Long-term gut colonization with ESBL-producing Escherichia coli in participants without known risk factors from the southeastern United States.

We evaluated gut carriage of extended spectrum beta lactamase producing Enterobacteriaceae (ESBL-E) in southeastern U.S. residents without recent in-patient healthcare exposure. Study enrollment was January 2021-February 2022 in Athens, Georgia, U.S. and included a diverse population of 505 adults plus 50 child participants (age 0-5). Based on culture-based screening of stool samples, 4.5% of 555 participants carried ESBL-Es. This is slightly higher than reported in studies conducted 2012-2015, which found carriage rates of 2.5-3.9% in healthy U.S. residents. All ESBL-E confirmed isolates (n=25) were identified as Escherichia coli. Isolates belonged to 11 sequence types, with 48% classified as ST131. Ninety six percent of ESBL-E isolates carried a blaCTX-M gene. Isolated ESBL-Es frequently carried virulence genes as well as multiple classes of antibiotic resistance genes. Long-term colonization was common, with 64% of ESBL-E positive participants testing positive when rescreened three months later. One participant yielded isolates belonging to two different E. coli sequence types that carried blaCTX-M-1 genes on near-identical plasmids, suggesting intra-gut plasmid transfer. Isolation of E. coli on media without antibiotics revealed that ESBL-E. coli typically made up a minor fraction of the overall gut E. coli population, although in some cases they were the dominant strain. ESBL-E carriage was not associated with a significantly different stool microbiome composition. However, some microbial taxa were differentially abundant in ESBL-E carriers. Together, these results suggest that a small subpopulation of US residents are long-term, asymptomatic carriers of ESBL-Es, and may serve as an important reservoir for community spread of these ESBL genes.

Draft genome sequence of multidrug-resistant Vibrio navarrensis strain DA9 isolated from a coastal canal in the Florida Keys (USA).

A presumptive Vibrio isolate with a multidrug resistance profile was isolated from surface seawater collected from a coastal canal in 2014 and identified as Vibrio navarrensis, designated as strain DA9. Here, we report a 5.1-Mb draft genome sequence of strain DA9 with a G + C content of 47.5%.

Vibrio alginolyticus growth kinetics and the metabolic effects of iron.

IMPORTANCE: Transmission of V. alginolyticus occurs opportunistically through direct seawater exposure and is a function of its abundance in the environment. Like other Vibrio spp., V. alginolyticus are considered conditionally rare taxa in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions, which remain poorly defined. Prior research has established the importance of temperature and salinity as the major determinants of Vibrio geographical and temporal range. However, bloom formation can be strongly influenced by other factors that may be more episodic and localized, such as changes in iron availability. Here we confirm the broad temperature and salinity tolerance of V. alginolyticus and demonstrate the importance of iron supplementation as a key factor for growth in the absence of thermal or osmotic stress. The results of this research highlight the importance of episodic iron input as a crucial metric to consider for the assessment of V. alginolyticus risk.

Distribution of Antibiotic Resistance in a Mixed-Use Watershed and the Impact of Wastewater Treatment Plants on Antibiotic Resistance in Surface Water.

The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.

Chronic alcohol use primes bronchial cells for altered inflammatory response and barrier dysfunction during SARS-CoV-2 infection.

