Insights into the effects of pulsed antimicrobials on the chicken resistome and microbiota from fecal metagenomes

Antimicrobial resistance has become a global problem that poses great threats to human health. Antimicrobials are widely used in broiler chicken production and consequently affect their gut microbiota and resistome. To better understand how continuous antimicrobial use in farm animals alters their microbial ecology, we used a metagenomic approach to investigate the effects of pulsed antimicrobial administration on the bacterial community, antibiotic resistance genes(ARGs) and ARG bacterial hosts in the feces of broiler chickens. Chickens received three 5-day courses of individual or combined antimicrobials, including amoxicillin, chlortetracycline and florfenicol. The florfenicol administration significantly increased the abundance of mcr-1 gene accompanied by floR gene, while amoxicillin significantly increased the abundance of genes encoding the AcrAB-tolC multidrug efflux pump(marA, soxS, sdiA, rob, evgS and phoP).These three antimicrobials all led to an increase in Proteobacteria. The increase in ARG host, Escherichia, was mainly attributed to the β-lactam, chloramphenicol and tetracycline resistance genes harbored by Escherichia under the pulsed antimicrobial treatments. These results indicated that pulsed antimicrobial administration with amoxicillin,chlortetracycline, florfenicol or their combinations significantly increased the abundance of Proteobacteria and enhanced the abundance of particular ARGs. The ARG types were occupied by the multidrug resistance genes and had significant correlations with the total ARGs in the antimicrobial-treated groups. The results of this study provide comprehensive insight into pulsed antimicrobial-mediated alteration of chicken fecal microbiota and resistome.

A review of the resistome within the digestive tract of livestock

Antimicrobials have been widely used to prevent and treat infectious diseases and promote growth in food-production animals.However,the occurrence of antimicrobial resistance poses a huge threat to public and animal health,especial y in less developed countries where food-producing animals often intermingle with humans.To limit the spread of antimicrobial resistance from food-production animals to humans and the environment,it is essential to have a comprehensive knowledge of the role of the resistome in antimicrobial resistance(AMR),The resistome refers to the col ection of al antimicrobial resistance genes associated with microbiota in a given environment.The dense microbiota in the digestive tract is known to harbour one of the most diverse resistomes in nature.Studies of the resistome in the digestive tract of humans and animals are increasing exponential y as a result of advancements in next-generation sequencing and the expansion of bioinformatic resources/tools to identify and describe the resistome.In this review,we outline the various tools/bioinformatic pipelines currently available to characterize and understand the nature of the intestinal resistome of swine,poultry,and ruminants.We then propose future research directions including analysis of resistome using long-read sequencing,investigation in the role of mobile genetic elements in the expression,function and transmission of AMR.This review outlines the current knowledge and approaches to studying the resistome in food-producing animals and sheds light on future strategies to reduce antimicrobial usage and control the spread of AMR both within and from livestock production systems.

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.

Biogeography and diversity patterns of antibiotic resistome in the sediments of global lakes

Lakes act as one of the reservoirs and dispersal routes of antibiotic resistance genes(ARGs)and pathogenic resistant bacteria in aquatic environments.Previous studies reported the occurrence and distribution of ARGs in lakes worldwide;however,few investigated the biogeography and diversity patterns of antibiotic resistome in the environment.To fill this gap,a large-scale data set of sediment metagenomes was collected from globally distributed lakes and characterized comprehensively using metagenomic assembly-based analysis,aiming to shed light on the biogeography and diversity patterns of ARGs in lake ecosystems from a global perspective.Our analyses showed that abundant and diverse ARGs were found in the global lake sediments,including a set of emerging ARGs such as mcr-type and carbapenem-resistant Enterobacteriaceae related genes.Most of the identified ARGs were generally associated with the commonly used antibiotics,suggesting the role of increasing antibiotic consumptions on the resistome prevalence.Spatially,the composition and diversity of ARGs varied across geographical distances and exhibited a scale-dependent distancedecay relationship.Notably,the composition of ARGs was largely shaped by bacterial community structure,and their diversities were co-governed by stochastic process(∼48%)and deterministic process(∼52%).Findings provide a valuable insight to better understand ecological mechanisms of ARGs in lake ecosystems and have important implication for the prevention and control of resistome risk.

