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Bacteria are ubiquitous in the environment and critical to human health and disease, yet only a small fraction can be identified through standard culture methods. Advances in next-generation sequencing techniques have improved bacterial identification, but these DNA-based methods cannot distinguish live bacteria from relic DNA. Recently, DNA-labeling dyes (e.g., 5-bromo-2′-deoxyuridine [BrdU] and propidium monoazide [PMA]) have been used to detect metabolically active bacteria in different sample types. Here, we compare BrdU and PMA in combination with 16SrRNA gene sequencing to characterize metabolically active bacteria in two different sample types: (1) manufactured products (n = 78; cigarettes, hookah, and little cigar) and (2) natural samples (n = 186; rainwater, soil, and produce). Metabolically active bacterial communities identified in BrdU-labeled samples had lower alpha diversity than that of PMA-treated and non-treated samples. Pseudomonas, Sphingomonas, Enterobacter, and Acinetobacter were observed in all the samples tested. Irrespective of sample type, Pseudomonas was predominant in BrdU-treated samples, while Acinetobacter was more abundant in non-treated samples compared to PMA-treated samples. We also observed that PMA-treated samples tend to overestimate the metabolically active bacterial fraction compared to BrdU-treated samples. Overall, our study highlights how different labeling techniques influence bacterial community analysis findings, underscoring the need for careful selection of labeling approaches when assessing environmental samples. 

ABSTRACT Antibiotics are often used to treat severeVibrioinfections, 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) andVibrio 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%) ofV. parahaemolyticusisolates 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 resistantV. vulnificusisolates 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 amongV. parahaemolyticusandV. vulnificusrecovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibriospp. 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, someVibrioisolates 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 withVibrio vulnificusinfections, 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 severeVibriospp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion. 

Abstract 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 ofFirmicutesand a lower relative abundance ofProteobacteria, when compared to non-users. Non-users had a higher relative abundance ofActinomyces, Granulicatella, Haemophilus, Neisseria, Oribacterium, Prevotella, Pseudomonas, Rothia, andVeillonellain 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 includingNeisseria subflava, Bulleidia mooreiandPorphyromonas endodontalis. Overall, our results provide a more holistic understanding of the structure of oral bacterial communities in tobacco users compared to non-users. 

Abstract 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 . Graphical abstract 

Abstract Climate change presents huge challenges to the already-complex decisions faced by U.S. agricultural producers, as seasonal weather patterns increasingly deviate from historical tendencies. Under USDA funding, a transdisciplinary team of researchers, extension experts, educators, and stakeholders is developing a climate decision support Dashboard for Agricultural Water use and Nutrient management (DAWN) to provide Corn Belt farmers with better predictive information. DAWN’s goal is to provide credible, usable information to support decisions by creating infrastructure to make subseasonal-to-seasonal forecasts accessible. DAWN uses an integrated approach to 1) engage stakeholders to coproduce a decision support and information delivery system; 2) build a coupled modeling system to represent and transfer holistic systems knowledge into effective tools; 3) produce reliable forecasts to help stakeholders optimize crop productivity and environmental quality; and 4) integrate research and extension into experiential, transdisciplinary education. This article presents DAWN’s framework for integrating climate–agriculture research, extension, and education to bridge science and service. We also present key challenges to the creation and delivery of decision support, specifically in infrastructure development, coproduction and trust building with stakeholders, product design, effective communication, and moving tools toward use. 

Abstract Advanced treated municipal wastewater is an important alternative water source for agricultural irrigation. However, the possible persistence of chemical and microbiological contaminants in these waters raise potential safety concerns with regard to reusing treated wastewater for food crop irrigation. Two low-cost and environmentally-friendly filter media, biochar (BC) and zero-valent iron (ZVI), have attracted great interest in terms of treating reused water. Here, we evaluated the efficacy of BC-, nanosilver-amended biochar- (Ag-BC) and ZVI-sand filters, in reducing contaminants of emerging concern (CECs),Escherichia coli (E. coli)and total bacterial diversity from wastewater effluent. Six experiments were conducted with control quartz sand and sand columns containing BC, Ag-BC, ZVI, BC with ZVI, or Ag-BC with ZVI. After filtration, Ag-BC, ZVI, BC with ZVI and Ag-BC with ZVI demonstrated more than 90% (> 1 log) removal ofE. colifrom wastewater samples, while BC, Ag-BC, BC with ZVI and Ag-BC with ZVI also demonstrated efficient removal of tested CECs. Lower bacterial diversity was also observed after filtration; however, differences were marginally significant. In addition, significantly (p < 0.05) higher bacterial diversity was observed in wastewater samples collected during warmer versus colder months. Leaching of silver ions occurred from Ag-BC columns; however, this was prevented through the addition of ZVI. In conclusion, our data suggest that the BC with ZVI and Ag-BC with ZVI sand filters, which demonstrated more than 99% removal of both CECs andE. coliwithout silver ion release, may be effective, low-cost options for decentralized treatment of reused wastewater. Graphical Abstract 

Abstract Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, includingVibriospp., 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-activeVibriospp.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-activeVibriospp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence ofV. cholerae,V. vulnificus, andV. parahaemolyticususing both methods, whileV. aesturianusandV. shiloniiwere detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach includingP. shigelloides,B. cereusandE. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted inVibriospp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-activeVibriospp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.