Alcohol use disorder (AUD) is a significant public health concern and people with AUD are more likely to develop severe acute respiratory distress syndrome (ARDS) in response to respiratory infections. To examine whether AUD was a risk factor for more severe outcome in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we examined early responses to infection using cultured differentiated bronchial epithelial cells derived from brushings obtained from people with AUD or without AUD. RNA-seq analysis of uninfected cells determined that AUD cells were enriched for expression of epidermal genes as compared with non-AUD cells. Bronchial epithelial cells from patients with AUD showed a significant decrease in barrier function 72 h postinfection, as determined by transepithelial electrical resistance. In contrast, barrier function of non-AUD cells was enhanced 72 h after SARS-CoV-2 infection. AUD cells showed claudin-7 that did not colocalize with zonula occludens-1 (ZO-1), indicative of disorganized tight junctions. However, both AUD and non-AUD cells showed decreased ß-catenin expression following SARS-CoV-2 infection. To determine the impact of AUD on the inflammatory response to SARS-CoV-2 infection, cytokine secretion was measured by multiplex analysis. SARS-CoV-2-infected AUD bronchial cells had enhanced secretion of multiple proinflammatory cytokines including TNFα, IL-1ß, and IFNγ as opposed to non-AUD cells. In contrast, secretion of the barrier-protective cytokines epidermal growth factor (EGF) and granulocyte macrophage-colony stimulating factor (GM-CSF) was enhanced for non-AUD bronchial cells. Taken together, these data support the hypothesis that AUD is a risk factor for COVID-19, where alcohol primes airway epithelial cells for increased inflammation and increased barrier dysfunction and increased inflammation in response to infection by SARS-CoV-2.NEW & NOTEWORTHY Alcohol use disorder (AUD) is a significant risk factor for severe acute respiratory distress syndrome. We found that AUD causes a phenotypic shift in gene expression in human bronchial epithelial cells, enhancing expression of epidermal genes. AUD cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had higher levels of proinflammatory cytokine secretion and barrier dysfunction not present in infected non-AUD cells, consistent with increased early COVID-19 severity due to AUD.

Twenty-year record of white pox disease in the Florida Keys: importance of environmental risk factors as drivers of coral health.

Declining coral populations worldwide place a special premium on identifying risks and drivers that precipitate these declines. Understanding the relationship between disease outbreaks and their drivers can help to anticipate when the risk of a disease pandemic is high. Populations of the iconic branching Caribbean elkhorn coral Acropora palmata have collapsed in recent decades, in part due to white pox disease (WPX). To assess the role that biotic and abiotic factors play in modulating coral disease, we present a predictive model for WPX in A. palmata using 20 yr of disease surveys from the Florida Keys plus environmental information collected simultaneously in situ and via satellite. We found that colony size was the most influential predictor for WPX occurrence, with larger colonies being at higher risk. Water quality parameters of dissolved oxygen saturation, total organic carbon, dissolved inorganic nitrogen, and salinity were implicated in WPX likelihood. Both low and high wind speeds were identified as important environmental drivers of WPX. While high temperature has been identified as an important cause of coral mortality in both bleaching and disease scenarios, our model indicates that the relative influence of HotSpot (positive summertime temperature anomaly) was low and actually inversely related to WPX risk. The predictive model developed here can contribute to enabling targeted strategic management actions and disease surveillance, enabling managers to treat the disease or mitigate disease drivers, thereby suppressing the disease and supporting the persistence of corals in an era of myriad threats.

Direct wastewater extraction as a simple and effective method for SARS-CoV-2 surveillance and COVID-19 community-level monitoring.

Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but preconcentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for 1 year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280 µl) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69-0.82). To compensate for the method's high limit of detection (approximately 106-107 copies l-1 in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100 000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

Coral Disease and Ingestion: Investigating the Role of Heterotrophy in the Transmission of Pathogenic Vibrio spp. using a Sea Anemone (Exaiptasia pallida) Model System.

Understanding disease transmission in corals can be complicated given the intricacy of the holobiont and difficulties associated with ex situ coral cultivation. As a result, most of the established transmission pathways for coral disease are associated with perturbance (i.e., damage) rather than evasion of immune defenses. Here, we investigate ingestion as a potential pathway for the transmission of coral pathogens that evades the mucus membrane. Using sea anemones (Exaiptasia pallida) and brine shrimp (Artemia sp.) to model coral feeding, we tracked the acquisition of the putative pathogens, Vibrio alginolyticus, V. harveyi, and V. mediterranei using GFP-tagged strains. Vibrio sp. were provided to anemones using 3 experimental exposures (i) direct water exposure alone, (ii) water exposure in the presence of a food source (non-spiked Artemia), and (iii) through a "spiked" food source (Vibrio-colonized Artemia) created by exposing Artemia cultures to GFP-Vibrio via the ambient water overnight. Following a 3 h feeding/exposure duration, the level of acquired GFP-Vibrio was quantified from anemone tissue homogenate. Ingestion of spiked Artemia resulted in a significantly greater burden of GFP-Vibrio equating to an 830-fold, 3,108-fold, and 435-fold increase in CFU mL-1 when compared to water exposed trials and a 207-fold, 62-fold, and 27-fold increase in CFU mL-1 compared to water exposed with food trials for V. alginolyticus, V. harveyi, and V. mediterranei, respectively. These data suggest that ingestion can facilitate delivery of an elevated dose of pathogenic bacteria in cnidarians and may describe an important portal of entry for pathogens in the absence of perturbing conditions. IMPORTANCE The front line of pathogen defense in corals is the mucus membrane. This membrane coats the surface body wall creating a semi-impermeable layer that inhibits pathogen entry from the ambient water both physically and biologically through mutualistic antagonism from resident mucus microbes. To date, much of the coral disease transmission research has been focused on mechanisms associated with perturbance of this membrane such as direct contact, vector lesions (predation/biting), and waterborne exposure through preexisting lesions. The present research describes a potential transmission pathway that evades the defenses provided by this membrane allowing unencumbered entry of bacteria as in association with food. This pathway may explain an important portal of entry for emergence of idiopathic infections in otherwise healthy corals and can be used to improve management practices for coral conservation.

