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.

Simplification of soil biota communities impairs nutrient recycling and enhances above- and belowground nitrogen losses.

Agriculture is a major source of nutrient pollution, posing a threat to the earth system functioning. Factors determining the nutrient use efficiency of plant-soil systems need to be identified to develop strategies to reduce nutrient losses while ensuring crop productivity. The potential of soil biota to tighten nutrient cycles by improving plant nutrition and reducing soil nutrient losses is still poorly understood. We manipulated soil biota communities in outdoor lysimeters, planted maize, continuously collected leachates, and measured N2 O- and N2 -gas emissions after a fertilization pulse to test whether differences in soil biota communities affected nutrient recycling and N losses. Lysimeters with strongly simplified soil biota communities showed reduced crop N (-20%) and P (-58%) uptake, strongly increased N leaching losses (+65%), and gaseous emissions (+97%) of N2 O and N2 . Soil metagenomic analyses revealed differences in the abundance of genes responsible for nutrient uptake, nitrate reduction, and denitrification that helped explain the observed nutrient losses. Soil biota are major drivers of nutrient cycling and reductions in the diversity or abundance of certain groups (e.g. through land-use intensification) can disrupt nutrient cycling, reduce agricultural productivity and nutrient use efficiency, and exacerbate environmental pollution and global warming.

Urbanized microbiota in infants, immune constitution, and later risk of atopic diseases.

BACKGROUND: Urbanization is linked with an increased burden of asthma and atopic traits. A putative mechanism is insufficient exposure to beneficial microbes early in life, leading to immune dysregulation, as was previously shown for indoor microbial exposures. OBJECTIVE: Our aim was to investigate whether urbanization is associated with the microbiota composition in the infants' body and early immune function, and whether these contribute to the later risk of asthma and atopic traits. METHODS: We studied the prospective Copenhagen Prospective Studies on Asthma in Childhood 20102010 mother-child cohort of 700 children growing up in areas with different degrees of urbanization. During their first year of life, airway and gut microbiotas, as well as immune marker concentrations, were defined. When the children were 6 years of age, asthma and atopic traits were diagnosed by pediatricians. RESULTS: In adjusted analyses, the risk of asthma and aeroallergen sensitization were increased in urban infants. The composition of especially airway but also gut microbiotas differed between urban and rural infants. The living environment-related structure of the airway microbiota was already associated with immune mediator concentrations at 1 month of age. An urbanized structure of the airway and gut microbiotas was associated with an increased risk of asthma coherently during multiple time points and also with the risks of eczema and sensitization. CONCLUSION: Our findings suggest that urbanization-related changes in the infant microbiota may elevate the risk of asthma and atopic traits, probably via cross talk with the developing immune system. The airways may facilitate this effect, as they are open for colonization by environmental airborne microbes and serve as an immune interface.

Microbial Key Players Involved in P Turnover Differ in Artificial Soil Mixtures Depending on Clay Mineral Composition.

Nutrient turnover in soils is strongly driven by soil properties, including clay mineral composition. One main nutrient is phosphorus (P), which is known to be easily immobilized in soil. Therefore, the specific surface characteristics of clay minerals might substantially influence P availability in soil and thus the microbial strategies for accessing P pools. We used a metagenomic approach to analyze the microbial potential to access P after 842 days of incubation in artificial soils with a clay mineral composition of either non-expandable illite (IL) or expandable montmorillonite (MT), which differ in their surface characteristics like soil surface area and surface charge. Our data indicate that microorganisms of the two soils developed different strategies to overcome P depletion, resulting in similar total P concentrations. Genes predicted to encode inorganic pyrophosphatase (ppa), exopolyphosphatase (ppx), and the pstSCAB transport system were higher in MT, suggesting effective P uptake and the use of internal poly-P stores. Genes predicted to encode enzymes involved in organic P turnover like alkaline phosphatases (phoA, phoD) and glycerophosphoryl diester phosphodiesterase were detected in both soils in comparable numbers. In addition, Po concentrations did not differ significantly. Most identified genes were assigned to microbial lineages generally abundant in agricultural fields, but some were assigned to lineages known to include oligotrophic specialists, such as Bacillaceae and Microchaetaceae.

Transitory microbial habitat in the hyperarid Atacama Desert.

Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity.

Biological Soil Crusts from Different Soil Substrates Harbor Distinct Bacterial Groups with the Potential to Produce Exopolysaccharides and Lipopolysaccharides.

Biological soil crusts (biocrusts) play an important role in improving soil stability and resistance to erosion by promoting aggregation of soil particles. During initial development, biocrusts are dominated by bacteria. Some bacterial members of the biocrusts can contribute to the formation of soil aggregates by producing exopolysaccharides and lipopolysaccharides that act as "glue" for soil particles. However, little is known about the dynamics of "soil glue" producers during the initial development of biocrusts. We hypothesized that different types of initial biocrusts harbor distinct producers of adhesive polysaccharides. To investigate this, we performed a microcosm experiment, cultivating biocrusts on two soil substrates. High-throughput shotgun sequencing was used to obtain metagenomic information on microbiomes of bulk soils from the beginning of the experiment, and biocrusts sampled after 4 and 10 months of incubation. We discovered that the relative abundance of genes involved in the biosynthesis of exopolysaccharides and lipopolysaccharides increased in biocrusts compared with bulk soils. At the same time, communities of potential "soil glue" producers that were highly similar in bulk soils underwent differentiation once biocrusts started to develop. In the bulk soils, the investigated genes were harbored mainly by Betaproteobacteria, whereas in the biocrusts, the major potential producers of adhesive polysaccharides were, aside from Alphaproteobacteria, either Cyanobacteria or Chloroflexi and Acidobacteria. Overall, our results indicate that the potential to form exopolysaccharides and lipopolysaccharides is an important bacterial trait for initial biocrusts and is maintained despite the shifts in bacterial community composition during biocrust development.

Effect of the Nursing Mother on the Gut Microbiome of the Offspring During Early Mouse Development.

The development of the gut microbiome is influenced by several factors. It is acquired during and after birth and involves both maternal and environmental factors as well as the genetic disposition of the offspring. However, it is unclear if the microbiome development is directly triggered by the mode of delivery and very early contact with the mother or mostly at later stages of initial development mainly by breast milk provided by the mother. To investigate to what extent the gut microbiome composition of the offspring is determined by the nursing mother, providing breast milk, compared to the birth mother during early development, a cross-fostering experiment involving two genetically different mouse lines was developed, being prone to be obese or lean, respectively. The microbiome of the colon was analyzed by high-throughput 16S rRNA gene sequencing, when the mice were 3 weeks old. The nursing mother affected both α- and ß-diversity of the offspring's gut microbiome and shaped its composition. Especially bacterial families directly transferred by breast milk, like Streptococcaceae, or families which are strongly influenced by the quality of the breast milk like Rikenellaceae, showed a strong response. The core microbiome transferred from the obese nursing mother showed a higher robustness in comparison to the microbiome transferred from the lean nursing mother. Overall, the nursing mother impacts the gut microbial composition of the offspring during early development and might play an important role for health and disease of the animals at later stages of life.

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection.

The Impact of the Diurnal Cycle on the Microbial Transcriptome in the Rhizosphere of Barley.

While root exudation follows diurnal rhythms, little is known about the consequences for the microbiome of the rhizosphere. In this study, we used a metatranscriptomic approach to analyze the active microbial communities, before and after sunrise, in the rhizosphere of barley. We detected increased activities of many prokaryotic microbial taxa and functions at the pre-dawn stage, compared to post-dawn. Actinomycetales, Planctomycetales, Rhizobiales, and Burkholderiales were the most abundant and therefore the most active orders in the barley rhizosphere. The latter two, as well as Xanthomonadales, Sphingomonadales, and Caulobacterales showed a significantly higher abundance in pre-dawn samples compared to post-dawn samples. These changes in taxonomy coincide with functional changes as genes involved in both carbohydrate and amino acid metabolism were more abundant in pre-dawn samples compared to post-dawn samples. This study significantly enhances our present knowledge on how rhizospheric microbiota perceives and responds to changes in the soil during dark and light periods.

Development of a Stable Lung Microbiome in Healthy Neonatal Mice.

