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.

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.

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.

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.

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.

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.

Saharan dust nutrients promote Vibrio bloom formation in marine surface waters.

Vibrio is a ubiquitous genus of marine bacteria, typically comprising a small fraction of the total microbial community in surface waters, but capable of becoming a dominant taxon in response to poorly characterized factors. Iron (Fe), often restricted by limited bioavailability and low external supply, is an essential micronutrient that can limit Vibrio growth. Vibrio species have robust metabolic capabilities and an array of Fe-acquisition mechanisms, and are able to respond rapidly to nutrient influx, yet Vibrio response to environmental pulses of Fe remains uncharacterized. Here we examined the population growth of Vibrio after natural and simulated pulses of atmospherically transported Saharan dust, an important and episodic source of Fe to tropical marine waters. As a model for opportunistic bacterial heterotrophs, we demonstrated that Vibrio proliferate in response to a broad range of dust-Fe additions at rapid timescales. Within 24 h of exposure, strains of Vibrio cholerae and Vibrio alginolyticus were able to directly use Saharan dust-Fe to support rapid growth. These findings were also confirmed with in situ field studies; arrival of Saharan dust in the Caribbean and subtropical Atlantic coincided with high levels of dissolved Fe, followed by up to a 30-fold increase of culturable Vibrio over background levels within 24 h. The relative abundance of Vibrio increased from ∼1 to ∼20% of the total microbial community. This study, to our knowledge, is the first to describe Vibrio response to Saharan dust nutrients, having implications at the intersection of marine ecology, Fe biogeochemistry, and both human and environmental health.

Abundance and Multilocus Sequence Analysis of Vibrio Bacteria Associated with Diseased Elkhorn Coral (Acropora palmata) of the Florida Keys.

The critically endangered elkhorn coral (Acropora palmata) is affected by white pox disease (WPX) throughout the Florida Reef Tract and wider Caribbean. The bacterium Serratia marcescens was previously identified as one etiologic agent of WPX but is no longer consistently detected in contemporary outbreaks. It is now believed that multiple etiologic agents cause WPX; however, to date, no other potential pathogens have been thoroughly investigated. This study examined the association of Vibrio bacteria with WPX occurrence from August 2012 to 2014 at Looe Key Reef in the Florida Keys, USA. The concentration of cultivable Vibrio was consistently greater in WPX samples than in healthy samples. The abundance of Vibrio bacteria relative to total bacteria was four times higher in samples from WPX lesions than in adjacent apparently healthy regions of diseased corals based on quantitative PCR (qPCR). Multilocus sequence analysis (MLSA) was used to assess the diversity of 69 Vibrio isolates collected from diseased and apparently healthy A. palmata colonies and the surrounding seawater. Vibrio species with known pathogenicity to corals were detected in both apparently healthy and diseased samples. While the causative agent(s) of contemporary WPX outbreaks remains elusive, our results suggest that Vibrio spp. may be part of a nonspecific heterotrophic bacterial bloom rather than acting as primary pathogens. This study highlights the need for highly resolved temporal sampling in situ to further elucidate the role of Vibrio during WPX onset and progression.IMPORTANCE Coral diseases are increasing worldwide and are now considered a major contributor to coral reef decline. In particular, the Caribbean has been noted as a coral disease hot spot, owing to the dramatic loss of framework-building acroporid corals due to tissue loss diseases. The pathogenesis of contemporary white pox disease (WPX) outbreaks in Acropora palmata remains poorly understood. This study investigates the association of Vibrio bacteria with WPX.

Biodegradation of Poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) Plastic under Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbial Diversity.

Poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (poly(3HB- co-3HHx)) thermoplastics are a promising biodegradable alternative to traditional plastics for many consumer applications. Biodegradation measured by gaseous carbon loss of several types of poly(3HB- co-3HHx) plastic was investigated under anaerobic conditions and aerobic seawater environments. Under anaerobic conditions, the biodegradation levels of a manufactured sheet of poly(3HB- co-3HHx) and cellulose powder were not significantly different from one another over 85 days with 77.1 ± 6.1 and 62.9 ± 19.7% of the carbon converted to gas, respectively. However, the sheet of poly(3HB- co-3HHx) had significantly higher methane yield ( p ≤ 0.05), 483.8 ± 35.2 mL·g-1 volatile solid (VS), compared to cellulose controls, 290.1 ± 92.7 mL·g-1 VS, which is attributed to a greater total carbon content. Under aerobic seawater conditions (148-195 days at room temperature), poly(3HB- co-3HHx) sheets were statistically similar to cellulose for biodegradation as gaseous carbon loss (up to 83% loss in about 6 months), although the degradation rate was lower than that for cellulose. The microbial diversity was investigated in both experiments to explore the dominant bacteria associated with biodegradation of poly(3HB- co-3HHx) plastic. For poly(3HB- co-3HHx) treatments, Cloacamonales and Thermotogales were enriched under anaerobic sludge conditions, while Clostridiales, Gemmatales, Phycisphaerales, and Chlamydiales were the most enriched under aerobic seawater conditions.

Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species.

Vibrio species are both ubiquitous and abundant in marine coastal waters, estuaries, ocean sediment, and aquaculture settings worldwide. We report here the isolation, characterization, and genome sequence of a novel Vibrio species, Vibrio antiquarius, isolated from a mesophilic bacterial community associated with hydrothermal vents located along the East Pacific Rise, near the southwest coast of Mexico. Genomic and phenotypic analysis revealed V. antiquarius is closely related to pathogenic Vibrio species, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio vulnificus, but sufficiently divergent to warrant a separate species status. The V. antiquarius genome encodes genes and operons with ecological functions relevant to the environment conditions of the deep sea and also harbors factors known to be involved in human disease caused by freshwater, coastal, and brackish water vibrios. The presence of virulence factors in this deep-sea Vibrio species suggests a far more fundamental role of these factors for their bacterial host. Comparative genomics revealed a variety of genomic events that may have provided an important driving force in V. antiquarius evolution, facilitating response to environmental conditions of the deep sea.

Systematic Analysis of White Pox Disease in Acropora palmata of the Florida Keys and Role of Serratia marcescens.

White pox disease (WPD) affects the threatened elkhorn coral, Acropora palmata. Owing in part to the lack of a rapid and simple diagnostic test, there have been few systematic assessments of the prevalence of acroporid serratiosis (caused specifically by Serratia marcescens) versus general WPD signs. Six reefs in the Florida Keys were surveyed between 2011 and 2013 to determine the disease status of A. palmata and the prevalence of S. marcescens. WPD was noted at four of the six reefs, with WPD lesions found on 8 to 40% of the colonies surveyed. S. marcescens was detected in 26.9% (7/26) of the WPD lesions and in mucus from apparently healthy colonies both during and outside of disease events (9%; 18/201). S. marcescens was detected with greater frequency in A. palmata than in the overlying water column, regardless of disease status (P = 0.0177). S. marcescens could not be cultured from A. palmata but was isolated from healthy colonies of other coral species and was identified as pathogenic pulsed-field gel electrophoresis type PDR60. WPD lesions were frequently observed on the reef, but unlike in prior outbreaks, no whole-colony death was observed. Pathogenic S. marcescens was circulating on the reef but did not appear to be the primary pathogen in these recent WPD episodes, suggesting that other pathogens or stressors may contribute to signs of WPD. Results highlight the critical importance of diagnostics in coral disease investigations, especially given that field manifestation of disease may be similar, regardless of the etiological agent.

Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance.

Over a 1-year period, bi-monthly estuarine surface water and plankton samples (63-200 and > 200 µm fractions) were assayed by polymerase chain reaction for the prevalence of total Vibrio parahaemolyticus, V. vulnificus and V. cholerae and select genes associated with clinical strains found in each species. Neither temperature nor plankton abundance was a significant correlate of total V. parahaemolyticus; however, the prevalence of genes commonly associated with clinical strains (trh, tdh, ORF8) increased with temperature and copepod abundance (P < 0.05). The prevalence of total V. vulnificus and the siderophore-related viuB gene also increased with temperature and copepod and decapod abundance (P < 0.001). Temperature and copepod abundance also covaried with the prevalence of V. cholerae (P < 0.05), but there was no significant relationship with ctxA or other genes commonly found in clinical strains. Results show that genes commonly associated with clinical Vibrio strains were more frequently detected in association with chitinous plankton. We conclude that V. parahaemolyticus, V. vulnificus, V. cholerae and subpopulations that harbour genes common to clinical strains respond distinctly to seasonal changes in temperature as well as shifts in the taxonomic composition of discrete plankton fractions.

Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

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%.

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.

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.

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.

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.

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.

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.