Sequence capture across large phylogenetic scales by using pooled PCR‐generated baits: A case study of Lepidoptera

Sequence capture across large phylogenetic scales is not easy because hybridization capture is only effective when the genetic distance between the bait and target is small. Here, we propose a simple but effective strategy to tackle this issue: pooling DNA from a number of selected representative species of different clades to prepare PCR‐generated baits to minimize the genetic distance between the bait and target. To demonstrate the utility of this strategy, we newly developed a set of universal nuclear markers (including 94 nuclear protein‐coding genes) for Lepidoptera, a superdiverse insect group. We used a DNA pool from six lepidopteran species (representing six superfamilies) to prepare PCR baits for the 94 markers. These homemade PCR baits were used to capture sequence data from 43 species of 17 lepidopteran families, and 94% of the target loci were recovered. We constructed two data sets from the obtained data (one containing ~90 kb target coding sequences and the other containing ~120 kb target + flanking coding sequences). Both data sets yielded highly similar and well‐resolved trees with 90% of nodes having >95% bootstrap support. Our capture experiment indicated that using DNA mixtures pooled from different clade‐representative species of Lepidoptera to prepare PCR baits can reliably capture a large number of targeted nuclear markers across different Lepidoptera lineages. We hope that this newly developed nuclear marker set will serve as a new phylogenetic tool for Lepidoptera phylogenetics, and the PCR bait preparation strategy can facilitate the application of sequence capture techniques by researchers to accelerate data collection.     

A curated data resource of 214K metagenomes for characterization of the global antimicrobial resistome

The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have been transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources to analyze such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76∙10⁸ read fragments were assigned to ARGs and 3.21∙10⁹ to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs.

The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods and a deeper understanding of how resistance genes are transmitted around the world. This study presents a large-scale remapping of sequencing reads of publicly available metagenomic datasets that can be used to monitor the global prevalence of AMR genes.     

Dissecting microbial communities and resistomes for interconnected humans,soil, and livestock

A debate is currently ongoing as to whether intensive livestock farms may constitute reservoirs of clinically relevant antimicrobial resistance (AMR), thus posing a threat to surrounding communities. Here, combining shotgun metagenome sequencing, machine learning (ML), and culture-based methods, we focused on a poultry farm and connected slaughterhouse in China, investigating the gut microbiome of livestock, workers and their households, and microbial communities in carcasses and soil. For both the microbiome and resistomes in this study, differences are observed across environments and hosts. However, at a finer scale, several similar clinically relevant antimicrobial resistance genes (ARGs) and similar associated mobile genetic elements were found in both human and broiler chicken samples. Next, we focused on Escherichia coli, an important indicator for the surveillance of AMR on the farm. Strains of E. coli were found intermixed between humans and chickens. We observed that several ARGs present in the chicken faecal resistome showed correlation to resistance/susceptibility profiles of E. coli isolates cultured from the same samples. Finally, by using environmental sensing these ARGs were found to be correlated to variations in environmental temperature and humidity. Our results show the importance of adopting a multi-domain and multi-scale approach when studying microbial communities and AMR in complex, interconnected environments.Subject terms: Metagenomics, Microbiome, Bacterial genetics, Bacterial infection, Infectious-disease diagnostics     

Whole-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.     

Termite mound formation reduces the abundance and diversity of soil resistomes

Termites are pivotal ecosystem engineers in tropical and subtropical habitats, where they construct massive nests (‘mounds’) that substantially modify soil properties and promote nutrient cycling. Yet, little is known about the roles of termite nesting activity in regulating the spread of antimicrobial resistance (AMR), one of the major Global Health challenges. Here, we conducted a large-scale (> 1500 km) investigation in northern Australia and found distinct resistome profiles in termite mounds and bulk soils. By profiling a wide spectrum of ARGs, we found that the abundance and diversity of antibiotic resistance genes (ARGs) were significantly lower in termite mounds than in bulk soils (P < 0.001). The proportion of efflux pump ARGs was significantly lower in termite mound resistome than in bulk soil resistome (P < 0.001). The differences in resistome profiles between termite mounds and bulk soils may result from the changes in microbial interactions owing to the substantial increase in pH and nutrient availability induced by termite nesting activities. These findings advance our understanding of the profile of ARGs in termite mounds, which is a crucial step to evaluate the roles of soil faunal activity in regulating soil resistome under global environmental change.     

