Distribution of tetracyclines and sulfonamides resistance genes around a smallholder pig farm: Modeling and attribution.

Livestock farm is a major source of antibiotics and antibiotic resistance genes (ARGs) pollution. ARGs can directly enter the environment through runoff and air deposition. The impact extent and the driving factors require further investigation to inform effective policies and actions to mitigate their spread. This study investigated a smallholder pig farm and its surrounding areas to understand the spread of ARGs. Topsoil samples were collected from 56 different sites within one kilometer of the farm, and a comprehensive analysis was conducted to reveal effects of soil properties, antibiotic residues, microbiome, mobilome on the variation of typical ARGs. The results confirmed that the ARGs reduced exponentially with increasing distance from the farm, with a goodness of fit (R2) of 0.7 for total ARGs. For tetracyclines (TC) and sulfonamides (SA) resistance genes, the fitting R2 exceeded 0.9. Model estimates allowed for quantitative comparisons of in-farm increments, out-farm background levels, and spread abilities of ARGs with distinct resistance mechanisms. SA-specific resistance genes (SRGs, 0.097 copies/16S rRNA gene) and TC-specific resistance genes (TRGs, 0.036 copies/16S rRNA gene) showed higher within-farm increases compared to multidrug resistance genes (MDRGs, 0.020 copies/16S rRNA gene). MDRGs, however, had a higher background level and a greater impact distance (0.18 km, 4.4 times the farm radius). Additionally spread abilities of TRGs varied by resistance mechanism, with ribosome protection proteins showing greater spread than TC inactivating enzymes and TC efflux pumps, likely due to different fitness costs. Correlation analysis and structural equation modeling indicated that changes in bacterial community composition and mobilome are primary factors influencing ARGs variation during their spread. Abiotic factors like soil nutrients and antibiotics also selectively enriched ARGs within the farm. These findings provide insights into the ARGs dissemination and could inform strategies to prevent their spread from smallholder livestock farms.

Protist predation promotes antimicrobial resistance spread through antagonistic microbiome interactions.

None declared.Conflicts of interestAntibiotic resistance has grown into a major public health threat. In this study, we reveal predation by protists as an overlooked driver of antibiotic resistance dissemination in the soil microbiome. While previous studies have primarily focused on the distribution of antibiotic resistance genes, our work sheds light on the pivotal role of soil protists in shaping antibiotic resistance dynamics. Using a combination of metagenomics and controlled experiments in this study, we demonstrate that protists cause an increase in antibiotic resistance. We mechanistically link this increase to a fostering of antimicrobial activity in the microbiome. Protist predation gives a competitive edge to bacteria capable of producing antagonistic secondary metabolites, which secondary metabolites promote in turn antibiotic-resistant bacteria. This study provides insights into the complex interplay between protists and soil microbiomes in regulating antibiotic resistance dynamics. This study highlights the importance of top-down control on the spread of antibiotic resistance and directly connects it to cross-kingdom interactions within the microbiome. Managing protist communities may become an important tool to control outbreaks of antibiotic resistance in the environment.

Genome-centric metagenomes unveiling the hidden resistome in an anchialine cave.

BACKGROUND: Antibiotic resistance is a critical global concern, posing significant challenges to human health and medical treatments. Studying antibiotic resistance genes (ARGs) is essential not only in clinical settings but also in diverse environmental contexts. However, ARGs in unique environments such as anchialine caves, which connect both fresh and marine water, remain largely unexplored despite their intriguing ecological characteristics. RESULTS: We present the first study that comprehensively explores the occurrence and distribution of ARGs and mobile genetic elements (MGEs) within an anchialine cave. Utilizing metagenomic sequencing we uncovered a wide array of ARGs with the bacitracin resistance gene, bacA and multidrug resistance genes, being the most dominant. The cave's microbial community and associated resistome were significantly influenced by the salinity gradient. The discovery of novel ß-lactamase variants revealed the cave's potential as a reservoir for previously undetected resistance genes. ARGs in the cave demonstrated horizontal transfer potential via plasmids, unveiling ecological implications. CONCLUSIONS: These findings highlight the need for further exploration of the resistome in unique environments like anchialine caves. The interconnected dynamics of ARGs and MGEs within anchialine caves offer valuable insights into potential reservoirs and mechanisms of antibiotic resistance in natural ecosystems. This study not only advances our fundamental understanding but also highlights the need for a comprehensive approach to address antibiotic resistance in diverse ecological settings.

