Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

Oral microbiome dysbiosis among cigarette smokers and smokeless tobacco users compared to non-users.

Tobacco use significantly influences the oral microbiome. However, less is known about how different tobacco products specifically impact the oral microbiome over time. To address this knowledge gap, we characterized the oral microbiome of cigarette users, smokeless tobacco users, and non-users over 4 months (four time points). Buccal swab and saliva samples (n = 611) were collected from 85 participants. DNA was extracted from all samples and sequencing was carried out on an Illumina MiSeq, targeting the V3-V4 region of the 16S rRNA gene. Cigarette and smokeless tobacco users had more diverse oral bacterial communities, including a higher relative abundance of Firmicutes and a lower relative abundance of Proteobacteria, when compared to non-users. Non-users had a higher relative abundance of Actinomyces, Granulicatella, Haemophilus, Neisseria, Oribacterium, Prevotella, Pseudomonas, Rothia, and Veillonella in buccal swab samples, compared to tobacco users. While the most abundant bacterial genera were relatively constant over time, some species demonstrated significant shifts in relative abundance between the first and last time points. In addition, some opportunistic pathogens were detected among tobacco users including Neisseria subflava, Bulleidia moorei and Porphyromonas endodontalis. Overall, our results provide a more holistic understanding of the structure of oral bacterial communities in tobacco users compared to non-users.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCE: Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

Increased incidence of vibriosis in Maryland, U.S.A., 2006-2019.

BACKGROUND: Vibrio spp. naturally occur in warm water with moderate salinity. Infections with non-cholera Vibrio (vibriosis) cause an estimated 80,000 illnesses and 100 fatalities each year in the United States. Climate associated changes to environmental parameters in aquatic ecosystems are largely promoting Vibrio growth, and increased incidence of vibriosis is being reported globally. However, vibriosis trends in the northeastern U.S. (e.g., Maryland) have not been evaluated since 2008. METHODS: Vibriosis case data for Maryland (2006-2019; n = 611) were obtained from the COVIS database. Incidence rates were calculated using U.S. Census Bureau population estimates for Maryland. A logistic regression model, including region, age group, race, gender, occupation, and exposure type, was used to estimate the likelihood of hospitalization. RESULTS: Comparing the 2006-2012 and 2013-2019 periods, there was a 39% (p = 0.01) increase in the average annual incidence rate (per 100,000 population) of vibriosis, with V. vulnificus infections seeing the greatest percentage increase (53%, p = 0.01), followed by V. parahaemolyticus (47%, p = 0.05). The number of hospitalizations increased by 58% (p = 0.01). Since 2010, there were more reported vibriosis cases with a hospital duration ≥10 days. Patients from the upper eastern shore region and those over the age of 65 were more likely (OR = 6.8 and 12.2) to be hospitalized compared to other patients. CONCLUSIONS: Long-term increases in Vibrio infections, notably V. vulnificus wound infections, are occurring in Maryland. This trend, along with increased rates in hospitalizations and average hospital durations, underscore the need to improve public awareness, water monitoring, post-harvest seafood interventions, and environmental forecasting ability.

The predisposition of smokers to COVID-19 infection: A mini-review of global perspectives.

Both SARS-CoV-2 and smoking tobacco adversely impact the respiratory system, damaging the airways and impairing lung function. While some studies have identified a positive association between smoking and increased susceptibility to COVID-19 infections, a few papers have concluded that smokers may be protected against such infections. Given these contradictory findings, there is an ongoing debate in the scientific community about whether or not smokers have a stronger predisposition towards COVID-19 infections. Through this mini-review, we aimed to study the relationship between tobacco smoking and COVID-19 infections by conducting a comprehensive literature search of peer reviewed articles that reported on the effects of smoking on COVID-19 susceptibility and were published globally over the past two years (January 2020-April 2022). Our search identified 31 articles that demonstrated a positive or strong relationship between smoking and COVID-19, while 13 articles had contrasting results. Additionally, we evaluated mechanistic studies suggesting that, among smokers, angiotensin-converting enzyme-2 genes are upregulated, facilitating easier binding of SARS-CoV-2, thereby increasing the risk of COVID-19 infection. In conclusion, the majority of studies in this area to date provide evidence of a strong relationship between smoking and COVID-19 infection; however, the strength of this association may vary across the smoking behaviors of differing populations. Future work could involve a meta-analysis of studies focusing on susceptibility to COVID-19 infection for different types of tobacco product smokers, which would result in a more comprehensive understanding of the predisposition of smokers towards COVID-19 infections.

