Highlights d Cities possess a consistent ''core'' set of non-human microbes d Urban microbiomes echo important features of cities and city-life d Antimicrobial resistance genes are widespread in cities d Cities contain many novel bacterial and viral species

Background Microbial communities present in environmental waters constitute a reservoir for antibiotic-resistant pathogens that impact human health. For this reason, a diverse variety of water environments are being analyzed using metagenomics to uncover public health threats. However, the composition of these communities along the coastal environment of a whole city, where sewage and beach waters are mixed, is poorly understood. Results We shotgun-sequenced 20 coastal areas from the city of Montevideo (capital of Uruguay) including beach and sewage water samples to characterize bacterial communities and their virulence and antibiotic resistance repertories. As expected, we found that sewage and beach environments present significantly different bacterial communities. This baseline allowed us to detect a higher prevalence and a more diverse repertory of virulence and antibiotic-resistant genes in sewage samples. Many of these genes come from well-known enterobacteria and represent carbapenemases and extended-spectrum betalactamases reported in hospital infections in Montevideo. Additionally, we were able to genotype the presence of both globally disseminated pathogenic clones and emerging antibiotic-resistant bacteria in sewage waters. Conclusions Our study represents the first in using metagenomics to jointly analyze beaches and the sewage system from an entire city, allowing us to characterize antibiotic-resistant pathogens circulating in urban waters. The data generated in this initial study represent a baseline metagenomic exploration to guide future longitudinal (time-wise) studies, whose systematic implementation will provide useful epidemiological information to improve public health surveillance. Electronic supplementary material The online version of this article (10.1186/s40168-019-0648-z) contains supplementary material, which is available to authorized users.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high‐throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non‐Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside‐modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D β‐lactamase (blaOXA‐205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase ( aadA6 ), and the other is a qacG2 gene.

Plasmids are mobile genetic elements important for bacterial adaptation. The study of plasmids from sequencing data is challenging because short reads produce fragmented assemblies, requiring of subsequent discrimination between chromosome and plasmid sequences. Although circularized assemblies are now possible using long-read data, there is still a need to differentiate plasmids from other circular elements. Here, we present plaSquid, a dockerized tool developed in Nextflow that expands plasmid detection and improves replicon typing and mobility groups classification schemes, outperforming previously available methods in both precision and sensitivity. When applied to ~10.5 million metagenomic contigs, plaSquid revealed a 2.7-fold increase in plasmid phylogenetic diversity. Also, we used plaSquid to uncover a significant role of plasmids in the widespread distribution of clinically-relevant antimicrobial resistance genes in the built environment, from cities to spacecraft. Together, we present an improved approach to study plasmid biology from fragmented or circularized genomic and metagenomic assemblies.

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Analysis of environmental samples for bacterial antibiotic resistance genes may have different objectives and analysis strategies. In some cases, the purpose was to study diversity and evolution of genes that could be grouped within a mechanism of antibiotic resistance. Different protocols have been designed for detection and confirmation that a functional gene was found. In this study, we present a sequence-based screening of candidate genes encoding beta-lactamases in 14 metagenomes of Antarctic microbial mats. The samples were obtained from different sites, representing diverse biogeographic regions of maritime and continental Antarctica. A protocol was designed based on generation of Hidden Markov Models from the four beta-lactamase classes by Ambler classification, using sequences from the Comprehensive Antibiotic Resistance Database (CARD). The models were used as queries for metagenome analysis and recovered contigs were subsequently annotated using RAST. According to our analysis, 14 metagenomes analyzed contain A, B and C beta-lactamase genes. Class D genes, however, were identified in 11 metagenomes. The most abundant was class C (46.8%), followed by classes B (35.5%), A (14.2%) and D (3.5%). A considerable number of sequences formed clusters which included, in some cases, contigs from different metagenomes. These assemblies are clearly separated from reference clusters, previously identified using CARD beta-lactamase sequences. While bacterial antibiotic resistance is a major challenge of public health worldwide, our results suggest that environmental diversity of beta-lactamase genes is higher than that currently reported, although this should be complemented with gene function analysis.

41Background: Microbial communities present in environmental waters constitute a reservoir for antibiotic-42 resistant pathogens that impact human health. For this reason a diverse variety of water environments are 43 being analyzed using metagenomics to uncover public health threats. However, the composition of these 44 communities along the coastal environment of a whole city where sewage and beach waters are mixed, is 45 poorly understood. 47Results: We shotgun-sequenced 20 coastal areas from the city of Montevideo (capital of Uruguay) including 48 beach and sewage water samples to characterize bacterial communities and their virulence and antibiotic 49 resistance repertories. We found that sewage and beach environments presented significantly different 50 bacterial communities. Sewage waters harbored a higher prevalence and a more diverse repertory of virulence 51 and antibiotic resistant genes mainly from well-known enterobacteria, including carbapenemases and 52 extended-spectrum betalactamases reported in hospital infections in Montevideo. Additionally, we were able 53 to genotype the presence of both globally-disseminated pathogenic clones as well as emerging antibiotic-54 resistant bacteria in sewage waters. 56Conclusions: Our study represents the first in using metagenomics to jointly analyze beaches and the sewage 57 system from an entire city, allowing us to characterize antibiotic-resistant pathogens circulating in urban 58 waters. The data generated in this initial study represent a baseline metagenomic exploration to guide future 59 longitudinal (time-wise) studies, whose systematic implementation will provide useful epidemiological 60 information to improve public health surveillance. 61 62

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