biofilm.

Quantum dots (QDs) of two different surface chemistries (carboxyl [COOH] and polyethylene glycol [PEG] modified) were utilized to determine the impact of surface functionality on QD mobility and distribution in Pseudomonas aeruginosa PAO1 biofilms. Confocal laser scanning microscopy was utilized to evaluate QD association with biofilm components (proteins, cells, and polysaccharides). Quantum dots did not preferentially associate with cell surfaces compared but did colocalize with extracellular proteins in the biofilm matrix. Neither PEG nor COOH QDs were found to be internalized by individual bacterial cells. Neither QD functionality nor flow rate of QD application (0.3 mL min(-1) or 3.0 mL min(-1)) resulted in a marked difference in QD association with P. aeruginosa biofilms. However, center of density determinations indicated COOH QDs could more easily penetrate the biofilm matrix by diffusion than PEG QDs. Biofilms with PEG QDs associated had rougher polysaccharide layers and rougher cell distribution than biofilms with COOH QDs. This work suggests natural biofilms may serve as deposition locations in natural and engineered environmental systems, and biofilm structural parameters may change based on exposure to nanomaterials of varied physical characteristics.

Development and Demonstration of a Method to Evaluate Bio-Sampling Strategies Using Building Simulation and Sample Planning Software.

In an effort to validate and demonstrate response and recovery sampling approaches and technologies, the U.S. Department of Homeland Security (DHS), along with several other agencies, have simulated a biothreat agent release within a facility at Idaho National Laboratory (INL) on two separate occasions in the fall of 2007 and the fall of 2008. Because these events constitute only two realizations of many possible scenarios, increased understanding of sampling strategies can be obtained by virtually examining a wide variety of release and dispersion scenarios using computer simulations. This research effort demonstrates the use of two software tools, CONTAM, developed by the National Institute of Standards and Technology (NIST), and Visual Sample Plan (VSP), developed by Pacific Northwest National Laboratory (PNNL). The CONTAM modeling software was used to virtually contaminate a model of the INL test building under various release and dissemination scenarios as well as a range of building design and operation parameters. The results of these CONTAM simulations were then used to investigate the relevance and performance of various sampling strategies using VSP. One of the fundamental outcomes of this project was the demonstration of how CONTAM and VSP can be used together to effectively develop sampling plans to support the various stages of response to an airborne chemical, biological, radiological, or nuclear event. Following such an event (or prior to an event), incident details and the conceptual site model could be used to create an ensemble of CONTAM simulations which model contaminant dispersion within a building. These predictions could then be used to identify priority area zones within the building and then sampling designs and strategies could be developed based on those zones.

Trophic transfer of nanoparticles in a simplified invertebrate food web.

The unique chemical and physical properties of engineered nanomaterials that make them attractive for numerous applications also contribute to their unexpected behaviour in the environment and biological systems. The potential environmental risks, including their impact on aquatic organisms, have been a central argument for regulating the growth of the nanotechnology sector. Here we show in a simplified food web that carboxylated and biotinylated quantum dots can be transferred to higher trophic organisms (rotifers) through dietary uptake of ciliated protozoans. Quantum dot accumulation from the surrounding environment (bioconcentration) was limited in the ciliates and no quantum dot enrichment (biomagnification) was observed in the rotifers. Our findings indicate that dietary uptake of nanomaterials should be considered for higher trophic aquatic organisms. However, limited bioconcentration and lack of biomagnification may impede the detection of nanomaterials in invertebrate species.

Antecedent growth conditions alter retention of environmental Escherichia coli isolates in transiently wetted porous media.