The antibiotic resistome： gene flow in environments, animals and human beings

The antibiotic resistance is natural in bacteria and predates the human use of antibiotics. Numerous antibiotic resistance genes （ARGs） have been discovered to confer resistance to a wide range of antibiotics. The ARGs in natural environments are highly integrated and tightly regulated in specific bacterial metabolic networks. However, the antibiotic selection pressure conferred by the use of antibiotics in both human medicine and agriculture practice leads to a significant increase of antibiotic resistance and a steady accumulation of ARGs in bacteria. In this review, we summarized, with an emphasis on an ecological point of view, the important research progress regarding the collective ARGs （antibiotic resistome） in bacterial communities of natural environments, human and animals, i.e., in the one health settings. We propose that the resistance gene flow in nature is ＂from the natural environments＂ and ＂to the natural environments＂; human and animals, as intermediate recipients and disseminators, contribute greatly to such a resistance gene ＂circulation.＂

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that shortterm antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ～3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.

Culturomics and metagenomics: In understanding of environmental resistome

Pharmaceutical residues, mainly antibiotics, have been called "emerging contaminants" in the environment because of their increasing frequency of detection in aquatic and terrestrial systems and their sublethal ecological effects. Most of them are undiscovered. Both human and veterinary pharmaceuticals, including antibiotics, are introduced into the environment via many different routes, including discharges from municipal wastewater treatment plants and land application of animal manure and biosolids to fertilize croplands. To gain a comprehensive understanding of the widespread problem of antibiotic resistance, modem and scientific approaches have been developed to gain knowledge of the entire antibiotic-resistant microbiota of various ecosystems, which is called the resistome. In this review, two omics methods, i.e. culturomics, a new approach, and metagenomics, used to study antibiotic resistance in environmental samples, are described. Moreover, we discuss how both omics methods have become core scientific tools to characterize microbiomes or resistomes, study natural communities and discover new microbes and new antibiotic resistance genes from environments. The combination of the method for get better outcome of both culturomics and metagenomics will significantly advance our understanding of the role of microbes and their specific properties in the environment.

Antibiotic resistome of Salmonella typhi:molecular determinants for the emergence of drug resistance

Resistome is a cluster of microbial genes encoding proteins with necessary functions to resist the action of antibiotics.Resistome governs essential and separate biological functions to develop resistance against antibiotics.The widespread clinical and nonclinical uses of antibiotics over the years have combined to select antibiotic-resistant determinants and develop resistome in bacteria.At present,the emergence of drug resistance because of resistome is a significant problem faced by clinicians for the treatment of Salmonella infection.Antibiotic resistome is a dynamic and ever-expanding component in Salmonella.The foundation of resistome in Salmonella is laid long before;therefore,the antibiotic resistome of Salmonella is reviewed,discussed,and summarized.We have searched the literature using PubMed,MEDLINE,and Google Scholar with related key terms(resistome,Salmonella,antibiotics,drug resistance)and prepared this review.In this review,we summarize the status of resistance against antibiotics in S.typhi,highlight the seminal work in the resistome of S.typhi and the genes involved in the antibiotic resistance,and discuss the various methods to identify S.typhi resistome for the proactive identification of this infection and quick diagnosis of the disease.

Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production

Subject Code:C18 With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Shen Jianzhong(沈建忠)from the College of Veterinary Medicine,China Agricultural University and Prof.Timothy R.Walsh from Cardiff University demonstrates the

Responses of nitrification performance,triclosan resistome and diversity of microbes to continuous triclosan stress in activated sludge system