Biofilm enhanced the mitigations of antibiotics and resistome in sulfadiazine and trimethoprim co-contaminated soils.

Reducing antibiotic levels in soil ecosystems is vital to curb the dissemination of antimicrobial resistance genes (ARGs) and mitigate global health threats. However, gaps persist in understanding how antibiotic resistome can be suppressed during antibiotic degradation. Herein, we investigate the efficacy of a biochar biofilm incorporating antibiotics-degrading bacterial strain (Arthrobacter sp. D2) to mitigate antibiotic resistome in non-manured and manure-amended soils with sulfadiazine (SDZ) and trimethoprim (TMP) contamination. Results show that biofilm enhanced SDZ degradation by 83.0% within three days and increased TMP attenuation by 55.4% over 60 days in non-manured soils. In the non-manured black soil, the relative abundance of ARGs increased initially after biofilm inoculation. However, by day 30, it decreased by 20.5% compared to the controls. Moreover, after 7 days, biofilm reduced TMP by 38.5% in manured soils and decreased the total ARG abundance by 19.0%. Thus, while SDZ degradation did not increase sulfonamide resistance genes, TMP dissipation led to a proliferation of insertion sequences and related TMP resistance genes. This study underscores the importance of antibiotic degradation in reducing related ARGs while cautioning against the potential proliferation and various ARGs transfer by resistant microorganisms.

Emergence of multidrug-resistant Providencia rettgeri clone in food-producing animals: A public health threat.

The occurrence of carbapenemases encoding genes in Providencia rettgeri is a critical public health concern since this species has intrinsic resistance to several antimicrobials, including polymyxins. The identification of this multidrug-resistant (MDR) pathogen outside the hospital setting has become increasingly frequent, and raises an alert for the global health agencies, as they indicate a possible spread of such pathogens. Herein, we described three MDR P. rettgeri isolates carrying a diversity of antimicrobial resistance genes (ARGs) isolated from stool samples of swine and bovine in Brazil. Molecular analysis revealed that all isolates belonged to the same clone. The whole genome sequencing (WGS) of a representative isolate (PVR-188) was performed by MiSeq Illumina® platform, while the assembling and annotation was achieved using SPAdes and Prooka, respectively. The WGS analyses indicated the presence of ARGs that confer resistance to ß-lactams (bla NDM-1, bla CTX-M-2), quinolones (qnrD1), aminoglycosides (aadA2, aadA1, aph(3')-Via), phenicol (catB2), sulfonamides (sul1, sul2), and trimethoprim (dfrA12, dfrA1). The presence of three plasmid replicons (Col3M, IncQ1, and IncT) was detected, but no phage sequences were found. The phylogenetic analyses confirmed the genomic relationship of the PVR-188 with P. rettgeri isolates recovered from animals and humans in the USA and Malaysia. In conclusion, we report the occurrence of MDR P. rettgeri clone colonizing the gut microbiota of food-producing animals in Brazil, revealing the spread of this pathogen beyond hospital boundaries.

Detection of Antimicrobial Proteins/Peptides and Bacterial Proteins Involved in Antimicrobial Resistance in Raw Cow's Milk from Different Breeds.

