Online dashboards for SARS-CoV-2 wastewater data need standard best practices: An environmental health communication agenda.

The COVID-19 pandemic has highlighted the benefits of wastewater surveillance to supplement clinical data. Numerous online information dashboards have been rapidly, and typically independently, developed to communicate environmental surveillance data to public health officials and the public. In this study, we review dashboards presenting SARS-CoV-2 wastewater data and propose a path toward harmonization and improved risk communication. A list of 127 dashboards representing 27 countries was compiled. The variability was high and encompassed aspects including the graphics used for data presentation (e.g., line/bar graphs, maps, and tables), log versus linear scale, and 96 separate ways of labeling SARS-CoV-2 wastewater concentrations. Globally, dashboard presentations also differed by region. Approximately half of the dashboards presented clinical case data, and 25% presented variant monitoring. Only 30% of dashboards provided downloadable source data. While any single dashboard is likely useful in its own context and locality, the high variation across dashboards at best prevents optimal use of wastewater surveillance data on a broader geographical scale and at worst could lead to risk communication issues and the potential for public health miscommunication. There is a great opportunity to improve scientific communication through the adoption of uniform data presentation conventions, standards, and best practices in this field.

Enhancing untargeted metabolomics using metadata-based source annotation.

Human untargeted metabolomics studies annotate only ~10% of molecular features. We introduce reference-data-driven analysis to match metabolomics tandem mass spectrometry (MS/MS) data against metadata-annotated source data as a pseudo-MS/MS reference library. Applying this approach to food source data, we show that it increases MS/MS spectral usage 5.1-fold over conventional structural MS/MS library matches and allows empirical assessment of dietary patterns from untargeted data.

Standards to support an enduring capability in wastewater surveillance for public health: Where are we?

The COVID-19 pandemic highlighted a wide range of public health system challenges for infectious disease surveillance. The discovery that the SARS-CoV-2 virus was shed in feces and can be characterized using PCR-based testing of sewage samples offers new possibilities and challenges for wastewater surveillance (WWS). However, WWS standardization of practices is needed to provide actionable data for a public health response. A workshop was convened consisting of academic, federal government, and industry stakeholders. The objective was to review WWS sampling protocols, testing methods, analyses, and data interpretation approaches for WWS employed nationally and identify opportunities for standardizing practices, including the development of documentary standards or reference materials in the case of SARS-CoV-2 surveillance. Other WWS potential future threats to public health were also discussed. Several aspects of WWS were considered and each offers the opportunity for standards development. These areas included sampling strategies, analytical methods, and data reporting practices. Each of these areas converged on a common theme, the challenge of results comparability across facilities and jurisdictions. For sampling, the consensus solution was the development of documentary standards to guide appropriate sampling practices. In contrast, the predominant opportunity for analytical methods was reference material development, such as PCR-based standards and surrogate recovery controls. For data reporting practices, the need for establishing the minimal required metadata, a metadata vocabulary, and standardizing data units of measure including measurement threshold definitions was discussed. Beyond SARS-CoV-2 testing, there was general agreement that the WWS platform will continue to be a valuable tool for a wide range of public health threats and that future cross-sector engagements are needed to guide an enduring WWS capability.

Toward absolute viability measurements for bacteria.

We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ1 , τ2 and p1 ), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach.

Standards efforts and landscape for rapid microbial testing methodologies in regenerative medicine.

The Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery (SCB) supports the development and commercialization of regenerative medicine products by identifying and addressing industry-wide challenges through standards. Through extensive stakeholder engagement, the implementation of rapid microbial testing methods (RMTMs) was identified as a high-priority need that must be addressed to facilitate more timely release of products. Since 2017, SCB has coordinated efforts to develop standards for this area through surveys, weekly meetings, workshops, leadership in working groups and participation in standards development organizations. This article describes the results of these efforts and discusses the current landscape of RMTMs for regenerative medicine products. Based on discussions with stakeholders across the field, an overview of traditional culture-based methods and limitations, alternative microbial testing technologies and current challenges, fit-for-purpose rapid microbial testing and case studies, risk-based strategies for selection of novel rapid microbial test methods and ongoing standards efforts for rapid microbial testing are captured here. To this end, SCB is facilitating several initiatives to address challenges associated with rapid microbial testing for regenerative medicine products. Two documentary standards are under development: an International Organization for Standardization standard to provide the framework for a risk-based approach to selecting fit-for-purpose assays primarily intended for cell and gene therapy products and an ASTM standard guide focused on sampling methods for microbial testing methods in tissue-engineered medical products. Working with the National Institute of Standards and Technology, SCB expects to facilitate the process of developing publicly available microbial materials for inter-laboratory testing. These studies will help collect the data necessary to facilitate validation of novel rapid methods. Finally, SCB has been working to increase awareness of, dialog about and participation in efforts to develop standards in the regenerative medicine field.

