Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes. - Related Documents




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334701.0000Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes. BACKGROUND: Antibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate. RESULTS: We evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and β-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1 × e(-5) and query coverage of ≥ 60% were employed in the Resfams database, the novel β-lactamases bla(HRV-1) and bla(HRVM-1) were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 β-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring bla(HRV-1) and bla(HRVM-1) and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended-spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages. CONCLUSION: Our results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Video abstract.202032482165
456010.9998High-resolution genomic surveillance elucidates a multilayered hierarchical transfer of resistance between WWTP- and human/animal-associated bacteria. BACKGROUND: Our interconnected world and the ability of bacteria to quickly swap antibiotic resistance genes (ARGs) make it particularly important to establish the epidemiological links of multidrug resistance (MDR) transfer between wastewater treatment plant (WWTP)- and human/animal-associated bacteria, under the One Health framework. However, evidence of ARGs exchange and potential factors that contribute to this transfer remain limited. RESULTS: Here, by combining culture-based population genomics and genetic comparisons with publicly available datasets, we reconstructed the complete genomes of 82 multidrug-resistant isolates from WWTPs and found that most WWTP-associated isolates were genetically distinct from their closest human/animal-associated relatives currently available in the public database. Even in the minority of lineages that were closely related, WWTP-associated isolates were characterized by quite different plasmid compositions. We identified a high diversity of circular plasmids (264 in total, of which 141 were potentially novel), which served as the main source of resistance, and showed potential horizontal transfer of ARG-bearing plasmids between WWTP- and humans/animal-associated bacteria. Notably, the potentially transferred ARGs and virulence factors (VFs) with different genetic backgrounds were closely associated with flanking insertion sequences (ISs), suggesting the importance of synergy between plasmids and ISs in mediating a multilayered hierarchical transfer of MDR and potentiating the emergence of MDR-hypervirulent clones. CONCLUSION: Our findings advance the current efforts to establish potential epidemiological links of MDR transmission between WWTP- and human/animal-associated bacteria. Plasmids play an important role in mediating the transfer of ARGs and the IS-associated ARGs that are carried by conjugative plasmids should be prioritized to tackle the spread of resistance. Video Abstract.202235078531
346020.9998Bioprospecting for β-lactam resistance genes using a metagenomics-guided strategy. Emergence of new antibiotic resistance bacteria poses a serious threat to human health, which is largely attributed to the evolution and spread of antibiotic resistance genes (ARGs). In this work, a metagenomics-guided strategy consisting of metagenomic analysis and function validation was proposed for rapidly identifying novel ARGs from hot spots of ARG dissemination, such as wastewater treatment plants (WWTPs) and animal feces. We used an antibiotic resistance gene database to annotate 76 putative β-lactam resistance genes from the metagenomes of sludge and chicken feces. Among these 76 candidate genes, 25 target genes that shared 40~70% amino acid identity to known β-lactamases were cloned by PCR from the metagenomes. Their resistances to four β-lactam antibiotics were further demonstrated. Furthermore, the validated ARGs were used as the reference sequences to identify novel ARGs in eight environmental samples, suggesting the necessity of re-examining the profiles of ARGs in environmental samples using the validated novel ARG sequences. This metagenomics-guided pipeline does not rely on the activity of ARGs during the initial screening process and may specifically select novel ARG sequences for function validation, which make it suitable for the high-throughput screening of novel ARGs from environmental metagenomes.201728584911
334530.9998Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening. A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum β-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important β-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel β-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.201931487611
345840.9997MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage. Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.201729163399