The lower respiratory tract has been previously considered sterile in a healthy state, but advances in culture-independent techniques for microbial identification and characterization have revealed that the lung harbors a diverse microbiome. Although research on the lung microbiome is increasing and important questions were already addressed, longitudinal studies aiming to describe developmental stages of the microbial communities from the early neonatal period to adulthood are lacking. Thus, little is known about the early-life development of the lung microbiome and the impact of external factors during these stages. In this study, we applied a barcoding approach based on high-throughput sequencing of 16S ribosomal RNA gene amplicon libraries to determine age-dependent differences in the bacterial fraction of the murine lung microbiome and to assess potential influences of differing "environmental microbiomes" (simulated by the application of used litter material to the cages). We could clearly show that the diversity of the bacterial community harbored in the murine lung increases with age. Interestingly, bacteria belonging to the genera Delftia and Rhodococcus formed an age-independent core microbiome. The addition of the used litter material influenced the lung microbiota of young mice but did not significantly alter the community composition of adult animals. Our findings elucidate the dynamic nature of the early-life lung microbiota and its stabilization with age. Further, this study indicates that even slight environmental changes modulate the bacterial community composition of the lung microbiome in early life, whereas the lung microbes of adults demonstrate higher resilience towards environmental variations.

CRISPR adaptive immune systems of Archaea.

CRISPR adaptive immune systems were analyzed for all available completed genomes of archaea, which included representatives of each of the main archaeal phyla. Initially, all proteins encoded within, and proximal to, CRISPR-cas loci were clustered and analyzed using a profile-profile approach. Then cas genes were assigned to gene cassettes and to functional modules for adaptation and interference. CRISPR systems were then classified primarily on the basis of their concatenated Cas protein sequences and gene synteny of the interference modules. With few exceptions, they could be assigned to the universal Type I or Type III systems. For Type I, subtypes I-A, I-B, and I-D dominate but the data support the division of subtype I-B into two subtypes, designated I-B and I-G. About 70% of the Type III systems fall into the universal subtypes III-A and III-B but the remainder, some of which are phyla-specific, diverge significantly in Cas protein sequences, and/or gene synteny, and they are classified separately. Furthermore, a few CRISPR systems that could not be assigned to Type I or Type III are categorized as variant systems. Criteria are presented for assigning newly sequenced archaeal CRISPR systems to the different subtypes. Several accessory proteins were identified that show a specific gene linkage, especially to Type III interference modules, and these may be cofunctional with the CRISPR systems. Evidence is presented for extensive exchange having occurred between adaptation and interference modules of different archaeal CRISPR systems, indicating the wide compatibility of the functionally diverse interference complexes with the relatively conserved adaptation modules.

Bacillus subtilis-597 induces changes in lung pathology and inflammation during influenza A virus infection in pigs.

In recent years, it has become apparent that imbalances in the gastrointestinal system can impact organs beyond the intestine such as the lungs. Given the established ability of probiotics to modulate the immune system by interacting with gastrointestinal cells, our research aimed to investigate whether administering the probiotic strain Bacillus subtilis-597 could mitigate the outcome of influenza virus infection in pigs. Pigs were fed a diet either with or without the probiotic strain B. subtilis-597 for 14 days before being intranasally inoculated with a swine influenza A H1N2 strain (1 C.2 lineage). Throughout the study, we collected fecal samples, blood samples, and nasal swabs to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination. Our findings indicate that the administration of B. subtilis-597 exhibit potential in reducing lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis-597 appeared to be unrelated to the gastrointestinal microbiota, as the composition remained unaltered by both the influenza infection and the administration of B. subtilis-597.

Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition.

BACKGROUND: In environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age. RESULTS: We conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs. CONCLUSIONS: We found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance. Video Abstract.

MAGinator enables accurate profiling of de novo MAGs with strain-level phylogenies.

Metagenomic sequencing has provided great advantages in the characterisation of microbiomes, but currently available analysis tools lack the ability to combine subspecies-level taxonomic resolution and accurate abundance estimation with functional profiling of assembled genomes. To define the microbiome and its associations with human health, improved tools are needed to enable comprehensive understanding of the microbial composition and elucidation of the phylogenetic and functional relationships between the microbes. Here, we present MAGinator, a freely available tool, tailored for profiling of shotgun metagenomics datasets. MAGinator provides de novo identification of subspecies-level microbes and accurate abundance estimates of metagenome-assembled genomes (MAGs). MAGinator utilises the information from both gene- and contig-based methods yielding insight into both taxonomic profiles and the origin of genes and genetic content, used for inference of functional content of each sample by host organism. Additionally, MAGinator facilitates the reconstruction of phylogenetic relationships between the MAGs, providing a framework to identify clade-level differences.

