Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing. - Related Documents




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322001.0000Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing. Understanding the complex microbiota of agricultural irrigation water is vital to multiple sectors of sustainable agriculture and public health. To date, microbiome characterization methods have provided comprehensive profiles of aquatic microbiotas, but have not described which taxa are likely metabolically-active. Here, we combined 5‑bromo‑2'-deoxyuridine (BrdU) labeling with 16S rRNA and shotgun sequencing to identify metabolically-active bacteria in reclaimed and agricultural pond water samples (n = 28) recovered from the Mid-Atlantic United States between March 2017 and January 2018. BrdU-treated samples were significantly less diverse (alpha diversity) compared to non-BrdU-treated samples. The most abundant taxa in the metabolically-active fraction of water samples (BrdU-treated samples) were unclassified Actinobacteria, Flavobacterium spp., Pseudomonas spp. and Aeromonas spp. Interestingly, we also observed that antimicrobial resistance and virulence gene profiles seemed to be more diverse and more abundant in non-BrdU-treated water samples compared to BrdU-treated samples. These findings raise the possibility that these genes may be associated more with relic (inactive) DNA present in the tested water types rather than viable, metabolically-active microorganisms. Our study demonstrates that the coupled use of BrdU labeling and sequencing can enhance understanding of the metabolically-active fraction of bacterial communities in alternative irrigation water sources. Agricultural pond and reclaimed waters are vital to the future of sustainable agriculture, and thus, the full understanding of the pathogenic potential of these waters is important to guide mitigation strategies that ensure appropriate water quality for intended purposes.202032726735
772510.9996Inter-facility characterization of bacteria in seafood processing plants: Exploring potential reservoirs of spoilage organisms and the resistome. A study was conducted in fish processing facilities to investigate the microbial composition, microbial metabolic potential, and distribution of antibiotic resistance genes. Whole metagenomic sequencing was used to analyze microbial communities from different processing rooms, operators and fish products. Taxonomic analyses identified the genera Pseudomonas and Psychrobacter as the most prevalent bacteria. A Principal Component Analysis revealed a distinct separation between fish product and environmental samples, as well as differences between fish product samples from companies processing either Gadidae or Salmonidae fish. Some particular bacterial genera and species were associated with specific processing rooms and operators. Metabolic analysis of metagenome assembled genomes demonstrated variations in microbiota metabolic profiles of microbiota across rooms and fish products. The study also examined the presence of antibiotic-resistance genes in fish processing environments, contributing to the understanding of microbial dynamics, metabolic potential, and implications for fish spoilage.202439071556
316420.9996Variations 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.202236194536
315530.9996In silico mapping of microbial communities and stress responses in a porcine slaughterhouse and pork products through its production chain, and the efficacy of HLE disinfectant. The use of shotgun metagenomic sequencing to understand ecological-level spread of microbes and their genes has provided new insights for the prevention, surveillance and control of microbial contaminants in the slaughterhouse environment. Here, microbial samples were collected from products and surrounding areas though a porcine slaughter process; shotgun metagenomic DNA-sequencing of these samples revealed a high community diversity within the porcine slaughterhouse and pork products, in zones originating from animal arrival through to the sale zones. Bacteria were more prevalent in the first zones, such as arrival- and anesthesia-zones, and DNA viruses were prevalent in the scorching-and-whip zone, animal products and sale zone. Data revealed the dominance of Firmicutes and Proteobacteria phyla followed by Actinobacteria, with a clear shift in the relative abundance of lactic acid bacteria (mainly Lactobacillus sp.) from early slaughtering steps to Proteobacteria and then to viruses suggesting site-specific community compositions occur in the slaughterhouse. Porcine-type-C oncovirus was the main virus found in slaughterhouse, which causes malignant diseases in animals and humans. As such, to guarantee food safety in a slaughterhouse, a better decipher of ecology and adaptation strategies of microbes becomes crucial. Analysis of functional genes further revealed high abundance of diverse genes associated with stress, especially in early zones (animal and environmental surfaces of arrival zone with 57,710 and 40,806 genes, respectively); SOS responsive genes represented the most prevalent, possibly associated with genomic changes responsible of biofilm formation, stringent response, heat shock, antimicrobial production and antibiotic response. The presence of several antibiotic resistance genes suggests horizontal gene transfer, thus increasing the likelihood for resistance selection in human pathogens. These findings are of great concern, with the suggestion to focus control measures and establish good disinfection strategies to avoid gene spread and microbial contaminants (bacteria and viruses) from the animal surface into the food chain and environment, which was achieved by applying HLE disinfectant after washing with detergent.202032846568