325350.9997Metagenome-assembled genomes indicate that antimicrobial resistance genes are highly prevalent among urban bacteria and multidrug and glycopeptide resistances are ubiquitous in most taxa. INTRODUCTION: Every year, millions of deaths are associated with the increased spread of antimicrobial resistance genes (ARGs) in bacteria. With the increasing urbanization of the global population, the spread of ARGs in urban bacteria has become a more severe threat to human health. METHODS: In this study, we used metagenome-assembled genomes (MAGs) recovered from 1,153 urban metagenomes in multiple urban locations to investigate the fate and occurrence of ARGs in urban bacteria. Additionally, we analyzed the occurrence of these ARGs on plasmids and estimated the virulence of the bacterial species. RESULTS: Our results showed that multidrug and glycopeptide ARGs are ubiquitous among urban bacteria. Additionally, we analyzed the deterministic effects of phylogeny on the spread of these ARGs and found ARG classes that have a non-random distribution within the phylogeny of our recovered MAGs. However, few ARGs were found on plasmids and most of the recovered MAGs contained few virulence factors. DISCUSSION: Our results suggest that the observed non-random spreads of ARGs are not due to the transfer of plasmids and that most of the bacteria observed in the study are unlikely to be virulent. Additional research is needed to evaluate whether the ubiquitous and widespread ARG classes will become entirely prevalent among urban bacteria and how they spread among phylogenetically distinct species.202336760505
965360.9997Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics. Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.201627767072
659770.9997Exploiting 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
346180.9997Metagenomics 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
991890.9997Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR. The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.202134282723
3450100.9997Global Distribution and Diversity of Prevalent Sewage Water Plasmidomes. Sewage water from around the world contains an abundance of short plasmids, several of which harbor antimicrobial resistance genes (ARGs). The global dynamics of plasmid-derived antimicrobial resistance and functions are only starting to be unveiled. Here, we utilized a previously created data set of 159,332 assumed small plasmids from 24 different global sewage samples. The detailed phylogeny, as well as the interplay between their protein domains, ARGs, and predicted bacterial host genera, were investigated to understand sewage plasmidome dynamics globally. A total of 58,429 circular elements carried genes encoding plasmid-related features, and MASH distance analyses showed a high degree of diversity. A single (yet diverse) cluster of 520 predicted Acinetobacter plasmids was predominant among the European sewage water. Our results suggested a prevalence of plasmid-backbone gene combinations over others. This could be related to selected bacterial genera that act as bacterial hosts. These combinations also mirrored the geographical locations of the sewage samples. Our functional domain network analysis identified three groups of plasmids. However, these backbone domains were not exclusive to any given group, and Acinetobacter was the dominant host genus among the theta-replicating plasmids, which contained a reservoir of the macrolide resistance gene pair msr(E) and mph(E). Macrolide resistance genes were the most common in the sewage plasmidomes and were found in the largest number of unique plasmids. While msr(E) and mph(E) were limited to Acinetobacter, erm(B) was disseminated among a range of Firmicutes plasmids, including Staphylococcus and Streptococcus, highlighting a potential reservoir of antibiotic resistance for these pathogens from around the globe. IMPORTANCE Antimicrobial resistance is a global threat to human health, as it inhibits our ability to treat infectious diseases. This study utilizes sewage water plasmidomes to identify plasmid-derived features and highlights antimicrobial resistance genes, particularly macrolide resistance genes, as abundant in sewage water plasmidomes in Firmicutes and Acinetobacter hosts. The emergence of macrolide resistance in these bacteria suggests that macrolide selective pressure exists in sewage water and that the resident bacteria can readily acquire macrolide resistance via small plasmids.202236069451
3909110.9997Biosolids as a Source of Antibiotic Resistance Plasmids for Commensal and Pathogenic Bacteria. Antibiotic resistance (AR) is a threat to modern medicine, and plasmids are driving the global spread of AR by horizontal gene transfer across microbiomes and environments. Determining the mobile resistome responsible for this spread of AR among environments is essential in our efforts to attenuate the current crisis. Biosolids are a wastewater treatment plant (WWTP) byproduct used globally as fertilizer in agriculture. Here, we investigated the mobile resistome of biosolids that are used as fertilizer. This was done by capturing resistance plasmids that can transfer to human pathogens and commensal bacteria. We used a higher-throughput version of the exogenous plasmid isolation approach by mixing several ESKAPE pathogens and a commensal Escherichia coli with biosolids and screening for newly acquired resistance to about 10 antibiotics in these strains. Six unique resistance plasmids transferred to Salmonella typhimurium, Klebsiella aerogenes, and E. coli. All the plasmids were self-transferable and carried 3-6 antibiotic resistance genes (ARG) conferring resistance to 2-4 antibiotic classes. These plasmids-borne resistance genes were further embedded in genetic elements promoting intracellular recombination (i.e., transposons or class 1 integrons). The plasmids belonged to the broad-host-range plasmid (BHR) groups IncP-1 or PromA. Several of them were persistent in their new hosts when grown in the absence of antibiotics, suggesting that the newly acquired drug resistance traits would be sustained over time. This study highlights the role of BHRs in the spread of ARG between environmental bacteria and human pathogens and commensals, where they may persist. The work further emphasizes biosolids as potential vehicles of highly mobile plasmid-borne antibiotic resistance genes.202133967971