Translating Bait Preference to Capture Success of Northern White-Tailed Deer

ABSTRACT Wildlife management and research have depended upon trapping as an essential tool for decades. Although deer (Odocoileus spp.) capture by Clover traps remains a basic technique that has changed little over time, researchers use it as an integral part of field operations to support increasingly sophisticated and costly project objectives. Despite reports of deer preference for certain baits, no study has determined if bait preference can effectively increase capture success of free-ranging deer. By supplementing corn bait with salt, peanut butter, or molasses, we tested effects of these bait treatments on capture success of free-ranging white-tailed deer (O. virginianus), as well as levels of nontarget animal disturbance in Clover traps, during February-March 2005. With 1,446 adjusted trap-nights and a 6.5% capture success rate, the probability of capture increased over time and varied among 4 study sites (df = 3, P < 0.005); however, we did not detect a significant effect of bait supplementation on capture success (df = 3, P > 0.8). Nontarget animal activity in the trap varied by site (df = 3, P < 0.001), bait treatment (df = 3, P=0.04), and Julian date (df=3, P < 0.001). Our results are the first to suggest that bait preference may not translate into actual improved capture success of free-ranging deer. Future research should focus on testing additional baits or bait supplements to determine if an increase in trapping success and a minimization of trap disturbance by nontarget species occurs.     

Co-occurrence of antibiotic,biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim’s Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment.     

Laboratory comparison of American lobster,Homarus americanus,foraging responses to invasive green crab,Carcinus maenas,and two traditional finfish baits

The American Lobster (Homarus americanus) is trapped primarily with finfish baits that are becoming more costly and difficult to acquire as stocks dwindle. Thus, alternative bait options should be considered. We made video recordings of lobster foraging responses in the laboratory to compare the attractiveness of the invasive green crab (Carcinus maenas) bait, two traditional finfish baits and an inert control. Solitary lobsters approached and handled all three biological baits with similar latencies and spent similar time in contact with the biological baits. In contrast, lobsters took longer to approach and handle, and spent significantly less time in contact with control bait. As green crabs were statistically indistinguishable in attractiveness from traditional baits in our laboratory setting, we suggest field trials should test this alternate bait in circumstances more directly relevant to the fishery.     

Testing target‐specific bait delivery for controlling feral pigs in a tropical rainforest

Mitigation of feral pig (Sus scrofa) impacts in Australia’s Wet Tropics World Heritage Area has been impeded by the lack of a target‐specific method for delivering toxic baits in the region. This study evaluated methods to reduce bait‐take by susceptible nontarget species without inhibiting bait‐take by pigs, to enable more effective pig management. We predicted that dingoes would not select an unprocessed corn bait and that other potential nontarget bait consumers would be unable to access the same bait presented under a lightweight cover. Neither of these methods was expected to reduce bait selection or access by pigs. We tested these predictions by monitoring animal interactions with covered and uncovered corn baits, and covered corn and manufactured baits. Use of corn as a bait substrate effectively prevented bait‐take by dingoes. Covering baits substantially reduced bait‐take by other nontarget species and completely prevented nontarget bait‐take when uncovered feed was provided simultaneously. The corn bait preparation was highly acceptable and accessible to feral pigs. We conclude that the methods evaluated here could enable the consideration of poison baiting as a viable method for controlling feral pigs in the World Heritage Area, where it has previously been unavailable.     

Use of Rhodamine B to Reveal Patterns of Interspecific Competition and Bait Acceptance in Raccoons