659640.9995Shotgun metagenomic sequencing of bulk tank milk filters reveals the role of Moraxellaceae and Enterobacteriaceae as carriers of antimicrobial resistance genes. In the present context of growing antimicrobial resistance (AMR) concern, understanding the distribution of AMR determinants in food matrices such as milk is crucial to protect consumers and maintain high food safety standards. Herein, the resistome of different dairy farms was investigated through a shotgun metagenomic sequencing approach, taking advantage of in-line milk filters as promising tools. The application of both the reads-based and the assembly-based approaches has allowed the identification of numerous AMR determinants, enabling a comprehensive resolution of the resistome. Notably most of the species harboring AMR genes were predicted to be Gram-negative genera, namely Enterobacter, Acinetobacter, Escherichia, and Pseudomonas, pointing out the role of these bacteria as reservoirs of AMR determinants. In this context, the use of de novo assembly has allowed a more holistic AMR detection strategy, while the reads-based approach has enabled the detection of AMR genes from low abundance bacteria, usually undetectable by assembly-based methods. The application of both reads-based and assembly-based approaches, despite being computationally demanding, has facilitated the comprehensive characterization of a food chain resistome, while also allowing the construction of complete metagenome assembled genomes and the investigation of mobile genetic elements. Our findings suggest that milk filters can successfully be used to investigate the resistome of bulk tank milk through the application of the shotgun metagenomic sequencing. In accordance with our results, raw milk can be considered a source of AMR bacteria and genes; this points out the importance of properly informing food business operators about the risk associated with poor hygiene practices in the dairy production environment and consumers of the potential microbial food safety risks derived from raw milk products consumption. Translating these findings as risk assessment outputs heralds the next generation of food safety controls.202235840264
322550.9995Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics. Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.202438637066
324360.9995Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach. The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using "universal" primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.201121167876
346170.9995Metagenomics insights into bacterial diversity and antibiotic resistome of the sewage in the city of Belém, Pará, Brazil. INTRODUCTION: The advancement of antimicrobial resistance is a significant public health issue today. With the spread of resistant bacterial strains in water resources, especially in urban sewage, metagenomic studies enable the investigation of the microbial composition and resistance genes present in these locations. This study characterized the bacterial community and antibiotic resistance genes in a sewage system that receives effluents from various sources through metagenomics. METHODS: One liter of surface water was collected at four points of a sewage channel, and after filtration, the total DNA was extracted and then sequenced on an NGS platform (Illumina® NextSeq). The sequenced data were trimmed, and the microbiome was predicted using the Kraken software, while the resistome was analyzed on the CARD webserver. All ecological and statistical analyses were performed using the. RStudio tool. RESULTS AND DISCUSSION: The complete metagenome results showed a community with high diversity at the beginning and more restricted diversity at the end of the sampling, with a predominance of the phyla Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria. Most species were considered pathogenic, with an emphasis on those belonging to the Enterobacteriaceae family. It was possible to identify bacterial groups of different threat levels to human health according to a report by the U.S. Centers for Disease Control and Prevention. The resistome analysis predominantly revealed genes that confer resistance to multiple drugs, followed by aminoglycosides and macrolides, with efflux pumps and drug inactivation being the most prevalent resistance mechanisms. This work was pioneering in characterizing resistance in a sanitary environment in the Amazon region and reinforces that sanitation measures for urban sewage are necessary to prevent the advancement of antibiotic resistance and the contamination of water resources, as evidenced by the process of eutrophication.202439629213