3469120.9997Antibiotic resistance genes of emerging concern in municipal and hospital wastewater from a major Swedish city. The spread of antibiotic resistance among bacterial pathogens is to a large extent mediated by mobile antibiotic resistance genes (ARGs). The prevalence and geographic distribution of several newly discovered ARGs, as well as some clinically important ARGs conferring resistance to last resort antibiotics, are largely unknown. Targeted analysis of wastewater samples could allow estimations of carriage in the population connected to the sewers as well as release to the environment. Here we quantified ARGs conferring resistance to linezolid (optrA and cfr(A)) and colistin (mcr-1, -2, -3, -4 and -5) and the recently discovered gar (aminoglycoside ARG) and sul4 (sulphonamide ARG) in raw hospital and municipal wastewater as well as treated municipal wastewater during five years in a low antibiotic resistance prevalence setting (Gothenburg, Sweden). Additionally, variations in bacterial composition of the wastewaters characterized by 16S rRNA sequencing were related to the variations of the ARGs in an attempt to reveal if the presence of known or suspected bacterial host taxa could explain the presence of the ARGs in wastewater. The mcr-1, mcr-3, mcr-4, mcr-5, sul4 and gar genes were detected regularly in all types of wastewater samples while optrA and cfr(A) were detected only in hospital wastewater. The most abundant genes were mcr-3 and mcr-5, especially in municipal wastewater. The detection of optrA was restricted to a peak during one year. Most of the ARGs correlated with taxa previously described as bacterial hosts and associated with humans. Although some of the tentative hosts may include bacteria also thriving in wastewater environments, detection of the ARGs in the wastewaters could reflect their presence in the gut flora of the contributing populations. If so, they could already today or in the near future hinder treatment of bacterial infections in a setting where they currently are rarely targeted/detected during clinical surveillance.202234748849
4666130.9997Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes. Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.201930808697
4550140.9997Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals, humans and environment in livestock farming. Anthropogenic environments such as those created by intensive farming of livestock, have been proposed to provide ideal selection pressure for the emergence of antimicrobial-resistant Escherichia coli bacteria and antimicrobial resistance genes (ARGs) and spread to humans. Here, we performed a longitudinal study in a large-scale commercial poultry farm in China, collecting E. coli isolates from both farm and slaughterhouse; targeting animals, carcasses, workers and their households and environment. By using whole-genome phylogenetic analysis and network analysis based on single nucleotide polymorphisms (SNPs), we found highly interrelated non-pathogenic and pathogenic E. coli strains with phylogenetic intermixing, and a high prevalence of shared multidrug resistance profiles amongst livestock, human and environment. Through an original data processing pipeline which combines omics, machine learning, gene sharing network and mobile genetic elements analysis, we investigated the resistance to 26 different antimicrobials and identified 361 genes associated to antimicrobial resistance (AMR) phenotypes; 58 of these were known AMR-associated genes and 35 were associated to multidrug resistance. We uncovered an extensive network of genes, correlated to AMR phenotypes, shared among livestock, humans, farm and slaughterhouse environments. We also found several human, livestock and environmental isolates sharing closely related mobile genetic elements carrying ARGs across host species and environments. In a scenario where no consensus exists on how antibiotic use in the livestock may affect antibiotic resistance in the human population, our findings provide novel insights into the broader epidemiology of antimicrobial resistance in livestock farming. Moreover, our original data analysis method has the potential to uncover AMR transmission pathways when applied to the study of other pathogens active in other anthropogenic environments characterised by complex interconnections between host species.202235333870
3910150.9997Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages. Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.202438935220
4987160.9997The Human Health Implications of Antibiotic Resistance in Environmental Isolates from Two Nebraska Watersheds. One Health field-based approaches are needed to connect the occurrence of antibiotics present in the environment with the presence of antibiotic resistance genes (ARGs) in Gram-negative bacteria that confer resistance to antibiotics important in for both veterinary and human health. Water samples from two Nebraska watersheds influenced by wastewater effluent and agricultural runoff were tested for the presence of antibiotics used in veterinary and human medicine. The water samples were also cultured to identify the bacteria present. Of those bacteria isolated, the Gram-negative rods capable of causing human infections had antimicrobial susceptibility testing and whole-genome sequencing (WGS) performed to identify ARGs present. Of the 211 bacterial isolates identified, 37 belonged to pathogenic genera known to cause human infections. Genes conferring resistance to beta-lactams, aminoglycosides, fosfomycins, and quinolones were the most frequently detected ARGs