Proteins involved in antibiotic resistance (resistome) and with antimicrobial activity are present in biological specimens. This study aims to explore the presence and abundance of antimicrobial peptides (AMPs) and resistome proteins in bovine milk from diverse breeds and from intensive (Pezzata rossa, Bruna alpina, and Frisona) and non-intensive farming (Podolica breeds). Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) profiling, bottom-up proteomics, and metaproteomics were used to comprehensively analyze milk samples from various bovine breeds in order to identify and characterize AMPs and to investigate resistome proteins. LAP-MALDI MS coupled with linear discriminant analysis (LDA) machine learning was employed as a rapid classification method for Podolica milk recognition against the milk of other bovine species. The results of the LAP-MALDI MS analysis of milk coupled with the linear discriminant analysis (LDA) demonstrate the potential of distinguishing between Podolica and control milk samples based on MS profiles. The classification accuracy achieved in the training set is 86% while it reaches 98.4% in the test set. Bottom-up proteomics revealed approximately 220 quantified bovine proteins (identified using the Bos taurus database), with cathelicidins and annexins exhibiting higher abundance levels in control cows (intensive farming breeds). On the other hand, the metaproteomics analysis highlighted the diversity within the milk's microbial ecosystem with interesting results that may reflect the diverse environmental variables. The bottom-up proteomics data analysis using the Comprehensive Antibiotic Resistance Database (CARD) revealed beta-lactamases and tetracycline resistance proteins in both control and Podolica milk samples, with no relevant breed-specific differences observed.

Monitoring of Pseudomonas aeruginosa mutational resistome dynamics using an enrichment panel for direct sequencing of clinical samples.

BACKGROUND: Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples. METHODS: A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample. FINDINGS: In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel ß-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo. INTERPRETATION: This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections. FUNDING: Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Metformin increases gut multidrug resistance genes in type 2 diabetes, potentially linked to Escherichia coli.

Metformin is the most commonly prescribed medication for treating type 2 diabetes (T2D). It is known that metformin can alter the gut microbiome, which influences the effectiveness of metformin treatment. We posited that if the gut microbiome, a reservoir of the resistome, is altered, then the resistome should change as well. To test this hypothesis, we reanalyzed microbiome data generated by Wu et al. (Nat Med 23(7):850-858, 2017), identifying antibiotic resistance genes (ARGs) and bacterial species. Through read-based analysis, we observed that the abundance of ARGs indeed changed in many samples treated with metformin. Moreover, the altered pattern was sufficiently heterogeneous across individual samples to allow subcategorization. We also found a strong correlation between the abundance of multidrug-resistant ARGs (MDR-ARGs) and the presence of E. coli. The contig-based analysis led to the same conclusion: an increase in MDR-ARGs due to metformin was associated with an increase in E. coli. In relation to this, we were able to confirm that the majority of MDR-ARGs are likely to originate from E. coli. These results suggest that metformin may have the potential side effect of increasing E. coli carrying ARGs, particularly MDR-ARGs, which could be a concern in T2D therapy that relies on metformin.

Emergence and transmission of the high-risk ST78 clone of OXA-48-producing Enterobacter hormaechei in a single hospital in Taiwan.

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes blaKPC-2, blaIMP-8, and predominantly blaOXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a blaOXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harbouring a blaIMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel blaKPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

Metagenomic Profiling of Internationally Sourced Sewage Influents and Effluents Yields Insight into Selecting Targets for Antibiotic Resistance Monitoring.

It has been debated whether wastewater treatment plants (WWTPs) primarily act to attenuate or amplify antibiotic resistance genes (ARGs). However, ARGs are highly diverse with respect to their resistance mechanisms, mobilities, and taxonomic hosts and therefore their behavior in WWTPs should not be expected to be universally conserved. We applied metagenomic sequencing to wastewater influent and effluent samples from 12 international WWTPs to classify the behavior of specific ARGs entering and exiting WWTPs. In total, 1079 different ARGs originating from a variety of bacteria were detected. This included ARGs that could be mapped to assembled scaffolds corresponding to nine human pathogens. While the relative abundance (per 16S rRNA gene) of ARGs decreased during treatment at 11 of the 12 WWTPs sampled and absolute abundance (per mL) decreased at all 12 WWTPs, increases in relative abundance were observed for 40% of the ARGs detected at the 12th WWTP. Also, the relative abundance of mobile genetic elements (MGE) increased during treatment, but the fraction of ARGs known to be transmissible between species decreased, thus demonstrating that increased MGE prevalence may not be generally indicative of an increase in ARGs. A distinct conserved resistome was documented in both influent and effluent across samples, suggesting that well-functioning WWTPs generally attenuate influent antibiotic resistance loads. This work helps inform strategies for wastewater surveillance of antibiotic resistance, highlighting the utility of tracking ARGs as indicators of treatment performance and relative risk reduction.