732580.9995Profiling the bacterial microbiome diversity and assessing the potential to detect antimicrobial resistance bacteria in wastewater in Kimberley, South Africa. Wastewater treatment plants (WWTPs) are hotspots for pathogens, and can facilitate horizontal gene transfer, potentially releasing harmful genetic material and antimicrobial resistance genes into the environment. Little information exists on the composition and behavior of microbes in WWTPs, especially in developing countries. This study used environmental DNA (eDNA) techniques to examine the microbiome load of wastewater from WWTPs. The DNA was isolated from wastewater samples collected from the treatment trains of three WWTPs in Kimberley, South Africa, and the microbial diversity and composition was compared through 16 S rRNA gene sequencing. The microbes detected were of the Kingdom Bacteria, and of these, 48.27% were successfully identified to genus level. The majority of reads from the combined bacterial data fall within the class Gammaproteobacteria, which is known to adversely impact ecological and human health. Arcobacteraceae constituted 19% of the bacterial reads, which is expected as this family is widespread in aquatic environments. Interestingly, the most abundant bacterial group was Bacteroides, which contain a variety of antibiotic-resistant members. Overall, various antibiotic-resistant taxa were detected in the wastewater, indicating a concerning level of antibiotic resistance within the bacterial community. Therefore, eDNA analysis can be a valuable tool in monitoring and assessing the bacterial microbiome in wastewater, thus providing important information for the optimization and improvement of wastewater treatment systems and mitigate public health risks.202439500921
327090.9995High-Throughput Shotgun Metagenomics of Microbial Footprints Uncovers a Cocktail of Noxious Antibiotic Resistance Genes in the Winam Gulf of Lake Victoria, Kenya. Background: A diverse range of pollutants, including heavy metals, agrochemicals, pharmaceutical residues, illicit drugs, personal care products, and other anthropogenic contaminants, pose a significant threat to aquatic ecosystems. The Winam Gulf of Lake Victoria, heavily impacted by surrounding human activities, faces potential contamination from these pollutants. However, studies exploring the presence of antibiotic resistance genes (ARGs) in the lake remain limited. In the current study, a shotgun metagenomics approach was employed to identify ARGs and related pathways. Genomic DNA was extracted from water and sediment samples and sequenced using the high-throughput Illumina NovaSeq platform. Additionally, phenotypic antibiotic resistance was assessed using the disk diffusion method with commonly used antibiotics. Results: The analysis of metagenomes sequences from the Gulf ecosystem and Comprehensive Antibiotic Resistance Database (CARD) revealed worrying levels of ARGs in the lake. The study reported nine ARGs from the 37 high-risk resistant gene families previously documented by the World Health Organization (WHO). Proteobacteria had the highest relative abundance of antibiotic resistance (53%), Bacteriodes (4%), Verrucomicrobia (2%), Planctomycetes Chloroflexi, Firmicutes (2%), and other unclassified bacteria (39%). Genes that target protection, replacement, change, and antibiotic-resistant efflux were listed in order of dominance. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed antibiotic resistance to beta-lactamase and vancomycin. Phenotypic resistance to vancomycin, tetracycline, sulfamethoxazole, erythromycin, trimethoprim, tetracycline, and penicillin was reported through the zone of inhibition. Conclusions: This study highlights that the Winam Gulf of Lake Victoria in Kenya harbors a diverse array of antibiotic-resistant genes, including those conferring multidrug resistance. These findings suggest that the Gulf could be serving as a reservoir for more antibiotic-resistant genes, posing potential risks to both human health and aquatic biodiversity. The insights gained from this research can guide policy development for managing antibiotic resistance in Kenya.202439741524