associated with horizontal gene transfer (HGT) in the watersheds. WGS also suggest recent HGT events involving ARGs transferred between watershed isolates and bacteria of human and animal origins. The results of this study demonstrate the linkage of antibiotics and bacterial ARGs present in the environment with potential human and/or veterinary health impacts. IMPORTANCE One health is a transdisciplinary approach to achieve optimal health for humans, animals, plants and their shared environment, recognizing the interconnected nature of health in these domains. Field based research is needed to connect the occurrence of antibiotics used in veterinary medicine and human health with the presence of antibiotic resistance genes (ARGs). In this study, the presence of antibiotics, bacteria and ARGs was determined in two watersheds in Nebraska, one with agricultural inputs and the other with both agricultural and wastewater inputs. The results presented in this study provide evidence of transfer of highly mobile ARG between environment, clinical, and animal-associated bacteria.202235311538
3459170.9997Diversity of antibiotic resistance gene variants at subsequent stages of the wastewater treatment process revealed by a metagenomic analysis of PCR amplicons. Wastewater treatment plants have been recognised as point sources of various antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) which are considered recently emerging biological contaminants. So far, culture-based and molecular-based methods have been successfully applied to monitor antimicrobial resistance (AMR) in WWTPs. However, the methods applied do not permit the comprehensive identification of the true diversity of ARGs. In this study we applied next-generation sequencing for a metagenomic analysis of PCR amplicons of ARGs from the subsequent stages of the analysed WWTP. The presence of 14 genes conferring resistance to different antibiotic families was screened by PCR. In the next step, three genes were selected for detailed analysis of changes of the profile of ARG variants along the process. A relative abundance of 79 variants was analysed. The highest diversity was revealed in the ermF gene, with 52 variants. The relative abundance of some variants changed along the purification process, and some ARG variants might be present in novel hosts for which they were currently unassigned. Additionally, we identified a pool of novel ARG variants present in the studied WWTP. Overall, the results obtained indicated that the applied method is sufficient for analysing ARG variant diversity.202338274111
3456180.9997Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil. Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (bla(TEM), bla(CTX-M-1) group, bla(CTX-M-9) group, bla(OXA-48), bla(VIM), mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered.201829567433
3477190.9997Whole-genome sequencing characterization of silver-resistant bacteria from the outfall of wastewater treatment plants and effluent-receiving rivers. The excessive use of silver compounds has led to the environmental dissemination of silver resistance genes. However, little is known about the epidemiology of silver-resistant bacteria in the environment. Wastewater treatment plants (WWTPs) link the clinical settings with the natural environment and serve as a major pathway for silver entering the natural environment. However, their role in the dissemination of silver resistance genes remains unclear. This study investigated the characteristics of silver-resistant bacteria in the vicinity of four WWTPs to assess their environmental impact. Water and sediment samples were collected from the WWTP outfalls and downstream rivers. Among 22 silver-resistant strains obtained through plate screening, the majority were Klebsiella spp., followed by Escherichia spp. and Kluyvera spp. Notably, ST23 and ST2464 were the predominant sequence types (multilocus sequence typing) identified among the Klebsiella pneumoniae isolates. Antimicrobial susceptibility testing and whole-genome sequencing were performed to identify environmental heavy metal and antibiotic-resistant genes. Whole-genome sequencing revealed the presence of the sil and pco operons, which together formed the copper homeostasis and silver resistance island. The silver resistance gene sequences varied. Various heavy metal resistance genes, including mer and ars, were detected in the strains, as were a diverse array of plasmid types, including IncFIB(K) and repB(R1701), and fosA and β-lactamase encoding genes. Taken together, the findings underscore the coexistence of silver resistance genes with multiple heavy metal resistance genes in wastewater bacteria, highlighting the environmental implications of silver usage. Efforts should be directed toward restricting silver usage, improving WWTP purification methods to safeguard human and environmental health. IMPORTANCE: The misuse of silver compounds has led to an increasing presence of silver-resistant microorganisms in the environment, which cannot be completely eliminated in wastewater treatment plants, allowing them to enter the environment and pose risks to environmental safety and human health. However, research on the epidemiology of silver-resistant bacteria in wastewater and their whole-genome sequencing remains limited. Our findings explain that silver-resistant bacteria from the environment often possess resistance to other heavy metals, share genetic similarities, and possess the potential for widespread transmission. Furthermore, these bacteria may enter clinical settings through environmental pathways, posing a risk to human health.202540762474