Workshop report: Toward the development of a human whole stool reference material for metabolomic and metagenomic gut microbiome measurements.

INTRODUCTION: To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements. OBJECTIVES: The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control. METHODS: The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material. RESULTS: This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research. CONCLUSIONS: Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

How we are assessing the developing antibacterial resin-based dental materials? A scoping review.

OBJECTIVES: To identify antibacterial additives and screening/assessment approaches used to evaluate the antibacterial activity of resin-based restorative dental materials containing these additives. DATA: In vitro studies that compared the antibacterial effects of resin-based restorative dental materials with and without antibacterial additives were screened. Risk bias was assessed, and the following data were extracted: antibacterial additive, parental dental material, curing mode, bacterial growth outcome assessment, samples used as a substrate for bacterial growth, inoculum complexity, and culture time as an indicator of biofilm maturity. SOURCE: Arksey and O'Malley's five stages framework using Medline (OVID), EMBASE, and Scopus (Elsevier) databases guided this review. STUDY SELECTION: From 6503 studies initially identified, 348 studies were considered eligible for full-text screening, and 153 were included for data extraction. Almost all studies have a high sampling bias related to both sample size and blindness. Quaternary ammonium monomers were the most investigated additive (45 %), and the most prevailing parental material was resin composite (49 %). There was extensive methodological heterogeneity among the studies for outcome assessment with the majority using resin composite disks (78 %), mono-species Streptococcus mutans as the inoculum (54 %), and a relatively short period of biofilm growth (≤24 h). CONCLUSION: The findings herein present the urgent need for improved biological efficacy studies in this important and exciting field. There is a need for efforts to improve study designs to mimic the oral environment in vivo and to develop standardized methods to help understand and optimize these materials. CLINICAL SIGNIFICANCE: Most studies that incorporate antibacterial additives into resin-based materials claim promising results by bacterial reduction. However, these results should be interpreted with caution due to significant variation in the methods applied for quantifying bacterial growth, the frequent lack of complexity in the biofilms, and the often-short duration of biofilm growth.

Chemical and Physical Transformations of Silver Nanomaterial Containing Textiles After Modeled Human Exposure.

The antimicrobial properties of silver nanomaterials (AgNM) have been exploited in various consumer applications, including textiles such as wound dressings. Understanding how these materials chemically transform throughout their use is necessary to predict their efficacy during use and their behavior after disposal. The aim of this work was to evaluate chemical and physical transformations to a commercial AgNM-containing wound dressing during modeled human exposure to synthetic sweat (SW) or simulated wound fluid (WF). Scanning electron microscopy with energy dispersive X-ray spectroscopy (EDS) revealed the formation of micrometer-sized structures at the wound dressing surface after SW exposure while WF resulted in a largely featureless surface. Measurements by X-ray photoelectron spectroscopy (XPS) revealed a AgCl surface (consistent with EDS) while X-ray diffraction (XRD) found a mixture of zero valent silver and AgCl suggesting the AgNM wound dressings surface formed a passivating AgCl surface layer after SW and WF exposure. For WF, XPS based findings revealed the addition of an adsorbed protein layer based on the nitrogen marker which adsorbed released silver at prolonged exposures. Silver release was evaluated by inductively coupled plasma mass spectrometry which revealed a significant released silver fraction in WF and minimal released silver in SW. Analysis suggests that the protein in WF sequestered a fraction of the released silver which continued with exposure time, suggesting additional processing at the wound dressing surface even after the initial transformation to AgCl. To evaluate the impact on antimicrobial efficacy, zone of inhibition (ZOI) testing was conducted which found no significant change after modeled human exposure compared to the pristine wound dressing. The results presented here suggest AgNM-containing wound dressings transform chemically in simulated human fluids resulting in a material with comparable antimicrobial properties with pristine wound dressings. Ultimately, knowing the resulting chemical properties of the AgNM wound dressings will allow better predictive models to be developed regarding their fate.