3875100.9995Ecological insights into the microbiology of food using metagenomics and its potential surveillance applications. A diverse array of micro-organisms can be found on food, including those that are pathogenic or resistant to antimicrobial drugs. Metagenomics involves extracting and sequencing the DNA of all micro-organisms on a sample, and here, we used a combination of culture and culture-independent approaches to investigate the microbial ecology of food to assess the potential application of metagenomics for the microbial surveillance of food. We cultured common foodborne pathogens and other organisms including Escherichia coli, Klebsiella/Raoultella spp., Salmonella spp. and Vibrio spp. from five different food commodities and compared their genomes to the microbial communities obtained by metagenomic sequencing following host (food) DNA depletion. The microbial populations of retail food were found to be predominated by psychrotrophic bacteria, driven by the cool temperatures in which the food products are stored. Pathogens accounted for a small percentage of the food metagenome compared to the psychrotrophic bacteria, and cultured pathogens were inconsistently identified in the metagenome data. The microbial composition of food varied amongst different commodities, and metagenomics was able to classify the taxonomic origin of 59% of antimicrobial resistance genes (ARGs) found on food to the genus level, but it was unclear what percentage of ARGs were associated with mobile genetic elements and thus transferable to other bacteria. Metagenomics may be used to survey the ARG burden, composition and carriage on foods to which consumers are exposed. However, food metagenomics, even after depleting host DNA, inconsistently identifies pathogens without enrichment or further bait capture.202539752189
6598110.9995Shallow shotgun sequencing of healthcare waste reveals plastic-eating bacteria with broad-spectrum antibiotic resistance genes. The burgeoning crises of antimicrobial resistance and plastic pollution are converging in healthcare settings, presenting a complex challenge to global health. This study investigates the microbial populations in healthcare waste to understand the extent of antimicrobial resistance and the potential for plastic degradation by bacteria. Our metagenomic analysis, using both amplicon and shallow shotgun sequencing, provided a comprehensive view of the taxonomic diversity and functional capacity of the microbial consortia. The viable bacteria in healthcare waste samples were analyzed employing full-length 16S rRNA sequencing, revealing a diverse bacterial community dominated by Firmicutes and Proteobacteria phyla. Notably, Proteus mirabilis VFC3/3 and Pseudomonas sp. VFA2/3 were detected, while Stenotrophomonas maltophilia VFV3/2 surfaced as the predominant species, holding implications for the spread of hospital-acquired infections and antimicrobial resistance. Antibiotic susceptibility testing identified multidrug-resistant strains conferring antimicrobial genes, including the broad-spectrum antibiotic carbapenem, underscoring the critical need for improved waste management and infection control measures. Remarkably, we found genes linked to the breakdown of plastic that encoded for enzymes of the esterase, depolymerase, and oxidoreductase classes. This suggests that specific bacteria found in medical waste may be able to reduce the amount of plastic pollution that comes from biological and medical waste. The information is helpful in formulating strategies to counter the combined problems of environmental pollution and antibiotic resistance. This study emphasises the importance of monitoring microbial communities in hospital waste in order to influence waste management procedures and public health policy. The findings highlight the need for a multidisciplinary approach to mitigate the risks associated with antimicrobial resistance and plastic waste, especially in hospital settings where they intersect most acutely.202539551377
6597120.9995Exploiting a targeted resistome sequencing approach in assessing antimicrobial resistance in retail foods. BACKGROUND: With the escalating risk of antimicrobial resistance (AMR), there are limited analytical options available that can comprehensively assess the burden of AMR carried by clinical/environmental samples. Food can be a potential source of AMR bacteria for humans, but its significance in driving the clinical spread of AMR remains unclear, largely due to the lack of holistic-yet-sensitive tools for surveillance and evaluation. Metagenomics is a culture-independent approach well suited for uncovering genetic determinants of defined microbial traits, such as AMR, present within unknown bacterial communities. Despite its popularity, the conventional approach of non-selectively sequencing a sample's metagenome (namely, shotgun-metagenomics) has several technical drawbacks that lead to uncertainty about its effectiveness for AMR assessment; for instance, the low discovery rate of resistance-associated genes due to their naturally small genomic footprint within the vast metagenome. Here, we describe the development of a targeted resistome sequencing method and demonstrate its application in the characterization of the AMR gene profile of bacteria associated with several retail foods. RESULT: A targeted-metagenomic sequencing workflow using a customized bait-capture system targeting over 4,000 referenced AMR genes and 263 plasmid replicon sequences was validated against both mock and sample-derived bacterial community preparations. Compared to shotgun-metagenomics, the targeted method consistently provided for improved recovery of resistance gene targets with a much-improved target detection efficiency (> 300-fold). Targeted resistome analyses conducted on 36 retail-acquired food samples (fresh sprouts, n = 