Temporal Stability of Salmonella enterica and Listeria monocytogenes in Surface Waters Used for Irrigation in the Mid-Atlantic United States.

Enteric bacterial pathogen levels can influence the suitability of irrigation water sources for fruits and vegetables. We hypothesize that stable spatial patterns of Salmonella enterica and Listeria monocytogenes levels may exist across surface water sources in the Mid-Atlantic U.S. Water samples were collected at four streams and two pond sites in the mid-Atlantic U.S. over 2 years, biweekly during the fruit and vegetable growing seasons, and once a month during nongrowing seasons. Two stream sites and one pond site had significantly different mean concentrations in growing and nongrowing seasons. Stable spatial patterns were determined for relative differences between the site concentrations and average concentration of both pathogens across the study area. Mean relative differences were significantly different from zero at four of the six sites for S. enterica and three of six sites for L. monocytogenes. There was a similarity between the mean relative difference distribution between sites over growing season, nongrowing season, and the entire observation period. Mean relative differences were determined for temperature, oxidation-reduction potential, specific electrical conductance, pH, dissolved oxygen, turbidity, and cumulative rainfall. A moderate-to-strong Spearman correlation (rs > 0.657) was found between spatial patterns of S. enterica and 7-day rainfall, and between relative difference patterns of L. monocytogenes and temperature (rs = 0.885) and dissolved oxygen (rs = -0.885). Persistence in ranking sampling sites by the concentrations of the two pathogens was also observed. Finding spatially stable patterns in pathogen concentrations highlights spatiotemporal dynamics of these microorganisms across the study area can facilitate the design of an effective microbial water quality monitoring program for surface irrigation water.

Conventional tobacco products harbor unique and heterogenous microbiomes.

While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2'-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.

Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study.

Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.

Bacterial communities of hookah tobacco products are diverse and differ across brands and flavors.

Young adults are increasingly using non-cigarette products, such as hookahs, since they are perceived as healthier alternatives to cigarette smoking. However, hookah users are exposed to not only carcinogenic compounds but also microorganisms that may play an active role in the development of both infectious and chronic diseases among users. Nevertheless, existing hookah research in this area has focused only on microorganisms that may be transferred to users through the smoking apparatus and not on bacterial communities associated with hookah tobacco. To address this knowledge gap, we conducted time-series experiments on commercially available hookah brands (Al Fakher (flavors: two apple, mint, and watermelon) and Fumari (flavors: white gummy bear, ambrosia, and mint chocolate chill)) stored under three different temperature and relative humidity conditions over 14 days. To characterize bacterial communities, the total DNA was extracted on days 0, 5, 9, and 14, PCR-amplified for the V3V4 region of the bacterial 16S rRNA gene, sequenced on the Illumina HiSeq platform, and analyzed using R. Diversity (alpha and beta) analyses revealed that the microbiotas of Fumari and Al Fakher products differed significantly and that flavor had a significant effect on the hookah microbiota. Overall, Pseudomonas, Bacillus, Sphingomonas, and Methylobacterium were the predominant bacterial taxa across all products. Additionally, we observed compositional differences between hookah brands across the 14-day incubation. These data suggest that the bacterial communities of hookah tobacco are diverse and differ across brands and flavors, which may have critical implications regarding exposures to specific bacteria among hookah users. KEY POINTS: • Commercial hookah products harbor diverse bacterial communities. • Brands and flavors impact the diversity of these communities. • Research on their viability and transmission to users' respiratory tracts is needed.

Extended Spectrum ß-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum ß-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected ß-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and ß-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum ß-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of ß-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

The effect of a first flush rainwater harvesting and subsurface irrigation system on E. coli and pathogen concentrations in irrigation water, soil, and produce.