Identification of representative species-specific genes for abundance measurements.

Motivation: Metagenomic binning facilitates the reconstruction of genomes and identification of Metagenomic Species Pan-genomes or Metagenomic Assembled Genomes. We propose a method for identifying a set of de novo representative genes, termed signature genes, which can be used to measure the relative abundance and used as markers of each metagenomic species with high accuracy. Results: An initial set of the 100 genes that correlate with the median gene abundance profile of the entity is selected. A variant of the coupon collector's problem was utilized to evaluate the probability of identifying a certain number of unique genes in a sample. This allows us to reject the abundance measurements of strains exhibiting a significantly skewed gene representation. A rank-based negative binomial model is employed to assess the performance of different gene sets across a large set of samples, facilitating identification of an optimal signature gene set for the entity. When benchmarked the method on a synthetic gene catalog, our optimized signature gene sets estimate relative abundance significantly closer to the true relative abundance compared to the starting gene sets extracted from the metagenomic species. The method was able to replicate results from a study with real data and identify around three times as many metagenomic entities. Availability and implementation: The code used for the analysis is available on GitHub: https://github.com/trinezac/SG_optimization. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Biological soil crusts on agricultural soils of mesic regions promote microbial cross-kingdom co-occurrences and nutrient retention.

Introduction: Biological soil crusts (biocrusts) are known as biological hotspots on undisturbed, nutrient-poor bare soil surfaces and until now, are mostly observed in (semi-) arid regions but are currently poorly understood in agricultural systems. This is a crucial knowledge gap because managed sites of mesic regions can quickly cover large areas. Thus, we addressed the questions (i) if biocrusts from agricultural sites of mesic regions also increase nutrients and microbial biomass as their (semi-) arid counterparts, and (ii) how microbial community assemblage in those biocrusts is influenced by disturbances like different fertilization and tillage regimes. Methods: We compared phototrophic biomass, nutrient concentrations as well as the abundance, diversity and co-occurrence of Archaea, Bacteria, and Fungi in biocrusts and bare soils at a site with low agricultural soil quality. Results and Discussion: Biocrusts built up significant quantities of phototrophic and microbial biomass and stored more nutrients compared to bare soils independent of the fertilizer applied and the tillage management. Surprisingly, particularly low abundant Actinobacteria were highly connected in the networks of biocrusts. In contrast, Cyanobacteria were rarely connected, which indicates reduced importance within the microbial community of the biocrusts. However, in bare soil networks, Cyanobacteria were the most connected bacterial group and, hence, might play a role in early biocrust formation due to their ability to, e.g., fix nitrogen and thus induce hotspot-like properties. The microbial community composition differed and network complexity was reduced by conventional tillage. Mineral and organic fertilizers led to networks that are more complex with a higher percentage of positive correlations favoring microbe-microbe interactions. Our study demonstrates that biocrusts represent a microbial hotspot on soil surfaces under agricultural use, which may have important implications for sustainable management of such soils in the future.

Differential responses of the gut microbiome and resistome to antibiotic exposures in infants and adults.

Despite their crucial importance for human health, there is still relatively limited knowledge on how the gut resistome changes or responds to antibiotic treatment across ages, especially in the latter case. Here, we use fecal metagenomic data from 662 Danish infants and 217 young adults to fill this gap. The gut resistomes are characterized by a bimodal distribution driven by E. coli composition. The typical profile of the gut resistome differs significantly between adults and infants, with the latter distinguished by higher gene and plasmid abundances. However, the predominant antibiotic resistance genes (ARGs) are the same. Antibiotic treatment reduces bacterial diversity and increased ARG and plasmid abundances in both cohorts, especially core ARGs. The effects of antibiotic treatments on the gut microbiome last longer in adults than in infants, and different antibiotics are associated with distinct impacts. Overall, this study broadens our current understanding of gut resistome dynamics and the impact of antibiotic treatment across age groups.

