Phylodynamics of a regional SARS-CoV-2 rapid spreading event in Colorado in late 2020.

Since the initial reported discovery of SARS-CoV-2 in late 2019, genomic surveillance has been an important tool to understand its transmission and evolution. Here, we sought to describe the underlying regional phylodynamics before and during a rapid spreading event that was documented by surveillance protocols of the United States Air Force Academy (USAFA) in late October-November of 2020. We used replicate long-read sequencing on Colorado SARS-CoV-2 genomes collected July through November 2020 at the University of Colorado Anschutz Medical campus in Aurora and the United States Air Force Academy in Colorado Springs. Replicate sequencing allowed rigorous validation of variation and placement in a phylogenetic relatedness network. We focus on describing the phylodynamics of a lineage that likely originated in the local Colorado Springs community and expanded rapidly over the course of two months in an outbreak within the well-controlled environment of the United States Air Force Academy. Divergence estimates from sampling dates indicate that the SARS-CoV-2 lineage associated with this rapid expansion event originated in late October 2020. These results are in agreement with transmission pathways inferred by the United States Air Force Academy, and provide a window into the evolutionary process and transmission dynamics of a potentially dangerous but ultimately contained variant.

The influence of perceived stress on the human microbiome.

OBJECTIVE: Microbial dysbiosis, a shift from commensal to pathogenic microbiota, is often associated with mental health and the gut-brain axis, where dysbiosis in the gut may be linked to dysfunction in the brain. Many studies focus on dysbiosis induced by clinical events or traumatic incidents; however, many professions in austere or demanding environments may encounter continuously compounded stressors. This study seeks to explore the relationship between microbial populations and stress, both perceived and biochemical. RESULTS: Eight individuals enrolled in the study to provide a longitudinal assessment of the impact of stress on gut health, with four individuals providing enough samples for analysis. Eleven core microbial genera were identified, although the relative abundance of these genera and other members of the microbial population shifted over time. Although our results indicate a potential relationship between perceived stress and microbial composition of the gut, no association with biochemical stress was observed. Increases in perceived stress seem to elucidate a change in potentially beneficial Bacteroides, with a loss in Firmicutes phyla. This shift occurred in multiple individuals, whereas using cortisol as a stress biomarker showed contradictory responses. These preliminary data provide a potential mechanism for gut monitoring, while identifying targets for downstream modulation.

Engineering and characterization of dehalogenase enzymes from Delftia acidovorans in bioremediation of perfluorinated compounds.

Per- and Polyfluorinated alkyl substances (PFAS) are a broad class of synthetic compounds that have fluorine substituted for hydrogen in several or all locations and are globally categorized as PFCs (perfluorochemicals; commonly called fluorinated chemicals). These compounds have unique chemical and physical properties that enable their use in non-stick surfaces, fire-fighting efforts, and as slick coatings. However, recent concerns over the health effects of such compounds, specifically perfluorooctanoic acid and perfluorooctane sulfonic acid (PFOA, PFOS; PFOA/S), have led to increased attention and research by the global community into degradation methods. In this study, soil samples from PFAS-contamination sites were cultured and screened for microbes with PFOA/S degradation potential, which led to the identification of Delftia acidovorans. It was found that D. acidovorans isolated from PFAS-contaminated soils was capable of growth in minimal media with PFOA as a sole carbon resource, and an observable fluoride concentration increase was observed when cells were exposed to PFOA. This suggests potential activity of a dehalogenase enzyme that may be of use in PFOA or PFAS microbial remediation efforts. Several associated haloacid dehalogenases have been identified in the D. acidovorans genome and have been engineered for expression in Escherichia coli for rapid production and purification. These enzymes have shown potential for enzymatic defluorination, a significant step in biological degradation and removal of PFOA/S from the environment. We hypothesize that bioremediation of PFAS using naturally occurring microbial degradation pathways may represent a novel approach to remove PFAS contamination.