Climate change is stressing irrigation water sources, necessitating the evaluation of alternative waters such as harvested rainwater to determine if they meet water quality and food safety standards. We collected water, soil, and produce samples between June and August 2019 from two vegetable rain garden (VRG) sites in Frederick, Maryland that harvest rainwater using a first flush system, and deliver this water to produce through subsurface irrigation. The raised VRG beds include layers of gravel, sand, and soil that act as filters. We recorded the average surface soil moisture in each bed as well as antecedent precipitation. All water (n = 29), soil (n = 55), and produce (n = 57) samples were tested for generic E. coli using standard membrane filtration, and water samples were also tested for Salmonella spp. and Listeria monocytogenes by selective enrichment. No Salmonella spp. or L. monocytogenes isolates were detected in any water samples throughout the study period. Average E. coli levels from all harvested rainwater samples at both sites (1.2 and 24.4 CFU/100 mL) were well below the Good Agricultural Practices food safety guidelines. Only 7 % (3/44) of produce samples from beds irrigated with harvested rainwater were positive for E. coli. E. coli levels in soil samples were positively associated with average surface soil moisture (r2 = 0.13, p = 0.007). Additionally, E. coli presence in water samples was marginally associated with a shorter length of antecedent dry period (fewer days since preceding rainfall) (p = 0.058). Our results suggest that harvested rainwater collected through a first flush system and applied to produce through subsurface irrigation meets current food safety standards. Soil moisture monitoring could further reduce E. coli contamination risks from harvested rainwater-irrigated produce. First flush and subsurface irrigation systems could help mitigate climate change-related water challenges while protecting food safety and security.

Viable bacteria abundant in cigarettes are aerosolized in mainstream smoke.

Multiple studies have demonstrated that cigarettes harbor bacterial pathogens. Yet, to our knowledge, there are no published data to date on whether or not these microorganisms can be aerosolized and transmitted to the respiratory tract of users. To address this knowledge gap, we characterized cigarette bacterial communities and evaluated whether or not they could be aerosolized in mainstream smoke. Filtered and unfiltered cigarettes were tested. Non-smoked tobacco leaf, enriched non-smoked tobacco leaf extract and enriched mainstream smoke extract samples (n = 144) were incubated on trypticase soy agar, and resulting bacterial colonies were sequenced. Total DNA was also extracted, followed by PCR amplification of the 16S rRNA gene, sequencing and analysis using UCHIME, QIIME and R packages. The predominant bacterial genera cultured from the mainstream smoke of unfiltered cigarettes were Bacillus, Terribacillus, Paenibacillus and Desulfotomaculum. Culturable bacteria were not recovered from the smoke of filtered products. However, sequencing data demonstrated no significant differences in bacterial community diversity in the smoke of filtered versus unfiltered cigarettes, suggesting that other non-culturable bacteria may be aerosolized in mainstream smoke as well. Our study provides novel evidence that tobacco-associated bacterial communities are viable, can be aerosolized in mainstream smoke, and could potentially be transferred to the oral cavity and respiratory tract of smokers.

Effects of season and water type on the distribution and antimicrobial resistance of Enterococcus faecalis and Ent. faecium from surface and reclaimed water.

AIMS: To evaluate the safety of irrigation water sources based on phenotypic antimicrobial resistance (AMR) in Enterococcus spp., a potential environmental reservoir for AMR determinants. METHODS AND RESULTS: Eleven sites representing fresh and brackish water rivers, ponds and reclaimed water, were sampled over 2 years. Samples (n = 333) yielded 198 unique isolates of Ent. faecalis and Ent. faecium which were tested for antimicrobial susceptibility by microbroth dilution. Species distribution was influenced by water type and season. Enterococcus faecalis was more likely found in freshwater rivers and in summer, and Ent. faecium in reclaimed water and in spring. Only 11% of isolates were pansusceptible, while 48.5% and 26.3% were single (SDR) and multidrug resistant (MDR), respectively. MDR was more likely detected in Ent. faecium than Ent. faecalis. Winter isolates were more likely than summer isolates to exhibit MDR than SDR. CONCLUSIONS: Enterococcus faecalis and Ent. faecium in surface and reclaimed water exhibited diverse phenotypic AMR and a low-level resistance to clinically important antimicrobials such as ampicillin, vancomycin and linezolid. SIGNIFICANCE AND IMPACT OF THE STUDY: Single and multidrug resistance in E. faecalis and E. faecium varied by season but not water type. Antimicrobial resistance prevalence can assist decisions on the safety of irrigation water sources for fresh produce crops.

Impact of irrigation water type and sampling frequency on Microbial Water Quality Profiles required for compliance with U.S. Food Safety Modernization Act Produce Safety Rule standards.