The interplay between mobilome and resistome in Staphylococcus aureus.

Antibiotic resistance genes (ARGs) in Staphylococcus aureus can disseminate vertically through successful clones, but also horizontally through the transfer of genes conveyed by mobile genetic elements (MGEs). Even though underexplored, MGE/ARG associations in S. aureus favor the emergence of multidrug-resistant clones, which are challenging therapeutic success in both human and animal health. This study investigated the interplay between the mobilome and the resistome of more than 10,000 S. aureus genomes from human and animal origin. The analysis revealed a remarkable diversity of MGEs and ARGs, with plasmids and transposons being the main carriers of ARGs. Numerous MGE/ARG associations were identified, suggesting that MGEs play a critical role in the dissemination of resistance. A high degree of similarity was observed in MGE/ARG associations between human and animal isolates, highlighting the potential for unrestricted spread of ARGs between hosts. Our results showed that in parallel to clonal expansion, MGEs and their associated ARGs can spread across different strain types sequence types (STs), favoring the evolution of these clones and their adaptation in selective environments. The high variability of MGE/ARG associations within individual STs and their spread across several STs highlight the crucial role of MGEs in shaping the S. aureus resistome. Overall, this study provides valuable insights into the complex interplay between MGEs and ARGs in S. aureus, emphasizing the need to elucidate the mechanisms governing the epidemic success of MGEs, particularly those implicated in ARG transfer.IMPORTANCEThe research presented in this article highlights the importance of understanding the interactions between mobile genetic elements (MGEs) and antibiotic resistance genes (ARGs) carried by Staphylococcus aureus, a versatile bacterium that can be both a harmless commensal and a dangerous pathogen for humans and animals. S. aureus has a great capacity to acquire and disseminate ARGs, enabling efficient adaption to various environmental or clinical conditions. By analyzing a large data set of S. aureus genomes, we highlighted the substantial role of MGEs, particularly plasmids and transposons, in disseminating ARGs within and between S. aureus populations, bypassing host barriers. Given that multidrug-resistant S. aureus strains are classified as a high-priority pathogen by global health organizations, this knowledge is crucial for understanding the complex dynamics of transmission of antibiotic resistance in this species.

Trajectory of antibiotic resistome response to antibiotics gradients: A comparative study from pharmaceutical and associated wastewater treatment plants to receiving river.

Pharmaceutical wastewater often contains significant levels of antibiotic residues, which continuously induce and promote antibiotic resistance during the sewage treatment process. However, the specific impact of antibiotics on the emergence of antibiotic resistance genes (ARGs), microbiomes, and mobile genetic elements (MGEs), as well as the dose-response relationship remain unclear. Herein, through metagenomic sequencing and analysis, we investigated the fate, transmission, and associated risk of ARGs over a ten-year period of exposure to a gradient of sulfonamide antibiotics at a pharmaceutical wastewater treatment plant (PWWTP), an associated wastewater treatment plant (WWTP), and the receiving river. Through abundance comparison and principal co-ordinates analysis (PCoA), our results revealed distinct ARG, microbiome, and MGE profiles across different antibiotic concentrations. Notably, there was a decreasing trend in the abundance of ARGs and MGEs as the antibiotic concentrations were attenuated (p < 0.05). Further partial least squares path modeling analysis, Procrustes analysis and network analysis indicated that variation in MGEs and microbiomes were the driving forces behind the distribution of ARGs. Based on these findings, we proposed an antibiotic-microbiome-MGE-ARG dissemination paradigm, in which integrons as key drivers were closely associated with prevalent ARGs such as sul1, sul2, and aadA. With a focus on human pathogenic bacteria and the associated health risks of ARGs, we conducted pathogen source analysis and calculated the antibiotic resistome risk index (ARRI). Our findings highlighted potential risks associated with the transition from PWWTP to WWTP, raising concerns regarding risk amplification due to the mixed treatment of antibiotic-laden industrial wastewater and domestic sewage. Overall, the results of our study provide valuable information for optimizing wastewater treatment practices to better manage antibiotic resistance.

