Antibiotic resistance, including multiresistance acquisition and dissemination by pathogens, is a critical healthcare issue threatening our management of infectious diseases [1-3]. Rapid accumulation of resistance phenotypes implies a reservoir of transferable antibiotic resistance gene determinants (ARGDs) selected in response to inhibition of antibiotic concentrations, as found in hospitals [1, 3-5]. Antibiotic resistance genes were found in environmental isolates, soil DNA [4-6], secluded caves [6, 7], and permafrost DNA [7, 8]. Antibiotics target essential and ubiquitous cell functions, and resistance is a common characteristic of environmental bacteria [8-11]. Environmental ARGDs are an abundant reservoir of potentially transferable resistance for pathogens [9-12]. Using metagenomic sequences, we show that ARGDs can be detected in all (n=71) environments analyzed. Soil metagenomes had the most diverse pool of ARGDs. The most common types of resistances found in environmental metagenomes were efflux pumps and genes conferring resistance to vancomycin, tetracycline, or β-lactam antibiotics used in veterinary and human healthcare. Our study describes the diverse and abundant antibiotic resistance genes in nonclinical environments and shows that these genes are not randomly distributed among different environments (e.g., soil, oceans or human feces).

SummarySoil is a large reservoir of microbial diversity and the majority of antimicrobial compounds used today in human and veterinary health care have been isolated from soil microorganisms. The Darwinian hypothesis of an 'arms-shields race' between antibiotic producers and resistant strains is often cited to explain antibiotic resistance gene determinants (ARGD) origins and diversity. ARGD abundance and antibiotic molecule exposure are, however, not systematically linked, and many other factors can contribute to resistance gene emergence, selection and dissemination in the environment. Soil is a heterogeneous habitat and represents a broad spectrum of different ecological niches. Soil harbours a large genetic diversity at small spatial scale, favouring exchange of genetic materials by means of horizontal gene transfer (HGT) that will contribute to ARGD dissemination between bacteria and eventually acquisition by pathogen genomes, therefore threatening antibiotic therapies. Our current knowledge on the extent of the soil resistome abundance and diversity has been greatly enhanced since the metagenomic revolution and help of high-throughput sequencing technologies. Different ecological hypotheses explaining their high prevalence in soil and questioning their transfer rate to pathogens, in respect to these recent experimental results, will be discussed in the present review.

25Wastewater treatment plants (WWTPs) have long been suggested as reservoirs and sources of 26 antibiotic resistance genes (ARGs) in the environment. In a WWTP ecosystem, human enteric and 27 environmental bacteria are mixed and exposed to pharmaceutical residues, potentially favoring 28 genetic exchange and thus ARG transmission. However, the contribution of microbial communities 29 in WWTP to ARG dissemination remains poorly understood. Here, we examined for the first time 30 plasmid permissiveness of an activated sludge microbial community, by utilizing an established 31 fluorescent bioreporter system. The activated sludge microbial community was challenged in 32 standardized filter matings with one of the three multi-drug resistance plasmids (pKJK5, pB10 and 33 RP4) harbored by Escherichia coli or Pseudomonas putida. Different donor-plasmid combinations 34 had distinct transfer frequencies, ranging from 3 to 50 conjugation events per 100,000 cells of the 35 WWTP microbial community. In addition, transfer was observed to a broad phylogenetic range of 36 13 bacterial phyla with several taxa containing potentially pathogenic species. Preferential transfer 37 to taxa belonging to the predicted evolutionary host range of the plasmids was not observed. 38Overall, the ARG dissemination potential uncovered in WWTP communities calls for a thorough 39 risk assessment of ARG transmission across the wastewater system, before identifying possible 40 mitigation strategies. 41 42 43 44 45 46 47 48 3 Introduction 49