Abstract: Passive treatment of raccoons (Procyon lotor) through distribution of vaccine-laden baits recently has emerged as a potential solution to address health and economic conflicts associated with raccoon rabies and may have applications in the management of other pathogens carried by raccoons if frequent bait deployments are used. Consumption of baits by nontarget species reduces the efficiency in which baits can be used to manage wildlife disease, although no study has explicitly evaluated the influence of bait competitor density on the ability to treat raccoons. Our objectives were to use the biomarker Rhodamine B (RB) to 1) evaluate patterns of raccoon bait acceptance as a function of competition with Virginia opossums (Didelphis virginiana), the dominant bait competitor; 2) characterize attributes of opossum bait acceptance to improve efficiency of raccoon treatment; and 3) evaluate the effect of repeated bait exposure on rates of bait acceptance as may be required in the management of wildlife disease issues beyond rabies. Identifying bait consumption by individuals based on the presence of an RB mark in a sample of whiskers, we used logistic regression to model raccoon and opossum bait acceptance as a function of bait availability, previous exposure to baits, demographic attributes, and an index of time spent in the baited area (residency index). For both raccoons and opossums, the best measure of bait availability was the variable number of baits per opossum. The most parsimonious logistic regression model for raccoon bait acceptance included the variables baits per opossum, exposure history, and residency index. The strength of the variable baits per opossum relative to competing measures of bait availability indicated bait consumption by opossums significantly limited the ability to treat raccoons. The most parsimonious model for opossum acceptance was composed of the variables baits per opossum, sex, weight, residency index, baits per opossum X sex, and weight X sex. Patterns of opossum bait acceptance likely were driven by effects of bait availability and sex-dependent differences in movement. Our results call attention to the importance of bait competition in limiting the ability to effectively treat raccoon populations through distribution of baits and suggest managers incorporate information on density of bait competitors, particularly opossums, in allocation of baits.     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

Levamisole can reduce bait monopolization in wild red foxes Vulpes vulpes

1. Baits are used worldwide to deliver vaccines, contraceptives and poison to wild animal species. In carnivores, multiple bait uptake and bait monopolization by dominant individuals may reduce baiting effectiveness. This study investigated whether a conditioned‐taste‐aversion agent, levamisole, can be used to decrease bait consumption by individual wild foxes Vulpes vulpes. 2. The results indicated that after consuming levamisole‐treated baits, foxes avoided eating treated baits but consumed untreated baits. We concluded that the reduction in bait consumption was achieved through learned aversion to levamisole rather than via conditioned‐taste‐aversion to baits. Adding levamisole to baits could potentially be used to reduce bait monopolization by individual wild foxes.     

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

Impact of enrichment conditions on cross‐species capture of fresh and degraded DNA

By combining high‐throughput sequencing with target enrichment (‘hybridization capture’), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination‐rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter – hybridization temperature – on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect^? arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on‐target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments.     

The response of German cockroaches to toxic baits:strain differences and the effects of selection pressure

Strain differences in the response of German cockroaches to toxic baits and their potential to evolve resistance to them was evaluated in choice tests. Percentage bait consumption (amount of bait consumed divided by total food consumed) was used to estimate relative attractancy/repellency of the baits. One strain apparently had low level resistance to abamectin, but no evidence of resistance was found in other strains tested with either Roach Ender or a gel bait (abamectin-based baits). Incipient behavioral resistance occurred in two strains selected for three generations with Roach Ender; a stronger response in a strain selected with the gel bait is attributed to the development of behavioral resistance. Baygon bait was highly repellent. Low level resistance to propoxur caused a large decrease in mortality compared with susceptible strains. Strong behavioral resistance developed in strains selected with Stapleton's Magnetic Roach Food.     

Extracting ‘legacy loci’ from an invertebrate sequence capture data set

Sequence capture studies result in rich data sets comprising hundreds to thousands of targeted genomic regions that are superseding Sanger-based data sets comprised of a few well-known loci with historical uses in phylogenetics (‘legacy loci’). However, integrating sequence capture and Sanger-based data sets is of interest as legacy loci can include different types of loci (e.g. mitochondrial and nuclear) across a potentially larger sample of species from past studies. Sequence capture data sets include nontargeted sequences, and there has been recent interest in extracting legacy loci from invertebrate data sets. Here, we use published legacy data from leaf-footed bugs (Hemiptera: Coreoidea) to recover 15 mitochondrial and seven nuclear legacy loci from off-target sequences in a sequence capture data set, explore approaches to improve legacy locus recovery, and combine these loci with sequence capture data for phylogenetic analysis. Two nuclear loci were determined to already be targeted by sequence capture baits. Most of the remaining loci were successfully recovered from off-target sequences, but this recovery varied greatly. Additionally, complementing complete mitogenomes with additional reference mitochondrial sequences from a genetic depository did not offer improvement for most of our taxa; however, supplementing these reference sequences with extracted legacy loci offered ≥6% improvement across taxa for a given mitochondrial locus (negligible improvement for nuclear loci). Phylogenetic analysis of legacy and sequence capture data produced a topology generally congruent with recent studies, but support was lower. Thus, future studies may employ the approaches used in this study to integrate legacy data with newly generated sequence capture data sets without added expenses.     