Clinical presentation and microbiological characteristics of community-acquired Staphylococcus aureus bacteraemia at a tertiary hospital in Costa Rica.

Introduction. Staphylococcus aureus is a leading agent in community-acquired bacteraemia (CAB) and has been linked to elevated mortality rates and methicillin resistance in Costa Rica.Gap statement and aim. To update and enhance previous data obtained in this country, we analysed the clinical manifestations of 54 S. aureus CAB cases in a tertiary hospital and delineated the sequence types (STs), virulome, and resistome of the implicated isolates.Methodology. Clinical information was retrieved from patient files. Antibiotic susceptibility profiles were obtained with disc diffusion and automated phenotypic tests. Genomic data were exploited to type the isolates and for detection of resistance and virulence genes.Results. Primary infections predominantly manifested as bone and joint infections, followed by skin and soft tissue infections. Alarmingly, 70% of patients continued to exhibit positive haemocultures beyond 48 h of treatment modification, with nearly a quarter requiring mechanical ventilation or developing septic shock. The 30-day mortality rate reached an alarming 40%. More than 60% of the patients were found to have received suboptimal or inappropriate antibiotic treatment, and there was an alarming tendency towards the overuse of third-generation cephalosporins as empirical treatment. Laboratory tests indicated elevated creatinine levels, leukocytosis, and bandaemia within the first 24 h of hospitalization. However, most showed improvement after 48 h. The isolates were categorized into 13 STs, with a predominance of representatives from the clonal complexes CC72 (ST72), CC8 (ST8), CC5 (ST5, ST6), and CC1 (ST188). Twenty-four isolates tested positive for mecA, with ST72 strains accounting for 20. In addition, we detected genes conferring acquired resistance to aminoglycosides, MLSB antibiotics, trimethoprim/sulfamethoxazole, and mutations for fluoroquinolone resistance in the isolate collection. Genes associated with biofilm formation, capsule synthesis, and exotoxin production were prevalent, in contrast to the infrequent detection of enterotoxins or exfoliative toxin genes.Conclusions. Our findings broaden our understanding of S. aureus infections in a largely understudied region and can enhance patient management and treatment strategies.

Multi-omics analysis reveals the genetic and environmental factors in shaping the gut resistome of a keystone rodent species.

Understanding the emergence and spread of antibiotic resistance genes (ARGs) in wildlife is critical for the health of humans and animals from a "One Health" perspective. The gut microbiota serve as a reservoir for ARGs; however, it remains poorly understood how environmental and host genetic factors influence ARGs by affecting the gut microbiota. To elucidate this, we analyzed whole-genome resequencing data from 79 individuals of Brandt's vole in two geographic locations with different antibiotics usage, together with metabolomic data and shotgun sequencing data. A high diversity of ARGs (851 subtypes) was observed in vole's gut, with a large variation in ARG composition between individuals from Xilingol and Hulunbuir in China. The diversity and composition of ARGs were strongly correlated with variations in gut microbiota community structure. Genome-wide association studies revealed that 803 loci were significantly associated (P<5.05×10-9) with 31 bacterial species, and bipartite networks identified 906 bacterial species-ARGs associations. Structural equation modeling analysis showed that host genetic factors, air temperature, and presence of pollutants (Bisphenol A) significantly affected gut microbiota community structure, which eventually regulated the diversity of ARGs. The present study advances our understanding of the complex host-environment interactions that underlie the spread of ARGs in the natural environments.

