CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids.

Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes.

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions.

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

Fungi and bacteria in the beds of rural and urban infants correlate with later risk of atopic diseases.

INTRODUCTION: Rural children have a lower risk of asthma and atopic diseases than urban children. However, whether indoor microbiota in non-farming rural homes provides protection is unclear. METHODS: Here, we examine if microbes in the beds of rural and urban infants are associated with later development of atopic diseases. We studied fungi and bacteria in the beds of 6-month-old infants (n = 514) in association with the risk of asthma, allergic rhinitis, eczema and aeroallergen sensitization at 6 years of age in the prospective COPSAC2010 cohort. RESULTS: Both fungal and bacterial diversity were lower in the beds of children, who later developed allergic rhinitis (-0.22 [-0.43,-0.01], padj = .04 and -.24 [-0.42,-0.05], padj = .01 respectively) and lower bacterial richness was discovered in beds of children later developing asthma (-41.34 [-76.95,-5.73], padj = .02) or allergic rhinitis (-45.65 [-81.19,-10.10], padj = .01). Interestingly, higher fungal diversity and richness were discovered in the beds of children developing eczema (0.23 [0.02,0.43], padj = .03 and 29.21 [1.59,56.83], padj = .04 respectively). We defined a limited set of fungal and bacterial genera that predicted rural/urban environment. Some rural-associated bacterial genera such as Romboutsia and Bacillus and fungal genera Spegazzinia and Physcia were also associated with reduced risk of diseases, including eczema. These fungal and bacterial fingerprints predicting the living environment were associated with asthma and allergic rhinitis, but not eczema, with rural compositions being protective. The bed dust bacteria mediated 27% of the protective association of a rural living environment for allergic rhinitis (p = .04). CONCLUSIONS: Bed dust microbes can be differentially associated with airway- and skin-related diseases. The differing bed dust microbiota between rural and urban infants may influence their later risk of asthma and allergic rhinitis.

Streamlining CRISPR spacer-based bacterial host predictions to decipher the viral dark matter.

Thousands of new phages have recently been discovered thanks to viral metagenomics. These phages are extremely diverse and their genome sequences often do not resemble any known phages. To appreciate their ecological impact, it is important to determine their bacterial hosts. CRISPR spacers can be used to predict hosts of unknown phages, as spacers represent biological records of past phage-bacteria interactions. However, no guidelines have been established to standardize host prediction based on CRISPR spacers. Additionally, there are no tools that use spacers to perform host predictions on large viral datasets. Here, we developed a set of tools that includes all the necessary steps for predicting the hosts of uncharacterized phages. We created a database of >11 million spacers and a program to execute host predictions on large viral datasets. Our host prediction approach uses biological criteria inspired by how CRISPR-Cas naturally work as adaptive immune systems, which make the results easy to interpret. We evaluated the performance using 9484 phages with known hosts and obtained a recall of 49% and a precision of 69%. We also found that this host prediction method yielded higher performance for phages that infect gut-associated bacteria, suggesting it is well suited for gut-virome characterization.

CRISPRloci: comprehensive and accurate annotation of CRISPR-Cas systems.

CRISPR-Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR-Cas system characterization that offers annotation quality previously available only after manual inspection.

Casboundary: automated definition of integral Cas cassettes.

MOTIVATION: CRISPR-Cas are important systems found in most archaeal and many bacterial genomes, providing adaptive immunity against mobile genetic elements in prokaryotes. The CRISPR-Cas systems are encoded by a set of consecutive cas genes, here termed cassette. The identification of cassette boundaries is key for finding cassettes in CRISPR research field. This is often carried out by using Hidden Markov Models and manual annotation. In this article, we propose the first method able to automatically define the cassette boundaries. In addition, we present a Cas-type predictive model used by the method to assign each gene located in the region defined by a cassette's boundaries a Cas label from a set of pre-defined Cas types. Furthermore, the proposed method can detect potentially new cas genes and decompose a cassette into its modules. RESULTS: We evaluate the predictive performance of our proposed method on data collected from the two most recent CRISPR classification studies. In our experiments, we obtain an average similarity of 0.86 between the predicted and expected cassettes. Besides, we achieve F-scores above 0.9 for the classification of cas genes of known types and 0.73 for the unknown ones. Finally, we conduct two additional study cases, where we investigate the occurrence of potentially new cas genes and the occurrence of module exchange between different genomes. AVAILABILITY AND IMPLEMENTATION: https://github.com/BackofenLab/Casboundary. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Airway Microbiota Modulates Effect of Azithromycin Treatment for Episodes of Recurrent Asthma-like Symptoms in Preschool Children: A Randomized Clinical Trial.

Rationale: Childhood asthma is often preceded by recurrent episodes of asthma-like symptoms, which can be triggered by both viral and bacterial agents. Recent randomized controlled trials have shown that azithromycin treatment reduces episode duration and severity through yet undefined mechanisms. Objectives: To study the influence of the airway microbiota on the effect of azithromycin treatment during acute episodes of asthma-like symptoms. Methods: Children from the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) cohort with recurrent asthma-like symptoms aged 12-36 months were randomized during acute episodes to azithromycin or placebo as previously reported. Before randomization, hypopharyngeal aspirates were collected and examined by 16S ribosomal RNA gene amplicon sequencing. Measurements and Main Results: In 139 airway samples from 68 children, episode duration after randomization was associated with microbiota richness (7.5% increased duration per 10 additional operational taxonomic units [OTUs]; 95% confidence interval, 1-14%; P = 0.025), with 15 individual OTUs (including several Neisseria and Veillonella), and with microbial pneumotypes defined from weighted UniFrac distances (longest durations in a Neisseria-dominated pneumotype). Microbiota richness before treatment increased the effect of azithromycin by 10% per 10 additional OTUs, and more OTUs were positively versus negatively associated with an increased azithromycin effect (82 vs. 58; P = 0.0032). Furthermore, effect modification of azithromycin was found for five individual OTUs (three OTUs increased and two OTUs decreased the effect; q < 0.05). Conclusions: The airway microbiota in acute episodes of asthma-like symptoms is associated with episode duration and modifies the effect of azithromycin treatment of the episodes in preschool children with recurrent asthma-like symptoms. Clinical trial registered with www.clinicaltrials.gov (NCT01233297).