10; ground meat, n = 26) and their corresponding bacterial enrichment cultures (n = 36) reveals in-depth features regarding the identity and diversity of AMR genes, most of which were otherwise undetected by the whole-metagenome shotgun sequencing method. Furthermore, our findings suggest that foodborne Gammaproteobacteria could be the major reservoir of food-associated AMR genetic determinants, and that the resistome structure of the selected high-risk food commodities are, to a large extent, dictated by microbiome composition. CONCLUSIONS: For metagenomic sequencing-based surveillance of AMR, the target-capture method presented herein represents a more sensitive and efficient approach to evaluate the resistome profile of complex food or environmental samples. This study also further implicates retail foods as carriers of diverse resistance-conferring genes indicating a potential impact on the dissemination of AMR.202336991496
3730130.9995Antibiotic-resistance and virulence-related genes in commercially bottled natural mineral waters. BACKGROUND: To date, the presence of antibiotics resistant genes (ARGs) and virulence-related genes (VRGs) has been evidenced in several surface waters, including natural surface water and wastewater, as well as drinking water. Bottled natural mineral waters, which are by law labelled as microbiologically pure at source, from underground aquifers, natural resurgence deposits or well suction pumps, do not undergo purification treatment, and do not experience any chemical decontamination or disinfection treatment, as in the case of drinking water from municipal conduits. The present study aimed to evaluate the presence of ARGs and VRGs, as well as the composition of microbial communities, in commercially bottled natural mineral drinking water by molecular methods. The study involved the analysis of bottled drinking water from four commercial brands. Moreover, an investigation was conducted into the potential association of known mobile elements or insertion sequences with the highlighted ARGs and VRGs. METHODS: Four commercial brands of drinking mineral bottled water were selected for analysis. A volume of 100 L from each brand was filtered to recover the microbes present in the water. The microbes successfully recovered on the filter, in conjunction with eventually other particles with a diameter of 0.22 μm or greater, or associated nucleic acids, underwent a process of DNA extraction using specific extraction kit. The extracted cell-DNA was subjected to shotgun sequencing. RESULTS: Sequence analysis revealed the presence of microbial communities associated with the water samples analyzed. Furthermore, several ARGs and VRGs were identified and, for some of them, a putative taxonomic assignment at genus level was defined. CONCLUSIONS: The results indicated that bottled drinking water may represent a potential reservoir of antibiotic resistance and virulence genes, which could persist and be transferred to other bacteria commonly found in the same water sample, as well as to microorganisms colonizing the human consumer. The use of the new molecular methods, such as next generation sequencing (NGS), could be useful for improving current methodologies for drinking water analysis, also considering their potential role of reservoir of antibiotic resistance and virulence genes, as well as the presence of potentially pathogenic microbes that cannot be detected by conventional cultural methods.202540993512
7728140.9995Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities. The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators' hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.202439095404
3223150.9995A cross-sectional comparison of gut metagenomes between dairy workers and community controls. BACKGROUND: As a nexus of routine antibiotic use and zoonotic pathogen presence, the livestock farming environment is a potential hotspot for the emergence of zoonotic diseases and antibiotic resistant bacteria. Livestock can further facilitate disease transmission by serving as intermediary hosts for pathogens before a spillover event. In light of this, we aimed to characterize the microbiomes and resistomes of dairy workers, whose exposure to the livestock farming environment places them at risk for facilitating community transmission of antibiotic resistant genes and emerging zoonotic diseases. RESULTS: Using shotgun sequencing, we investigated differences in the taxonomy, diversity and gene presence of 10 dairy farm workers and 6 community controls' gut metagenomes, contextualizing these samples with additional publicly available gut metagenomes. We found no significant differences in the prevalence of resistance genes, virulence factors, or taxonomic composition between the two groups. The lack of statistical significance may be attributed, in part, to the limited sample size of our study or the potential similarities in exposures between the dairy workers and community controls. We did, however, observe patterns warranting further investigation including greater abundance of tetracycline resistance genes and prevalence of cephamycin