A Novel Dental Sealant Containing Dimethylaminohexadecyl Methacrylate Suppresses the Cariogenic Pathogenicity of Streptococcus mutans Biofilms.

Cariogenic oral biofilms are strongly linked to dental caries around dental sealants. Quaternary ammonium monomers copolymerized with dental resin systems have been increasingly explored for modulation of biofilm growth. Here, we investigated the effect of dimethylaminohexadecyl methacrylate (DMAHDM) on the cariogenic pathogenicity of Streptococcus mutans (S. mutans) biofilms. DMAHDM at 5 mass% was incorporated into a parental formulation containing 20 mass% nanoparticles of amorphous calcium phosphate (NACP). S. mutans biofilms were grown on the formulations, and biofilm inhibition and virulence properties were assessed. The tolerances to acid stress and hydrogen peroxide stress were also evaluated. Our findings suggest that incorporating 5% DMAHDM into 20% NACP-containing sealants (1) imparts a detrimental biological effect on S. mutans by reducing colony-forming unit counts, metabolic activity and exopolysaccharide synthesis; and (2) reduces overall acid production and tolerance to oxygen stress, two major virulence factors of this microorganism. These results provide a perspective on the value of integrating bioactive restorative materials with traditional caries management approaches in clinical practice. Contact-killing strategies via dental materials aiming to prevent or at least reduce high numbers of cariogenic bacteria may be a promising approach to decrease caries in patients at high risk.

Evaluating changes to Ralstonia pickettii in high-purity water to guide selection of potential calibration materials for online water bioburden analyzers.

Online water bioburden analyzers (OWBAs) can provide real-time feedback on viable bacteria in high-purity water (HPW) systems for pharmaceutical manufacturers. To calibrate and validate OWBAs, which detect bacteria using scattered light and bacterial autofluorescence, standards are needed that mimic the characteristics of bacteria in HPW. To guide selection of potential standards, e.g., fluorescent microspheres, a relevant bacterial contaminant, Ralstonia pickettii, was characterized for size, count, viability, and autofluorescence after exposure for 24 h to HPW or a nutrient environment. The cells exposed to HPW showed smaller sizes, with lower counts and autofluorescence intensities, but similar spectral features. The cell characteristics are discussed in comparison with a set of fluorescent microspheres, considering factors relevant to OWBAs. These studies suggest that fluorescent microspheres should be relatively small (< 1 µm diameter) and dim, while covering a broad emission range from ≈ (420 to 600) nm to best mimic the representative R. pickettii.

Structural insights into DNA-stabilized silver clusters.

Despite their great promise as fluorescent biological probes and sensors, the structure and dynamics of Ag complexes derived from single stranded DNA (ssDNA) are less understood than their double stranded counterparts. In this work, we seek new insights into the structure of single AgNssDNA clusters using analytical ultracentrifugation (AUC), nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular dynamics simulations (MD) of a fluorescent (AgNssDNA)8+ nanocluster. The results suggest that the purified (AgNssDNA)8+ nanocluster is a mixture of predominantly Ag15 and Ag16 species that prefer two distinct long-lived conformational states: one extended, the other approaching spherical. However, the ssDNA strands within these clusters are highly mobile. Ag(i) interacts preferentially with the nucleobase rather than the phosphate backbone, causing a restructuring of the DNA strand relative to the bare DNA. Infrared spectroscopy and MD simulations of (AgNssDNA)8+ and model nucleic acid homopolymers suggest that Ag(i) has a higher affinity for cytosine over guanine bases, little interaction with adenine, and virtually none with thymine. Ag(i) shows a tendency to interact with cytosine N3 and O2 and guanine N7 and O6, opening the possibility for a Ag(i)-base bifurcated bond to act as a nanocluster nucleation and strand stabilizing site. This work provides valuable insight into nanocluster structure and dynamics which drive stability and optical properties, and additional studies using these types of characterization techniques are important for the rational design of single stranded AgDNA nanocluster sensors.