The challenges of defining the human nasopharyngeal resistome

The nasopharynx is an important microbial reservoir for the emergence and spread of antibiotic-resistant organisms. The nasopharyngeal resistome is an extensive, adaptable reservoir of antibiotic-resistance genes (ARGs) within this niche. Metagenomic sequencing decodes the genetic material of all organisms within a sample using next-generation technologies, permitting unbiased discovery of novel ARGs and associated mobile genetic elements (MGEs). The challenges of sequencing a low-biomass bacterial sample have limited exploration of the nasopharyngeal resistome. Here, we explore the current understanding of the nasopharyngeal resistome, particularly the role of MGEs in propagating antimicrobial resistance (AMR), explore the advantages and limitations of metagenomic sequencing technologies and bioinformatic pipelines for nasopharyngeal resistome analysis, and highlight the key outstanding questions for future research.     

Bait Uptake and Caching by Red Foxes and Nontarget Species in Urban Reserves

Abstract: The management of biodiversity in urban areas provides a challenge for conservation managers who are interested in the recovery of native species by controlling exotic species. Exotic-animal control programs using poisons can be contentious in terms of the health and safety of nontarget species, including people. Managing exotic predators in urban areas must be effective at 2 levels: controlling the target species and minimizing impacts to nontarget species. We investigated the feasibility of instigating a poison-baiting program to control nonindigenous European red foxes (Vulpes vulpes) in city conservation reserves (Perth, Australia). We selected 3 reserves to assess the safety and efficacy of baiting for foxes by maximizing bait uptake by the target species, and minimizing uptake by nontarget species. We tested 2 types of meat bait using 4 bait presentation methods (untethered, uncovered; untethered, covered; tethered, uncovered; tethered, covered). Bait uptake by foxes was highest in urban reserves compared to that in a peri-urban reserve. Bait type and presentation method equally explained bait uptake by foxes. Untethered and uncovered baits were removed 10% more often by foxes, and untethered baits had been cached more often than tethered baits. Baits cached by foxes for up to 1 week were not removed by species other than foxes. Domestic dogs and native birds were common nontarget species to remove baits. Dogs showed no aversion to removing any bait type, nor did bait presentation method influence bait removal. Birds removed fewer baits that had been tethered and covered. We provide an evidence base to demonstrate that bait uptake by nontarget species can be minimized, although we suggest that a fox control program is likely to be more of an organizational challenge to change public attitudes toward responsible dog ownership rather than a technical challenge to poison foxes.     

The biological and chemical contents of atmospheric particulate matter and implication of its role in the transmission of bacterial pathogenesis

Atmospheric particulate matter (APM) is an environmental hazard that endangers human health and causes a variety of diseases. In this work, the microbial community composition, chemical element composition and antimicrobial resistance gene (ARG) prevalence, along with their relationships with environmental parameters were analysed using APM samples collected in Jinan, China. Pathogenic Klebsiella and Aeromonas were found to be significantly correlated with PM_2.5 and temperature, suggesting their proliferation on APM. PM_2.5 and PM₁₀ have similar microbial community compositions but different chemical element compositions, suggesting they have different origins, which have little impact on microbial community structures. This finding, together with analysis of the timing of microbial community structure changes, suggests that microbial community composition is impacted by anthropic activities. Further investigations showed that rare metals including lanthanides are significantly negatively correlated with pathogens in APM, suggesting their inhibitory role. ARGs were observed for every class of antibiotic except for carbapenems in APM, suggesting high ARG prevalence in APM, and APM functions in transmission of antimicrobial resistance. Results obtained in this study suggest that APM can act as a transmission vehicle for pathogenic bacteria and ARGs and lead to the implication of a new transmission route for bacterial pathogenesis by APM.