A longitudinal study to examine the influence of farming practices and environmental factors on pathogen prevalence using structural equation modeling.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome's Simpson's Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

Use and Evaluation of a pES213-Derived Plasmid for the Constitutive Expression of gfp Protein in Pathogenic Vibrios: a Tagging Tool for In Vitro Studies.

Insertion of green fluorescent protein (GFP) into bacterial cells for constitutive expression is a powerful tool for the localization of species of interest within complex mixtures. Here, we demonstrate and evaluate the efficacy of the pES213-derived donor plasmid pVSV102 (gfp Knr) as a conjugative tool for the tagging of Vibrio and related species (termed vibrios). Using a triparental mating assay assisted by the helper plasmid pEVS104 (tra trb Knr), we successfully tagged 12 species within the Vibrionaceae family representing 8 of the proposed clades. All transconjugant strains demonstrated bright fluorescence and were readily differentiable within complex mixtures of nontagged cells. Plasmid retention was assessed using persistence and subculture experimentation. Persistence experiments evaluated plasmid loss over time for nonsubcultured samples inoculated into antibiotic-free media and sterile artificial seawater, whereas subculture trials evaluated plasmid loss following one to four subculture passages. Strong plasmid retention (≥80%) was observed in persistence experiments for all transconjugant strains for up to 48 h in both antibiotic-free media and artificial seawater with the exception of Vibrio cholerae, which showed a substantial decline in media after 24 h. Subculturing experiments also demonstrated strong plasmid stability, with all transconjugant strains showing ≥80% retention after four subculture passages. The results of this research suggest that pVSV102 is a stable GFP plasmid for the tagging of a broad range of vibrios. IMPORTANCE Prior research has suggested that the use of Aliivibrio fischeri-derived donor plasmids with the pES213 origin of replication may provide increased plasmid stability for the tagging of vibrios compared to Escherichia coli-derived p15A plasmids. Here, we present a structured protocol for conjugation-based tagging of vibrios using the pES213-derived plasmid pVSV102 and evaluate the plasmid stability of tagged strains. These methods and the resulting transconjugant strains provide important standardized tools to facilitate experimentation requiring the use of traceable vibrio strains. Furthermore, the determination of the species-specific plasmid stability provides an estimation of the anticipated level of plasmid loss under the given set of culture conditions. This estimation can be used to reduce the occurrence of experimental biases introduced by plasmid drift.

A Review of Asteroid Biology in the Context of Sea Star Wasting: Possible Causes and Consequences.

AbstractSea star wasting-marked in a variety of sea star species as varying degrees of skin lesions followed by disintegration-recently caused one of the largest marine die-offs ever recorded on the west coast of North America, killing billions of sea stars. Despite the important ramifications this mortality had for coastal benthic ecosystems, such as increased abundance of prey, little is known about the causes of the disease or the mechanisms of its progression. Although there have been studies indicating a range of causal mechanisms, including viruses and environmental effects, the broad spatial and depth range of affected populations leaves many questions remaining about either infectious or non-infectious mechanisms. Wasting appears to start with degradation of mutable connective tissue in the body wall, leading to disintegration of the epidermis. Here, we briefly review basic sea star biology in the context of sea star wasting and present our current knowledge and hypotheses related to the symptoms, the microbiome, the viruses, and the associated environmental stressors. We also highlight throughout the article knowledge gaps and the data needed to better understand sea star wasting mechanistically, its causes, and potential management.