Effect of dental monomers and initiators on Streptococcus mutans oral biofilms.

OBJECTIVE: Resin-based composites are known to elute leachables that include unincorporated starting materials. The objective of this work was to determine the effect of common dental monomers and initiators on Streptococcus mutans biofilm metabolic activity and biomass. METHODS: S. mutans biofilms were inoculated in the presence of bisphenol A glycerolate dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), camphorquinone (CQ), and ethyl 4-(dimethylamino)benzoate (4E) at 0.01µg/mL up to 500µg/mL, depending on the aqueous solubility of each chemical. Biofilms were evaluated at 4h and 24h for pH (n=3-8), biomass via crystal violet (n=12), metabolic activity via tetrazolium salt (n=12), and membrane permeability for selected concentrations via confocal microscopy (n=6). Parametric and non-parametric statistics were applied. RESULTS: 500µg/mL TEGDMA reduced 24h metabolic activity but not biomass, similar to prior results with leachables from undercured BisGMA-TEGDMA polymers. 50µg/mL BisGMA reduced biofilm biomass and activity, slightly delayed the pH drop, and decreased the number of cells with intact membranes. 100µg/mL CQ delayed the pH drop and metabolic activity at 4h but then significantly increased the 24h metabolic activity. 4E had no effect up to 10µg/mL. SIGNIFICANCE: Monomers and initiators that leach from resin composites affect oral bacterial biofilm growth in opposite ways. Leachables, which can be released for extended periods of time, have the potential to alter oral biofilm biomass and activity and should be considered in developing and evaluating new dental materials.

pH-Sensitive Compounds for Selective Inhibition of Acid-Producing Bacteria.

Stimuli-responsive compounds that provide on-site, controlled antimicrobial activity promise an effective approach to prevent infections, reducing the need for systemic antibiotics. We present a novel pH-sensitive quaternary pyridinium salt (QPS), whose antibacterial activity is boosted by low pH and controlled by adjusting the pH between 4 and 8. Particularly, this compound selectively inhibits growth of acid-producing bacteria within a multispecies community. The successful antibacterial action of this QPS maintains the environmental pH above 5.5, a threshold pH, below which demineralization/erosion takes place. The design, synthesis, and characterization of this QPS and its short-chain analogue are discussed. In addition, their pH-sensitive physicochemical properties in aqueous and organic solutions are evaluated by UV-vis spectroscopy, dynamic light scattering, and NMR spectroscopy. Furthermore, the mechanism of action reveals a switchable assembly that is triggered by acid-base interaction and formed by tightly stacked π-conjugated systems and base moieties. Finally, a model is proposed to recognize the correlated but different mechanisms of pH sensitivity and acid-induced, pH-controlled antibacterial efficacy. We anticipate that successful application of these QPSs and their derivatives will provide protections against infection and erosion through targeted treatments to acid-producing bacteria and modulation of environmental pH.

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.

Biofilm over teeth and restorations: What do we need to know?

OBJECTIVE: The goal of this manuscript is to provide an overview of biofilm attributes and measurement approaches in the context of studying biofilms on tooth and dental material surfaces to improve oral health. METHODS: A historical perspective and terminology are presented, followed by a general description of the complexity of oral biofilms. Then, an approach to grouping measurable biofilm properties is presented and considered in relation to biofilm-material interactions and material design strategies to alter biofilms. Finally, the need for measurement assurance in biofilm and biofilm-materials research is discussed. RESULTS: Biofilms are highly heterogeneous communities that are challenging to quantify. Their characteristics can be broadly categorized into constituents (identity), quantity, structure, and function. These attributes can be measured over time and in response to substrates and external stimuli. Selecting the biofilm attribute(s) of interest and appropriate measurement methods will depend on the application and, in the case of antimicrobial therapies, the strategic approach and expected mechanism of action. To provide measurement assurance, community accepted protocols and guidelines for minimum data and metadata should be established and broadly applied. Consensus standards may help to streamline testing and demonstration of product claims. SIGNIFICANCE: Understanding oral biofilms and their interactions with tooth and dental material surfaces holds great promise for enabling improvements in oral and overall human health. Both substrate and biofilm properties should be considered to develop a more thorough understanding of the system.