Triclosan(TCS)is commonly found in wastewater treatment plants,which often affects biological treatment processes.The responses of nitrification,antibiotic resistome and microbial community under different TCS concentrations in activated sludge system were evaluated in this study.The experiment was conducted in a sequencing batch reactor(SBR)for 240 days.Quantitative PCR results demonstrated that the abundance of ammonium oxidizing bacteria could be temporarily inhibited by 1 mg/L TCS and then gradually recovered.And the abundances of nitrite oxidizing bacteria(NOB)under 2.5 and 4 mg/L TCS were three orders of magnitude lower than that of seed sludge,which accounted for partial nitrification.When the addition of TCS was stopped,the abundance of NOB increased.The mass balance experiments of TCS demonstrated that the primary removal pathway of TCS changed from adsorption to biodegradation as TCS was continuously added into the SBR system.Moreover,TCS increased the abundance of mexB,indicating the efflux pump might be the main TCS-resistance mechanism.As a response to TCS,bacteria could secrete more protein(PN)than polysaccharide.Three-dimensional excitation-emission matrix revealed that tryptophan PN-like substances might be the main component in PN to resist TCS.High-throughput sequencing found that the relative abundances of Paracoccus,Pseudoxanthomonas and Thauera increased,which could secrete extracellular polymeric substances(EPS).And Sphingopyxis might be the main TCS-degrading bacteria.Overall,TCS could cause partial nitrification and increase the relative abundances of EPS-secreting bacteria and TCS-degrading bacteria.

Metagenomic insights into the profile of antibiotic resistomes in sediments of aquaculture wastewater treatment system

To meet the rapidly growing global demand for aquaculture products,large amounts of antibiotics were used in aquaculture,which might accelerate the evolution of antibiotic-resistant bacteria(ARB)and the propagation of antibiotic genes(ARGs).In our research,we revealed the ARGs profiles,their co-occurrence with mobile genetic elements(MGEs),and potential hosts in sediments of a crab pond wastewater purification system based on metagenomic analysis.The residual antibiotic seems to increase the propagation of ARGs in the crab pond,but there was no clear relationship between a given antibiotic type and the corresponding resistance genes.The effect of aquaculture on sediment was not as profound as that of other anthropogentic activities,but increased the relative abundance of sulfon-amide resistance gene.A higher abundance of MGEs,especially plasmid,increased the po-tential ARGs dissemination risk in crab and purification ponds.Multidrug and sulfonamide resistance genes had greater potential to transfer because they were more frequently carried by MGEs.The horizontal gene transfer was likely to occur among a variety of microorgan-isms,and various ARGs hosts including Pseudomonas,Acinetobacter,Escherichia,and Klebsiella were identified.Bacterial community influenced the composition of ARG hosts,and Pro-teobacteria was the predominant hosts.Overall,our study provides novel insights into the environmental risk of ARGs in sediments of aquaculture wastewater treatment system.

Profiling the antibiotic resistome in soils between pristine and human-affected sites on the Tibetan Plateau

With increasing pressure from anthropogenic activity in pristine environments,the comprehensive profiling of antibiotic resistance genes(ARGs)is essential to evaluate the potential risks from human-induced antibiotic resistance in these under-studied places.Here,we characterized the microbial resistome in relatively pristine soil samples collected from four distinct habitats on the Tibetan Plateau,using a Smart chip based high-throughput q PCR approach.We compared these to soils from the same habitats that had been subjected to various anthropogenic activities,including residential sewage discharge,animal farming,atmospheric deposition,and tourism activity.Compared to pristine samples,an average of23.7%more ARGs were detected in the human-affected soils,and the ARGs enriched in these soils mainly encoded resistances to aminoglycoside and beta-lactam.Of the four habitats studied,soils subjected to animal farming showed the highest risks of ARG enrichment and dissemination.As shown,the number of ARGs enriched(a total of 42),their fold changes(17.6 fold on average),and the co-occurrence complexity between ARGs and mobile genetic elements were all the highest in fecal-polluted soils.As well as antibiotics themselves,heavy metals also influenced ARG distributional patterns in Tibetan environments.However,compared to urban areas,the Tibetan Plateau had a low potential for ARG selection and exhibited low carriage of ARGs by mobile genetic elements,even in environments impacted by humans,suggesting that these ARGs have a limited capacity to disseminate.The present study examined the effects of multiple anthropogenic activities on the soil resistomes in relatively pristine environments.