Non-point source fecal contamination from aging wastewater infrastructure is a primary driver of antibiotic resistance in surface waters.

Antibiotic resistance is a global threat to human health. Many surface water resources are environmental hotspots of antibiotic resistant gene (ARG) transfer, with agricultural runoff and human waste highlighted as common sources of ARGs to aquatic systems. Here we quantified fecal marker genes and ARGs in 992 stream water samples collected seasonally during a 5-year period from 115 sites across the Upper Oconee watershed (Georgia, USA), an area characterized by gradients of agricultural and urban development. Widespread fecal contamination was found from humans (48% of samples), ruminants (55%), and poultry (19%), and 73% of samples tested positive for at least one of the six targeted ARGs (ermB, tet(B), blaCTX-M-1, blaKPC, blaSHV, and qnrS). While ARGs were strongly correlated with human fecal markers, many highly contaminated samples were not associated with sewage outfalls, an expected source of fecal and ARG pollution. To determine sources of contamination, we synthesized ARG and fecal marker data with geospatial data on land use/land cover and wastewater infrastructure across the watershed. This novel analysis found strong correlations between ARGs and measures of sewer density, sewer length, and septic system age within sample watersheds, indicating non-point sources of fecal contamination from aging wastewater infrastructure can be critical disseminators of anthropogenic ARGs in the environment.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Toward an Integrated System of Climate Change and Human Health Indicators: A Conceptual Framework.

Environmental health indicators are helpful for tracking and communicating complex health trends, informing science and policy decisions, and evaluating public health actions. When provided on a national scale, they can help inform the general public, policy makers, and public health professionals about important trends in exposures and how well public health systems are preventing those exposures from causing adverse health outcomes. There is a growing need to understand national trends in exposures and health outcomes associated with climate change and the effectiveness of climate adaptation strategies for health. To date, most indicators for health implications of climate change have been designed as independent, individual metrics. This approach fails to take into account how exposure-outcome pathways for climate-attributable health outcomes involve multiple, interconnected components. We propose reframing climate change and health indicators as a linked system of indicators, which can be described as follows: upstream climate drivers affect environmental states, which then determine human exposures, which ultimately lead to health outcomes; these climate-related risks are modified by population vulnerabilities and adaptation strategies. We apply this new conceptual framework to three illustrative climate-sensitive health outcomes and associated exposure-outcome pathways: pollen allergies and asthma, West Nile Virus infection, and vibriosis.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Free-Living Aquatic Turtles as Sentinels of Salmonella spp. for Water Bodies.

Reptile-associated human salmonellosis cases have increased recently in the United States. It is not uncommon to find healthy chelonians shedding Salmonella enterica. The rate and frequency of bacterial shedding are not fully understood, and most studies have focused on captive vs. free-living chelonians and often in relation to an outbreak. Their ecology and significance as sentinels are important to understanding Salmonella transmission. In 2012-2013, Salmonella prevalence was determined for free-living aquatic turtles in man-made ponds in Clarke and Oconee Counties, in northern Georgia (USA) and the correlation between species, basking ecology, demographics (age/sex), season, or landcover with prevalence was assessed. The genetic relatedness between turtle and archived, human isolates, as well as, other archived animal and water isolates reported from this study area was examined. Salmonella was isolated from 45 of 194 turtles (23.2%, range 14-100%) across six species. Prevalence was higher in juveniles (36%) than adults (20%), higher in females (33%) than males (18%), and higher in bottom-dwelling species (31%; common and loggerhead musk turtles, common snapping turtles) than basking species (15%; sliders, painted turtles). Salmonella prevalence decreased as forest cover, canopy cover, and distance from roads increased. Prevalence was also higher in low-density, residential areas that have 20-49% impervious surface. A total of 9 different serovars of two subspecies were isolated including 3 S. enterica subsp. arizonae and 44 S. enterica subsp. enterica (two turtles had two serotypes isolated from each). Among the S. enterica serovars, Montevideo (n = 13) and Rubislaw (n = 11) were predominant. Salmonella serovars Muenchen, Newport, Mississippi, Inverness, Brazil, and Paratyphi B. var L(+) tartrate positive (Java) were also isolated. Importantly, 85% of the turtle isolates matched pulsed-field gel electrophoresis patterns of human isolates, including those reported from Georgia. Collectively, these results suggest that turtles accumulate Salmonella present in water bodies, and they may be effective sentinels of environmental contamination. Ultimately, the Salmonella prevalence rates in wild aquatic turtles, especially those strains shared with humans, highlight a significant public health concern.