The current epidemic of antibiotic resistance has been facilitated by the wide and rapid horizontal dissemination of antibiotic resistance genes (ARGs) in microbial communities. Indeed, ARGs are often located on plasmids, which can efficiently shuttle genes across diverse taxa. While the existence conditions of plasmids have been extensively studied in a few model bacterial populations, their fate in complex bacterial communities is poorly understood. Here, we coupled plasmid transfer assays with serial growth experiments to investigate the persistence of the broad-host-range IncP-1 plasmid pKJK5 in microbial communities derived from a sewage treatment plant. The cultivation conditions combined different nutrient and oxygen levels, and were non-selective and non-conducive for liquid-phase conjugal transfer. Following initial transfer, the plasmid persisted in almost all conditions during a 10-day serial growth experiment (equivalent to 60 generations), with a transient transconjugant incidence up to 30%. By combining cell enumeration and sorting with amplicon sequencing, we mapped plasmid fitness effects across taxa of the microbial community. Unexpected plasmid fitness benefits were observed in multiple phylotypes of Aeromonas, Enterobacteriaceae, and Pseudomonas, which resulted in community-level plasmid persistence. We demonstrate, for the first time, that plasmid fitness effects across community members can be estimated in high-throughput without prior isolation. By gaining a fitness benefit when carrying plasmids, members within complex microbial communities might have a hitherto unrecognised potential to maintain plasmids for long-term community-wide access.

Background Aquaculture is on the rise worldwide, and the use of antibiotics is fostering higher production intensity. However, recent findings suggest that the use of antibiotics comes at the price of increased antibiotic resistance. Yet, the effect of the oral administration of antibiotics on the mobility of microbial resistance genes in the fish gut is not well understood. In the present study, Piaractus mesopotamicus was used as a model to evaluate the effect of the antimicrobial florfenicol on the diversity of the gut microbiome as well as antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) using a metagenomic approach. Results The total relative abundance of ARGs and MGEs significantly increased during the antibiotic exposure. Additionally, phage integrases, transposases, and transposons flanking ARGs accumulated in the gut microbiome of P. mesopotamicus because of the antibiotic exposure. MGEs co-occurring with ARGs showed a significant positive correlation with the total ARGs found. Furthermore, shifts in the gut microbiome towards well-known putative pathogens such as Salmonella , Plesiomonas , and Citrobacter were observed following florfenicol treatment. Mainly Plesiomonas and Citrobacter harbored genes that code for multidrug and phenicol efflux pumps. Moreover, several genes related to RNA processing and modification, cell motility, SOS response, and extracellular structure were enriched due to the antibiotic application. The observed effects were visible during the complete application phase and disappeared at the post-exposure phase. Conclusions Our findings suggest that the oral administration of antibiotics increases the potential for MGE-mediated exchange of ARGs in the gut of fish and could contribute to the enrichment and dispersion of ARGs in aquaculture systems. Importantly, this increase in the potential for ARGs exchange could be an effect of changes in community structure and/or ARG mobilization. Electronic supplementary material The online version of this article (10.1186/s40168-019-0632-7) contains supplementary material, which is available to authorized users.

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Climate change alters frequencies and intensities of soil drying-rewetting and freezing-thawing cycles. These fluctuations affect soil water availability, a crucial driver of soil microbial activity. While these fluctuations are leaving imprints on soil microbiome structures, the question remains if the legacy of one type of weather fluctuation (e.g., drying-rewetting) affects the community response to the other (e.g., freezing-thawing). As both phenomenons give similar water availability fluctuations, we hypothesized that freezing-thawing and drying-rewetting cycles have similar effects on the soil microbiome. We tested this hypothesis by establishing targeted microcosm experiments. We created a legacy by exposing soil samples to a freezing-thawing or drying-rewetting cycle (phase 1), followed by an additional drying-rewetting or freezing-thawing cycle (phase 2). We measured soil respiration and analyzed soil microbiome structures. Across experiments, larger CO2 pulses and changes in microbiome structures were observed after rewetting than thawing. Drying-rewetting legacy affected the microbiome and CO2 emissions upon the following freezing-thawing cycle. Conversely, freezing-thawing legacy did not affect the microbial response to the drying-rewetting cycle. Our results suggest that drying-rewetting cycles have stronger effects on soil microbial communities and CO2 production than freezing-thawing cycles and that this pattern is mediated by sustained changes in soil microbiome structures.