The occurrence of banana Fusarium wilt aggravates antibiotic resistance genes dissemination in soil.

The spread of antibiotic resistance genes (ARGs) and subsequent soil-borne disease outbreaks are major threats to soil health and sustainable crop production. However, the relationship between occurrences of soil-borne diseases and the transmission of soil ARGs remains unclear. Here, soil ARGs, mobile genetic elements and microbial communities from co-located disease suppressive and conducive banana orchards were deciphered using metagenomics and metatranscriptomics approaches. In total, 23 ARG types, with 399 subtypes, were detected using a metagenomics approach, whereas 23 ARG types, with 452 subtypes, were discovered using a metatranscriptomics method. Furthermore, the metagenomics analysis revealed that the ARG total abundance levels were greater in rhizospheres (0.45 ARGs/16S rRNA on average) compared with bulk (0.32 ARGs/16S rRNA on average) soils. Interestingly, metatranscriptomics revealed that the total ARG abundances were greater in disease-conducive (8.85 ARGs/16S rRNA on average) soils than disease suppressive (1.45 ARGs/16S rRNA on average) soils. Mobile genetic elements showed the same trends as ARGs. Network and binning analyses indicated that Mycobacterium, Streptomyces, and Blastomonas are the main potential hosts of ARGs. Furthermore, Bacillus was significantly and negatively correlated with Fusarium (P < 0.05, r = -0.84) and hosts of ARGs (i.e., Mycobacterium, Streptomyces, and Blastomonas). By comparing metagenomic and metatranscriptomic analyses，this study demonstrated that metatranscriptomics may be more sensitive in indicating ARGs activities in soil. Our findings enable the more accurate assessment of the transmission risk of ARGs. The data provide a new perspective for recognizing soil health, in which soil-borne disease outbreaks appear to be associated with ARG spread, whereas beneficial microbe enrichment may mitigate wilt disease and ARG transmission.

Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum.

Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103-106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results. IMPORTANCE: High-throughput sequencing (HTS) is one of the crucial new technologies that gives us insights into previously hidden parts of microbial communities. The DNA extraction method is an important step that can have a major impact on the results, and understanding this impact is of paramount importance for their reliable interpretation. Our results are of great value for the interpretation of sputum microbiome and resistome results obtained by targeted HTS. Our findings allow for a more rational design of future microbiome studies, which would lead to higher repeatability of results and easier comparison between different laboratories. This could also facilitate the introduction of targeted HTS in clinical microbiology for reliable identification of pathogenic bacteria and testing for antimicrobial resistance (AMR). As AMR is a major threat to public health, the improved methods for determining AMR would bring great benefits to both the healthcare system and society as a whole.

Integrative genome-centric metagenomics for surface water surveillance: Elucidating microbiomes, antimicrobial resistance, and their associations.

Surface water ecosystems are intimately intertwined with anthropogenic activities and have significant public health implications as primary sources of irrigation water in agricultural production. Our extensive metagenomic analysis examined 404 surface water samples from four different geological regions in Chile and Brazil, spanning irrigation canals (n = 135), rivers (n = 121), creeks (n = 74), reservoirs (n = 66), and ponds (n = 8). Overall, 50.25 % of the surface water samples contained at least one of the pathogenic or contaminant bacterial genera (Salmonella: 29.21 %; Listeria: 6.19 %; Escherichia: 35.64 %). Furthermore, a total of 1,582 antimicrobial resistance (AMR) gene clusters encoding resistance to 25 antimicrobial classes were identified, with samples from Brazil exhibiting an elevated AMR burden. Samples from stagnant water sources were characterized by dominant Cyanobacteriota populations, resulting in significantly reduced biodiversity and more uniform community compositions. A significant association between taxonomic composition and the resistome was supported by a Procrustes analysis (p < 0.001). Notably, regional signatures were observed regarding the taxonomic and resistome profiles, as samples from the same region clustered together on both ordinates. Additionally, network analysis illuminated the intricate links between taxonomy and AMR at the contig level. Our deep sequencing efforts not only mapped the microbial landscape but also expanded the genomic catalog with newly characterized metagenome-assembled genomes (MAGs), boosting the classification of reads by 12.85 %. In conclusion, this study underscores the value of metagenomic approaches in surveillance of surface waters, enhancing our understanding of microbial and AMR dynamics with far-reaching public health and ecological ramifications.