Experimental and statistical methods to evaluate antibacterial activity of a quaternary pyridinium salt on planktonic, biofilm-forming, and biofilm states.

Robust evaluation and comparison of antimicrobial technologies are critical to improving biofilm prevention and treatment. Herein, a multi-pronged experimental framework and statistical models were applied to determine the effects of quaternary pyridinium salt, 4-acetyl-1-hexadecylpyridin-1-ium iodide (QPS-1), on Streptococcus mutans in the planktonic, biofilm-forming and biofilm cell states. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined via common methods with novel application of statistical approaches combining random effects models and interval censored data to estimate uncertainties. The MICs and MBCs for planktonic and biofilm-forming states ranged from 3.12 to 12.5 µg ml-1, with biofilm values only ≈ 8 times higher. Potent anti-biofilm activity and reactive structural features make QPS-1 a promising antibacterial additive for dental and potentially other biomedical devices. Together, the experimental framework and statistical models provide estimates and uncertainties for effective antimicrobial concentrations in multiple cell states, enabling statistical comparisons and improved characterization of antibacterial agents.

Primer containing dimethylaminododecyl methacrylate kills bacteria impregnated in human dentin blocks.

Antibacterial dimethylaminododecyl methacrylate (DMADDM) was recently synthesized. The objectives of this study were to: (1) investigate antibacterial activity of DMADDM-containing primer on Streptococcus mutans impregnated into dentin blocks for the first time, and (2) compare the antibacterial efficacy of DMADDM with a previous quaternary ammonium dimethacrylate (QADM). Scotchbond Multi-Purpose (SBMP) bonding agent was used. DMADDM and QADM were mixed into SBMP primer. Six primers were tested: SBMP control primer P, P+2.5% DMADDM, P+5% DMADDM, P+7.5% DMADDM, P+10% DMADDM, and P+10% QADM. S. mutans were impregnated into human dentin blocks, and each primer was applied to dentin to test its ability to kill bacteria in dentinal tubules. Bacteria in dentin were collected via a sonication method, and the colony-forming units (CFU) and inhibition zones were measured. The bacterial inhibition zone of P+10% DMADDM was 10 times that of control primer (P<0.05). CFU in dentin with P+10% DMADDM was reduced by three orders of magnitude, compared with control. DMADDM had a much stronger antibacterial effect than QADM, and antibacterial efficacy increased with increasing DMADDM concentration. Dentin shear bond strengths were similar among all groups (P>0.1). In conclusion, antibacterial DMADDM-containing primer was validated to kill bacteria inside dentin blocks, possessing a much stronger antibacterial potency than the previous QADM. DMADDM-containing bonding agent was effective in eradicating bacteria in dentin, and its efficacy was directly proportional to DMADDM mass fraction. Therefore, DMADDM may be promising for use in bonding agents as well as in other restorative and preventive materials to inhibit bacteria.

Automation of antimicrobial activity screening.

Manual and automated methods were compared for routine screening of compounds for antimicrobial activity. Automation generally accelerated assays and required less user intervention while producing comparable results. Automated protocols were validated for planktonic, biofilm, and agar cultures of the oral microbe Streptococcus mutans that is commonly associated with tooth decay. Toxicity assays for the known antimicrobial compound cetylpyridinium chloride (CPC) were validated against planktonic, biofilm forming, and 24 h biofilm culture conditions, and several commonly reported toxicity/antimicrobial activity measures were evaluated: the 50 % inhibitory concentration (IC50), the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC). Using automated methods, three halide salts of cetylpyridinium (CPC, CPB, CPI) were rapidly screened with no detectable effect of the counter ion on antimicrobial activity.