Site Fidelity is Associated with Food Provisioning and Salmonella in an Urban Wading Bird.

Food provisioning can change wildlife pathogen dynamics by altering host susceptibility via nutrition and/or through shifts in foraging behavior and space use. We used the American white ibis (Eudocimus albus), a wading bird increasingly observed in urban parks, as a model to study synergistic relationships between food provisioning and infection risk across an urban gradient in South Florida. We tested whether Salmonella prevalence was associated with changes in ibis diet (stable isotope analysis), space use (site fidelity via GPS tracking), and local density (flock size). We compared the relative importance of these mechanisms by ranking candidate models using logistic regression. We detected Salmonella in 27% of white ibises (n = 233) sampled at 15 sites. Ibises with diets higher in anthropogenic food exhibited higher site fidelity. Salmonella prevalence was higher at sites where ibises exhibited greater site fidelity and Salmonella was more prevalent in soil and water. Overlap in Salmonella serotypes between ibises and soil or water also was more likely at sites where ibises exhibited higher site fidelity. Our results suggest that repeated use of foraging areas may increase Salmonella exposure for birds if foraging areas are contaminated from animal feces, human waste, or other bacterial sources. Limiting wildlife feeding in parks-perhaps best achieved through understanding the motivations for feeding, education, and enforcement-may reduce health risks for wildlife and the public.

SARS-CoV-2 Wastewater Surveillance for Public Health Action.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has garnered extensive public attention during the coronavirus disease pandemic as a proposed complement to existing disease surveillance systems. Over the past year, methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage have advanced, and concentrations in wastewater have been shown to correlate with trends in reported cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, bridging the communication and knowledge gaps between researchers and public health responders is needed. We describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions, including establishing ethics consideration for monitoring. Although wastewater surveillance to assess community infections is not a new idea, the coronavirus disease pandemic might be the initiating event to make this emerging public health tool a sustainable nationwide surveillance system, provided that these barriers are addressed.

Reused poultry litter microbiome with competitive exclusion potential against Salmonella Heidelberg.

The success of poultry litter reuse in U.S. poultry production can be attributed to the efficient treatment methods used by producers during downtimes (the time lapse between consecutive flocks, during which the broiler house is empty). During this period, reused litter may be decaked, tilled/windrowed, or treated with acid-based amendments to reduce ammonia and bacteria levels. Competitive exclusion, pH, and temperature are proposed factors that influence the level of pathogens and the overall litter microbiome during downtimes. We previously reported on the bacterial genetic factors associated with the fitness of two strains of Salmonella enterica serovar Heidelberg (SH) incubated for 14 d in reused litter. Here, we investigated the physicochemical parameters and the microbiome of the litter correlating with SH abundance during this period. We used 16S ribosomal RNA gene sequencing to determine the litter microbiome and whole genome sequencing to characterize bacteria with competitive exclusion potential against SH. The ß diversity of the litter microbiome was significantly affected by the duration of incubation, microcosm, and microcosm plus Heidelberg strain combinations. In addition, ß diversity was significantly affected by litter parameters, including NH4 , pH, moisture, water activity, and aluminum. The major phyla observed in the reused litter throughout the 14-d incubation experiment were Firmicutes and Actinobacteria, although their abundance differed by microcosm and time. Amplicon-specific variants homologous to the members of the genera Nocardiopsis and Lentibacillus and the family Bacillaceae_2 were found to significantly correlate with the abundance of Salmonella. A consortium of Bacillus subtilis strains isolated from the litter microcosms reduced the growth of SH in vitro.