Dissemination and persistence of antimicrobial resistance (AMR) along the wastewater-river continuum.

Antimicrobial resistance (AMR) is a global health hazard. Although clinical and agricultural environments are well-established contributors to the evolution and dissemination of AMR, research on wastewater treatment works (WwTWs) has highlighted their potential role as disseminators of AMR in freshwater environments. Using metagenomic sequencing and analysis, we investigated the changes in resistomes and associated mobile genetic elements within untreated wastewater influents and treated effluents of five WwTWs, and sediments collected from corresponding river environments in Oxfordshire, UK, across three seasonal periods within a year. Our analysis demonstrated a high diversity and abundance of antimicrobial resistance genes (ARGs) in untreated wastewater influents, reflecting the varied anthropogenic and environmental origins of wastewater. WwTWs effectively reduced AMR in the final effluent, with an average 87 % reduction in normalised ARG abundance and an average 63 % reduction in richness. However, wastewater effluents significantly impacted the antimicrobial resistome of the receiving rivers, with an average 543 % increase in ARG abundance and a 164 % increase in richness from upstream sediments to downstream sediments. The normalised abundance of the human gut-associated bacteriophage crAssphage was highly associated with both ARG abundance and richness. We observed seasonal variation in the resistome of raw influent which was not found in the effluent-receiving sediments. We illustrate the potential of WwTWs as focal points for disseminating ARGs and resistance-selecting chemicals, contributing to the elevation of environmental AMR. Our study emphasises the need for a comprehensive understanding of the anthropogenic impacts on AMR evolution and dissemination in wastewater and river environments, informing efforts to mitigate this growing public health crisis.

Horizontal plasmid transfer promotes antibiotic resistance in selected bacteria in Chinese frog farms.

The emergence and dissemination of antibiotic resistance genes (ARGs) in the ecosystem are global public health concerns. One Health emphasizes the interconnectivity between different habitats and seeks to optimize animal, human, and environmental health. However, information on the dissemination of antibiotic resistance genes (ARGs) within complex microbiomes in natural habitats is scarce. We investigated the prevalence of antibiotic resistant bacteria (ARB) and the spread of ARGs in intensive bullfrog (Rana catesbeiana) farms in the Shantou area of China. Antibiotic susceptibilities of 361 strains, combined with microbiome analyses, revealed Escherichia coli, Edwardsiella tarda, Citrobacter and Klebsiella sp. as prevalent multidrug resistant bacteria on these farms. Whole genome sequencing of 95 ARB identified 250 large plasmids that harbored a wide range of ARGs. Plasmid sequences and sediment metagenomes revealed an abundance of tetA, sul1, and aph(3″)-Ib ARGs. Notably, antibiotic resistance (against 15 antibiotics) highly correlated with plasmid-borne rather than chromosome-borne ARGs. Based on sequence similarities, most plasmids (62%) fell into 32 distinct groups, indicating a potential for horizontal plasmid transfer (HPT) within the frog farm microbiome. HPT was confirmed in inter- and intra-species conjugation experiments. Furthermore, identical mobile ARGs, flanked by mobile genetic elements (MGEs), were found in different locations on the same plasmid, or on different plasmids residing in the same or different hosts. Our results suggest a synergy between MGEs and HPT to facilitate ARGs dissemination in frog farms. Mining public databases retrieved similar plasmids from different bacterial species found in other environmental niches globally. Our findings underscore the importance of HPT in mediating the spread of ARGs in frog farms and other microbiomes of the ecosystem.