Antibiotic resistome mostly relates to bacterial taxonomy along a suburban transmission chain

Antibiotic resistance genes comprising antibiotic resistome are of great concern due to their increase in the environment.Recent evidence of shared resistomes between soils and animal husbandry has imposed potential risks to human health.However,the correlation between a given community’s resistome and bacterial taxonomic composition is controversial.Here,a transmission chain of resistomes from swine manure to compost and compost-amended soil were analyzed in five suburban areas of Beijing,China,with unamended agricultural soils as control soils.Antibiotic resistomes and bacterial taxonomic compositions were distinct between(I)manure and compost;and(II)compost-amended and control soils.In manure,compost,and compost-amended soils,theβ-diversity of the resistome and bacterial taxonomic composition was significantly correlated,while no correlation was detected in control soils.Bacterial taxonomic composition explained 36.0%of total variations of the resistome composition,much higher than environmental factors.Together,those results demonstrated that antibiotic resistome was closely related to bacterial taxonomic composition along the suburban transmission chain.

uCARE Chem Suite and uCAREChemSuiteCLI: Tools for bacterial resistome prediction

In the era of antibiotic resistance,in silico prediction of bacterial resistome pro-files,likely to be associated with inactivation of new potential antibiotics is of utmost impor-tance.Despite this,to the best of our knowledge,no tool exists for such prediction.Therefore,under the rationale that drugs with similar structures have similar resistome profiles,we devel-oped two models,a deterministic model and a stochastic model,to predict the bacterial re-sistome likely to neutralize uncharacterized but potential chemical structures.The current version of the tool involves the prediction of a resistome for Escherichia coli and Pseudomonas aeruginosa.The deterministic model on omitting two diverse but relatively less characterized drug classes,polyketides and polypeptides showed an accuracy of 87%,a sensitivity of 85%,and a precision of 89%,whereas the stochastic model predicted antibiotic classes of the test set compounds with an accuracy of 72%,a sensitivity of 75%,and a precision of 83%.The models have been implemented in both a standalone package and an online server,uCAREChemSuite-CLI and uCARE Chem Suite,respectively.In addition to resistome prediction,the online version of the suite enables the user to visualize the chemical structure,classify compounds in 19 pre-defined drug classes,perform pairwise alignment,and cluster with database compounds using a graphical user interface.

Three-Year Consecutive Field Application of Erythromycin Fermentation Residue Following Hydrothermal Treatment:Cumulative Effect on Soil Antibiotic Resistance Genes

Fermentation-based antibiotic production results in abundant nutrient-rich fermentation residue with high potential for recycling,but the high antibiotic residual concentration restricts its usefulness(e.g.,in land application as organic fertilizer).In this study,an industrial-scale hydrothermal facility for the treatment of erythromycin fermentation residue(EFR)was investigated,and the potential risk of the long-term soil application of treated EFR promoting environmental antibiotic resistance development was evaluated.The treatment effectively removed bacteria and their DNA,and an erythromycin removal ratio of up to approximately 98%was achieved.The treated EFR was utilized as organic fertilizer for consecutive field applications from 2018 to 2020,with dosages ranging from 3750 to 15000 kg·hm^(-2),resulting in subinhibitory levels of erythromycin(ranging from 0.83-76.00μg·kg^(-1))in soils.Metagenomic shotgun sequencing was then used to characterize the antibiotic resistance genes(ARGs),mobile genetic elements(MGEs),and bacterial community composition of the soils.The soil ARG abundance and diversity did not respond to the treated EFR application in the first year,but gradually changed in the second and third year of application.The highest fold change in relative abundance of macrolide-lincosamide-streptogramin(MLS)and total ARGs were 12.59 and 2.75 times,compared with the control(CK;without application),respectively.The soil MGEs and taxonomic composition showed similar temporal trends to those of the ARGs,and appeared to assist in driving increasing ARG proliferation,as revealed by correlation analysis and structural equation models(SEMs).The relative abundance of particular erm resistance genes(RNA methyltransferase genes)increased significantly in the third year of treated EFR application.The close association of erm with MGEs suggested that horizontal gene transfer played a critical role in the observed erm gene enrichment.Metagenomic binning results demonstrated that the proliferation of mac genecarrying hosts was responsible for the increased abundance of mac genes(efflux pump genes).This study shows that sub-inhibitory levels of erythromycin in soils had a cumulative effect on soil ARGs over time and emphasizes the importance of long-term monitoring for assessing the risk of soil amendment with treated industrial waste.