Campus Reset: Dynamic Planning and Response to SARS-CoV-2 Infections at the US Air Force Academy.

Predominantly asymptomatic infections, such as those for SARS-CoV-2, require robust surveillance testing to identify people who are unknowingly spreading the virus. The US Air Force Academy returned to in-person classes for more than 4000 cadets aged 18-26 years during the fall 2020 semester to meet graduation and leadership training requirements. To enable this sustained cadet footprint, the institution developed a dynamic SARS-CoV-2 response plan using near-real-time data to inform decisions and trigger policies. A surveillance testing program based on mathematical modeling and a policy-driven campus reset option provided a scaled approach to react to SARS-CoV-2 conditions. This program adequately controlled the spread of the virus for the first 2 months of the academic semester but failed to predict or initially mitigate a significant outbreak in the second half of the semester. Although this approach did not completely eliminate SARS-CoV-2 infections in the population, it served as an early warning system to alert public health authorities to potential issues, which allowed timely responses while containment was still possible.

Efficiency of pooled surveillance testing in academic labs to detect and inhibit COVID-19 outbreaks.

Robust surveillance testing is a key strategic plan to prevent COVID-19 outbreaks and slow the spread of the SARS-CoV-2 pandemic; however, limited resources, facilities and time often impair the implementation of a widespread surveillance effort. To mitigate these resource limitations, we employed a strategy of pooling samples, reducing reagent cost and processing time. Through utilizing academic faculty and labs, successful pooled surveillance testing was conducted throughout Fall 2020 semester to detect positive SARS-CoV-2 infections in a population of 4400 students. During the semester, over 25,000 individual COVID status evaluations were made by pooling eight individual samples into one quantitative reverse transcription polymerase chain reaction. This pooled surveillance strategy was highly effective at detecting infection and significantly reduced financial burden and cost by $3.6 million.

Characterization of human norovirus binding to gut-associated bacterial ligands.

OBJECTIVE: Research suggests human norovirus binding to histo-blood group antigen (HBGA)-like molecules on enteric bacteria may enhance viral pathogenesis; however, the properties of these bacterial ligands are not well known. Previous work identified, but did not characterize, seven norovirus-binding bacteria. To further examine this bacteria-virus binding interaction, enteric bacteria were analyzed via Western blot with anti-HBGA antibodies and lectins targeting HBGA-associated sugar components. Virus overlay assays using capsids from six different human norovirus strains further identified responsible ligands and strain dependent binding properties. RESULTS: Each bacterial species possessed varying degrees of HBGA-like activity, and lectin binding further elucidated potential sugar residues involved (N-acetyl-galactosamine, α-D-galactose or α-L-fucose). Both GI and GII norovirus capsids bound specific bacterial ligand sizes, and generally corresponded to anti-HBGA Western blot patterns. A 35-kDa band reacted with all HBGA antibodies, bound all six of the noroviruses tested, and had a high affinity for the lectins. Collectively, this work characterizes the varying carbohydrate residues potentially responsible for norovirus-bacteria interactions and provides a basis for future ligand identification.

Human norovirus binding to select bacteria representative of the human gut microbiota.