The U.S. Food Safety Modernization Act (FSMA) Produce Safety Rule (PSR) requires that farmers generate a Microbial Water Quality Profile (MWQP) from 20 samples per agricultural water source, taken over 2-4 years and five annual samples thereafter. Farmers must use the MWQP to ascertain a geometric mean (GM) of ≤126 CFU/100 mL and statistical threshold value (STV) of ≤410 CFU/100 mL of generic Escherichia coli. Farmers are responsible for collecting samples and paying for testing, incurring a financial and time burden. To determine if testing frequency can be reduced without compromising accuracy, water samples (n = 279) were collected from twelve sites in the U.S. Mid-Atlantic region from 2016 to 2018 comprising tidal brackish river, non-tidal fresh river, pond, vegetable processing, and reclaimed water. The GM and STV were calculated for all sites and water types using all samples, and for multiple sub-samples of <20 from each site and water type. A Monte Carlo simulation was used to determine the proportion of sub-sample sizes that yielded the same determination as the entire sample size of PSR standard compliance. Four sites, two pond and two reclaimed water sites, complied with PSR GM and STV requirements when using the entire sample set. When a water source's calculated GM and STV using the entire sample set hovered close to the PSR thresholds, sub-sample sizes approached the recommended 20 samples to reach a congruent compliance determination. However, 99% agreement was obtained with a sub-sample of five when the absolute difference between the GM and STV from total samples and the PSR thresholds was ≥2.6 and 4.5 log CFU/100 mL E. coli, respectively. These findings suggest that under certain conditions the MWQP may be generated with well below 20 samples, reducing the economic burden on farmers while still maintaining a representative MWQP.

Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland.

Irrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland's Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM. IMPORTANCE In the last several decades, Maryland's Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

Climate change, extreme events, and increased risk of salmonellosis: foodborne diseases active surveillance network (FoodNet), 2004-2014.

BACKGROUND: Infections with nontyphoidal Salmonella cause an estimated 19,336 hospitalizations each year in the United States. Sources of infection can vary by state and include animal and plant-based foods, as well as environmental reservoirs. Several studies have recognized the importance of increased ambient temperature and precipitation in the spread and persistence of Salmonella in soil and food. However, the impact of extreme weather events on Salmonella infection rates among the most prevalent serovars, has not been fully evaluated across distinct U.S. regions. METHODS: To address this knowledge gap, we obtained Salmonella case data for S. Enteriditis, S. Typhimurium, S. Newport, and S. Javiana (2004-2014; n = 32,951) from the Foodborne Diseases Active Surveillance Network (FoodNet), and weather data from the National Climatic Data Center (1960-2014). Extreme heat and precipitation events for the study period (2004-2014) were identified using location and calendar day specific 95th percentile thresholds derived using a 30-year baseline (1960-1989). Negative binomial generalized estimating equations were used to evaluate the association between exposure to extreme events and salmonellosis rates. RESULTS: We observed that extreme heat exposure was associated with increased rates of infection with S. Newport in Maryland (Incidence Rate Ratio (IRR): 1.07, 95% Confidence Interval (CI): 1.01, 1.14), and Tennessee (IRR: 1.06, 95% CI: 1.04, 1.09), both FoodNet sites with high densities of animal feeding operations (e.g., broiler chickens and cattle). Extreme precipitation events were also associated with increased rates of S. Javiana infections, by 22% in Connecticut (IRR: 1.22, 95% CI: 1.10, 1.35) and by 5% in Georgia (IRR: 1.05, 95% CI: 1.01, 1.08), respectively. In addition, there was an 11% (IRR: 1.11, 95% CI: 1.04-1.18) increased rate of S. Newport infections in Maryland associated with extreme precipitation events. CONCLUSIONS: Overall, our study suggests a stronger association between extreme precipitation events, compared to extreme heat, and salmonellosis across multiple U.S. regions. In addition, the rates of infection with Salmonella serovars that persist in environmental or plant-based reservoirs, such as S. Javiana and S. Newport, appear to be of particular significance regarding increased heat and rainfall events.

Perceptions on the use of recycled water for produce irrigation and household tasks: A comparison between Israeli and Palestinian consumers.