Antimicrobial usage and resistance in beef production

Antimicrobials are critical to contemporary high-intensity beef production. Many different antimicrobials are approved for beef cattle, and are used judiciously for animal welfare, and controversially, to promote growth and feed efficiency. Antimicrobial administration provides a powerful selective pressure that acts on the microbial community, selecting for resistance gene determinants and antimicrobial-resistant bacteria resident in the bovine flora. The bovine microbiota includes many harmless bacteria, but also opportunistic pathogens that may acquire and propagate resistance genes within the microbial community via horizontal gene transfer. Antimicrobial-resistant bovine pathogens can also complicate the prevention and treatment of infectious diseases in beef feedlots,threatening the efficiency of the beef production system. Likewise, the transmission of antimicrobial resistance genes to bovine-associated human pathogens is a potential public health concern. This review outlines current antimicrobial use practices pertaining to beef production, and explores the frequency of antimicrobial resistance in major bovine pathogens. The effect of antimicrobials on the composition of the bovine microbiota is examined, as are the effects on the beef production resistome. Antimicrobial resistance is further explored within the context of the wider beef production continuum, with emphasis on antimicrobial resistance genes in the food chain, and risk to the human population.

Soil microbial ecology through the lens of metatranscriptomics

Metatranscriptomics is a cutting-edge technology for exploring the gene expression by,and functional activities of,the microbial community across diverse ecosystems at a given time,thereby shedding light on their metabolic responses to the prevailing environmental conditions.The double-RNA approach involves the simultaneous analysis of rRNA and mRNA,also termed structural and functional metatranscriptomics.By contrast,mRNA-centered metatranscriptomics is fully focused on elucidating community-wide gene expression profiles,but requires either deep sequencing or effective rRNA depletion.In this review,we critically assess the challenges associated with various experimental and bioinformatic strategies that can be applied in soil microbial ecology through the lens of functional metatranscriptomics.In particular,we demonstrate how recent methodological advancements in soil metatranscriptomics catalyze the development and expansion of emerging research fields,such as rhizobiomes,antibiotic resistomes,methanomes,and viromes.Our review provides a framework that will help to design advanced metatranscriptomic research in elucidating the functional roles and activities of microbiomes in soil ecosystems.

Genome-resolved metagenomics of Venice Lagoon surface sediment bacteria reveals high biosynthetic potential and metabolic plasticity as successful strategies in an impacted environment

Bacteria living in sediments play essential roles in marine ecosystems and deeper insights into the ecology and biogeochemistry of these largely unexplored organisms can be obtained from‘omics’approaches.Here,we characterized metagenome-assembled-genomes(MAGs)from the surface sediment microbes of the Venice Lagoon(northern Adriatic Sea)in distinct sub-basins exposed to various natural and anthropogenic pressures.MAGs were explored for biodiversity,major marine metabolic processes,anthropogenic activity-related functions,adaptations at the microscale,and biosynthetic gene clusters.Starting from 126 MAGs,a non-redundant dataset of 58 was compiled,the majority of which(35)belonged to(Alpha-and Gamma-)Proteobacteria.Within the broad microbial metabolic repertoire(including C,N,and S metabolisms)the potential to live without oxygen emerged as one of the most important features.Mixotrophy was also found as a successful lifestyle.Cluster analysis showed that different MAGs encoded the same metabolic patterns(e.g.,C fixation,sulfate oxidation)thus suggesting metabolic redundancy.Antibiotic and toxic compounds resistance genes were coupled,a condition that could promote the spreading of these genetic traits.MAGs showed a high biosynthetic potential related to antimicrobial and biotechnological classes and to organism defense and interactions as well as adaptive strategies for micronutrient uptake and cellular detoxification.Our results highlighted that bacteria living in an impacted environment,such as the surface sediments of the Venice Lagoon,may benefit from metabolic plasticity as well as from the synthesis of a wide array of secondary metabolites,promoting ecosystem resilience and stability toward environmental pressures.