Recent reports describe the ability of select bacterial strains to bind human norovirus, although the specificity of such interactions is unknown. The purpose of this work was to determine if a select group of bacterial species representative of human gut microbiota bind to human norovirus, and if so, to characterize the intensity and location of that binding. The bacteria screened included naturally occurring strains isolated from human stool (Klebsiella spp., Citrobacter spp., Bacillus spp., Enterococcus faecium and Hafnia alvei) and select reference strains (Staphylococcus aureus and Enterobacter cloacae). Binding in PBS was evaluated to three human norovirus strains (GII.4 New Orleans 2009 and Sydney 2012, GI.6) and two surrogate viruses (Tulane virus and Turnip Crinkle Virus (TCV)) using a suspension assay format linked to RT-qPCR for quantification. The impact of different overnight culture media prior to washing on binding efficiency in PBS was also evaluated, and binding was visualized using transmission electron microscopy. All bacteria tested bound the representative human norovirus strains with high efficiency (<1 log10 of input virus remained unbound or <10% unbound and >90% binding efficiency) (p>0.05); there was selective binding for Tulane virus and no binding observed for TCV. Binding efficiency was highest when bacteria were cultured in minimal media (<1 log10 of input virus remained unbound, so >90% bound), but notably decreased when cultured in enriched media (1-3 log10 unbound or 0.01 -<90% bound)) (p<0.05). The norovirus-bacteria binding occurred around the outer cell surfaces and pili structures, without apparent localization. The findings reported here further elucidate and inform the dynamics between human noroviruses and enteric bacteria with implications for norovirus pathogenesis.

Virus-Bacteria Interactions: An Emerging Topic in Human Infection.

Bacteria and viruses often occupy the same niches, however, interest in their potential collaboration in promoting wellness or disease states has only recently gained traction. While the interaction of some bacteria and viruses is well characterized (e.g., influenza virus), researchers are typically more interested in the location of the infection than the manner of cooperation. There are two overarching types of bacterial-virus disease causing interactions: direct interactions that in some way aid the viruses, and indirect interactions aiding bacteria. The virus-promoting direct interactions occur when the virus exploits a bacterial component to facilitate penetration into the host cell. Conversely, indirect interactions result in increased bacterial pathogenesis as a consequence of viral infection. Enteric viruses mainly utilize the direct pathway, while respiratory viruses largely affect bacteria in an indirect fashion. This review focuses on some key examples of how virus-bacteria interactions impact the infection process across the two organ systems, and provides evidence supporting this as an emerging theme in infectious disease.

Norovirus Binding to Ligands Beyond Histo-Blood Group Antigens.

Histo-blood group antigens (HBGAs) are commonly accepted as the cellular receptors for human norovirus. However, some human noroviruses have been found not to bind any HBGA ligand, suggesting potential additional co-factors. Some ligands have been found to bind noroviruses and have the potential to be additional cellular receptors/attachment factors for human norovirus or inhibitors of the HBGA interaction. The studies identifying these mostly characterize different chemical, human, food, or bacterial components and their effect on norovirus binding and infection, although the mechanism of interaction is unknown in many cases. This review seeks to supplement the already well-covered HBGA-norovirus literature by covering non-HBGA human norovirus ligands and inhibitors to provide investigators with a more comprehensive view of norovirus ligands.

Capture and concentration of viral and bacterial foodborne pathogens using apolipoprotein H.

The need for improved pathogen separation and concentration methods to reduce time-to-detection for foodborne pathogens is well recognized. Apolipoprotein H (ApoH) is an acute phase human plasma protein that has been previously shown to interact with viruses, lipopolysaccharides (LPS) and bacterial proteins. The purpose of this study was to determine if ApoH was capable of binding and efficiently capturing two representative human norovirus strains (GI.1 and GII.4), a cultivable surrogate, and four bacterial pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus). Experiments were carried out using an ApoH-conjugated magnetic bead-based capture followed by pathogen detection using nucleic acid amplification. For all three viruses studied, >10% capture efficiency (<1 Log10 loss in RT-qPCR amplifiable units) was observed. The same capture efficiencies were observed for the bacterial pathogens tested, with the exception of E. coli O157:H7 (approximately 1% capture efficiency, or 2 Log10 loss in CFU equivalents). The efficiency of the capture steps did not vary as a consequence of input target concentration or in the presence of an abundance of background microflora. A complementary plate-based capture assay showed that ApoH bound to a variety of human norovirus virus-like particles. ApoH has the potential to be a broadly reactive ligand for separating and concentrating representative foodborne pathogens, both bacteria and viruses.

Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.

Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential relationship between these two enzymes remains to be elucidated.