Water scarcity has resulted in extensive wastewater recycling for agricultural irrigation in both Israel and the Palestinian Territories. However, minimal data have been collected regarding perceptions about wastewater recycling between the populations in these two areas. While geographically close and economically linked, these two populations differ in terms of governance, income, and access to technology for wastewater recycling. To address the data gap pertaining to perceptions of wastewater recycling, a survey was administered among a convenience sample of subjects (n = 236) recruited from Eilat, Israel and Bethlehem, West Bank, from May to November 2018. The survey included questions addressing knowledge of water sources, water scarcity, and recycled water; willingness to use recycled water for produce irrigation and household tasks; and demographics. Israeli willingness to use recycled water for various purposes ranged from 8.3% to 55.1%, and more than half of Israeli respondents were willing to serve both raw and cooked produce irrigated with recycled water. Willingness to use recycled water ranged from 28.9% to 41.7% among the Palestinian respondents, and Palestinian respondents were more willing to engage in high-contact uses (i.e. drinking and cooking) than Israeli respondents. Among the Israeli respondents, experience or familiarity with wastewater recycling and water contamination were frequently significantly associated with willingness to use recycled water. In contrast, among Palestinian respondents, personal water contamination experience, home water safety testing, and trust in authorities to monitor recycled wastewater reuse were frequently significantly associated with willingness to use recycled water. Given the likely increasing water stress in both Israel and the Palestinian Territories, as well as the continued evolution of wastewater treatment technologies and the substantial amount of agricultural trade ongoing between Israel and the Palestinian Territories, it is important to identify effective and appropriate outreach and communication strategies to enable successful and acceptable water recycling.

Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed.

Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2'-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

"Zooming" Our Way through Virtual Undergraduate Research Training: A Successful Redesign of the CONSERVE Summer Internship Program.

The COVID-19 pandemic has had an enormous impact on education globally, forcing the teaching community to think outside the box and create innovative educational plans to benefit students at home. Here, we narrate how the undergraduate, laboratory-based Summer Internship Program of our CONSERVE Center of Excellence, which focuses heavily on engaging women and underrepresented minorities in STEM programming, took a turn from an in-person research experience to a fully virtual one. We share our challenges and how we overcame them. Additionally, we provide a description of our virtual internship professional development curriculum, as well as the creative research projects that our seven interns were able to achieve in an 8-week virtual internship, including projects focused on the microbiological water quality of recycled irrigation water; social media promotion, enhancement and marketing of online educational resources focused on water, microbial contamination, and food crop irrigation; decision support systems for using recycled water in agricultural settings; and the effectiveness of zero-valent iron sand filtration in improving agricultural water quality, to name a few. Upon evaluating our internship program, we observed that more than 80% of our interns were either very satisfied or satisfied with the overall virtual internship experience. Through this experience, both the educators and the interns learned that although a virtual laboratory internship cannot completely replace in-person learning, it can still result in a very meaningful educational experience.

Nicotine concentration and mentholation affect bacterial community diversity in SPECTRUM research cigarettes.

Previous studies have characterized bacterial communities in menthol versus non-menthol cigarettes. However, these studies evaluated commercial cigarettes, for which levels of chemical constituents are largely unknown, and therefore, could not assess the impact of varying nicotine and menthol concentrations on tobacco bacterial communities. To address this knowledge gap, we performed time-series experiments using SPECTRUM research cigarettes with varying nicotine and menthol levels. Cigarettes were incubated under three storage conditions for 14 days. Cigarette tobacco was then sub-sampled (n = 288), DNA extracted, and subjected to PCR amplification of the V3V4 region of the 16S rRNA gene, followed by Illumina HiSeq sequencing. Sequences were analyzed using QIIME and R. Incubation under varying conditions did not affect bacterial diversity. However, significant differences in bacterial communities were observed across varying nicotine concentrations in menthol and non-menthol products. For example, Pseudomonas spp. was negatively correlated with nicotine concentrations in menthol cigarettes. A significantly higher relative abundance of P. veronii and P. viridiflava was observed in menthols versus non-menthols, while a significantly higher relative abundance of Bacillus foraminis and B. coagulans was found in non-menthols versus menthols. Additional bacteria (e.g., Staphylococcus spp., Jeotgalicoccus psychrophilus, and B. flexus) significantly changed in relative abundance between days 0 and 14. Our findings demonstrate that nicotine and menthol levels have a significant impact on the relative abundance of potential bacterial pathogens present in cigarettes. Future work is needed to demonstrate whether these tobacco-associated bacteria could be transferred to users while smoking, ultimately contributing to adverse respiratory impacts. KEY POINTS: • Varying nicotine levels changes bacterial composition of research cigarettes. • Mentholation affects the tobacco bacterial microbiome. • SPECTRUM research cigarettes are dominated by Pseudomonas and Bacillus.