The physical transport of Escherichia coli in terrestrial environments may require control to prevent its dissemination from potential high-density sources, such as confined animal feedlot operations. Biobarriers, wherein convective flows carrying pathogens pass through a porous matrix with high retentive capacity, may present one such approach. Eight environmental E. coli isolates were selected to conduct operational retention tests (ORT) with potential biobarrier materials Pyrax or dolomite, or silica glass as control. The conditions in the ORT were chosen to simulate conditioning by manure solutes, a pulse application of a bacterial load followed by rainfall infiltration, and natural drainage. Removal was limited, and likely caused by the relatively high velocities during drainage, and the conditioning of otherwise favorable adhesion sites. Flagella-mediated motility showed the strongest correlation to biobarrier retention. Significant variability was observed across the E. coli isolates, but consistently higher retention was observed for cells with external versus intestinal pregrowth histories. E. coli O157:H7 was retained the least with all examined matrices, while E. coli K-12 displayed moderate retention and may not serve as representative model strain. Pyrax is a good candidate biobarrier material given its superior removal ability across the tested E. coli strains.

Intestinal versus external growth conditions change the surficial properties in a collection of environmental Escherichia coli isolates.

Predicting the fate of microorganisms in the environment is increasingly warranted, especially for pathogenic strains. A major habitat of Escherichia coli, which encompasses commensal as well as pathogenic strains, is the gastrointestinal tract with conditions very different from the environment it encounters after shedding from the host or during cultivation in the laboratory. We developed two relevant growth conditions representative of intestinal (host-associated) and external (postshedding) environments to investigate the surficial properties and behaviors of a diverse subset of E. coli feedlot isolates. Surficial properties may determine an isolate's physical fate. A pronounced increase in cell hydrophobicity and concomitant biofilm mass formation was observed for isolates grown under external conditions. Isolates that exhibited the highest surface hydrophobicity also formed visible suspended aggregates under external conditions. Other than hydrophobicity, flagella-mediated motility was determinant in affecting E. coli biofilm formation under external conditions, with all four nonmotile E. coli isolates characterized as thin-biofilm formers. The majority (88%) of Ag43+ (outer membrane protein, antigen 43) isolates formed thick biofilms, whereas the majority (75%) of Ag43- isolates formed thin biofilms. The tested E. coli O157:H7 strain behaved differently from the environmental E. coli isolates: it displayed a low electrostatic charge, a small decrease in hydrophobicity upon shifts to external conditions, and very little biofilm formation. On the other hand, the commonly used laboratory strain E. coli K-12 displayed low hydrophobicity both intestinally and externally, but it formed significant biofilm mass under external conditions. Clearly, various E. coli strains manifest significant variability in surficial behavior. This variability is further modulated by growth conditions. The interacting strain-inherent and cultivation-dependent effects on surficial behavior may have broad consequences for the fate and ecology of pathogenic and commensal E. coli strains.

Requirements for the Development of Bacillus Anthracis Spore Reference Materials Used to Test Detection Systems.

Bacillus anthracis spores have been used as biological weapons and the possibility of their further use requires surveillance systems that can accurately and reliably detect their presence in the environment. These systems must collect samples from a variety of matrices, process the samples, and detect the spores. The processing of the sample may include removal of inhibitors, concentration of the target, and extraction of the target in a form suitable for detection. Suitable reference materials will allow the testing of each of these steps to determine the sensitivity and specificity of the detection systems. The development of uniform and well-characterized reference materials will allow the comparison of different devices and technologies as well as assure the continued performance of detection systems. This paper discusses the special requirements of reference materials for Bacillus anthracis spores that could be used for testing detection systems. The detection of Bacillus anthracis spores is based on recognition of specific characteristics (markers) on either the spore surface or in the nucleic acids (DNA). We have reviewed the specific markers and their relevance to characterization of reference materials. We have also included the approach for the characterization of candidate reference materials that we are developing at the NIST laboratories. Additional applications of spore reference materials would include testing sporicidal treatments, techniques for sampling the environment, and remediation of spore-contaminated environments.

Plasmid introduction in metal-stressed, subsurface-derived microcosms: plasmid fate and community response.

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 micro M CdCl(2)), permitting long-term community monitoring. The broad-host-range IncPalpha plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd(r) or Ni(r) density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni(r) transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.