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids.

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

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.

FANCD2 binding identifies conserved fragile sites at large transcribed genes in avian cells.

Common Chromosomal Fragile Sites (CFSs) are specific genomic regions prone to form breaks on metaphase chromosomes in response to replication stress. Moreover, CFSs are mutational hotspots in cancer genomes, showing that the mutational mechanisms that operate at CFSs are highly active in cancer cells. Orthologs of human CFSs are found in a number of other mammals, but the extent of CFS conservation beyond the mammalian lineage is unclear. Characterization of CFSs from distantly related organisms can provide new insight into the biology underlying CFSs. Here, we have mapped CFSs in an avian cell line. We find that, overall the most significant CFSs coincide with extremely large conserved genes, from which very long transcripts are produced. However, no significant correlation between any sequence characteristics and CFSs is found. Moreover, we identified putative early replicating fragile sites (ERFSs), which is a distinct class of fragile sites and we developed a fluctuation analysis revealing high mutation rates at the CFS gene PARK2, with deletions as the most prevalent mutation. Finally, we show that avian homologs of the human CFS genes despite their fragility have resisted the general intron size reduction observed in birds suggesting that CFSs have a conserved biological function.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding.

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo.

Characterizing leader sequences of CRISPR loci.

MOTIVATION: The CRISPR-Cas system is an adaptive immune system in many archaea and bacteria, which provides resistance against invading genetic elements. The first phase of CRISPR-Cas immunity is called adaptation, in which small DNA fragments are excised from genetic elements and are inserted into a CRISPR array generally adjacent to its so called leader sequence at one end of the array. It has been shown that transcription initiation and adaptation signals of the CRISPR array are located within the leader. However, apart from promoters, there is very little knowledge of sequence or structural motifs or their possible functions. Leader properties have mainly been characterized through transcriptional initiation data from single organisms but large-scale characterization of leaders has remained challenging due to their low level of sequence conservation. RESULTS: We developed a method to successfully detect leader sequences by focusing on the consensus repeat of the adjacent CRISPR array and weak upstream conservation signals. We applied our tool to the analysis of a comprehensive genomic database and identified several characteristic properties of leader sequences specific to archaea and bacteria, ranging from distinctive sizes to preferential indel localization. CRISPRleader provides a full annotation of the CRISPR array, its strand orientation as well as conserved core leader boundaries that can be uploaded to any genome browser. In addition, it outputs reader-friendly HTML pages for conserved leader clusters from our database. AVAILABILITY AND IMPLEMENTATION: CRISPRleader and multiple sequence alignments for all 195 leader clusters are available at http://www.bioinf.uni-freiburg.de/Software/CRISPRleader/ CONTACT: costa@informatik.uni-freiburg.de or backofen@informatik.uni-freiburg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci.

MOTIVATION: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection of leader regions, the identification of target sites (protospacers) on invading genetic elements and the characterization of protospacer-adjacent motifs. RESULTS: We present a fast and accurate tool to determine the crRNA-encoding strand at CRISPR loci by predicting the correct orientation of repeats based on an advanced machine learning approach. Both the repeat sequence and mutation information were encoded and processed by an efficient graph kernel to learn higher-order correlations. The model was trained and tested on curated data comprising >4500 CRISPRs and yielded a remarkable performance of 0.95 AUC ROC (area under the curve of the receiver operator characteristic). In addition, we show that accurate orientation information greatly improved detection of conserved repeat sequence families and structure motifs. We integrated CRISPRstrand predictions into our CRISPRmap web server of CRISPR conservation and updated the latter to version 2.0. AVAILABILITY: CRISPRmap and CRISPRstrand are available at http://rna.informatik.uni-freiburg.de/CRISPRmap. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus.

Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB. Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference-mediated by one type IIIB module Cmr-α, and a Csx1 protein, efficiently eliminated plasmid vectors carrying matching protospacers but lacking PAM motifs. Moreover, Cmr-α-mediated interference was dependent on directional transcription of the protospacer, in contrast to the transcription-independent activities of the type IA and type IIIA DNA interference. We infer that the interference mechanism involves transcription-dependent DNA targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S. islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.

A novel single-tailed fusiform Sulfolobus virus STSV2 infecting model Sulfolobus species.

A newly isolated single-tailed fusiform virus, Sulfolobus tengchongensis spindle-shaped virus STSV2, from Hamazui, China, is characterised. It contains a double-stranded modified DNA genome of 76,107 bp and is enveloped by a lipid membrane structure. Virions exhibit a single coat protein that forms oligomers when isolated. STSV2 is related to the single-tailed fusiform virus STSV1 and, more distantly, to the two-tailed bicaudavirus ATV. The virus can be stably cultured over long periods in laboratory strains of Sulfolobus and no evidence was found for cell lysis under different stress conditions. Therefore, it constitutes an excellent model virus for archaeal virus-host studies.

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.

Escherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.

CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results favor the concept that E. coli's CRISPR-Cas is an antiprophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.

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.

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.