resistance genes as well as lower average gene diversity (even after accounting for differential sequencing depth) in dairy workers' metagenomes. We also found evidence of commensal organism association with tetracycline resistance genes in both groups (including Faecalibacterium prausnitzii, Ligilactobacillus animalis, and Simiaoa sunii). CONCLUSIONS: This study highlights the utility of shotgun metagenomics in examining the microbiomes and resistomes of livestock workers, focusing on a cohort of dairy workers in the United States. While our study revealed no statistically significant differences between groups in taxonomy, diversity and gene presence, we observed patterns in antibiotic resistance gene abundance and prevalence that align with findings from previous studies of livestock workers in China and Europe. Our results lay the groundwork for future research involving larger cohorts of dairy and non-dairy workers to better understand the impact of occupational exposure to livestock farming on the microbiomes and resistomes of workers.202439033279
3234160.9995Global profiling of antibiotic resistomes in maize rhizospheres. The spreading of antimicrobial resistance (AMR) in crops and food products represents a global concern. In this study, we conducted a survey of resistomes in maize rhizosphere from Michigan, California, the Netherlands, and South Africa, and investigated potential associations with host bacteria and soil management practices in the crop field. For comparison, relative abundance of antibiotic resistance genes (ARGs) is normalized to the size of individual metagenomes. Michigan maize rhizosphere metagenomes showed the highest abundance and diversity of ARGs, with the detection of blaTEM-116, blaACT-4/-6, and FosA2, exhibiting high similarity (≥ 99.0%) to those in animal and human pathogens. This was probably related to the decade-long application of manure/composted manure from antibiotic-treated animals. Moreover, RbpA, vanRO, mtrA, and dfrB were prevalently found across most studied regions, implying their intrinsic origins. Further analysis revealed that RbpA, vanRO, and mtrA are mainly harbored by native Actinobacteria with low mobility since mobile genetic elements were rarely found in their flanking regions. Notably, a group of dfrB genes are adjacent to the recombination binding sites (attC), which together constitute mobile gene cassettes, promoting the transmission from soil bacteria to human pathogens. These results suggest that maize rhizosphere resistomes can be distinctive and affected by many factors, particularly those relevant to agricultural practices.202336781495
4644170.9995Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes. Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.202235982656
7723180.9995Karst spring microbiome: Diversity, core taxa, and community response to pathogens and antibiotic resistance gene contamination. Karst aquifers are important water resources for drinking water supplies worldwide. Although they are susceptible to anthropogenic contamination due to their high permeability, there is a lack of detailed knowledge on the stable core microbiome and how contamination may affect these communities. In this study, eight karst springs (distributed across three different regions in Romania) were sampled seasonally for one year. The core microbiota was analysed by 16S rRNA gene amplicon sequencing. To identify bacteria carrying antibiotic resistance genes and mobile genetic elements, an innovative method was applied, consisting of high-throughput antibiotic resistance gene quantification performed on potential pathogen colonies cultivated on Compact Dry™ plates. A taxonomically stable bacterial community consisting of Pseudomonadota, Bacteroidota, and Actinomycetota was revealed. Core analysis reaffirmed these results and revealed primarily freshwater-dwelling, psychrophilic/psychrotolerant species affiliated to Rhodoferax, Flavobacterium, and Pseudomonas genera. Both sequencing and cultivation methods indicated that more than half of the springs were contaminated with faecal bacteria and pathogens. These samples contained high levels of sulfonamide, macrolide, lincosamide and streptogramins B, and trimethoprim resistance genes spread primarily by transposase and insertion sequences. Differential abundance analysis found Synergistota, Mycoplasmatota, and Chlamydiota as suitable candidates for pollution monitoring in karst springs. This is the first study highlighting the applicability of a combined approach based on high-throughput SmartChip™ antibiotic resistance gene quantification and Compact Dry™ pathogen cultivation for estimating microbial contaminants in karst springs and other challenging low biomass environments.202337364839
4643190.9995Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes. Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs (as measured through PERMANOVA, P < 0.001) and that the relative abundance of many individual AMR genes/variants increased over time (as measured with MaAsLin2, P(adj) < 0.05). Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.202336455460