Expanding known viral diversity in the healthy infant gut.

The gut microbiome is shaped through infancy and impacts the maturation of the immune system, thus protecting against chronic disease later in life. Phages, or viruses that infect bacteria, modulate bacterial growth by lysis and lysogeny, with the latter being especially prominent in the infant gut. Viral metagenomes (viromes) are difficult to analyse because they span uncharted viral diversity, lacking marker genes and standardized detection methods. Here we systematically resolved the viral diversity in faecal viromes from 647 1-year-olds belonging to Copenhagen Prospective Studies on Asthma in Childhood 2010, an unselected Danish cohort of healthy mother-child pairs. By assembly and curation we uncovered 10,000 viral species from 248 virus family-level clades (VFCs). Most (232 VFCs) were previously unknown, belonging to the Caudoviricetes viral class. Hosts were determined for 79% of phage using clustered regularly interspaced short palindromic repeat spacers within bacterial metagenomes from the same children. Typical Bacteroides-infecting crAssphages were outnumbered by undescribed phage families infecting Clostridiales and Bifidobacterium. Phage lifestyles were conserved at the viral family level, with 33 virulent and 118 temperate phage families. Virulent phages were more abundant, while temperate ones were more prevalent and diverse. Together, the viral families found in this study expand existing phage taxonomy and provide a resource aiding future infant gut virome research.

Donor-dependent fecal microbiota transplantation efficacy against necrotizing enterocolitis in preterm pigs.

The development of necrotizing enterocolitis (NEC), a life-threatening inflammatory bowel disease affecting preterm infants, is connected with gut microbiota dysbiosis. Using preterm piglets as a model for preterm infants we recently showed that fecal microbiota transplantation (FMT) from healthy suckling piglet donors to newborn preterm piglets decreased the NEC risk. However, in a follow-up study using donor stool from piglets recruited from another farm, this finding could not be replicated. This allowed us to study donor-recipient microbiota dynamics in a controlled model system with a clear difference in NEC phenotype. Preterm piglets (n = 38) were randomly allocated to receive control saline (CON), or rectal FMT using either the ineffective (FMT1) or the effective donor stool (FMT2). All animals were followed for four days before necropsy and gut pathological evaluation. Donor and recipient colonic gut microbiota (GM) were analyzed by 16 S rRNA gene amplicon sequencing and shotgun metagenomics. As expected, only FMT2 recipients were protected against NEC. Both FMT groups had shifted GM composition relative to CON, but FMT2 recipients had a higher lactobacilli relative abundance compared to FMT1. Limosilactobacillus reuteri and Lactobacillus crispatus strains of FMT recipients showed high phylogenetic similarity with their respective donors, indicating engraftment. Moreover, the FMT2 group had a higher lactobacilli replication rate and harbored specific glycosaminoglycan-degrading Bacteroides. In conclusion, subtle species-level donor differences translate to major changes in engraftment dynamics and the ability to prevent NEC. This could have implications for proper donor selection in future FMT trials for NEC prevention.

Metagenomic evidence for co-occurrence of antibiotic, biocide and metal resistance genes in pigs.

Antibiotic-resistant pathogens constitute an escalating public health concern. Hence a better understanding of the underlying processes responsible for this expansion is urgently needed. Co-selection of heavy metal/biocide and antibiotic resistance genes (ARGs) has been suggested as one potential mechanism promoting the proliferation of antimicrobial resistance (AMR). This paper aims to elucidate this interplay and exploit differences in antibiotic usage to infer patterns of co-selection by the non-antibiotic factors metals and biocides in the context of pig farming. We examined 278 gut metagenomes from pigs with continuous antibiotic exposure, only at weaning and at no exposure. Metals as growth promoters and biocides as disinfectants are currently used with little restrictions in stock farming. The pigs under continuous antibiotic exposure displayed the highest co-occurrence of ARGs and other genetic elements while the pigs under limited use of antibiotics still showed abundant co-occurrences. Pathogens belonging to Enterobacteriaceae displayed increased co-occurrence phenomena, suggesting that this maintenance is not a random selection process from a mobilized pool but pertains to specific phylogenetic clades. These results suggest that metals and biocides displayed strong selective pressures on ARGs exerted by intensive farming, regardless of the current use of antibiotics.